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An evaluation of cystic fibrosis screening programmes for implementation in British Columbia Scriabin, Jannie Martine 1982

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EVALUATION OF CYSTIC FIBROSIS SCREENING PROGRAMMES FOR IMPLEMENTATION IN BRITISH COLUMBIA by JANNIE MARTINE SCRIABIN B.Sc, The University of British Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology, Faculty of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1982 (c) Jannie Martine Scriabin, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) ABSTRACT Four methods were investigated to determine t h e i r s u i t a b i l i t y for use i n a CF screening programme f or the province of B r i t i s h Columbia. A f e c a l t r y p s i n method which measured t r y p s i n a c t i v i t y by incubating dry s t o o l samples on f i l t e r paper cards with the substrate p - t o s y l - a r g i n i n e methyl ester (TAME) and a pH s e n s i t i v e dye was shown to be n o n - s p e c i f i c and therefore u n s a t i s f a c t o r y . An attempt to combine a f e c a l albumin screen with a more s p e c i f i c q u a n t i t a t i v e immunodiffusion technique f o r albumin and alpha-1 a n t i t r y p s i n was unsuccessful. A meconium albumin assay using the Boehringer-Mannheim Corporation (BMC) t e s t - s t r i p and a more s p e c i f i c f e c a l t r y p s i n assay which uses the substrate b e n z o y l - a r g i n i n e - p - n i t r o a n i l i d e (BAPNA) were incorporated i n t o two p i l o t p r o j e c t s at Children's H o s p i t a l i n Vancouver. The BMC t e s t - s t r i p was simple to use, r e l i a b l e and inexpensive. Of 8,891 i n f a n t s tested, 3 p o s i t i v e s were diagnosed as s u f f e r i n g from c y s t i c f i b r o s i s and 1 CF pa t i e n t tested negative. False p o s i t i v e s were obtained on 1.3% of i n f a n t s . The incidence of CF as determined by t h i s screen was 1 i n 2000. The meconium albumin screen was s a t i s f a c t o r y as a l o c a l p i l o t p r o j e c t but the disadvantages of t e s t i n g the unstable meconium specimens make the screen unsuitable f o r a province-wide a p p l i c a t i o n . The BAPNA f e c a l t r y p s i n method devised by Crossley was used to t e s t 4085 dry s t o o l specimens c o l l e c t e d i n the h o s p i t a l and at home. Out of a t o t a l number of 190 p o s i t i v e r e s u l t s , none was diagnosed as having CF, giv i n g a f a l s e . p o s i t i v e rate of 5.0% for the h o s p i t a l c o l l e c t e d specimens and 3.4% for the specimens collected at home. The false positive rate in the hospital collected specimens was due mostly to the large proportion of young infants (under 3 days). The false positive rate of the home collected specimens appeared to be due mostly to the thinner spread of stool sample on the card. Because the quantity of stool sample per test was significantly lower in the home than the hospital collected specimens a new cut-off point for the home collected specimens was considered. Its application/ however did not lower the false positive rate s u f f i c i e n t l y . As a result, the high incidence of false positives and the d i f f i c u l t i e s encountered as a result of this incidence also makes the fecal trypsin screen unsuitable for the province of B.C. Di f f i c u l t i e s encountered during the follow-up of positive results obtained in the two p i l o t projects are discussed and recommendations are made regarding the e f f i c i e n t and adequate implementation of a follow-up system. i i i ACKNOWLEDGEMENTS I would p a r t i c u l a r l y l i k e to thank Dr. Derek A. Applegarth for h i s expert guidance, for h i s patience through obstacles and delays and for h i s f a i t h i n the eventual p o s i t i v e conclusion of t h i s research. Thanks a l s o to Dr. A.G.F. Davidson, for allowing me to get involved i n t h i s i n t e r e s t i n g research, for h i s guidance and help i n e s t a b l i s h i n g the CF screen p i l o t p r o j e c t s and pursuing the follow-ups. To Dr. P.E. Reid many thanks for h i s thorough review of my t h e s i s d r a f t and for h i s con s t r u c t i v e suggestions. S p e c i a l thanks to S h i r l e y T u r t l e for the many hours of meticulous t e c h n i c a l assistance and h e l p f u l discussions i n the laboratory. Thanks f i n a l l y to my husband Michael who u n s e l f i s h l y understood my need to devote many hours to t h i s research and gave me h i s support through d i f f i c u l t times. i v CONTENTS ABSTRACT H ACKNOWLEDGEMENTS <' iv LIST OF TABLES « ix LIST OF FIGURES x INTRODUCTION 1 Cystic Fibrosis Thesis Objective The Need for a Neonatal Cystic Fibrosis Screen C r i t e r i a for a Successful Screening Programme Age to be Screened EXISTING NEONATAL SCREENING PROGRAMMES . 10 Sweat Electrolytes Tests for Detecting Pancreatic Insufficiency Meconium Albumin Meconium Albumin and Alb:Alpha-l Antitrypsin Ratio Fecal Albumin and Alb:Alpha-l Antitrypsin Ratio Fecal Trypsin SELECTION AND DEVELOPMENT OF TESTING PROCEDURES 19 BMS TEST-STRIP FOR MECONIUM ABLUMIN 21 Testing Procedure Collection of Samples BMC Methodology Stability Investigation Results Meconium Sample Collection Sensitivity Specificity Discussion Sample Collection Sensitivity Specificity Incidence of CF in the Population Screened ROBINSON AND ELLIOTT FECAL TRYPSIN SCREEN 32 Testing Procedure Collection of Samples Trypsin Methodology Investigation into Inconsistent Results and Non-Specificity v Results Sensitivity and Specificity Investigation into Possible Causes of Inconsistent Results and Non-Specificity Discussion FECAL ALBUMIN:ALPHA-1 ANTITRYPSIN RATIO 48 Testing Procedure Albumin:Alpha-l Antitrypsin Ratio and Pancreatic Insufficiency Elution of Stool Samples from F i l t e r Paper Cards Qualitative Albumin Method Results Relationship of Ratio to Pancreatic Insufficiency Elution of Stool Samples from F i l t e r Paper Cards Qualitative Albumin Method Discussion Relationship of Ratio to Pancreatic Insufficiency Elution of Stool Samples from F i l t e r Paper Cards Qualitative Albumin Method CROSSLEY FECAL TRYPSIN METHOD 61 Testing Procedure Collection of Fecal Samples Trypsin Methodology Investigation to Establish Procedure Background Interference Doubling the Substrate Concentration Validity of Visual Evaluation of Yellow Intensity Results Investigation to Establish Procedure Background Interference Doubling the Substrate Concentration Validity of Visual Evaluation of Yellow Intensity Sensitivity and Specificity Age of Infant When Hospital Specimen was Collected Hospital and Home Collected Specimens from the Same Infant Comparison of Hospital and Home Collected Specimens Discussion Investigation to Establish Procedure Background Interference Doubling the Substrate Concentration Validity of Visual Evaulation of Yellow Intensity Sensitivity and Specificity Sample Collection Hospital and Home Collected Specimens Establishment of New Cut-Off Point vi Protocol Request for Stool Samples for Chymotrypsin Quantitative Chymotrypsin Analysis Sweat Electrolytes C l i n i c a l Follow-Up Only Review of the Fi l e s at Medical Records Results Meconium Albumin CF Screen Diagnostic Follow-Up Medical Records Investigation Fecal Trypsin CF Screen Diagnostic Follow-Up Medical Records Investigation Discussion Meconium Albumin CF Screen Diagnostic Follow-Up Medical Records Investigation Fecal Trypsin Screen Diagnostic Follow-Up Medical Records Investigation D i f f i c u l t i e s Encountered with the Follow-Up Procedure SUMMARY AND CONCLUSIONS 133 ADDENDUM — AN UPDATE 140 APPENDICES 145 A. Screening Card for Collection of Stool Sample for Fecal Trypsin Screen B. Letter to Parents: Request for Collection of Infant's Stool Sample for CF Screening Programme C. Calculation of % Error Resulting from Use of Crossley's Fecal Pigment Correction Prodcedure D. 1. Letter to Physician: Request for Stool Sample for Quantitative Chymotrypsin Analysis 2. Instructions for Stool Sample Collection E. C l i n i c a l Questionnaire F. Letter to Physician: Request for C l i n i c a l Follow-up to Physician Who Felt Laboratory Follow-up was not warranted G. Chi-Square Test for Significance of Difference Between False Positive Rates for Hospital and Home Collected Specimens in Fecal Trypsin CF Screen H. Chi-Square Test for Significance of Difference Between False Positive Rates for Less than 3-Day Old Infants and At least 3-Day Old Infants I. Chi-Square Test for Significance of Changes in Results for Hospital and Home Collected Specimens on the Same Infant J. 1. Chi-Square Test for Significance of Difference Between Distributions of Net Absorbance Readings of Hospital and Home Collected Specimens i v i i 2. Test for Significance of Difference Between Means of Hospital and Home Collected Specimens Net Abosrbance Values K. 1. Comparison of Screening Results from Freezer and Room Temperature Stored Specimens 2. Comparison of Specimen Results Before and After Mailing 3. Combined Data for Room Temperature Stored Specimens L. Chi-Square Test for Significance of Differences Between Distributions of Absorbance Values at 460nm of Hospital and Home Collected Specimens M. Comparison of the Precision of the Results of Hospital and Home Collected Specimens N. Correlation Between Positive Meconium Screen Results and Presence of Necrotizing Enterocolitis 0. 1. Rank Test for Significance of the Effect of Doubling the BAPNA Substrate Concentration 2. Test for Correlation Between % Increase in Absorbance Due to Doubling Substrate Concentration and the Original Absorbance Value P. Conditions Under Which an Equal Percent Reduction in Absorbance at 410 and 460 Nanometers from Hospital to Home Collected Specimens can Occur. Q. Calculation of Confidence that Child with Positive Fecal Trypsin Screen Result has CF. BIBLIOGRAPHY 182 v i i i LIST OF TABLES TABLE PAGE I. Incidence of CF in North America and Western Europe 2 II. Meconium Albumin Screening Test for CF 26 III. Fecal Trypsin Control Specimen Results Robinson and E l l i o t t Method 40 IV. Albumin and Alpha-1 Antitrypsin Content of Feces Immunodiffusion Method 55 V. Fecal Pigment Background Interference, Crossley Method 70 VI. Tests and Their Corresponding Test Blanks in the Crossley Method 72 VII. Low Trypsin Activity Samples Tested with Crossley Method Using 0.25 and 0.50 mg L-BAPNA per Test 74 VIII. Comparison of Visual with Spectrophotometric Evaluations of 522 Tests: Crossley Method 77 IX. Fecal Trypsin Screening Test for CF: Hospital Collected Specimens 78 X. Fecal Trypsin Screening Test for CF: Home Collected Specimens 78 XI. Fecal Trypsin Screening Test for CF: Hospital and Home Collected Specimens on the Same Infant . . . . 80 XII. CF Screening P i l o t Projects 113 XIII. Summary of Autopsy Reports Meconium Screening Test Positives 114 ix LIST OF FIGURES FIGURE PAGE I. Trypsin Activity with Two Different BAPNA Substrate Concentrations 76 II. Comparison of Net Absorbance Values from F i r s t Assay of Hospital and Home Collected Specimens 81 III. Comparison of Net Absorbance Values at 460 nm from Hospital and Home Collected Specimens 84 IV. CF Screening and Follow-Up Protocol Flow Chart .105 V. Results of the Diagnostic Follow-Up on Positives from the Meconium Screen 116 VI. Results from an Investigation for Possible Causes of Increased Meconium Albumin in Non-CF Infants 119 VII. Results of the Diagnostic Follow-Up on Positives from the Fecal Trypsin Screen 120 x - 1 -INTRODUCTION C y s t i c F i b r o s i s C y s t i c F i b r o s i s i s the most common l e t h a l genetic disease i n Canada.^ x It i s an autosomal recessive t r a i t , the disease expressing i t s e l f only i n the homozygous s t a t e . The metabolic defect i s unknown. Present s t a t i s t i c s i n d i c a t e that 1 i n 20 to 1 i n 30 white Canadians 33 carry the gene for c y s t i c f i b r o s i s (CF). The incidence of CF i n newborn whites i s c a l c u l a t e d to be somewhere between 1 i n 1,600 and 1 i n 3,600.7' / 5 /' < ?° T h e incidence i s very low i n the Negro and O r i e n t a l population.* 5^' 0 3" Table I l i s t s the incidence of CF as determined by various i n v e s t i g a t o r s i n North America, Europe, A u s t r a l i a and New Zealand. The genetic disorder r e s u l t s i n a general dysfunction of a number of exocrine glands. The mucous glands produce an extremely t h i c k , viscous and s t i c k y mucous which obstructs the ducts of various organs. This a f f e c t s the pancreas, the lungs, the l i v e r and the g a s t r o - i n t e s t i n a l t r a c t . The CF disease state g e n e r a l l y a t t r a c t s the phsycians' a t t e n t i o n with f a i l u r e of the c h i l d to gain weight, steatorrhea and other symptoms of pancreatic i n s u f f i c i e n c y or chronic r e s p i r a t o r y t r a c t i n f e c t i o n . -- 2 -TABLE I. INCIDENCE OF CF IN NORTH AMERICA AND WESTERN EUROPE Screening Investigators Incidence 0 No. Centre Screened MECONIUM ALBUMIN SCREEN South Wales Uppsala, Sweden Milwaukee, USA Philadelphia, USA Western Europe, 16 countries Leeds, England Prosser et a l Hellsing anc^ Kollberg Bruns et a l Holsclaw, Keith and V5\53. Palmer •/7 European Working Group for Neonatal Screening Evans et al 3° 99 1:1,556 1:2,943 1:8,114 1:3,361 1:1,936 1:2,247 34,228 8,830 16,227* 20,171+ 199,475 15,734 FECAL TRYPSIN SCREEN New Zealand New Zealand Australia Robinson and E l l i o t t Crossley, Berryman and E l l i o t t ' h Forrest, Wilcken and Turner 3 2 1:2,198 1:2,250 1:4,000 6,595 4,500 20,000 * r a c i a l distribution: 80% white, 19% black, 1% others + r a c i a l distribution: 75% white, 19.4% black, 2.2% others, mainly Oriental, 2.4% no information recorded ° incidence of cases which tested positive in the screen and were confirmed to be CF - 3 -The sweat glands are al s o a f f e c t e d and e x h i b i t a defect i n the tubular reabsorption of e l e c t r o l y t e s r e s u l t i n g i n an increased concentration of sodium c h l o r i d e i n the sweat. The disease i s found i n a wide range of s e v e r i t y , some cases being only m i l d l y a f f e c t e d i n e i t h e r the pancreas or lungs, often not presenting i t s e l f u n t i l l a t e i n childhood. Others are severely a f f e c t e d from b i r t h although as f a r as i t i s known the lungs are s t i l l healthy at b i r t h . 9 ' V 7 > 9 E a r l y Treatment E a r l y treatment i s h e l p f u l i n a l l cases and can prevent the often ~ "~- f a t a l complications of the disease. J This has been indi c a t e d i n various ways: 1. The average l i f e span of CF vi c t i m s has increased considerably over the past 20 years, mostly due to the improvement i n treatment but al s o as a r e s u l t of the recogni t i o n of milder cases and as the r e s u l t of e a r l i e r d e t e c t i o n . 2. Studies of s i b l i n g s i n d i c a t e that e a r l y treatment leads to better prognosis. In these studies the second c h i l d to have CF i n a family i s diagnosed at an e a r l i e r stage because of the family doctor being aware of the genetic t r a i t . The younger s i b l i n g s received the same treatment but showed s i g n i f i c a n t l y better prognosis a f t e r 7 years of age than the older s i b l i n g who had been diagnosed much l a t e r i n the development of the disease. - 4 -Thesis Objective The o b j e c t i v e of t h i s t h e s i s i s to evaluate e x i s t i n g and i f necessary to develop new neonatal screening programmes for CF i n an attempt to implement one i n the province of B r i t i s h Columbia. The d e c i s i o n to conduct CF screening has been made by others and i s beyond the scope of t h i s t h e s i s . Nevertheless, the b e n e f i t s and p o t e n t i a l r i s k s of a CF screening programme are b r i e f l y reviewed i n the next s e c t i o n for the i n t e r e s t e d reader. The Need for a Neonatal C y s t i c F i b r o s i s Screen A* B e n e f i t s of Screening i) The incidence of CF i s s u f f i c i e n t l y high to j u s t i f y the a p p l i c a t i o n of a neonatal screening programme. i i ) E a r l y presymptomatic diagnosis by screening followed by appropriate treatment w i l l improve the g a s t r o i n t e s t i n a l problems and slow down the progressive bronchopulmonary disease which a f f e c t most pa t i e n t s with CF sooner or l a t e r . a) A f f e c t e d c h i l d r e n can be treated promptly with enzyme supplement and a n t i b i o t i c s . b) P r o phylactic pulmonary treatment can be i n i t i a t e d p r i o r to the development of i r r e v e r s i b l e pulmonary complications. The e a r l y lung changes i n CF often occur within the f i r s t three months of l i f e and are f e l t to be r e v e r s i b l e . No p a t h o l o g i c a l abnormality can be shown i n the lungs of a CF baby at b i r t h . - 5 -i i i ) The l i f e expectancy of the C.F. p a t i e n t i s increased i f diagnosed and treated e a r l y . 37 The study by Shwachman S3 i n d i c a t e s that over a 2 0 year period the l i f e expectancy has improved from approximately 1 to 2 0 years of age i n a group diagnosed e a r l y i n l i f e . Warwick's s t a t i s t i c a l study of data obtained from the CF Foundation 95 Registry agrees with t h i s conclusion. iv) E a r l y diagnosis i n a c h i l d can r e s u l t i n parents r e c e i v i n g genetic c o u n s e l l i n g before they consider another pregnancy. Since amniocentesis f o r i d e n t i f i c a t i o n and subsequent abortion of a f f e c t e d fetuses i s not yet a v a i l a b l e , neonatal screening i s the only source of genetic information f o r prospective parents who are not aware that they carry the CF gene. v) Cost of the screening programme i s minimal considering the p o t e n t i a l savings to the community through prevention of complications. The screening c o s t a l s o compares favourably with other screening programmes. ' Ea r l y treatment r e s u l t s i n savings since i t leads to a reduction i n i n p a t i e n t care, but against t h i s would need to be set the cost of drugs and outpatient care, which would presumably be administered for a longer time period. vi) A r e l i a b l e confirmation t e s t , the sweat e l e c t r o l y t e determination, i s a v a i l a b l e . I t does not require s o p h i s t i c a t e d equipment and f a c i l i t i e s . B. P o t e n t i a l Risks of Screening i) There i s a disagreement over how best to manage the e a r l y diagnosed babies, with some physicians advocating non-intervention u n t i l - 6 -symptoms eventually appear. The o b j e c t i o n r a i s e d to t r e a t i n g symptom free c h i l d r e n i s i n regards to the p o s s i b l e hazards of prolonged a n t i b i o t i c therapy which can lead to complications such as the c o l o n i s a t i o n of the r e s p i r a t o r y t r a c t by organisms i n s e n s i t i v e to treatment. i i ) The e f f e c t i v e n e s s of genetic c o u n s e l l i n g may not warrant screening. Experience i n New York has shown that almost h a l f of the f a m i l i e s who had produced CF a f f e c t e d c h i l d r e n went to further pregnancies a f t e r c o u n s e l l i n g despite the 1:4 r i s k of a further CF a f f e c t e d c h i l d with each conception. 7 i i i ) Concern has been expressed that harm could r e s u l t from parents' knowledge that t h e i r c h i l d i s going to s u f f e r from CF i n the time period before the c h i l d manifests the disease. ' J iv) Since CF i s not curable, the c o s t - b e n e f i t has been questioned. E a r l y diagnosis does not reduce the cost to the community as d r a s t i c a l l y as i n the case of e.g. PKU where inadequately treated or l a t e diagnosed p a t i e n t s are a 40 to 60 year burden to the community. v) Psychological s t r e s s may be caused to the parents of infants with f a l s e p o s i t i v e screening r e s u l t s due to low s p e c i f i c i t y of the t e s t i n g method. The Research Group on E t h i c a l , S o c i a l and Legal Issues on Counselling suggests that screening programmes be e s t a b l i s h e d only for those genetic disorders that have a f a i r l y high incidence and that the programme be structured on the basis of one or more c l e a r l y i d e n t i f i e d 7C goals. Three d i s t i n g u i s h a b l e categories of goals are that the programme: - 7 -1) contributes to improving the health of persons who s u f f e r from the genetic disorder, 2) allows c a r r i e r s of a given v a r i a n t gene to make informed choices regarding reproduction, * 3) move towards a l l e v i a t i n g the a n x i e t i e s of f a m i l i e s and communities faced with the prospect of serious genetic disease. It would appear that a neonatal screen for CF, a genetic disorder whose incidence i s r e l a t i v e l y high, meets at l e a s t the f i r s t two of these o b j e c t i v e s since e a r l y d e t e c t i o n w i l l improve and increase the l i f e span of the CF c h i l d as a r e s u l t of e a r l i e r treatment and genetic c o u n s e l l i n g w i l l be a v a i l a b l e f or the parents. Without screening, approximately 60% of CF p a t i e n t s i n Canada are diagnosed during the f i r s t year of l i f e and therefore receive treatment at t h i s f a i r l y e a r l y stage. 9 1 9 Unequivocal proof that e a r l i e r treatment yet (presymptomatic) would further improve t h e i r c o n d i t i o n s i g n i f i c a n t l y i s not a v a i l a b l e as yet but the evidence seems to poi n t i n that d i r e c t i o n . * The remaining CF p a t i e n t s , the 40% that are diagnosed a f t e r 1 year of l i f e , would gain the most from a neonatal screen for the presence of CF may not be suspected c l i n i c a l l y f o r some time and i r r e v e r s i b l e lung damage may occur. Respiratory and g a s t r o i n t e s t i n a l problems present themselves * Lubin and Bonner found that the heights and weights of i n f a n t s with CF diagnosed before 6 months of age f e l l i n the top 25 percent of these measures for those diagnosed a f t e r 6 months of age. While Palmer t : i" reported that e a r l y diagnosis and treatment was associated with a reduced number of h o s p i t a l admissions, enhanced growth and improved c l i n i c a l c o n d i t i o n . - 8 -e a r l y i n most of these undiagnosed p a t i e n t s . If detected by a CF screen, these p a t i e n t s could be treated accordingly and years of inappropriate treatment could be avoided. C r i t e r i a f o r a Successful Screening Programme 3 As recommended by the Committee of the National Academy of Sciences the following points were taken i n t o c o n s i d e r a t i o n i n evaluating CF screening programmes: i) the v a l i d i t y , r e l i a b i l i t y and safety of the screening t e s t , i i ) costs i i i ) acceptance of the screening t e s t by the community, inc l u d i n g both consumers and p r a c t i s i n g physicians. When a t e s t i n g procedure appeared s u i t a b l e f o r a CF screening programme, a p i l o t p r o j e c t was set up i n order to a s c e r t a i n that the programme's goals were a t t a i n a b l e and that acceptable l e v e l s of s p e c i f i c i t y and s e n s i t i v i t y could be reached. The CF p i l o t screen was operated from Children's H o s p i t a l where the CF Assessment C l i n i c was located. The c l i n i c had experience with CF and had e f f e c t i v e communication with the screening lab and the p r a c t i s i n g p h y s i c i a n who provided primary care to the p a t i e n t . Presumptive p o s i t i v e s were r e f e r r e d to t h i s c l i n i c which was capable of confirming the diagnosis, i n i t i a t i n g and monitoring the therapy and counselling the family. The screening laboratory included i n the routine of the t e s t i n g procedure f o r C.F. the following recommendations made by the Committee: i) the t e s t was performed under s t r i c t q u a l i t y c o n t r o l procedures with standards and c o n t r o l specimens inserted as unknowns i n the d a i l y runs; - 9 -i i ) a second sample was tested before considering the screening t e s t p o s i t i v e since the p o s s i b i l i t i e s of a t e s t e r r o r were great as a r e s u l t of the nature of the screening t e s t i t s e l f and the number of specimens being handled; i i i ) the r e s u l t s were reviewed p e r i o d i c a l l y to determine i f the procedure needed to be a l t e r e d or the c u t - o f f point needed to be changed; iv) the specimens were saved under con d i t i o n s that maximized s t a b i l i t y i n order to enable r e t e s t i n g of specimens should a f a l s e negative appear; v) permanent records were kept and tabulated p e r i o d i c a l l y to determine changes i n the frequency of both true and f a l s e p o s i t i v e s and the i n t e r v a l i t took for the h o s p i t a l to send specimens was checked p e r i o d i c a l l y i n order to c o r r e c t delays. Age to be Screened Diagnosis, even i n the e a r l y symptomatic phase, does not exclude the 33 p o s s i b i l i t y that the lung may be damaged i r r e v e r s i b l y . Since evidence to date i n d i c a t e s that the lung i s normal at b i r t h , the i d e a l time to detect the homozygote for the CF gene would be before lung damage has had time to s t a r t . Mass screening should therefore be performed on the newborn i n f a n t , as soon as p o s s i b l e a f t e r d e l i v e r y . - 10 -EXISTING NEONATAL SCREENING PROGRAMMES C y s t i c f i b r o s i s i s c h a r a c t e r i z e d by what i s known as the " c l i n i c a l t r i a d " of chronic pulmonary disease, pancreatic i n s u f f i c i e n c y and abnormally high concentration of e l e c t r o l y t e s i n sweat. The d e t e c t i o n of the l a s t two are p o s s i b i l i t i e s f o r a screening t e s t . I. SWEAT ELECTROLYTES The p r i n c i p a l d i a g n o s t i c t e s t f o r CF i s the sweat t e s t f o r the presence of abnormally high l e v e l s of sodium or c h l o r i d e . Between 98 and 79 99% of the CF p a t i e n t s d i s p l a y an increased l e v e l of sodium c h l o r i d e and t h i s abnormality appears to be present from b i r t h . Although there are reports of i s o l a t e d cases where an i n f a n t has the c l i n i c a l expression of the disease and a negative sweat t e s t , " the abnormal sweat e l e c t r o l y t e r e s u l t s are the most constant symptom. Attempts have therefore been made to adapt the sweat t e s t to mass CF screening. To date a l l methods have proved i n e f f e c t i v e f o r the following reasons: ' ' 1. The young i n f a n t provides serious problems with sweat c o l l e c t i o n f o r many newborns produce l i t t l e or no sweat. 2. The standard sweat sodium and/or c h l o r i d e determination i s too time consuming to apply as a rountine screening method e s p e c i a l l y since - 11 -r e l i a b l e r e s u l t s require s t i m u l a t i o n of sweat production to ensure maximal secretory r a t e s . Other methods which are more e f f i c i e n t are too imprecise and u n r e l i a b l e . 3. Since the t e s t i s not r e l i a b l e when performed on the newborn, there i s a low degree of p a t i e n t compliance because the i n f a n t must be brought to the h o s p i t a l for the t e s t at a l a t e r date. As a r e s u l t screening by measuring sweat e l e c t r o l y t e s i s l i m i t e d to h i g h - r i s k i n d i v i d u a l s such as s i b l i n g s and f i r s t cousins of CF p a t i e n t s . Several i n v e s t i g a t o r s have however attempted to overcome these problems and reports on t h e i r successes and f a i l u r e s may be found i n the l i t e r a t u r e . *>9Z.97t*o, The most common and most r e l i a b l e method used to analyse sweat i s the standard q u a n t i t a t i v e p i l o c a r p i n e iontophoresis t e s t of Gibson and Cooke.^ Other methods are c o n d u c t i v i t y measurements,* , J , o f osmolality measurements,^ i o n - s e l e c t i v e electrode,V>' J'^° or the i n d i r e c t method of analysing n a i l - c l i p p i n g s ( s a l t i n n a i l s comes from sweat glands) I I . TESTS FOR DETECTING PANCREATIC INSUFFICIENCY Several t e s t s for detecting pancreatic i n s u f f i c i e n c y are a v a i l a b l e and have been implemented i n mass CF screening programmes. A l l of them suf f e r from one main disadvantage, the f a i r l y high f a l s e negative rate as a r e s u l t of the i n c o n s i s t e n t presence of pancreatic i n s u f f i c i e n c y i n CF c h i l d r e n . - 12 -Ten to f i f t e e n percent of CF in f a n t s do not d i s p l a y pancreatic i n s u f f i c i e n c y . * 1 2 ' 2 3 ' ' 5 5 ' 8 0 As a r e s u l t , t h i s percentage range i s quoted by most i n v e s t i g a t o r s as the expected f a l s e negative rate for CF screening programmes. There are i n d i c a t i o n s l a t e l y , however, that these f i g u r e s may be a l i t t l e h i g h . / t > ' 7 5 t 8 3 I t appears that pancreatic functions t e s t s were not always performed promptly on the in f a n t s that gave a f a l s e negative. As a r e s u l t there i s confusion as to whether the pancreatic d e f i c i e n c y develops i n most cases as the c h i l d gets o l d e r ^ or whether most CF c h i l d r e n have the d e f i c i e n c y at b i r t h with a few developing normal pancreatic function l a t e r i n l i f e . ' 6 I f the former holds then the % of f a l s e negatives could be expected to be higher than 10 to 15%. I f the l a t t e r holds then the number of f a l s e negatives found in a neonatal screening programme as a r e s u l t of normal pancreatic function should be lower than the 10 to 15% frequently quoted. The two most frequently used CF screening t e s t s which detect pancreatic i n s u f f i c i e n c y are the a n a l y s i s of meconium for albumin and the a n a l y s i s of feces for t r y p s i n a c t i v i t y . Two other l e s s commonly used screens are a l s o discussed. A. Meconium Albumin Meconium i s a dark green mucilagenous material present i n the i n t e s t i n e of the f u l l term f e t u s . I t i s a mixture of the secretions of the i n t e s t i n a l glands and some amniotic f l u i d . - 13 -- An increased albumin l e v e l i s found i n the meconium from CF in f a n t s ? ' H e l l s i n g and Kollberg* 5^ who tested the meconium from 1,000 healthy newborns using a s i n g l e r a d i a l immunodiffusion technique report that 59% contained l e s s than 0.3 mg of albumin per g of d r i e d meconium 52. and 99.6% contained l e s s than 4.0 rag per g. A further study i n which Kollberg and H e l l s i n g used the same method on a screening s e r i e s of 8830 healthy i n f a n t s reports that 99.8% of the meconium specimens had an albumin concentration below 20 mg per g dry weight meconium. The lowest albumin concentration i n 15 c y s t i c f i b r o s i s specimens tested concurrently with t h e i r f i r s t study was 35 mg per g o f d r i e d meconium while most newborn CF i n f a n t s had a meconium albumin l e v e l i n the region of 80 mg per g. The albumin i n meconium i s probably derived from amniotic f l u i d swallowed by the fetus i n utero. I t accumulates i n the CF meconium i f the pancreatic enzymes are absent or decreased i n concentration. False p o s i t i v e s are found i n premature i n f a n t s because many of these i n f a n t s have low p r o t e o l y t i c a c t i v i t y i n the meconium. The presence of blood i n the specimen and the presence of a contaminent such as g l y c e r i n e from a r e c t a l suppository or baby ointments containing p r o t e i n may also be the cause of f a l s e p o s i t i v e s . ^ The s u l f o s a l i c y l i c a c i d t e s t , A l b u s t i x and the BMC t e s t - s t r i p have been used to screen meconium albumin. The l a s t i s the most common method used. - 14 -BMC T e s t - S t r i p s The Boehringer-Mannheim (BMC) t e s t - s t r i p * was designed to detect an albumin content above 20 mg per g dry wt. of meconium. The BMC t e s t - s t r i p s were used i n the following studies: 1. P r o s s e r * 8 i n South Wales and North S t a f f o r s h i r e , 1974-1978. Prosser e t a l tested 34,228 samples using the s u l f o s a l i c y l i c a c i d t e s t , A l b u s t i x * * , immunodiffusion technique and the BMC t e s t - s t r i p s for comparative purposes. However, only 2,106 of the samples were tested with the BMC t e s t . One CF pa t i e n t was detected i n t h i s sample and a f a l s e p o s i t i v e rate of 0.6% was obtained. 2. Stephan 5* 0 i n Europe, 1975. This study reports the r e s u l t s from the t e s t i n g of 69,000 infants with the BMC t e s t - s t r i p . The r e s u l t s that were compiled for t h i s study were from 16 European centres (plus 2 American c e n t r e s ) . Since some h i g h - r i s k groups and two American centres had also been included i n the study the European incidence could not be determined from the 60 p o s i t i v e s detected i n the 69,000. However, i n a s t r i c t l y random group of 34,300 neonates from European centres, 19 p o s i t i v e r e s u l t s were obtained g i v i n g a frequency of 1 i n 1,800. The f a l s e negative rate was reported as 0.5%. 99 In 1976 a report was published by the European Working Group for Neonatal C y s t i c F i b r o s i s Screening based on an extension of the study * Boehringer-Mannheim Corporation, Mannheim, West Germany ** Ames Company (D i v i s i o n of Miles Laboratory) E l k h a r t , IN, USA. - 15 -by Stephan. A t o t a l of 199,475 newborns had been screened. The o v e r a l l ' incidence of CF i n t h i s group was 1 i n 1,936. The f a l s e p o s i t i v e rate was 0.5%, and the f a l s e negative rate was 15%. As a r e s u l t of t h i s study, the t e s t - s t r i p became mandatory as a screening t e s t f o r a l l neonates i n West Germany. 3. Bruns'° i n Milwaukee, 1977. This centre tested 16,224 newborns born i n the Milwaukee area h o s p i t a l s . Two CF i n f a n t s were diagnosed c o r r e c t l y by the t e s t and two were missed. The f a l s e p o s i t i v e rate was 0.9%. 4. Holsclaw* 8 i n P h i l a d e l p h i a , 1978. This group screened 20,171 i n f a n t s . Four p o s i t i v e r e s u l t s were confirmed as CF, 2 CF were missed. A f a l s e p o s i t i v e rate of 0.3% was obtained. 30 5. Evans i n Leeds, 1980. Between October 1975 and January 1980, 15,734 babies were screened. Seven of the BMC p o s i t i v e s were diagnosed as having CF, with no missed cases. The f a l s e p o s i t i v e rate was 0.83%. Eight f a l s e p o s i t i v e s were associated with the presence of blood i n the meconium. B. Meconium Albumin and Alb:Alpha-l A n t i t r y p s i n Ratio R y l e y ^ 7 ^ i n an attempt to reduce the number of f a l s e p o s i t i v e s obtained with the BMC t e s t - s t r i p , combined the meconium albumin screening t e s t with a q u a n t i t a t i v e Immunoelectrophoresis technique f or albumin and alpha-1 a n t i t r y p s i n . - 16 -Alpha-1 a n t i t r y p s i n i s normally present i n meconium specimens from healthy i n f a n t s since t h i s p r o t e i n i s r e s i s t a n t to p r o t e o l y s i s by the pancreatic proteases. I t s concentration i n meconium specimens from CF c h i l d r e n i s almost the same as the normal range. Albumin, on the other hand, i s found i n very low concentrations i n meconium from the healthy i n f a n t s f o r t h i s p r o t e i n i s l i a b l e to p r o t e o l y s i s and i s present i n increased amounts i n specimens from CF c h i l d r e n with pancreatic i n s u f f i c i e n c y . This r e s u l t s i n a s i g n i f i c a n t l y higher albumin:alpha-l a n t i t r y p s i n r a t i o i n meconium specimens from CF c h i l d r e n with pancreatic i n s u f f i c i e n c y as compared to healthy c h i l d r e n . The r a t i o of albumin to alpha-1 a n t i t r y p s i n was therefore used by Ryley to exclude pancreatic i n s u f f i c i e n c y i n the healthy i n f a n t s who gave ^ p o s i t i v e r e s u l t with the BMC t e s t . These healthy i n f a n t s had increased l e v e l s i n both the albumin and the alpha-1 a n t i t r y p s i n i n the meconium r e s u l t i n g i n a normal albumin:alpha-1 a n t i t r y p s i n r a t i o . Meconium specimens from a t o t a l of 2,325 babies were examined. Approximately 0.2% of healthy i n f a n t s gave a p o s i t i v e r e a c t i o n with the BMC t e s t . It was p o s s i b l e to conclude that these were f a l s e p o s i t i v e s and that there was very l i t t l e evidence of pancreatic i n s u f f i c i e n c y despite the BMC p o s i t i v e r e s u l t on the basis of the normal albumin:alpha-1 a n t i t r y p s i n r a t i o . C. Fecal Albumin and Albumin:Alpha-1 A n t i t r y p s i n Ratio Although albumin i s not present i n as high a concentration i n feces as i n meconium, there i s a s i g n i f i c a n t d i f f e r e n c e i n the f e c a l albumin - 17 -concentration from healthy and CF c h i l d r e n . 76 77 Electroimmunoassay was employed by Ryley ' to detect albumin and albumin:alpha-1 a n t i t r y p s i n i n feces. Feces from i n f a n t s with CF had an albumin content of more than 2.0 mg per g dry weight and an albumin:alpha-1 a n t i t r y p s i n r a t i o greater than 3.0. Ryley reports i t s usefulness i n d i s t i n g u i s h i n g between in f a n t s free of CF who gave a p o s i t i v e meconium screening t e s t from i n f a n t s with CF. Only a l i m i t e d number of specimens (51 non-CF and 9 CF) were tested i n the f i r s t report of t h i s s t u d y . 7 7 The second report 7* 5 d e t a i l s a 4 year routine screening program i n which 15/464 specimens were examined for r a i s e d meconium albumin l e v e l s by the BMC t e s t - s t r i p method and electro-immuno assay. The incidence of f a l s e p o s i t i v e r e s u l t s was 0.5% i n e i t h e r t e s t and t h i s incidence was reduced by 100% by determining the r a t i o of albumin: alpha-1 a n t i t r y p s i n i n subsequent f e c a l specimens. D. Fecal Trypsin A. TAME Method The f i r s t method to be developed for screening s t o o l samples for t r y p s i n was a semi-quantitative t e s t used on specimens c o l l e c t e d on swabs. The procedure was used i n a small t r i a l screen c a r r i e d out i n Auckland, New Zealand by Robinson and E l l i o t t i n 1974. 7 / This method was l a t e r modified and used i n a mass screening programme i n 1975. In the 7JL modified method s t o o l specimens were c o l l e c t e d on f i l t e r paper cards, allowed to dry and tested with the substrate p - t o s y l - L - a r g i n i n e methyl ester (TAME). - 18 -Robinson reports screening 6,595 newborn i n f a n t s and detecting 3 cases of CF with no known cases of missed CF and a f a l s e p o s i t i v e rate of 0.6%. B. BAPNA Method A more s p e c i f i c method which uses the substrate benzoyl-arginine-p-n i t r o a n i l i d e (BAPNA) was devised by Crossley i n New Zealand i n 1977. During overnight incubation, the presence of t r y p s i n enzyme causes the release of yellow p - n i t r o a n i l i n e from the c o l o u r l e s s substrate. Results were evaluated by eye and a l l samples with a weak colour or with a d i f f e r e n t t i n t were read spectrophotometrically. Dry s t o o l samples c o l l e c t e d on f i l t e r paper cards were a l s o used i n t h i s method. In a screen of 4,500 newborn i n f a n t s , the incidence of f a l s e p o s i t i v e r e s u l t s was 0.1%. Two in f a n t s with CF were detected i n t h i s study with no. evidence of f a l s e negative r e s u l t s . Crossley reports that the method was adopted by three centres i n New Zealand (Auckland, Wellington and Waikato) t e s t i n g 700 newborn in f a n t s per week. 3X F o r r e s t adopted Crossley's method for a mass screening study i n New South Wales, A u s t r a l i a where 20,000 infants were tested. Three CF were detected and 2 CF were missed, both with normal pancreatic function at the time of diagnosis. The f a l s e p o s i t i v e rate was 0.53%. - 19 -SELECTION AND DEVELOPMENT OF TESTING PROCEDURES The t e s t i n g procedure must meet s e v e r a l c r i t e r i a to be s u i t a b l e for a screening programme to detect c y s t i c f i b r o s i s i n newborns i n the province of B r i t i s h Columbia. Because of the number of t e s t s to be performed on a routine b a s i s , the Jiest must be inexpensive and r e l a t i v e l y simple. In a d d i t i o n the screening t e s t must be s u f f i c i e n t l y s e n s i t i v e , s p e c i f i c , p r e c i s e and accurate to avoid large numbers of f a l s e p o s i t i v e s and negatives. Since the screening programme w i l l eventually include samples from a l l the geographically dispersed populations i n B r i t i s h Columbia, the t e s t i n g procedure must meet one other c r i t e r i o n . It must be e f f e c t i v e at t e s t i n g "mailed-in" specimens i f a c e n t r a l screening laboratory i s incorporated i n t o the programme or a l t e r n a t e l y i t must be e a s i l y performed by a v a r i e t y of personnel i n a l l the various h o s p i t a l s throughout the province. According to reviews of a l l screening m e t h o d s r e p o r t e d at the time that the i n i t i a t i o n of a screening programme was being considered, the most promising i n terms of the above mentioned c r i t e r i a appeared to be a n a l y s i s of meconium f or increased l e v e l s of albumin. A p i l o t project,, t e s t i n g albumin i n meconium from the newborns at Vancouver General H o s p i t a l (VGH), was therefore i n i t i a t e d i n A p r i l 1975 and was i n operation u n t i l June 1979. The author became involved i n the meconium - 20 -p i l o t p r o j e c t i n June 1977. She was responsible for the follow-up on the majority of the p o s i t i v e s obtained with t h i s method from the i n i t i a t i o n of the meconium p i l o t screening programme to i t s termination. A neonatal screening method which measured the a c t i v i t y of t r y p s i n i n s t o o l samples was the next method to be evaluated by the author (although the c o l l e c t i o n of f e c a l specimens had been i n i t i a t e d p r i o r to her i n v e s t i g a t i o n ) . The t r y p s i n method inve s t i g a t e d was one developed by Robinson and E l l i o t t a n d s p e c i f i e s the use of the synthetic substrate p - t o s y l - L - a r g i n i n e methyl ester (TAME). This i n v e s t i g a t i o n was followed by the author's attempt to adopt the 77 general p r i n c i p a l of Ryley's f e c a l albumin: alpha-1 a n t i t r y p s i n r a t i o to a mail i n card system. A second t r y p s i n method, the more s p e c i f i c method of Crossley's which uses the substrate benzoyl-arginine-p-nitroanalide (BAPNA), was the l a s t method to be i n v e s t i g a t e d by the author. Crossley's procedure was incorporated i n t o a p i l o t p r o j e c t at VGH i n November 1977 and was i n operation concurrently with the meconium screen for comparative purposes u n t i l June 1979. As i n the case of the meconium screen, the author was responsible for the follow-up i n v e s t i g a t i o n of p o s i t i v e r e s u l t s from the Crossley t r y p s i n screen. D e t a i l s on the i n v e s t i g a t i o n s of these four t e s t i n g procedures follow i n the next four chapters. The p r o t o c o l , r e s u l t s and d i s c u s s i o n of the follow-up performed on i n f a n t s who tested p o s i t i v e i n the two p i l o t screening projects are presented i n the subsequent chapter. - 2 1 -BMC TEST-STRIP FOR MECONIUM ALBUMIN The Boehringer-Mannheira Corporation* (BMC) t e s t - s t r i p for meconium albumin was designed to detect an albumin content above 2 0 mg per g of d r i e d meconium.** Since t h i s s t r i p was reported to be r e l a t i v e l y easy to use, s e n s i t i v e and r e l i a b l e , ' i t appeared to meet B r i t i s h Columbia's s p e c i f i c requirements. I. TESTING PROCEDURE A. C o l l e c t i o n of Samples Nurses at the Vancouver General H o s p i t a l were i n s t r u c t e d to c o l l e c t the FIRST meconium passed by each i n f a n t . I f t h i s was not p o s s i b l e , a second meconium was to be c o l l e c t e d and l a b e l l e d as such. The meconium specimen was placed i n a styrofoam box, containing a frozen freezer-pack, immediately a f t e r c o l l e c t i o n . The specimens i n the styrofoam box were tr a n s f e r r e d d a i l y to the freezer and the freezer-pack i n the box was replaced with a new frozen pack. The frozen meconium specimens were * Boehringer - Mannheim Corporation, Mannheim, West Germany. ** as detected by a s i n g l e r a d i a l immunodiffusion technique." - 22 -transported once a week to Children's H o s p i t a l where they were stored i n a freezer u n t i l analyzed. B. BMC Methodology P r i n c i p l e : The BMC t e s t i s a simple d i p s t i c k method which incorporates the p r i n c i p l e of ascending chromatography. The t e s t s t r i p i s impregnated with tetrabromphenolphthalein e t h y l ester as the i n d i c a t o r . Procedure: 1. The meconium sample was w e l l mixed before t e s t i n g . Some samples were covered with a thick l i g h t brown gelatinous substance, or had a s o l i d mass of the gelatinous substance attached to one end of the meconium sample. This gelatinous substance contained a higher concentration of albumin and was therefore mixed i n thoroughly with the r e s t of the meconium specimen. 2. Using the t e s t - s t r i p as a spatula, a small sample of meconium was taken from the p l a s t i c container. The meconium was spread over the e n t i r e width of the lower part of the t e s t - s t r i p , approximately 5 to 10 mm along i t s length. 3. This t e s t - s t r i p was then placed immediately into a small p l a s t i c v i a l containing 3 to 5 drops of deionized water, making sure that part of the meconium layer was above water l e v e l . 4. An increased albumin concentration (more than 20 mg albumin per g meconium dry weight) produced, a f t e r 15 minutes, an intense blue colour - 23 -across the width of the s t r i p and at l e a s t half-way along the length of the s t r i p . This colour was not n e c e s s a r i l y of uniform i n t e n s i t y but was d e f i n i t e l y an intense blue. A p o s i t i v e c o n t r o l was tested concurrently and used for colour comparison. F a i n t blue colourations or t i n t s of blue on the rims of the t e s t - s t r i p were regarded as negative. When reading the t e s t s , a check was made to ensure that the water fr o n t had advanced up the t e s t - s t r i p w e l l beyond the meconium. I f not, i t was assumed that proper chromatographic movement had not occurred and the t e s t was repeated. The c h a r a c t e r i s t i c of the specimen was noted*, with s p e c i a l a t t e n t i o n to the presence of blood. The follow-up procedure, discussed l a t e r , was i n i t i a t e d on a l l p o s i t i v e r e s u l t s . C. S t a b i l i t y I n v e s t i g a t i o n "Early reports ' i n d i c a t e d that albumin i n meconium was r e l a t i v e l y s t a b l e and that the transport of non-refrigerated specimens through the post d i d not a f f e c t the r e s u l t s . On t h i s b a s i s , the BMC t e s t was i n i t i a l l y performed on meconium specimens stored temporarily at room temperature and/or r e f r i g e r a t o r temperature p r i o r to t h e i r transport to Children's H o s p i t a l . However some of these specimens a r r i v e d at * Af t e r gaining some experience, i t became p o s s i b l e to d i f f e r e n t i a t e v i s u a l l y between meconium, t r a n s i t i o n a l s t o o l and s t o o l specimens. - 24 -Children's H o s p i t a l with mould growth, some were too dry to t e s t and others gave an a t y p i c a l r e a c t i o n with the BMC t e s t - s t r i p . A s t a b i l i t y i n v e s t i g a t i o n was therefore performed at Children's H o s p i t a l s h o r t l y a f t e r the program was i n i t i a t e d * t e s t i n g known p o s i t i v e and negative meconium specimens and meconium specimens to which known q u a n t i t i e s of albumin had been added. These specimens were w e l l mixed and d i v i d e d i n t o s e v e r a l p o r t i o n s . A representative sample was stored at room temperature, 4°C and at -15°C for up to 5, 10 and 14 days. 95 Results i n d i c a t e d that the albumin was not s t a b l e at room temperature, specimens became dry and mould growth which i n t e r f e r e d with the r e a c t i o n appeared i n the p o s i t i v e c o n t r o l s . Reconstitution of the d r i e d specimens had a l s o been attempted but t h i s r e s u l t e d i n an a t y p i c a l r e a c t i o n . A t y p i c a l reactions a l s o occurred o c c a s i o n a l l y with the 4°C stored specimens. More d e t a i l e d studies were reported by other i n v e s t i g a t o r s ' v e r i f y i n g that storage at room temperature r e s u l t e d i n a large decrease i n albumin concentration (approximately 50%); storage at 4°C r e s u l t e d i n an approximate decrease of 16% and storage at -15°C r e s u l t e d i n a decrease of l e s s than 1%. The p r o t o c o l for c o l l e c t i o n of meconium samples at VGH was therefore changed i n May of 1976. A l l samples were frozen immediately and t r a n s f e r r e d to Children's H o s p i t a l i n the frozen state (as o u t l i n e d under the procedure). This was the c o l l e c t i o n procedure i n use when the author became involved i n the p r o j e c t i n June 1977. * P r i o r to the author's involvement. - 25 -I I . RESULTS A. Meconium Sample C o l l e c t i o n As noted i n Table XII on page 113, 8891 meconium samples were c o l l e c t e d out of a p o s s i b l e t o t a l of 10,091. A c t u a l l y , another 414 specimens were c o l l e c t e d by the nurses at VGH but these specimens were ei t h e r too dry or i n s u f f i c i e n t i n quantity to t e s t for albumin using the BMC t e s t - s t r i p . Approximately 12% of the specimens tested (1083 out of 8891) lacked the green colour and mucilaginous c h a r a c t e r i s t i c that are normally found with the f i r s t or even the second meconium specimens. These specimens were thought to be s t o o l specimens. B. S e n s i t i v i t y Three c h i l d r e n with CF were detected as having increased albumin i n the meconium by the BMC t e s t - s t r i p . One c h i l d with CF gave a normal r e s u l t with t h i s screening t e s t . The s e n s i t i v i t y of the meconium albumin t e s t i s therefore 75% (See Table I I ) . C. S p e c i f i c i t y In the meconium screen, 98.7% of the t o t a l number of infants that, to date, appear not to have CF were negative to the t e s t . A f a l s e p o s i t i v e rate of 1.31% was therefore obtained, t h i s number representing the 116 i n f a n t s with increased meconium albumin that were detected among the non-CF groups. TABLE I I . MECONIUM ALBUMIN SCREENING TEST FOR CF Diagnosis BMC T e s t - S t r i p Result CF Non-CF T o t a l P o s i t i v e 3 116 119 Negative 1 8,771 8,772 4 8,887 8,891 I I I . DISCUSSION The BMC t e s t - s t r i p method was shown to be a r e l i a b l e and simple t e s t . No t e c h n i c a l d i f f i c u l t i e s were encountered during the 4 years i t was used to t e s t approximately 9000 specimens. Results were easy to i n t e r p r e t for the dark blue end-point was d i s t i n c t . The method was best performed i n a c e n t r a l laboratory. I t was necessary to adhere s t r i c t l y to the procedure with a t t e n t i o n to d e t a i l s i n order to avoid f a l s e p o s i t i v e s and f a l s e negative r e s u l t s . This agrees with the g u i d e l i n e s set f o r t h by the Committee on Genetics of the 13 American Academy of P e d i a t r i c s who report that experience with PKU screening has revealed the necessity of a c e n t r a l i z e d and c a r e f u l l y standardized t e s t i n g programme to maximize both s p e c i f i c i t y and s e n s i t i v i t y . Less than 1% of the t o t a l number of meconium specimens contained a s o l i d s e c t i o n of l i g h t e r coloured gelatinous substance and required an extra thorough mixing as a r e s u l t of t h i s concentrated p r o t e i n mass. For the majority of specimens, the t e s t i n g procedure was a quick two step procedure, a p p l i c a b l e to a large s e r i e s of t e s t s i n a screening programme. Sample C o l l e c t i o n The c o l l e c t i o n rate was at an acceptable l e v e l . The s t a f f at the Vancouver General H o s p i t a l c o l l e c t e d meconium samples from 92.2% of the newborn i n f a n t population under study. The t e s t i n g rate of 81.1%, although an unfortunate decrease due to dry and i n s u f f i c i e n t quantity (>3 specimens, compares favourably with other screening centres. Since the concentration of albumin decreases with each meconium passed, i n both healthy and CF i n f a n t s , i t i s important that the specimen tested be the f i r s t or i n i t i a l meconium specimen. Testing second meconium specimens or t r a n s i t i o n a l specimens ( t r a n s i t i o n from meconium to stool) could lead to f a l s e negatives. The questionable meconium* rate of 12% obtained i n t h i s meconium screening programme i s high. Several attempts were made to lower t h i s percentage by asking the * Specimens that i n colour and consistency resembled s t o o l rather than meconium specimens. - 28 -co-operation of the nursing s t a f f but t h i s was not s u c c e s s f u l . Most of t h i s communication was however i n the form of memos with some telephone c a l l s placed to the wards. A personal v i s i t i n the form of a seminar to the nurses explaining the importance of c o l l e c t i n g the f i r s t meconium specimen might have r e s u l t e d i n a more e f f e c t i v e sample c o l l e c t i o n . I n terest i n the seminar could p o s s i b l y be stimulated by discussing CF as a disease, the purpose of the screening programme and i t s e f f e c t i v e n e s s . A f t e r the i n i t i a l specimen s t a b i l i t y problems were solved (by s t o r i n g specimens at 4°C immediately a f t e r c o l l e c t i o n and freezing the specimen w i t h i n a couple of hours) no more problems were encountered. Because of the sample i n s t a b i l i t y unless frozen however, the screen could not be adapted to a province-wide programme that incorporated t e s t i n g f a c i l i t i e s at a c e n t r a l laboratory. Previous investigators'"''* 8 had advised against using the BMC t e s t - s t r i p at bed-side. Our own experience agrees with t h i s . Our lack of t e c h n i c a l d i f f i c u l t i e s with t h i s t e s t was i n part due to the small number of laboratory personnel (2) performing the t e s t and the very good communication between the two people involved. S e n s i t i v i t y Since a negative r e s u l t was obtained with the BMC t e s t - s t r i p f or one of the four i n f a n t s who had been diagnosed as having CF, the s e n s i t i v i t y of the meconium albumin t e s t was c a l c u l a t e d to be 75%. This rate i s undesirably low and i s a d e f i n i t e disadvantage of the meconium screen. - 29 -The CF c h i l d whose meconium tested negative displayed a normal pancreatic function and as a r e s u l t a normal concentration of albumin was present i n the meconium specimen. The BMC t e s t - s t r i p r e s u l t was therefore not i n error and the problem was a p h y s i o l o g i c a l one due to the overlap between the d i s t r i b u t i o n s of pancreatic function l e v e l of CF p a t i e n t s and healthy i n f a n t s . This c h i l d represented one of the 10 to 15% of i n f a n t s with CF who have normal or only s l i g h t l y disturbed pancreatic function as discussed e a r l i e r on page 11. S p e c i f i c i t y Our VGH based, BMC meconium screen s p e c i f i c i t y of 98.7% i s at an apceptable l e v e l and i n agreement with s p e c i f i c i t i e s reported by screening programmes set up i n other countriea / O j ^ , ^ 3 j & 0 a l l of which reported a s p e c i f i c i t y of 99.1% or higher. Our s l i g h t l y lower s p e c i f i c i t y may have been due to the large proportion of samples that came from the high r i s k newborn nursery as i s evidenced by the large percentage of premature i n f a n t s which gave p o s i t i v e meconium r e s u l t s (see Figure VI on page 119). Incidence of CF i n the Population Screened The incidence of CF i n the newborn population screened i n Vancouver was 4 i n 8,891 or 1 i n 2222. - 30 -Of the 156,197 l i v e b i r t h s * * i n B.C. during the time period that the meconium screen was i n e f f e c t ( A p r i l 1975 to June 1979), 36* have been diagnosed as having C F . as of May 1981. Four of the 36 CF i n f a n t s were part of the population screened. The remaining 32 i n f a n t s l i v e d outside of the area serviced by the Vancouver General H o s p i t a l and were therefore not part of t h i s screening programme. Since CF i s more prevalent among Caucasians and rare among Negroes and O r i e n t a l s , the incidence as determined by the meconium screen for Caucasians could be adjusted to approximately 1 i n 2000, i f the 9% O r i e n t a l and Negro population of Vancouver** i s taken i n t o c o n s i d e r a t i o n . B r i t i s h Columbia's population as a whole c o n s i s t s of approximately 2.7% O r i e n t a l s and Negroes** From l i t e r a t u r e expectations and our incidence f i g u r e s , there may be about 39 C F . p a t i e n t s i n B.C. born between A p r i l 1975 and June 1979 who are s t i l l undiagnosed as of May 1981. The incidence as determined by t h i s p a r t i c u l a r screen appears to agree w e l l with incidence f i g u r e s given i n the l i t e r a t u r e and quoted i n Table I on Page 2. However, the s t a t i s t i c s used for the VGH based meconium screen include r e f e r r a l s from other h o s p i t a l s (outborn babies). These r e f e r r a l s a l t e r the percentage of high r i s k and often premature i n f a n t s present i n the t o t a l population. The presence of these i n f a n t s could influence the incidence of f a l s e p o s i t i v e s and the incidence of CF. * BC C y s t i c F i b r o s i s Foundation ** S t a t i s t i c s Canada, Vancouver O f f i c e - 31 -Out of the 4 CF infants in this programme one was in fact a referral patient (lowering the incidence to 1:2964). ROBINSON AND ELLIOTT FECAL TRYPSIN METHOD An i n v e s t i g a t i o n i n t o Robinson and E l l i o t t ' s technique of measuring s t o o l t y p s i n a c t i v i t y on dry specimens of feces from newborn c h i l d r e n at 4 to 5 days of age was st a r t e d i n June, 1977 and discontinued i n January, 1978. I. TESTING PROCEDURE A. C o l l e c t i o n of Samples - -The nurses at the VGH were i n s t r u c t e d to c o l l e c t a s t o o l sample from each i n f a n t on the t h i r d day a f t e r b i r t h or l a t e r . Three pea s i z e s t o o l samples were placed on f i l t e r paper by the nurse as per i n s t r u c t i o n s p r i n t e d on the screening card (Appendix A). The card was stamped with the baby's name, date of b i r t h , doctor and date of sampling. The cards were placed i n a styrofoam box containing a freezerpack and t r a n s f e r r e d d a i l y to the fr e e z e r . The s t o o l specimens were transported once a week to Children's H o s p i t a l where they were tested. - 33 -B. Trypsin Methodology P r i n c i p l e : The Robinson and E l l i o t t method detects t r y p t i c a c t i v i t y by measuring the a b i l i t y of the enzyme to release hydrogen ions from the substrate p - t o s y l - a r g i n i n e methyl ester (TAME) when the s t o o l sample i s incubated with t h i s substrate, a buffer and a pH s e n s i t i v e dye mixture. Reagents and M a t e r i a l : 1. Sample cards The sample cards were prepared from f i l t e r paper, Whatman 3, flow rate medium. Polyethylene f i l m (35 um) sleeves protected the samples. 2. B u f f e r , pH 8.2, 0.005 moles per l i t e r . The buffer contained 0.354 g TRIS-HC1, 0.334 g TRIS, 2.34 g sodium c h l o r i d e and 2.9 g calcium c h l o r i d e dihydrate d i s s o l v e d i n a l i t e r of d i s t i l l e d water. 3. Tame Substrate The substrate consisted of 2.07 g p - t o s y l - a r g i n i n e methyl ester* d i s s o l v e d i n 50 ml of b u f f e r . Sigma Chemical Company, St. Louis, Mo., USA. - 34 -4. Indicator Solution The i n d i c a t o r mixture contained 0.2% w/v bromothymol blue i n 50% ethanol and 0.2% w/v phenol red i n 50% w/v ethanol i n the following proportions: 5 ml of bromthymol blue s o l u t i o n , 5 ml of phenol red s o l u t i o n and 50 ml of b u f f e r . 5. Stock Trypsin standard.* The stock standard was prepared d a i l y by weighing 10 mg of t r y p s i n and d i s s o l v i n g i t i n 10 ml of b u f f e r . Procedure: 1. P r i o r to each run, a fresh mixture of i n d i c a t o r - b u f f e r - s u b s t r a t e was prepared i n the proportions of 6:2:1. The pH was adjusted to 8.2 with 0.1 M NaOH. 2. A 6 mm d i s c containing a representative sample of feces was punched (using a paper hole puncher) from each f i l t e r paper card into an appropriately numbered w e l l i n a disposable p l a s t i c sample t r a y . * * 3. Discs from four c o n t r o l specimens were included i n randomly placed p o s i t i o n s . The c o n t r o l sample cards were prepared at C h i l d r e n j s H o s p i t a l from s t o o l specimens containing a known concentration of chymotrypsin and/or t r y p s i n . Both normal and abnormal c o n t r o l s were included i n each run. * Sigma Chemical Company, St. Louis, Mo., USA, 16,000 units of a c t i v i t y per mg, 98% p r o t e i n . ** Clear p l a s t i c t r a y , 96 Wells, Linbro Co., USA - 35 -4. D i l u t i o n s of the stock t r y p s i n standard were made by p i p e t t i n g 0.4 ml of stock t r y p s i n standard (1 mg per ml) and of working d i l u t i o n s of stock standard (1:1000, 1:1500 and 1:2000) i n t o each of 4 wells i n the t r a y . 5. A 0.9 ml p o r t i o n of i n d i c a t o r - b u f f e r - s u b s t r a t e mixture was pipe t t e d i n t o each w e l l containing the d i s c s and the standard s o l u t i o n s . 6. The tray was mixed gently with a r o t a t i n g motion and f l o a t e d i n a water bath at 37°C. 7. When the second standard (1:1000 d i l u t i o n ) turned yellow the tray^ was removed from the water bath. At that point the colour sequence of JL JL the set of standards was: Std. 1 (stock std.) - b r i g h t yellow, 2 -yellow, 3 - orange and 4 - purple. 8. The colour of each t e s t was recorded. 9. The t e s t was considered to contain a normal t r y p s i n concentration i f the i n d i c a t o r colour converted to yellow during the incubation period. I f an abnormal r e s u l t was obtained, the t e s t was repeated i n d u p l i c a t e on d i s c s from the same screening card. I f the s t o o l sample smear appeared to be unusually l i g h t , the t e s t was repeated i n d u p l i c a t e using 2 d i s c s per w e l l . C. I n v e s t i g a t i o n Into Inconsistent Results and N o n - S p e c i f i c i t y The intentions were to request a s t o o l specimen for q u a n t i t a t i v e chymotrypsin analyses from i n f a n t s which gave a presumptive p o s i t i v e r e s u l t (an abnormally low t r y p s i n a c t i v i t y ) i n d i c a t i n g a high p r o b a b i l i t y of c y s t i c f i b r o s i s . I t was apparent immediately however that the method - 36 -was not performing to the same accuracy arid p r e c i s i o n as reported by Robinson and E l l i o t t . Several problems e x i s t e d : 1. Inconsistent r e s u l t s were obtained when both p o s i t i v e and negative c o n t r o l s were repeated from the same sample card. These r e s u l t s are reported on pages 39 and 40 and discussed on pages 45 and 46. 2. The r e a c t i o n rate of the standards v a r i e d g r e a t l y from one run to another (See r e s u l t s on page 41). 3. The number of f a l s e p o s i t i v e s was very high. (See r e s u l t s on page 39.) Because of t h i s v a r i a b i l i t y , the method was examined i n d e t a i l . Reagent preparation, sample preparation and storage, environmental cp n d i t i o n s , and n o n - s p e c i f i c i t y were i n v e s t i g a t e d . 1. Reagent Preparation a) Extra care was taken to prepare the stock standard and working standards i n order to minimize weighing and p i p e t t i n g ( d i l u t i o n ) e r r o r s . New t r y p s i n was purchased and two sets of standards, one new and one o l d , were included i n the run, one at the beginning and one at the end. b) The colour change of the standards obtained during the incubation period was d i f f e r e n t from that described i n Robinson and E l l i o t t ' s paper. They mention a d i s t i n c t colour change from purple to yellow as compared with a gradual change from purple to orange, to l i g h t yellow, to - 37 -b r i g h t yellow that was obtained i n our laboratory following the procedure o u t l i n e d above. Several weaker concentrations of the i n d i c a t o r s o l u t i o n s were tested i n order to determine whether the p r e c i p i t a t e of one of the dyes i n the mixture was responsible for the colour v a r i a t i o n . c) A new TRIS buffer with a concentration of 0.050 moles per l i t e r and a pH of 8.0 was prepared. Controls and p a t i e n t s specimens were tested and compared with previous r e s u l t s using the method as o u t l i n e d incorporating the new b u f f e r . 2. Sample Preparation and Storage Dry and wet specimens were prepared i n the laboratory as follows: Three sets of cards were prepared from 10 c o n t r o l s t o o l specimens. Each specimen was spread on the f i l t e r paper card by pressing on the outside of the p l a s t i c f i l m . One of the sets of sample cards was placed i n i n d i v i d u a l envelopes and placed i n the freezer immediately. The feces on the other two sets of sample cards were allowed to dry at room temperature for approximately 6 hours. The p l a s t i c f i l m was removed i n order for the feces to dry properly. Once the samples were dry, the cards were inse r t e d i n t o the p l a s t i c f i l m and placed i n i n d i v i d u a l envelopes. One set was stored i n the freezer, one set at room temperature. Results from a l l three sets were compared. 3. Environmental Conditions Although the t e s t s were r o u t i n e l y c a r r i e d out i n an area exposed to organic solvents (using an uncovered specimen t r a y ) , a p o r t i o n of the te s t s was repeated under c o n t r o l l e d environmental c o n d i t i o n s . The water - 38 -bath was placed i n a fume hood and a l l e f f o r t s were made to avoid environmental contamination of the specimens. A reagent blank, c o n s i s t i n g of a clean d i s c of f i l t e r paper and the reagents i n a t e s t w e l l , was included with every set of determinations i n order to detect n o n - s p e c i f i c colour change due to environmental contamination. 4. I n v e s t i g a t i o n Into N o n - S p e c i f i c i t y a) Three normal c o n t r o l s t o o l specimens, 3 CF s t o o l specimens and a specimen from a pa t i e n t with Schwachman Syndrome were autoclaved for 15 minutes at 18 pounds pressure. Both the o r i g i n a l specimens and the autoclaved specimens were tested with the Robinson and E l l i o t t method. b) The method was c a r r i e d out as o u t l i n e d but without the a d d i t i o n o f the substrate TAME on a se l e c t e d number of c o n t r o l s : 4 CF specimens, 1 Schwachman Syndrome, and 3 normal c o n t r o l s . 5. I n v e s t i g a t i o n of I n t e r f e r i n g Compound A l l of the c o n t r o l specimens and the specimens from newborn in f a n t s that had been tested with the Robinson and E l l i o t t method, were retested with the method s u b s t i t u t i n g buffer for TAME i n order to determine whether the unknown i n t e r f e r i n g compound was s p e c i f i c for c y s t i c f i b r o s i s . The f e c a l specimens that appeared to contain the compound were then exposed to ammonia and retested without TAME. - 39 -I I . RESULTS A. S e n s i t i v i t y and S p e c i f i c i t y The Robinson and E l l i o t t t r y p s i n method was used to t e s t s t o o l specimens from 513 i n f a n t s and r e s u l t e d i n 42 specimens d i s p l a y i n g low " t r y p s i n a c t i v i t y . The p o s i t i v e rate was therefore 8.3%. The s e n s i t i v i t y of the method was evaluated through the use of p o s i t i v e c o n t r o l specimens. A t o t a l of 4 specimens known to lack the t r y p s i n enzyme or known to contain an abnormally decreased a c t i v i t y of t r y p s i n were tested (3 CF and 1 Schwachman Syndrome). The number of f a l s e negatives obtained for these c o n t r o l specimens v a r i e d from day-to-day. Some of these abnormal co n t r o l s (controls with low t r y p s i n concentration) displayed a more ra p i d colour change than the normal c o n t r o l s . The r e s u l t s from these c o n t r o l specimens are reported i n Table I I I . TABLE I I I . FECAL TRYPSIN CONTROL SPECIMEN RESULTS ROBINSON AND ELLIOTT METHOD Control Diagnosis Chymotrypsin No. of Results No. A c t i v i t y 0 Determ. 132 CF n e g l i g i b l e 24 3Abn*, 21 N + 130 CF below normal 22 22N 111 CF n e g l i g i b l e 28 16 Abn, 12 N 129 Schwachman below normal 22 11 Abn, 11 N 112 healthy normal 24 24N 110 healthy normal 8 8N ° Pancreatic function assessment based upon q u a n t i t a t i v e f e c a l chymotrypsin a n a l y s i s * Abn: abnormally low l e v e l or lack of t r y p s i n a c t i v i t y . + N: normal l e v e l of t r y p s i n a c t i v i t y - 41 -B. I n v e s t i g a t i o n Into P o s s i b l e Causes of Inconsistent Results and No n - S p e c i f i c i t y 1. Reagent Preparation a) The re a c t i o n rate of the standards continued to vary g r e a t l y from one run to another despite the use of a new b o t t l e of t r y p s i n and the attempts made to minimize weighing and p i p e t t i n g e r r o r s when ' preparing the stock and working standards. The time period required to reach the s t i p u l a t e d colour change var i e d from 28 to 43 minutes. The v a r i a t i o n i n time taken for the t e s t s to reach the end-point (on repeating the same t e s t s i n d i f f e r e n t batches) d i d not p a r a l l e l that of the standards. The v a r i a t i o n was not apparent within the same batch but was a batch-to-batch discrepancy. b) When the concentration of both the bromthymol blue and phenol red were reduced to h a l f of the suggested values, the colour change obtained at the end of the incubation period was then a d i s t i n c t change from purple to yellow. The screening r e s u l t s were not a f f e c t e d by t h i s a l t e r a t i o n . — c) The a l t e r a t i o n i n buffer concentration and pH produced a l e s s d i s t i n c t colour change than was obtained with the o r i g i n a l buffer concentration of 0.005 moles per l i t e r , pH 8.2. The screening r e s u l t s -obtained when several c o n t r o l s , and newborns were tested incorporating the two d i f f e r e n t buffers were not s i g n i f i c a n t l y d i f f e r e n t . - 42 -2. Sample Preparation and Storage There was no s i g n i f i c a n t d i f f e r e n c e i n r e s u l t s between the 3 sets of c o n t r o l s : one set prepared i n a manner i d e n t i c a l to that used at the VGH r e s u l t i n g i n wet samples; one d r i e d at room temperature and stored i n the freezer and one set d r i e d at room temperature and stored at room temperature. 3. Environmental conditions The r e s u l t s for the c o n t r o l specimens and newborn infants d i d not improve when the t e s t was performed i n a fume hood i n another laboratory. The accuracy and p r e c i s i o n of the method remained e s s e n t i a l l y the same. The reagent blank c o n s i s t e n t l y displayed i n a c t i v i t y . C. I n v e s t i g a t i o n Into N o n - S p e c i f i c i t y a) Autoclaved C o n t r o l Specimens The three autoclaved normal c o n t r o l specimens no longer turned the i n d i c a t o r s o l u t i o n yellow within the required time period when tested with the Robinson and E l l i o t t method. In only one of the specimens, however, di d the i n d i c a t o r s o l u t i o n remain i t s o r i g i n a l purple colour. The other two specimens d i d cause some colour change of the i n d i c a t o r s o l u t i o n (to orange) even though the t r y p s i n should have been t o t a l l y i n a c t i v a t e d . - 43 -The 3 CF s t o o l specimens, Controls 111, 130 and 132 d i d not produce a c l e a r b r i g h t yellow t e s t s o l u t i o n , w i t h i n the required time period when the autoclaved specimens were tested. The colour of the in d i c a t o r s o l u t i o n d i d change however from purple to orange or to pale yellow during t h i s time period i n a l l 3 CF autoclaved c o n t r o l specimen t e s t s . (These unautoclaved specimens often gave f a l s e negative r e s u l t s * , producing a c l e a r b r i g h t yellow t e s t solution.) The s t o o l specimen from the patient with Schwachman syndrome (Control 129) which contained low t r y p s i n a c t i v i t y , displayed t o t a l i n a c t i v i t y a f t e r autoclaving. The i n d i c a t o r s o l u t i o n remained the o r i g i n a l purple colour. b) Testing with the Robinson and E l l i o t t Method Without TAME A l l of the 5 p o s i t i v e c o n t r o l s , s t o o l samples from CF patients known to have abnormally low pancreatic function, turned the i n d i c a t o r s o l u t i o n yellow within the c r i t i c a l time period when tested s u b s t i t u t i n g the buffer for the substrate TAME i n the Robinson and E l l i o t t method. In one of the CF c o n t r o l s (Control 130) the time taken to reach the yellow increased s i g n i f i c a n t l y , whereas i n the other 4 CF controls the time period was approximately the same when tested with or without the a d d i t i o n of TAME i n the t e s t s o l u t i o n . * Displayed t r y p s i n a c t i v i t y when they were lacking i n t r y p s i n enzyme. - 44 -The s t o o l samples from the patient with Schwachman syndrome p a t i e n t and the 2 normal patients d i d not produce a yellow colour i n the t e s t s o l u t i o n that were missing TAME. 5. I n v e s t i g a t i o n of I n t e r f e r i n g Compound When the 513 specimens from newborn in f a n t s and the c o n t r o l s were re - t e s t e d with the Robinson and E l l i o t t method s u b s t i t u t i n g the buffer for the substrate TAME, 10% of the newborn i n f a n t specimens as well as the 5 CF specimens tested e a r l i e r turned the i n d i c a t o r s o l u t i o n yellow within,the required time period. When these f e c a l specimens from newborn i n f a n t s , that appeared to contain an i n t e r f e r i n g compound, were exposed to ammonia and re-tested without TAME, a decrease of 8% occurred i n the number of p o s i t i v e s . However, a decrease of 6% occurred a l s o when the same specimens were repeated without exposure to ammonia. The r e s u l t s for the CF s t o o l specimens tested without TAME were the same before and a f t e r the specimens were exposed to ammonia. I I I . DISCUSSION The i n i t i a l attempt at s e t t i n g up a f e c a l t r y p s i n CF screen was made with the Robinson and E l l i o t t method. As described by the New Zealand authors the method appeared to be s u i t a b l e for a m a i l - i n screening programme for the province of B.C. Although 513 s t o o l samples from newborns at the Vancouver General H o s p i t a l were tested the pr o j e c t never reached the follow-up stage - 45 -because the method proved u n s a t i s f a c t o r y from the s t a r t . There was a great day-to-day v a r i a b i l i t y and both f a l s e negatives and f a l s e p o s i t i v e s occurred r e g u l a r l y with the c o n t r o l samples of known low and normal f e c a l t r y p s i n l e v e l s . A cause of the batch-to-batch v a r i a t i o n i n time taken f o r the standards to reach the end-point was not detected. Although the same type of batch-to-batch v a r i a t i o n appeared to be present i n the specimens, the two d i d not p a r a l l e l each other. As a r e s u l t , the r e a c t i o n rate of the standards was not a r e l i a b l e guide f o r s e t t i n g the length of the incubation time period as was suggested by Robinson and E l l i o t t . The a l t e r a t i o n s made to the buffer concentration d i d not improve the assay. The weaker concentration of i n d i c a t o r s o l u t i o n produced a colour change that resembled the one described by Robinson~ahd E l l i o t t . It was thought l i k e l y that the p r e c i p i t a t i o n of one of the dyes i n the mixture (probably bromothymol blue) was responsible for the d i f f e r e n t end-points obtained (using the concentration s p e c i f i e d by the method) as a f i n e p r e c i p i t a t e formed during the preparation of t h i s s o l u t i o n . Use of the weaker i n d i c a t o r s o l u t i o n solved t h i s problem. The screening r e s u l t s however remained imprecise and u n r e l i a b l e using the new i n d i c a t o r s o l u t i o n . Nor d i d the wet sample cards received from the VGH or the storage appear to be responsible for the imprecision. Since the screening r e s u l t s obtained under s t r i c t l y c o n t r o l l e d environmental conditions were not s i g n i f i c a n t l y d i f f e r e n t from the r e s u l t s obtained during routine determinations, environmental contamination with a c i d i c or a l k a l i n e vapours do not appear responsible for the lack of p r e c i s i o n . The c o n s i s t e n t l y i n a c t i v e reagent blank confirmed t h i s conclusion. - 46 -Because the r e s u l t s obtained were not reproducible and because only a small sample of newborns was tested, the s p e c i f i c i t y , presented as a f a l s e p o s i t i v e rate of 8.3%, i s inaccurate but i t does point to an unacceptably low s p e c i f i c i t y for a CF screening t e s t . The s e n s i t i v i t y as evaluated by the CF c o n t r o l specimens was a l s o at an unacceptable l e v e l . The f a l s e negative r e s u l t s , obtained for the CE c o n t r o l specimens, i n d i c a t e d the presence of a n o n - s p e c i f i c r e a c t i o n . This was confirmed by the development of the yellow end-point within the required time period when the CF c o n t r o l specimens were tested without the a d d i t i o n of the substrate TAME. The i n t e r f e r i n g compound implicated by t h i s n o n - s p e c i f i c r e a c t i o n i n the absence of TAME and present i n the 5 CF specimens appeared to be absent from s t o o l samples from 3 normal c o n t r o l s and 1 Schwachman syndrome pa t i e n t tested concurrently. The absence o f the compound i n the normal c o n t r o l specimens was probably only c o i n c i d e n t a l however for i t was present i n 10% of the random sample of newborns (n=513) tested l a t e r . The i n t e r f e r i n g compound was therefore not a s p e c i f i c c h a r a c t e r i s t i c of CF. Since i t d i d appear, however, i n a l l 5 CF specimens tested, the compound appears to be present i n the s t o o l samples of c y s t i c s i n a much greater percentage than i n the normal population. (The p r o b a b i l i t y that i t would show up i n a l l 5 CF specimens due to chance alone when i t i s present i n 10% of the population i s 1.0 x 10 5 ; e s s e n t i a l l y a n e g l i g i b l e chance.) There i s the p o s s i b i l i t y that the i n t e r f e r i n g compound i s present i n the s t o o l samples from a l l c y s t i c s and from that standpoint i t would be of i n t e r e s t to t e s t other CF specimens and to i d e n t i f y the compound or compounds i n question. - 47 -The compound (or compounds) was heat s e n s i t i v e as indic a t e d by the g r e a t l y decreased r e a c t i o n rate obtained upon t e s t i n g the autoclaved CF s t o o l specimens with the R & E method. This heat s e n s i t i v e compound was not present i n s i g n i f i c a n t amounts i n the 3 normal c o n t r o l specimens that were autoclaved and retested and i t appeared to be t o t a l l y absent from the s t o o l specimen obtained from the pa t i e n t with Schwachman syndrome. ' Since the absence of the i n t e r f e r i n g compound (capable of producing a colour change i n the absence of TAME) i n the 3 normal c o n t r o l specimens appeared to be c o i n c i d e n t a l i t i s doubtful that there i s any s i g n i f i c a n c e to i t s absence i n the specimen from the Schwachman syndrome p a t i e n t . I t was theorized that the i n t e r f e r i n g compound might be an organic a c i d and that the poor r e p r o d u c i b i l i t y might be r e l a t e d to the slow and va r i e d d i s s o l u t i o n rate of the acid(s) from the various s t o o l samples. This theory was not substantiated. The r e s u l t s obtained from the t e s t i n g of specimens before and a f t e r ammonia exposure were not s i g n i f i c a n t l y d i f f e r e n t . The decrease i n number of p o s i t i v e s that occurred when p o s i t i v e specimens were retested probably r e f l e c t e d the poor p r e c i s i o n of the method rather than any ac t i o n of ammonia on the i n t e r f e r i n g compound. Also the i n d i c a t i o n that the compound or at l e a s t one of the compounds i s heat s e n s i t i v e casts doubt on i t being an organic a c i d . As a r e s u l t no d e f i n i t e conclusions could be drawn as to the c h a r a c t e r i s t i c of t h i s i n t e r f e r i n g compound. Since the method f a i l e d to d i s t i n g u i s h between CF pat i e n t s and normals there seemed to be no purpose i n continuing the i n v e s t i g a t i o n . FECAL ALBUMIN:ALPHA—1 ANTITRYPSIN RATIO Since s t o o l samples from CF c h i l d r e n with pancreatic i n s u f f i c i e n c y were reported to contain a s i g n i f i c a n t l y higher albumin:alpha-1 a n t i t r y p s i n r a t i o as compared with healthy c h i l d r e n 7 7 an attempt was made to set up a screening programme that incorporated t h i s r a t i o . The plan was to analyse s t o o l samples q u a l i t a t i v e l y for albumin with a follow-up albumin:alpha-1 a n t i t r y p s i n r a t i o determination on those specimens that displayed an increased albumin concentration. T h e o r e t i c a l l y t h i s r a t i o could be considered an index for pancreatic i n s u f f i c i e n c y and should r e s u l t i n a lower f a l s e p o s i t i v e rate than obtained with the f e c a l albumin t e s t alone. To adopt the screening programme i n B.C, however, methods have to be designed that would be s u f f i c i e n t l y s e n s i t i v e and s p e c i f i c to measure the albumin and albumin:alpha-1 a n t i t r y p s i n r a t i o i n small samples of d r i e d feces spread on f i l t e r paper cards. I. TESTING PROCEDURE A. Albumin:Alpha-1 A n t i t r y p s i n Ratio and Pancreatic I n s u f f i c i e n c y F i r s t i t was necessary to v e r i f y the r e l a t i o n s h i p between albumin:alpha-1 a n t i t r y p s i n r a t i o s and pancreatic i n s u f f i c i e n c y . Albumin - 49 -and albumin:alpha-1 a n t i t r y p s i n r a t i o were determined using an immunodiffusion p l a t e method on s t o o l specimens from 8 CF c h i l d r e n with pancreatic i n s u f f i c i e n c y , 4 CF c h i l d r e n on enzyme therapy, a c h i l d with Schwachman syndrome, 7 healthy i n f a n t s with known normal pancreatic f u n c t i o n (normal q u a n t i t a t i v e chymotrypsin concentration) and 7 random samples from healthy c h i l d r e n assumed to have normal pancreatic f u n c t i o n . The specimens were numbered randomly and treated as " b l i n d c o n t r o l s " . The s t o o l specimens were frozen as soon as p o s s i b l e a f t e r c o l l e c t i o n . None was v i s i b l y contaminated with blood. P r i o r to a n a l y s i s a p o r t i o n of the specimen was thawed and d r i e d f or 48 hours i n a d e s s i c a t o r under vacuum. A 10% w/v s t o o l s o l u t i o n was prepared i n 0.05 moles per l i t r e TRIS acetate b u f f e r , pH 7.3, containing 0.3 moles per l i t e r NaCl and 0.1 moles per l i t e r EDTA.* The mixture was mixed f o r 30 minutes with a Vortex mixer, and cen t r i f u g e d a t 3000 x g f o r 30 minutes to remove the i n s o l u b l e m a t e r i a l . The supernatant was then applied to each of the s p e c i f i c immunodiffusion p l a t e s . a) Albumin 1. A 20 m i c r o l i t e r p o r t i o n of supernatant and 10 m i c r o l i t r e p o r t i o n * EDTA i s necessary for detection of albumin by the immunodiffusion technique. A f a i n t p r e c i p i t i n r i n g was obtained i n the absence of EDTA, a d i s t i n c t r i n g when buffer containing EDTA was used. EDTA removes po s s i b l e i n t e r f e r i n g c a t i o n s . - 50 -of buffer were applied i n two stages to the albumin p l a t e * , which has an assay range of 2.8 to 44.4 mg. per d l . 2. The diameters of the p r e c i p i t i n rings were measured and the concentrations were read o f f a c a l i b r a t i o n curve prepared at the same time. b) Alpha-1 A n t i t r y p s i n 1. A 20 m i c r o l i t e r p o r t i o n of supernatant was applied i n two stages to an alpha-1 a n t i t r y p s i n p l a t e * , which has an assay range of 0.8 to 12.5 mg per d l . 2. The i n t e n s i t y of the p r e c i p i t i n rings was increased by treatment with 3, 4-dihroxyphenylalanine (DOPA) s o l u t i o n as o u t l i n e d by Madhosingh 58 and Wood. B. E l u t i o n of St o o l Samples From F i l t e r Paper Cards. Dried s t o o l samples were prepared using the 23 s t o o l samples from CF and non-CF c h i l d r e n ( l i s t e d above) as follo w s : 1. A pea s i z e d sample of s t o o l specimen was placed on the f i l t e r paper card i n the center of a marked 2.5 mm c i r c l e . Three samples from one specimen were smeared on one card. 2. The card was covered with p l a s t i c f i l m . * LC Partigen Immunodiffusion p l a t e s , Behring I n s t i t u t e , Canadian Hoechst Ltd., Montreal - 51 -3. The outside of the p l a s t i c f i l m was pressed to spread the specimen evenly w i t h i n the c i r c l e . The p l a s t i c f i l m was removed immediately. 4. The specimen was l e f t at room temperature for approximately 4 hours to dry and then placed i n a paper envelope and stored i n the f r e e z e r . A method was devised to elute the s t o o l samples from the f i l t e r paper cards r e s u l t i n g i n a s o l u t i o n that contained approximately 10 g of feces per d l i n TRIS acetate buffer containing sodium c h l o r i d e and EDTA. This concentration of f e c a l s o l u t i o n , applied to the p l a t e s as o u t l i n e d e a r l i e r should have r e s u l t e d i n p r e c i p i t i n rings f o r the albumin present i n s t o o l s from CF c h i l d r e n and for the alpha-1 a n t i t r y p s i n present i n a l l 27 s t o o l specimens. However, neither the albumin nor the alpha-1 a n t i t r y p s i n was detected on any of the immunodiffusion p l a t e s . The e x t r a c t i o n procedure was therefore modified to provide a more concentrated f e c a l s o l u t i o n and employed on 8 CF s t o o l specimens known to contain an increased concentration of albumin as follows: 1. Two d i s c s of f i l t e r paper, each containing a representative sample of specimen, were c l i p p e d i n t o a small t e s t tube using a standard paper hole puncher. 2. A 0.5 ml sample of buffer was added and the s o l u t i o n was mixed on a vortex mixer p e r i o d i c a l l y f o r 30 minutes. 3. The f e c a l s o l u t i o n was cent r i f u g e d f or 15 to 20 minutes at 3000 x g for 20 minutes. - 52 -4. The fecal solution was then placed in a dessicator under vacuum for 48 hours u n t i l completely dry. 5. The residue was dissolved in 50 microliters of d i s t i l l e d water resulting in a 20% w/v feces solution. (Ten discs of f i l t e r paper containing dried samples of feces were weighed individually. The average weight of feces on a f i l t e r paper disc was 5 mg. The feces from two discs was extracted into 50 microlitres of solution.) These extracts were applied to the albumin immunodiffusion plate as specified e a r l i e r . V i s i b l e precipition rings were obtained for the 8 CF stool specimens and as a result the procedure was repeated on a l l of the 27 stool specimen cards and applied to both albumin and alpha-1 antitrypsin pJLates. The resultant precipitin rings on the albumin plates were smaller than expected and no rings were detected for any of the specimens on the alpha-1 antitrypsin plates. Since i t appeared that the extraction procedure was inadequate, various technical modifications were made such as altering the extraction time and mixing vigour, increasing the centrifugation speed and time. The eluants were changed substituting water for the buffer used i n i t i a l l y and buffer for the water used in the f i n a l extraction. These various modifications were tested for their effectiveness on stool specimens to which a known amount of albumin had been added. An attempt was also made to concentrate the f i n a l fecal solution applied to the immunodiffusion plate. The volume of d i s t i l l e d water used to dissolve the feces, however, could only be reduced within certain - 53 -l i m i t s . These l i m i t s were set by the necessity to have s u f f i c i e n t volume to wash the d r i e d feces o f f the side of the t e s t tube and to d i s s o l v e the feces so that a cl e a r suspension and homogenous s o l u t i o n was obtained for a p p l i c a t i o n to the immunodiffusion p l a t e . C. Q u a l i t a t i v e Albumin Method Concurrently with the above i n v e s t i g a t i o n s an attempt was made to f i n d a s u i t a b l e q u a l i t a t i v e albumin method to be used to screen the s t o o l samples. I d e a l l y , the albumin method should give a p o s i t i v e r e s u l t for s t o o l s containing albumin above 2.0 mg per g of dry weight and a negative r e s u l t 77 fpr normal s t o o l specimens which contain l e s s than 0.1 mg per g. Several methods were inve s t i g a t e d t e s t i n g a s e l e c t number of s t o o l specimens, c o n t r o l s and random specimens from healthy newborns. The S6 methods in v e s t i g a t e d were: Lowry, Brom-cresol green dye binding 73 method and A l b u s t i x . * I I . RESULTS A. Relationship of Ratio to Pancreatic I n s u f f i c i e n c y . A l l 8 of the s t o o l specimens from CF c h i l d r e n with pancreatic Ames Company (D i v i s i o n of Miles Laboratories) E l k h a r t , In, USA. d e f i c i e n c y produced a measurable r i n g on the albumin immunodiffusion p l a t e , i n d i c a t i n g an abnormally high concentration of albumin. Seventeen of the remaining s t o o l specimens (12 normal, 1 Schwachman syndrome and 4 from CF c h i l d r e n on enzyme therapy) contained a normal concentration of albumin. These samples contained albumin l e v e l s below the s e n s i t i v i t y of the immunodiffusion p l a t e i n d i c a t i n g a concentration of l e s s than 0.28 mg of albumin per g dry weight of feces. Two of the s t o o l specimens from healthy newborns contained an abnormally high concentration of albumin. Measurable p r e c i p i t i n r i n g s were obtained on the alpha-1 a n t i t r y p s i n p l a t e s for 13 out of the t o t a l 27 s t o o l specimens tested. The remaining 14 specimens contained alpha-1 a n t i t r y p s i n i n concentrations beyond the range of the c a l i b r a t i o n curve i n d i c a t i n g a concentration of more than 1.5 mg of alpha-1 a n t i t r y p s i n per g dry weight of feces. These were re-t e s t e d using 10 m i c r o l i t e r s of supernatant or l e s s instead of the 20 m i c r o l i t e r s s p e c i f i e d . The r e s u l t s of the albumin, alpha-1 a n t i t r y p s i n , and albumin:alpha-1 a n t i t r y p s i n r a t i o for the 27 s t o o l specimens inve s t i g a t e d are summarized i n Table IV. B. E l u t i o n of Stool Samples from F i l t e r Paper Cards P r i c i p i t i n rings appeared on the albumin immunodiffusion plates for the 8 CF s t o o l sample extracts prepared from the sample cards. The concentrations obtained for these samples were approximately 65% l e s s than when the determination was performed on the o r i g i n a l s t o o l sample. TABLE IV. ALBUMIN AND x ANTITRYPSIN CONTENT OF FECES IMMUNODIFFUSION METHOD No. of Albumin (mg/g dry wt.) Alpha-1 A n t i t r y p s i n (mg/g dry wt.) Albumin: alpha-1 a n t i t r y p s i n r a t i o Spec (n) mean + l i m i t s S.D. of values mean + S.D. l i m i t s of values mean + S.D. l i m i t s of values CF with pancreatic i n s u f f i c i e n c y 8 3.6 + 2.9 1.0-9.2 0.56 + .26 0.30-1.02 5.7 + 2.1 3.3-10.2 CF on enzyme therapy 4 0.28* 1.6 + .34 1.20-2.03 0.23 Schwachman 1 0.28 0.94 0.29 Known normal pancreatic function 6 1 0.28 1.6 0.95 + .62 3.42 0.31-1.82 0.90 0.49 Healthy c h i l d r e n 6 1 0.28 8.2 2.2 + 1.6 9.2 0.73-2.86 0.38 0.93 * with the method ou t l i n e d , the lowest concentration detectable i s 0.28 mg. albumin/g. dry wt. This poor e x t r a c t i o n of s t o o l constituents r e s u l t e d i n an i n a b i l i t y to detect the alpha-1 a n t i t r y p s i n . None of the specimen extractions r e s u l t e d i n v i s i b l e p r e c i p i t i n rings i n d i c a t i n g that the concentration of alpha-1 a n t i t r y p s i n i n the extracts was below 0.8 mg per d l of e x t r a c t , the lowest concentration detectable on the LC Partigen immunodiffusion p l a t e s . The percentage recovery d i d not increase with any of the modified e x t r a c t i o n procedures as i n d i c a t e d by the lack of a s i g n i f i c a n t increase i n the albumin concentration detected. Although i t was p o s s i b l e to increase the concentration of f e c a l e x t r a c t s l i g h t l y by reducing the volume of buffer used to d i s s o l v e the feces i n the f i n a l step of the e x t r a c t i o n procedure, i t was not p o s s i b l e to concentrate the e x t r a c t s u f f i c i e n t l y to detect the alpha-1 a n t i t r y p s i n on the immunodiffusion p l a t e . C. Q u a l i t a t i v e Albumin Method 1. Lowry Method The Lowry method detected p r o t e i n i n a l l of the 27 c o n t r o l s t o o l specimens. A q u a n t i t a t i v e value was not obtained for these specimens for the blue colour produced was much darker i n colour than the highest standard, beyond the range of photometric accuracy of the spectrophotometer and beyond the concentration range of adherence to Beer's law for the method. I t was however p o s s i b l e to assess the specimens q u a l i t a t i v e l y . The p r o t e i n concentrations of a l l of the s t o o l specimens were greater than 25 - 57 -rag per g of the dry weight of feces as compared with previous r e s u l t of 1.0 to 9.2 mg per g of dry wt. obtained for the CF specimens using the immunodiffusion method. The specimens that contained the highest concentration of p r o t e i n as detected by the Lowry method were the s t o o l specimens from normal patients which were previously determined to contain the lowest concentration of albumin, l e s s than 0.28 mg per g dry weight of feces, using the immunodiffusion method. I t was not worthwhile to repeat the determinations on the s t o o l samples using a smaller sample since the Lowry method obviously was measuring the t o t a l p r o t e i n present as w e l l as other i n t e r f e r i n g substances and not the albumin of i n t e r e s t . 2f Dye Binding Method The bromcresol green dye binding method produced an unusual colour change when used to t e s t the c o n t r o l s t o o l samples. The f e c a l pigments appeared to mask the e f f e c t of albumin on the bromcresol green s o l u t i o n . The r e s u l t a n t colour could not be r e l a t e d to albumin concentration. 3. A l b u s t i x I t was a l s o not po s s i b l e to detect the increased albumin concentrations i n the 8 CF s t o o l specimens with A l b u s t i x . A 10% w/v f e c a l s o l u t i o n was required to s a t i s f y the s e n s i t i v i t y of the A l b u s t i x reagent s t r i p of 20 mg albumin per d l of s o l u t i o n . The pr o t e i n and f e c a l pigments present i n a f e c a l e x t r a c t of t h i s concentration i n t e r f e r e d with the detection of albumin by the A l b u s t i x method. - 58 -I I I . DISCUSSION Relationship of Ratio to Pancreatic I n s u f f i c i e n c y The albumin concentration range of 1.0 to 9.2 mg per g dry weight of feces from i n f a n t s with CF agrees f a i r l y w e l l with that reported by Ryley (reported i n the next paragraph). The alpha-1 concentrations of both CF and non-CF s t o o l specimens appear to be s l i g h t l y lower than Ryley's. The d i f f e r e n c e i s however i n s i g n i f i c a n t when the small sample s i z e i s taken i n t o c o nsideration. As a r e s u l t of these lower alpha-1 a n t i t r y p s i n concentrations however, the albumin:alpha-1 a n t i t r y p s i n r a t i o s for inf a n t s with CF were a l s o s l i g h t l y lower than those reported by Ryley. R y l e y 7 7 reported a mean of 4.7 (+1.6) mg of albumin and a mean of 0.9 (+0.4) mg of alpha-1 a n t i t r y p s i n per g of dry weight of feces from CF i n f a n t s . For feces from non-CF in f a n t s Ryley reported a mean of 2.0 (+1.0) mg of alpha-1 a n t i t r y p s i n per g dry weight of feces. The albumin:alpha-1 a n t i t r y s p i n r a t i o reported by him for feces from CF inf a n t s was 6.7 (+3.6). Most of the remaining s t o o l specimens (17 out of 19) contained a normal concentration of albumin which according to Ryley i s l e s s than 0.1 mg of albumin per g dry weight of feces. The method as o u t l i n e d d i d not r e s u l t i n p r e c i p i t i n rings for these specimens because the albumin concentrations of these samples were below the s e n s i t i v i t y of the p l a t e . The data obtained, however, seemed s u f f i c i e n t for the purpose of t h i s i n v e s t i g a t i o n . - 59 -The r e l a t i o n s h i p between an abnormally high albumin:alpha-1 a n t i t r y p s i n r a t i o and pancreatic i n s u f f i c i e n c y was supported by the data obtained. An abnormally high albumin:alpha-1 a n t i t r y p s i n r a t i o , greater than 3.3, was obtained for a l l of the 8 CF s t o o l specimens from c h i l d r e n known to have pancreatic i n s u f f i c i e n c y . The remaining 19 specimens (16 non-CF c h i l d r e n and 4 CF c h i l d r e n on enzyme therapy) had albumin:alpha-1 a n t i t r y p s i n r a t i o s l e s s than 1.0. A normal albumin:alpha-1 a n t i t r y p s i n r a t i o was obtained for s t o o l specimens from 2 healthy c h i l d r e n despite the increased f e c a l albumin concentration i n both specimens. The specimens were probably more concentrated than normal causing an increase i n both the albumin and the alpha-1 a n t i t r y p s i n . As a r e s u l t a normal albumin:alpha-1 a n t i t r y p s i n r a t i o was obtained for these 2 specimens. I t appears therefore that the albumin:alpha-1 a n t i t r y p s i n r a t i o i s a more s p e c i f i c measurement for pancreatic i n s u f f i c i e n c y than the measurement of albumin alone. E l u t i o n of Stool Specimen from F i l t e r Paper The e x t r a c t i o n procedure devised to detect the abnormally high concentrations of albumin i n dry s t o o l samples on d i s c s of f i l t e r paper appeared to be adequate and s u i t a b l e for use with the immunodiffusion p l a t e . These same extracts d i d not contain s u f f i c i e n t alpha-1 a n t i t r y p s i n however to be detected with the alpha-1 a n t i t r y p s i n immunodiffusion plate method. The % recovery of alpha-1 a n t i t r y p s i n was too low r e s u l t i n g i n alpha-1 a n t i t r y p s i n l e v e l s i n the extra c t being below the s e n s i t i v i t y of the LC Partigen p l a t e s . - 60 -The a n a l y s i s of dry s t o o l specimens on f i l t e r paper cards f o r albumin presented another problem. Spurious r e s u l t s were obtained i n the q u a n t i t a t i v e albumin determinations. Since feces are not s t e r i l e when passed these r e s u l t s were probably due to the a c t i o n of b a c t e r i a l proteases. Q u a l i t a t i v e Albumin Method None of the methods in v e s t i g a t e d was s u i t a b l e f o r screening the dry s t o o l samples on f i l t e r paper cards f o r an abnormally high albumin concentration. The Lowry method presented s p e c i f i c i t y problems. The f a l s e l y high r e s u l t s obtained f o r s t o o l samples from both normal and CF pa t i e n t s were probably due to the measurement of a l l of the p r o t e i n present i n the sample rather than a s p e c i f i c albumin measurement as w e l l as other i n t e r f e r i n g substances. Interference from the f e c a l pigments appeared to be the major problem with the dye binding method, the bromcresol green method, and the A l b u s t i x reagent s t r i p s . The colour change that was to be r e l a t e d , i n each method, to the albumin concentration was masked i n these methods. A l i t e r a t u r e search d i d not uncover a q u a l i t a t i v e albumin method with the necessary s p e c i f i c i t y and s e n s i t i v i t y . f - 61 -CROSSLEY FECAL TRYPSIN METHOD Since the f e c a l t r y p s i n method of Crossley, Berryman and E l l i o t t was a l s o designed to t e s t d r i e d feces c o l l e c t e d on f i l t e r paper cards, i t seemed an obvious a l t e r n a t i v e to the unsuccessful TAME method discussed above. I. TESTING PROCEDURE A* C o l l e c t i o n of Fecal Samples a) The c o l l e c t i o n procedure previously e s t a b l i s h e d at the VGH for the Robinson and E l l i o t t TAME method was continued. The same screening cards (Appendix A) were used for c o l l e c t i n g d r i e d f e c a l samples for the Crossley method which was i n i t i a t e d i n November, 1977. b) Home c o l l e c t i o n s were, s t a r t e d i n September 1978. The mothers were given the f i l t e r paper card, p l a s t i c sleeve, envelope and a l e t t e r (Appendix B) at VGH on the day of discharge from the h o s p i t a l asking them to c o l l e c t the s t o o l specimen at home within the f i r s t two weeks of l i f e o f the c h i l d . The specimen was to be mailed to Children's H o s p i t a l on the day of c o l l e c t i o n . - 62 -B. Trypsin Methodology P r i n c i p l e : A c o l o u r l e s s substrate, benzoyl-arginine-p-nitroanalide (BAPNA) i s employed i n t h i s method for the determination of t r y p s i n i n feces. When t h i s substrate i s hydrolyzed by t r y p s i n , yellow p - n i t r o a n i l i n e i s released. Samples from i n f a n t s with CF, who lack t r y p s i n , give n e g l i g i b l e colour. Reagents: 1. Buffer The buffer contained 0.1M TRIS-HCl, 0.04 moles per l i t e r C a C l 2 and 0.08 moles per l i t e r NaCl and had a pH of 8.2 2. BAPNA Substrate The substrate was prepared from 0.65 mg of pure L-isomer BAPNA* per ml of d i s t i l l e d water. The s o l u t i o n was protected from l i g h t while i t was heated i n hot water for a few minutes to d i s s o l v e the BAPNA. I t was stab l e for seve r a l weeks i f stored at room temperature i n the dark. Merck, Sharpe and Dohm, Montreal, Canada. - 63 -3. Stock Trypsin Standard The stock standard was prepared every two weeks by d i s s o l v i n g 1 mg of porcine t r y p s i n * , i n 1.00 ml of 0.001 M HCl. This s o l u t i o n was stored at 4°C. The stock standard contained 15,680 u n i t s of t r y s p i n a c t i v i t y per ml of s o l u t i o n . 4. Working Trypsin Standards The stock standard was d i l u t e d 1:10, 1:100 and 1:10,00 with the b u f f e r . Procedure: 1. A 6 mm d i s c containing a representative sample of feces was punched with a standard paper punch from each screening card i n t o numbered 12 x 75 mm t e s t tubes. Care was taken to obtain c o n s i s t e n t sample thickness by v i s u a l l y s e l e c t i n g an appropriate area on the card. 2. A 10 m i c r o l i t r e p o r t i o n of each working standard was pipetted i n t o properly l a b e l l e d t e s t tubes r e s u l t i n g i n 1.0, 0.1 and 0.01 microgram t r y p s i n per t e s t tube representing 15.68, 1.568 and 0.1568 un i t s of t r y p s i n a c t i v i t y * r e s p e c t i v e l y . 3. Discs from c o n t r o l specimens, 2 normal and 2 untreated CF, were included i n the set of determinations, interspersed throughout the t e s t s at random. * Sigma Chemical Company, St. Louis, Mo, USA 16000 Units of a c t i v i t y per mg., 98% p r o t e i n i n that p a r t i c u l a r sample. Since each b o t t l e of purchased porcine t r y p s i n v a r i e s s l i g h t l y i n t r y p s i n a c t i v i t y , reference w i l l be made to the t r y p s i n standard i n terms of weight rather than a c t i v i t y . - 64 -4. A blank f i l t e r paper d i s c was placed i n a t e s t tube for the reagent blank. 5. An equal volume of BAPNA s o l u t i o n was mixed with an equal volume of buffer and 0.8 ml was added to each t e s t tube using a p i p e t t e dispenser. 6. The t e s t tubes were placed i n a dark cupboard for 15 to 20 minutes. Each tube was then vortexed b r i e f l y to loosen the p l a s t i c f i l m from the specimen and the tubes were returned to the dark cupboard to stand overnight. 7. In the morning, between 17 to 18 hours l a t e r , the t e s t tubes were removed from the cupboard and examined v i s u a l l y as follows: a) P a i r s of t e s t tubes were examined against a white background for a c l e a r fluorescent yellow colour that compares i n i n t e n s i t y to the 1.0 ug working standard (approximately 15 units of a c t i v i t y , depending on the p a r t i c u l a r l o t of porcine t r y p s i n ) . Samples that were equal to or greater than the yellow colour of the standard were considered to contain a normal concentration of t r y p s i n . b) Solutions that were paler i n colour than the 1.0 microgram standard or s o l u t i o n s with a d i f f e r e n t t i n t due to f e c a l background colour were placed aside for spectrophotometry examination along with the reagent blank and standards. 8. D i s t i l l e d water, 2.2 ml, was added to the reagent blank, standards and each of the abnormally coloured samples. The tubes were placed i n the dark for 15 to 20 minutes. 9. Samples that appeared t u r b i d or cloudy were centrifuged for 5 minutes to remove the f e c a l d e b r i s . - 65 -10. Spectrophotometrie absorbance readings at two wavelengths were taken of the reagent blank, standards and samples: a) The reagent blank was read against d i s t i l l e d water at a wavelength of 410 nm. The instrument was then zeroed on the reagent blank and the standards and t e s t s were read. b) The standards and t e s t s were read against d i s t i l l e d water at 460 nm. 11. The two absorbance readings for each s o l u t i o n were used to c a l c u l a t e a net absorbance according to the following formula suggested by Crossley (see a l s o explanation next page): Abs,, -2 (Abs) . = net absorbance 410 460 If a net absorbance value of 0.300 or l e s s was obtained, the assay was repeated, i n t r i p l i c a t e , on the same specimen. I f the fecal-smear on the t e s t card appeared to be unusually l i g h t , the t e s t was repeated i n t r i p l i c a t e using 2 d i s c s per t e s t tube. A specimen was considered to have an abnormally low t r y p s i n concentration i f the average of the t r i p l i c a t e net absorbance values was l e s s than 0.300. The follow-up procedure, discussed l a t e r , was then i n i t i a t e d . C. I n v e s t i g a t i o n to E s t a b l i s h Procedure Background Interference 1. F e c a l Pigments Soluble pigments present i n s t o o l samples add to the colour of the - 66 -t e s t s o l u t i o n s . These pigments, e s p e c i a l l y b i l e pigments, could contribute s i g n i f i c a n t l y to the absorbance reading at 410 nm and may lead to f a l s e negative r e s u l t s . Crossley's method attempts to c o r r e c t these f a l s e l y elevated A b s ^ ^ readings by reading the s o l u t i o n at 460 nm as wel l as 410 nm. According to Crossley, the absorbance reading obtained at 460 nm i s due mostly to f e c a l pigments with p - n i t r o a n i l i n e c o n t r i b u t i n g a small amount. To compensate for these pigments, the reading at 460 nm i s m u l t i p l i e d by 2 and subtracted from the 410 nm reading. Since the average mean r a t i o of Abs. 1 r t/Abs obtained by 4J.0 4oU Crossley was 1.38 (S.D. of 0.20) the background interference would appear to be overcompensated for i n some t e s t s . Because of a concern that t h i s overcompensation may lead to f a l s e p o s i t i v e s an i n v e s t i g a t i o n was made in t o t h i s background interference c o r r e c t i o n procedure. Various f e c a l samples, representing a wide range of background colours and a wide range of t r y p s i n concentrations were stud i e d . These samples, 48 pa t i e n t samples and 17 c o n t r o l samples, were tested with the Crossley method with and without the ad d i t i o n of the substrate, BAPNA. 2. Test Banks Since f e c a l pigments and t u r b i d i t y appear to i n t e r f e r e with obtaining a r e l i a b l e absorbance reading for the produced p - n i t r o a n i l i n e , a c o r r e c t i o n blank for each t e s t , or a TEST BLANK, was considered. These blanks, each containing a sample d i s c and 0.8 ml of b u f f e r , were - 67 -incubated overnight with t h e i r respective t e s t s o l u t i o n s ; d i l u t e d with 2.2 ml of d i s t i l l e d water and read spectrophotoraetrically. Both the t e s t s and the t e s t blanks were read only at 410 nm. At 410 nm the absorbance of the t e s t represents the yellow p - n i t r o a n i l i n e produced by the t r y p s i n enzyme and al s o any f e c a l pigment and t u r b i d i t y that may be present. The absorbance of the t e s t blank would be due to only the f e c a l pigment and t u r b i d i t y r e l a t e d to that p a r t i c u l a r f e c a l sample. The subtrac t i o n of the Test Blank Abs... from the Test , 4 1 0 A b s ^ g would therefore c o r r e c t for the presence of any f e c a l pigment and t u r b i d i t y . The 460 nm reading was therefore no longer required. Several t e s t blanks (2 or 3) were prepared for each t e s t i n order to check i n t o the r e p r o d u c i b i l i t y of the absorbance readings of the blanks. 3. T u r b i d i t y A f t e r the 17 to 18 hr. incubation period, the t e s t s were evaluated by eye and 2.2 ml of d i s t i l l e d water was added to those that needed to be read spectrophotometrically. The force of the water stream mixed the s o l u t i o n so that an even d i s t r i b u t i o n of colour r e s u l t e d . The s t o o l p a r t i c l e s themselves however were al s o f l o a t i n g i n the mixture and these i n t e r f e r e d with the spectrophotometrie reading. The tubes were therefore allowed to stand for 15 to 20 minutes, during which time most of the debris s e t t l e d to the bottom of the tube leaving a c l e a r s o l u t i o n . A few t e s t s , approximately 1%, remained t u r b i d and these were centrifuged to c l e a r the s o l u t i o n . - 68 -Doubling the Substrate Concentration Crossley performed the f i r s t assay using 0.25 mg L-BAPNA per t e s t . Re-assaying was done i n du p l i c a t e using 0.25 mg and 0.50 mg L-BAPNA per t e s t . She reports that by re-analyzing the p o s i t i v e samples with the more concentrated s o l u t i o n of BAPNA the frequencey of p o s i t i v e r e s u l t s was reduced from 0.4 to 0.1% This concept was evaluated: a) T h i r t y samples that gave net absorbance values of 0.300 or l e s s on a l l four assays ( p o s i t i v e s had been re-assayed i n t r i p l i c a t e ) were re-tested using 0.25 mg BAPNA and 0.50 mg BAPNA. The r e s u l t s of the 0.50 mg BAPNA were compared to the 0.25 mg BAPNA substrate r e s u l t s . b) Working standards of various concentrations of t r y p s i n were tested with the Crossley method using 0.25 and 0.50 mg L-BAPNA per t e s t i n order to in v e s t i g a t e the k i n e t i c s of the enzyme r e a c t i o n . V a l i d i t y of V i s u a l Evaluation of Yellow I n t e n s i t y In the method, as o u t l i n e d e a r l i e r on page 64 under Procedure, spectrophotmetrie readings are to be taken only on those t e s t s that appear l e s s intense i n yellow colour than the 1.0 microgram standard (representing approximately 15 units of t r y p s i n a c t i v i t y ) or that appear to have an unusual colour t i n t when examined by eye. This v i s u a l e valuation of the yellow i n t e n s i t y of the t e s t s was recommended by Crossley. - 69 -In order to e s t a b l i s h the v a l i d i t y of t h i s v i s u a l evaluation, a l l of the t e s t s were evaluated both v i s u a l l y and spectrophotometrically during t h i s p i l o t p r o j e c t . D e tailed comparisons were made of the v i s u a l and spectrophotometric readings on two sets of samples (approximately 500 samples per set) at d i f f e r e n t times, to e s t a b l i s h c o r r e l a t i o n between the v i s u a l evaluation, the 410 nm absorbance reading and the f i n a l r e s u l t . The time periods chosen were: one at the beginning of the pro j e c t (May, 1978) and one when a new techno l o g i s t was performing the t e s t (August, • 1978) . I I . RESULTS A. I n v e s t i g a t i o n to E s t a b l i s h Procedure Background Interference 1. F e c a l Pigments Results from the i n v e s t i g a t i o n i n t o a representative sample of f e c a l specimens with a wide v a r i e t y of background pigments and a wide range of t r y p s i n concentrations are l i s t e d i n Table V. The absorbance readings at wavelengths 410 and 460 nm are given for the samples tested with and without the substrate BAPNA. The absorbance readings at 410 nm for the t e s t s performed without BAPNA (Column b) represent the readings for the f e c a l background int e r f e r e n c e at that wavelength. The absorbance readings at 410 nm for the te s t s performed with BAPNA represent the absorbance due to the f e c a l TABLE V. FECAL PIGMENT BACKGROUND INTERFERENCE, CROSSLEY METHOD SAMPLE WITH BAPNA WITHOUT BAPNA DESCRIPTION* absorbance absorbance Colour of Test 410nm 460nm Net Colour of Test 410nm 460nm Ratio % Solution a Solution b c b/c Error Standard: 1.0 microgram YELLOW 1.55 .072 1.487° colourless 0 0 I " Stool Samples: brown, normal YELLOW 1.675 0.164 1.347+ pale yellow 0.106 0.066 1.61 1 '-3 dark brown YELLOW 1.756 0.132 1.492 pale yellow 0.141 0.081 1.74 1 -1 greyish brown hint of yellow 0.032 0.007 0.018 hint of yellow 0.014 0.008 1.75 1 -8 shiny, dark brown brownish yellow 1.400 0.450 0.500 brownish yellow 0.540 0.320 1.69 ]-21 light brown pale 0.150 0.012 0.126 colourless 0.005 0.003 1.67 1 " 2 greenish brown YELLOW 1.621 0.116 1.389 hint of yellow 0.037 0.015 2.47 I +2 green greenish yellow 1.636 0.108 1.420 light green 0.050 0.029 1.72 1 -1 green YELLOW 1.630 0.097 1.436 hint of yellow 0.027 0.020 1.35 ! _ 1 green hint of yellow 0.052 0.012 0.028 hint of yellow 0.020 0.011 1.82 1 -6 shiny, greenish brown greenish yellow 2.078 0.395 1.288 1 marked green 0.465 0.302 1.54 '-10 greenish yellow YELLOW 1.662 0.094 1.474 hint of yellow 0.042 0.020 2.10 ' 0 yellow YELLOW 1.595 0.087 1.421 1 hint of yellow 0.024 0.020 1.20 -1 yellowish brown YELLOW 1.676 0.138 1.400 light yellow 0.109 0.079 1.38 ! -3 In the Crossley t r y p s i n method, p - n i t r o a n i l i n e i s measured at 410nm and 2 A b s 4 6 0 i s subtracted from the A b s 4 i 0 to compensate for f e c a l background interference on the assumption that the pigments absorb twice as much at 410nm as at 460 nm. The % error caused by t h i s assumption, instead of using the actual r a t i o (b/c), i s reported i n the l a s t column. o + visual appearance of stool on f i l t e r paper screening cards, average readings of 5 determinations for the standard average readings of a minimum of 3 determinations of the stool samples. - 71 -background in t e r f e r e n c e and the p - n i t r o a n i l i n e produced as a r e s u l t of the enzyme degradation of BAPNA. Crossley claims that the 460 nm readings i n the t e s t s performed with BAPNA (Column a), when m u l t i p l i e d by 2, provides an estimate of the f e c a l background interferance at 410 nm present under normal conditions of the t e s t . As a r e s u l t , Table V includes a column that reports the % error " caused by using t h i s f a c t o r of 2 i n the f e c a l pigment c o r r e c t i o n for a l l s t o o l samples even though there i s considerable v a r i a t i o n i n pigment content among the samples. (See Appendix C for the c a l c u l a t i o n of the % e r r o r ) . In order to c a l c u l a t e the % error i t was necessary to compare the absorbance readings of the f e c a l pigments at 410 nm (Column b) to the aborbance of the f e c a l pigments at a wavelength of 460 nm (Column C), both i n the absence of p - n i t r o a n a l i n e . The r a t i o A b s 4 1 o / / A b s 4 6 0 f ° r 3 s e l e c t i o n of f e c a l pigments i s included i n Table V, Column b/c. This table includes only a few r e s u l t s of the 65 specimens tested. The r a t i o v a r i e d from 0.92 to 2.47 with an average of 1.48 and an S.D. of 0.385 for the 65 specimens tested with and without BAPNA. Four of the specimens had an A b s 4 1 Q / A b s 4 6 0 r a t i o over 2.0, ranging from 2.09 to 2.47. 2. Test Blanks Absorbance readings from various pigmented f e c a l samples, which produced a r e l a t i v e l y high f e c a l background i n t e r f e r e n c e , and t h e i r respective t e s t blanks are represented i n Table VI (Samples no. 1 to 4). The table also includes the absorbance readings for both t e s t and t e s t blanks for seve r a l c o n t r o l specimens (Samples No. 5 to 8). I - 72 -TABLE VI. TESTS AND THEIR CORRESPONDING TEST BLANKS IN THE CROSSLEY METHOD Sample Colour of Test Absorbance at 410 nm No. S o l u t i o n Test Test Blanks i i i i i i 1 c l e a r l y YELLOW 2.131 .290 .303 2 YELLOW 2.138 .170 .185 3 YELLOW 2.238 .356 .365 4 orange-yellow 0.087 .072 .043 5 greenish YELLOW 2.243 .022 .013 6 c l e a r l y YELLOW 1.640 .059 .041 .072 7 yellow 0.358 .016 .017 .016 8 h i n t of yellow 0.053 .022 .015 .013 - 73 -3. T u r b i d i t y C e n t r i f u g a t i o n c l e ared the t e s t s o l u t i o n s i n most s i t u a t i o n s . In those few t e s t s where the t e s t s o l u t i o n was s t i l l t u r b i d a f t e r c e n t r i f u g a t i o n , re-assay was necessary. I f the t u r b i d i t y was due to a heavy f e c a l smear on the screening card, the sample was re-assayed using a smaller s t o o l sample. Temperature a f f e c t e d the t u r b i d i t y of the t e s t s o l u t i o n s . This was noticed a f t e r incubating t e s t s at room temperature overnight for the usual 17 to 18 hours'in the middle of the summer. The nights were very warm and t h i s increase i n temperature caused an increase i n the number of t u r b i d t e s t s that d i d not c l e a r when cen t r i f u g e d . This problem may have been one of b a c t e r i a l growth and was avoided by incubating the t e s t s i n an environment where the temperature remained f a i r l y constant and close to normal room temperature (21°C). Doubling the Substrate Concentration The r e s u l t s from a random sample of 10, out of the 30 samples tested i n d u p l i c a t e with 0.25 mg and 0.50 mg L-BAPNA per t e s t are l i s t e d i n Table V I I . One of the samples i n Table VII, no. 8, shows a decreased net absorbance value for the 0.50 mg L-BAPNA t e s t r e s u l t as compared with the 0.25 mg L-BAPNA t e s t r e s u l t . There were a t o t a l of 4 samples i n the 30 retested that gave a s i m i l a r decreased net absorbance value when the an a l y s i s was repeated using the more concentrated substrate s o l u t i o n . The decrease ranged from 5.9% to 55% with a mean of 30%. - 74 -TABLE V I I . LOW TRYSPIN ACTIVITY SAMPLES* TESTED WITH CROSSLEY METHOD USING 0.25 AND 0.50 MG L-BAPNA PER TEST Sample No. Net Absorbance Values"*" % 0.25 mg 0.50 mg Increase 4 8 15 16 18 20 23 24 26 28 .248 .213 .107 .250 .244 .136 .285 .118 .212 .183 .689 .201 .129 .785 .491 .167 .464 .331 .317 .192 178 -5.9 21 214 101 23 63 181 50 4.9 * Samples that r e s u l t e d i n net absorbance values l e s s than 0.300 when i n i t i a l l y tested with the Crossley method as o u t l i n e d . + Results given represent the mean value of duplicate t e s t i n g - 75 -Results obtained when working standards of various concentrations of t r y p s i n were tested with the Crossley method using a 0.25 mg and 0.50 mg L-BAPNA per t e s t are p l o t t e d i n Figure I. V a l i d i t y of V i s u a l Evaluation of Yellow I n t e n s i t y The data from the study i n which two sets of spectrophotometric readings were compared to v i s u a l evaluations were e s s e n t i a l l y the same. As a ^ r e s u l t only one set of data i s presented. The second d e t a i l e d study (August, 1978) i s presented i n Table V I I I . In the d e t a i l e d study the v i s u a l evaluation of the t e s t s involved d i f f e r e n t i a t i n g between a c l e a r b r i g h t yellow as produced by the 1.0 /jg standard, representing approximately 15 un i t s of t r y p s i n a c t i v i t y (recorded as "YELLOW" i n Table V I I I , i n d i c a t i n g that the t e s t colour was considered to be equal to or greater than the 1.0 jag standard), "LIGHT" yellow when the i n t e n s i t y was l e s s than that of the standard but s t i l l an obvious yellow, "PALE" yellow when only a h i n t of the colour was present, "COLORLESS" and "TINTED". The l a t t e r r e f e r r e d to the presence of either a brownish or greenish f e c a l pigment or other abnormal t i n t . B. S e n s i t i v i t y and S p e c i f i c i t y The f e c a l t r y p s i n screen using Crossley's method d i d not detect any CF c h i l d r e n (Tables IX and X). The s e n s i t i v i t y of t h i s method has therefore not been est a b l i s h e d to date. 76 -FIGURE I. 2.8 Z.6 2.4 2.2 2.0 1.8 1.6 H 1.4 % 1.2 8 i . o 3 0.8 0.6 0.4 0.2 TRYPSIN ACTIVITY WITH TWO DIFFERENT BAPNA SUBSTRATE CONCENTRATIONS -0.25 mg BAPNA/Test -0.50 mg BAPNA/Test 2 4 6 8 10 12 14 UNITS OF TRYPSIN ACTIVITY The broken l i n e represents the r e l a t i o n s h i p between absorbance and t r y p s i n concentration using the Crossley method with 0.25 mg of L-BAPNA per t e s t . The s o l i d l i n e was determined using 0.50 mg of L-BAPNA per t e s t . Various concentrations of standards were used f o l l o w i n g the method as o u t l i n e d under the procedure. TABLE VIII. COMPARISON OF VISUAL WITH SPECTROPHOTOMETRIC EVALUATIONS OF 522 TESTS : CROSSLEY METHOD VISUAL EVALUATION SPECTROPHOTOMETRIC READINGS / / COLOUR No. % Abs. at 410nm No. Ab s410 ~ 2 20.300 (Abs 460) <0.300 / STD. 1.0 ug YELLOW 9 1.390-1.605 9 9 -TESTS YELLOW equal or greater than s t d . 375 71.8 1.390-2.078 0.561-1.389 247 128 247 128 -LIGHT yellow 59 11.3 0.365-1.099 0.331-0.336 57 2 57 2 PALE yellow 22 4.2 0.354 0.121-0.356 1 21 1 21 COLOURLESS 3 0.6 0.84-0.87 3 - 3 TINTED brownish or greenish 68 13.0 0.315-1.894 0.183-0.637 59 9 59 9 The Crossley method for f e c a l t r y p s i n i s designed so that only those t e s t s which appear to be a l i g h t e r yellow than the standard or have a d i f f e r e n t t i n t are read spectrophotometrically. If the net absorbance i s l e s s than 0.300, the specimen i s considered to have an abnormally low t r y p s i n concentration (35/522=6.7% p o s i t i v e on f i r s t assay i n t h i s small sample). - 78 -TABLE IX. FECAL TRYSPIN SCREENING TEST FOR CF: HOSPITAL COLLECTED SPECIMENS Diagnosis Crossley BAPNA Test Result CF Non-CF T o t a l P o s i t i v e 0 160 160 Negative 0 3,050 3,050 0 3,210 3,210 TABLE X. FECAL TRYPSIN SCREENING TEST FOR CF: HOME COLLECTED SPECIMENS Diagnosis Crossley BAPNA Test Result CF Non-CF T o t a l P o s i t i v e 0 30 30 Negative 0 845 845 0 875 875 The s p e c i f i c i t y of the f e c a l t r y p s i n screen was c a l c u l a t e d to be 95.0% for the h o s p i t a l (VGH) c o l l e c t e d specimens (Table IX) and 96.6% for the specimens mailed i n from home (Table X). The f a l s e p o s i t i v e rates were 5.0 and 3.4% r e s p e c t i v e l y and the chi-square t e s t i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e between these two rates ( c a l c u l a t i o n i n Appendix G) . - 79 -C. Age of Infant When Ho s p i t a l Specimen Was C o l l e c t e d A large proportion of mothers and t h e i r i n f a n t s were discharged from the h o s p i t a l on the 4th day a f t e r b i r t h and as a consequence of t h i s the nurses ran i n t o d i f f i c u l t i e s with obtaining a s t o o l specimen from the baby on day 4. Approximately 49% of the specimens received were from in f a n t s that were l e s s than 3 days o l d when the specimen was c o l l e c t e d . Of the CF screen " p o s i t i v e " population 78% were l e s s than 3 days o l d . There were however only 39% under 3 days of age i n the group that tested negative with the CF screen. This i s a d i f f e r e n c e of 39%; a high l y s i g n i f i c a n t d i f f e r e n c e i n d i c a t i n g that there i s a r e l a t i o n s h i p between the p o s i t i v e r e s u l t s and the high proportion of inf a n t s under 3 days of age (Appendix H) . D. H o s p i t a l and Home C o l l e c t e d Specimens From the Same Infant During a 10 month time period, 692 inf a n t s were tested by the f e c a l t r y p s i n screening method on both VGH and home c o l l e c t e d specimens. One in f a n t displayed low f e c a l t r y p s i n a c t i v i t y i n both specimens. Further follow-up however indicated that these r e s u l t s were both f a l s e p o s i t i v e s for the c h i l d d i d not appear to have CF. The f a l s e p o s i t i v e rates for these r e s u l t s from t e s t s performed on the same in f a n t s were 5.1% f o r the VGH c o l l e c t e d specimens and 2.9% for the specimens mailed i n from home (Table XI). - 80 -TABLE XI. FECAL TRYPSIN SCREENING TEST FOR CF: HOSPITAL AND HOME COLLECTED SPECIMENS ON THE SAME INFANT VGH + + 1 21 22 HOME 34 636 670 35 657 692 Results of a McNemar t e s t for the s i g n i f i c a n c e of changes (from p o s i t i v e to negative and v i c e versa between VGH and home c o l l e c t e d specimens) i n d i c a t e s no s i g n i f i c a n t d i f f e r e n c e between the two c o l l e c t i o n methods as far as s p e c i f i c i t y i s concerned ( c a l c u l a t i o n i n Appendix I ) . E. Comparison of Ho s p i t a l and Home C o l l e c t e d Specimens The net absorbance values of a representative sample of VGH c o l l e c t e d specimens were compared with representative sample of home c o l l e c t e d specimens covering the same time period. The d i s t r i b u t i o n s of the net absorbance readings are displayed i n Figure I I . An a p p l i c a t i o n of the Chi-square t e s t i n d i c a t e d that there was a s i g n i f i c a n t d i f f e r e n c e between the two populations of absorbance values and the means were al s o shown to be s i g n i f i c a n t l y d i f f e r e n t (Appendix J ) . - 81 -FIGURE I I . COMPARISON OF NET ABSORBANCE VALUES FROM FIRST ASSAY OP HOSPITAL AND HOME COLLECTED SPECIMENS .3 .6 .9 1.2 1.5 1.8 Net absorbance: Hospital c o l l e c t i o n .3 .6 .9 1.2 1.5 1.8 Net absorbance: Home co l l e c t i o n - 82 -The VGH group represents 215 p o s i t i v e r e s u l t s obtained on the f i r s t assay which decreases to 160 p o s i t i v e r e s u l t s when the sample i s retested i n t r i p l i c a t e . This i s a decrease of 25.6%. The home group decreases 47.4% from 57 p o s i t i v e s i n the f i r s t assay to 30 p o s i t i v e s a f t e r r e - t e s t i n g . Four p o s s i b l e reasons for the lower net absorbance values i n the home c o l l e c t e d specimens are: l o s s of enzyme a c t i v i t y i n the s t o o l sample due to the mailing process, increase i n f e c a l background interference i n s t o o l sample c o l l e c t e d from older c h i l d at home, uneven spread of specimen and/or thinner spread of specimen on home screening card. These four p o s s i b l e reasons for the s i g n i f i c a n t l y lower net absorbance values i n the home c o l l e c t e d specimens were in v e s t i g a t e d . 1. Comparison of Freezer to Room Temperature Stored and Maile d - i n Specimens a) Co n t r o l specimens (6) and newborn specimens (14) were stored at room temperature and i n the freezer for varying time periods from 3 to 7 days and analyzed by the Crossley method. Of the 20 specimens, 12 displayed a decrease i n enzyme a c t i v i t y a f t e r the storage at room temperature. The Sign t e s t was applied and ind i c a t e d that there was a 25.1% p r o b a b i l i t y that the decrease i n r e s u l t s on the room temperature stored specimens was due to random error alone (Section 1, Appendix K). b) As a check on the s t a b i l i t y of the t r y p s i n a c t i v i t y i n the mail, 25 specimen cards were prepared (controls and healthy newborn i n f a n t s specimens) and mailed to Children's H o s p i t a l from various areas i n - 83 -Greater Vancouver. Trypsin determinations using Crossley's screening method were performed before and a f t e r m a i l i n g . A decrease i n t r y p s i n a c t i v i t y was displayed by 14 out of the 25 "mailed-in" cards. The sign t e s t was applied to the r e s u l t s and ind i c a t e d that there was a 27.0% p r o b a b i l i t y that the decrease could be due to random error alone (Section 2, Appendix K). c) Since a decrease i n t r y p s i n a c t i v i t y was obtained i n both the room temperature stored specimens and the mailed-in specimens (also at room temperature), the data of these two groups were combined and the Sign t e s t was ap p l i e d . There was a 14.6% p r o b a b i l i t y that the decrease i n room temperature stored specimens was due to random error alone (Section 3, Appendix K). 2. Comparison of Fe c a l Pigment Interference i n H o s p i t a l and Home C o l l e c t e d Specimens I t was postulated that the lowered net absorbance values from the home c o l l e c t e d specimens could be p a r t i a l l y due to the presence of a lar g e r concentration of f e c a l pigment compared to the VGH c o l l e c t e d specimens. This increased amount could lead to lower net absorbance values when 2 Abs... i s subtracted from the Abs... 460 410 (overcompensation). To in v e s t i g a t e t h i s p o s s i b i l i t y the absorbance values at 460 nm from a representative sample of VGH c o l l e c t e d specimens and home c o l l e c t e d specimens were compared. The r e s u l t s from each of these samples are presented i n Figure I I I . These bar graphs v i s u a l l y i n d i c a t e that the home Ab s ^ g values appear to be smaller than the VGH Abs^,, values. S t a t i s t i c a l l y i t was shown that the home Abs A j ; f l - 84 -FIGURE III. COMPARISON OF ABSORBANCE VALUES AT 460 NM FROM HOSPITAL AND HOME COLLECTED SPECIMENS - 85 -values had a s i g n i f i c a n t l y lower mean and that the d i s t r i b u t i o n had s h i f t e d to the l e f t . The d i f f e r e n c e between the means, 0.0115, i s more than 3 standard deviations greater than 0.* That t h i s s h i f t i s highly s i g n i f i c a n t was v e r i f i e d by a Chi-Square t e s t (Appendix L ) . 3. P r e c i s i o n of Repeated A n a l y s i s on the Same Screening Card Poor preparation of screening cards at home with uneven thickness of the s t o o l sample could p o s s i b l y lead to wider d i s p e r s i o n of r e s u l t s compared to the VGH c o l l e c t e d r e s u l t s . This would a l s o r e s u l t i n poor p r e c i s i o n of the four determinations performed on each of the f i r s t assay p o s i t i v e specimens ( i . e . poor p r e c i s i o n when r e - t e s t i n g ) . The standard deviations of these four determinations on the f i r s t assay p o s i t i v e s of the home c o l l e c t e d specimens were compared with the VGH c o l l e c t e d specimens. Because there were i n d i c a t i o n s that the S.D. v a r i e s with the absorbance range, only those specimens that gave a net absorbance value between 0.250 and 0.300 were used for the comparison. Results from 37 VGH samples (4 determinations for each sample) were compared with 10 sets of r e s u l t s from home c o l l e c t e d samples and no s i g n i f i c a n t d i f f e r e n c e i s S.D.'s was detected (Appendix M) between the two groups. V i s u a l examination of the screening cards prepared at home al s o i n d i c a t e d that the cards prepared at home by the mothers appeared to be equal i n consistency of spread to those prepared by the nurses at VGH. * By the c e n t r a l l i m i t theorem, the S.D. of the d i f f e r e n c e between the means i s .00297 2 + .00245 2 = .00371 - 86 -4. Thinner Spread on Home C o l l e c t e d Cards There are two po s s i b l e reasons to suspect that the quantity of f e c a l sample on the screening cards from home may be l e s s than from VGH. a) A large proportion of the t o t a l number of s t o o l samples from VGH were t r a n s i t i o n a l s t o o l samples ( t r a n s i t i o n from meconium to stool) which are f a i r l y dense samples. The s t o o l samples passed by the s l i g h t l y older i n f a n t a t home tend to be more l i q u i d , and would therefore coat the screening card i n a thinner l a y e r . b) Mothers might be a l i t t l e more r e l u c t a n t than the nurse to spread a pea s i z e sample on the screening card. The d i f f e r e n t d i s t r i b u t i o n of home r e s u l t s could therefore be due to l e s s sample on a majority of the screening cards prepared at home. V i s u a l examination of the home c o l l e c t e d screening cards i n d i c a t e d that although the thickness of the s t o o l sample d i d not warrant the use of 2 d i s c s per t e s t , the majority of them d i d appear to be s l i g h t l y decreased iri thickness compared to the VGH cards. I I I . DISCUSSION The second method to be investigated for incorporation i n t o a f e c a l t r y p s i n CF screen, the Crossley et a l method, proved to be r e l i a b l e and became part of a p i l o t p r o j e c t at VGH a f t e r a thorough i n v e s t i g a t i o n of some of the problem areas and questionable steps i n the procedure. - 87 -In v e s t i g a t i o n to E s t a b l i s h Procedure 1. Background Interference C o r r e c t i o n Procedure a) F e c a l Pigments Crossley's background c o r r e c t i o n procedure, where 2Abs^g 0 i s subtracted from A b s 4 1 Q i s based on two of her f i n d i n g s : i) the endogenous absorbance at 410 nm was never more than twice that at 460 nm. i i ) p - n i t r o a n i l i n e had an absorbance at 460 nm about 10% of that at 410 nm. Our r e s u l t s v a r i e d s l i g h t l y from hers. The absorbance readings of p - n i t r o a n i l i n e are dependent upon the instrument and the chemical p u r i t y . The p - n i t r o a n i l i n e used i n our laboratory gave an average absorbance reading at 460 nm of approximately 4.4% of that at 410 nm. This means that when the c o r r e c t i o n procedure i s used that i n ad d i t i o n to subtracting the f e c a l pigment absorbance some of the absorbance due to the p - n i t r o a n i l i n e i s a l s o being subtracted ( i . e . 2 x 4.4% Abs^g) bi a s i n g the net absorbance. This a d d i t i o n a l s u b t r a c t i o n a l t e r s the net absorbance by a constant percentage however and i s therefore acceptable since i t does not a f f e c t the ranking of the r e s u l t s . A l l i t means i s that approximately 91% of the yellow colour due to the p - n i t r o a n i l i n e i s estimated i n a l l of the t e s t s . Most, but not a l l , of the r a t i o s of Abs.../Abs... of the various 410 4oU f e c a l pigments from 65 newborn s t o o l samples studied i n our laboratory were lower than the factor of 2 employed i n the c o r r e c t i o n procedure. Presumably using a factor of 2 should therefore c o r r e c t for a very high - 88 -percentage of pigments but not a l l . I f f a l s e negatives are to be avoided altogether however i t would appear that a larger factor (e.g. 2.5) should be used. This larger factor would however increase the p r o b a b i l i t y of f a l s e p o s i t i v e s i n samples with a lower Abs.,./Abs... r a t i o due to r 410 460 i t s p a r t i c u l a r f e c a l pigment i f t h i s i s combined with a t r y p s i n a c t i v i t y ( l e v e l at a low normal l e v e l . In the representative sample given i n Table V on page 70, 11 of the 13 samples had a r a t i o of A b s 4 1 Q / A b s 4 6 0 l e s s than 2. The percentage error reported for these 11 specimens i n d i c a t e that the use of the factor 2.0, instead of the lower factor determined for those p a r t i c u l a r pigments, r e s u l t e d i n overcompensation. In a l l 11 samples the % error i s on the negative side; i . e. the c a l c u l a t e d net absorbances a f t e r the f,ecal pigment c o r r e c t i o n were lower than they should have been. The degree of error of the net absorbance value obtained using Crossley's c o r r e c t i o n procedure i s dependent on s e v e r a l f a c t o r s : i . the larger the d e v i a t i o n between 2.0 and the a c t u a l r a t i o , the larger the % e r r o r . i i . the greater the concentration of f e c a l pigment the larger the error because the_ % error = 1 - 2.0 concentration of f e c a l pigment. true r a t i o i i i . the % error i s greater i n those s t o o l specimens with low t r y p s i n concentration because the proportion of f e c a l pigment to concentration of p - n i t r o a n a l i n e i s increased. ( i . e . the f e c a l pigment absorbance i s a larger f r a c t i o n of the t o t a l absorbance at 410 nm) - 89 -The e f f e c t s of the above mentioned f a c t o r s are evident i n the % error r e s u l t s reported i n Table V. A l l but two of the e r r o r s obtained i n that representative sample of 13 t e s t s were within a reasonable l e v e l , e s p e c i a l l y since the method i s not a q u a n t i t a t i v e one. The concern i s that the % error does not cause a low normal to drop below the c u t - o f f point and become a f a l s e p o s i t i v e ; A larger error such as the -21% error obtained i n the worst case i s however p o s s i b l e and could p o t e n t i a l l y lead to f a l s e p o s i t i v e r e s u l t s i n a specimen with a borde r l i n e t r y p s i n a c t i v i t y l e v e l . In the t o t a l number of pigmented samples tested with and without BAPNA (65) , an error of 10% or more due to overcompensation occurred i n 5 samples (7.7% of the samples). Since only those t e s t s o l u t i o n s which tyave an unusual t i n t or appear to be l e s s intense i n yellow colour than the 1.0 ug standard (approximately 15 units of t r y p s i n a c t i v i t y ) when examined by eye are read spectrophotometrically, the c o r r e c t i o n factor w i l l be used on only approximately 30% of the t o t a l number of specimens tested. Overcompensation i s however only a p o s s i b i l i t y i n those t e s t s o l u t i o n s which contain a considerable amount of pigment and these are present i n approximately 15% of the t o t a l number of tes t s (Table V I I I , page 77). A rough estimate of the p o t e n t i a l number of f a l s e p o s i t i v e s r e s u l t i n g from the use of t h i s c o r r e c t i o n procedure i s , therefore, 7.7% of 15% = 1.2% of the t o t a l number tested. Only those s t o o l specimens which d i s p l a y low normal l e v e l s of t r y p s i n are i n danger of becoming f a l s e p o s i t i v e s as a r e s u l t of t h i s overcompensation e r r o r . Approximately 50% of the specimens evaluated v i s u a l l y as " t i n t e d " had low absorbance readings at 410 nm. A conservative estimate, therefore, of - 90 -the percentage of t e s t s that could p o t e n t i a l l y r e s u l t i n a f a l s e p o s i t i v e as a consequence of the e r r o r i n the pigment c o r r e c t i o n procedure i s 0.6% This i s an acceptable l e v e l f or a CF screen. The i n v e s t i g a t i o n i n t o the f e c a l pigment c o r r e c t i o n procedure was performed on the h o s p i t a l c o l l e c t e d specimens. The home c o l l e c t e d specimens appear to contain a smaller number of strongly pigmented s t o o l specimens. As a r e s u l t , l e s s than a 0.6% f a l s e p o s i t i v e r a t e , due to the c o r r e c t i o n procedure e r r o r , i s expected for the home c o l l e c t e d specimens. b. Test Blanks and T u r b i d i t y The use of a Test Blank f o r each t e s t could t h e o r e t i c a l l y eliminate the e r r o r due to endogenous absorbances, which contribute to the t e s t absorbance at 410 nm. The f e c a l pigment and t u r b i d i t y may contribute s i g n i f i c a n t l y . The concept of incorporating a Test Blank i n t o the procedure was however discarded for two reasons: i . The Test Blank absorbance readings reported i n Table VI on page 72, appear to be f a i r l y imprecise as a r e s u l t of the semi-q u a l i t a t i v e sampling technique used. This method of c o r r e c t i n g can only be accurate i f the Test Blank d i s c contains the same amount of feces as the Test d i s c . Two Blanks prepared concurrently gave imprecise r e s u l t s due to the v a r i a b i l i t y of the thickness of feces on the screening card d i s c s . This method was therefore not an improvement with respect to % error to Crossley's c o r r e c t i o n procedure. i i . The i n t r o d u c t i o n of a t e s t blank c o n t r a d i c t s one of the e a r l i e r requirements for a screening t e s t procedure: that the procedure should be simple and require very l i t t l e time to set up. - 91 -Although a t e s t blank i s necessary only for those t e s t s that need to be read spectrophotometrically (30% of the t o t a l number tes t e d ) , the blank would need to be prepared for a l l of the t e s t s since there i s no way of p r e d i c t i n g which t e s t s would or would not have to be read on the spectrophotometer ahead of time. A l t e r n a t i v e l y , to set up a t e s t blank l a t e r would mean the t e s t would have to be repeated alongside of i t for the two need to be incubated under the same conditions and for the same length of time. Since i t i s p o s s i b l e to eliminate the t u r b i d i t y i n most t e s t s o l u t i o n s by temperature c o n t r o l during the incubation period and by allowing the t e s t s o l u t i o n s to stand for 15 to 20 minutes p r i o r to the spectrophotometric reading a Test Blank i s no longer necessary to solve the t u r b i d i t y problem. For the few s o l u t i o n s that remained t u r b i d despite adherance to the above mentioned precautions c e n t r i f u g a t i o n c l eared the s o l u t i o n . Since very few t e s t s o l u t i o n s needed c e n t r i f u g a t i o n (less than 1%) t h i s increased the workload, a n e g l i g i b l e amount. I t i s important to c l e a r the s o l u t i o n s p r i o r to the spectrophotometrie reading because t u r b i d i t y could t h e o r e t i c a l l y r e s u l t i n f a l s e p o s i t i v e s (again only on specimens with low normal t r y p s i n l e v e l s ) using the c o r r e c t i o n procedure o u t l i n e d . An error occurs with t u r b i d s o l u t i o n s when twice the absorbance at 460 nm i s subtracted from the absorbance at 410 nm because the s c a t t e r i n g of l i g h t at 410 nm and a t 460 nm due to i n s o l u b l e p a r t i c l e s i s e s s e n t i a l l y equal. - 92 -2. Doubling the Substrate Concentration The i n v e s t i g a t i o n i n t o the procedure of using a substrate with twice the concentration for re-assaying p o s i t i v e t e s t s i n d i c a t e d that i t was not a v a l i d procedure to employ even though Crossley reports that i t reduced her p o s i t i v e rate by 3 percentage p o i n t s . The r e s u l t s from the 30 samples tested with both 0.25 mg and 0.50 mg L-BAPNA displayed a general increase i n net-absorbance values with the more concentrated substrate. T e c h n i c a l e r r o r s appear to be the only explanation for the 5 samples that gave a decreased net absorbance value when re-assayed. Because of t h i s discrepancy a rank t e s t was applied to the absorbance values obtained for both assays and t h i s s t a t i s t i c a l t e s t i n d i c a t e d that a s i g n i f i c a n t increase i n values was obtained when the t e s t s were re-assayed with the 0.50 rag BAPNA (see Appendix 0, Section 1) . The % d i f f e r e n c e between the mean of the 0.25 mg BAPNA r e s u l t s and the 0.50 mg BAPNA r e s u l t s was c a l c u l a t e d for each sample and a l i n e a r regression a n a l y s i s performed. No s i g n i f i c a n t c o r r e l a t i o n was found between % increase and the absorbance reading (see Appendix 0, Section 2). The r e s u l t s obtained can be explained i n terms of the enzyme k i n e t i c s of the r e a c t i o n involved. Since higher absorbance values were obtained when the t e s t s were re-assayed with the more concentrated substrate, the enzyme r e a c t i o n could not have been i n zero order k i n e t i c s . This i s not s u r p r i s i n g since the r e a c t i o n i s allowed to proceed for 17 to 18 hours. The substrate concentration w i l l most l i k e l y not remain i n excess for most of the samples during t h i s time period. However, i t appears as i f the substrate may have been i n excess for the - 93 -lower t r y p s i n concentrations since a l i n e a r r e l a t i o n s h i p between absorbance and t r y p s i n concentration was obtained for the low concentrations of standards (Figure I, page 76). This r e l a t i o n s h i p appears to hold up to the 0.7 u n i t t r y p s i n standard using 0.25 mg BAPNA and the 1.4 u n i t t r y p s i n standard using 0.50 mg BAPNA. With the 0.25 mg BAPNA, the substrate therefore appears to be i n excess for t e s t s with net absorbance values under 0.300. This absorbance value v a r i e s considerably from run to run as a r e s u l t of wavelength e r r o r * and v a r i a t i o n i n incubation time and temperature, (e.g. + 0.100 for the 1.0 g standard). A safe estimate would be that the l i n e a r r e l a t i o n s h i p between t r y p s i n concentration and absorbance holds for those specimens that gave a. net absorbance reading up to at l e a s t 0.200. This i n f e r s zero order k i n e t i c s and as a consequence of t h i s , the re a c t i o n rate i s independent of substrate concentration. Doubling the substrate concentration would therefore not s i g n i f i c a n t l y increase the amount of p - n i t r o a n i l i n e formed for these specimens. This would a l s o explain why Crossley found that the net absorbances of 7 C.F. samples were not s i g n i f i c a n t l y increased. Presumably the t r y p s i n concentrations of these C.F. specimens were s u f f i c i e n t l y low that the enzyme r e a c t i o n was e s s e n t i a l l y i n zero order. The t e s t s o l u t i o n s with higher concentrations of t r y p s i n , however, would be a f f e c t e d by the increase i n BAPNA. These specimens would have the r e a c t i o n i n f i r s t order for a large p o r t i o n of the incubation period * Wavelength error occurs because the wavelength chosen to read the p - n i t r o a n i l i n e , 410 nm, f a l l s on a steep slope of the s p e c t r a l absorbance curve of p - n i t r o a n i l i n e with i t s wavelength maximum at 385 nm. - 94 -and the increased substrate would therefore cause an increased production of p - n i t r o a n i l i n e . This would r e s u l t i n a reduction i n the number of p o s i t i v e s since t e s t s with net absorbance values c l o s e to 0.300 would now read considerably higher. This s i g n i f i c a n t l y increased production of p - n i t r o a n i l i n e w i l l occur however with a l l specimens that give a net absorbance value of approximately 0.200 or higher when re-assayed with the double substrate concentration. C F . specimens with low r e s i d u a l t r y p s i n may al s o convert from a p o s i t i v e to a negative. I t i s therefore not advisable to repeat the assay using double substrate. I t e s s e n t i a l l y amounts to the same thing as lowering the c u t - o f f point for the re-assay r e s u l t s . This c u t - o f f point was es t a b l i s h e d at 0.300 on the basis of 0.25 mg BAPNA per t^est to avoid f a l s e negatives and cannot be used with a 0.50 mg BAPNA per t e s t . A more l o g i c a l procedure to eliminate a screening error as a r e s u l t of poor p r e c i s i o n i s to repeat the p o s i t i v e samples i n t r i p l i c a t e and base a f i n a l d e c i s i o n on the average of the 4 net absorbance values although t h i s would increase the cost and time of screening. 3. V a l i d i t y of V i s u a l Evaluation of Yellow I n t e n s i t y Since i t i s d i f f i c u l t to judge colour i n t e n s i t y by eye and since t h i s judgement i s dependent upon background colours and i n d i v i d u a l colour perception an i n v e s t i g a t i o n i n t o the consistency of the eye evaluation procedure suggested by Crossley seemed necessary. - 95 -A comparison of the v i s u a l evaluation and the spectrophotometry readings of 522 random samples i n d i c a t e s that the eye evaluation i s r e l i a b l e and can be incorporated i n t o the screening procedure as a time and labour saving measure. / Approximately 70% of the samples tested appeared v i s u a l l y to be equal to or greater i n i n t e n s i t y than the 1.0 ;ug standard (representing approximately 15 units of t r y p s i n a c t i v i t y ) . These te s t s would therefore be considered normal and would not be c a r r i e d through the procedure any fu r t h e r . Almost l / 3 r d of those thought to be equal to the standard i n colour v i s u a l l y were a c t u a l l y l e s s intense when measured spectrophotometrically. I n d i v i d u a l d i f f e r e n c e s would a l t e r t h i s proportion. One tec h n o l o g i s t , for instance, became quite expert at evaluating the yellow i n t e n s i t y . A l l the t e s t s evaluated by her as YELLOW f e l l w i t h i n a f a i r l y narrow absorbance range of 1.0 to 2.0 at 410 nm while the absorbance range obtained for YELLOW by another technologist reached as low as 0.561. The technique of the f i r s t t e c hnologist was therefore adopted and a l l t e s t s were evaluated comparing a maximum of 4 t e s t s with the 1.6 ug standard against a white background. The error that was made on 30% of the v i s u a l evaluations by f a l s e l y r a t i n g them YELLOW would not have r e s u l t e d i n f a l s e negative screening r e s u l t s i f they had only been examined v i s u a l l y because a f a i r l y large safety margin e x i s t s . Not u n t i l the 410 nm absorbance drops to 0.331 i n the LIGHT yellow group d i d the net absorbance drop to a value below 0.300 gi v i n g a p o s i t i v e r e s u l t . Out of the 59 LIGHT yellow t e s t s only 2 f e l l i n t h i s category. - 96 -If the i n i t i a l eye evaluation had been r e l i e d on for the 522 t e s t s i n the study presented i n Table VIII and only those t e s t s that were not evaluated as YELLOW had been read, then the 35 t e s t s that gave net absorbance below 0.300 would a l l have been read spectrophotometrically. The eye evaluation screening procedure would therefore have been s u c c e s s f u l i n detecting a l l of the p o s i t i v e s i n that p a r t i c u l a r random sample. This was al s o the s i t u a t i o n i n the other random sample of approximately 500 t e s t s studied i n d e t a i l . Further evidence to v e r i f y the v a l i d i t y of the i n i t i a l eye evaluation s e l e c t i o n procedure was made by comparing the eye evaluation to the absorbance readings of a l l the p o s i t i v e t e s t s obtained i n t h i s f e c a l CF screen p i l o t p r o j e c t . On the f i r s t assay, 272 t e s t s out of the t o t a l 4081 specimen cards tested had a net absorbance of l e s s than 0.3 and were therefore presumptive p o s i t i v e s . Three of these t e s t s were considered to be YELLOW when evaluated v i s u a l l y . These 3 would therefore not have been read on the spectrophotometer and would have been considered normal leading to f a l s e negatives r e s u l t s . The three discrepancies were due to err o r s that were detected when the specimens were re-assayed. In one of the t e s t s the v i s u a l evaluation di d not agree with the absorbance readings simply because the absorbance readings were i n e r r o r . When the t e s t was repeated i n duplicate the v i s u a l and spectrophotometric readings agreed. The other two discrepancies were due to i n c o r r e c t v i s u a l evaluations. In both, the moderately high concentration of yellow p - n i t r o a n i l i n e noted d i d not agree with net absorbance values of around 0.2. When the t e s t s were repeated, i t was noticed that both t e s t - 97 -s o l u t i o n s had an unusual t i n t and that the l a t t e r had not been noticed on the f i r s t assay. These discrepancies occurred within two weeks a f t e r the procedure was i n i t i a t e d . I t drew a t t e n t i o n to the importance of v i s u a l l y examining the t e s t s o l u t i o n s for background pigments. These pigments i n t e r f e r e with the v i s u a l evaluation of the yellow i n t e n s i t y and i f present the t e s t must be read spectrophotometrically. S e n s i t i v i t y and S p e c i f i c i t y Since none of the i n f a n t s screened by the f e c a l t r y p s i n screen have been diagnosed as having CF, the s e n s i t i v i t y of the method has not been est a b l i s h e d to date. The method d i d however c o n s i s t e n t l y detect the abnormal c o n t r o l s prepared with s t o o l specimens from known CF i n f a n t s that displayed pancreatic i n s u f f i c i e n c y . The s p e c i f i c i t i e s of the h o s p i t a l and home c o l l e c t e d specimens were e s s e n t i a l l y the same with 95.0 and 96.6% r e s p e c t i v e l y . The two screening populations were therefore evaluated as one u n i t . The f a l s e p o s i t i v e rate on the f i r s t assay for t r y p s i n on the dry s t o o l specimens was 6.7% which was lowered to 4.7% when the t e s t was repeated on the same sample. The s p e c i f i c i t y of the Crossley method on both VGH and home c o l l e c t e d specimens combined was 95.3%. A f a l s e p o s i t i v e rate of 4.7% i s too high from both a f i n a n c i a l and e t h i c a l standpoint. Follow-up problems r e s u l t i n g from such a high f a l s e p o s i t i v e rate are discussed on page 128. - 98 -Sample C o l l e c t i o n The c o l l e c t i o n rate of f e c a l specimens at the Vancouver General H o s p i t a l was 68.7% which was much lower than that of the mecononium CF screen. The s t o o l specimen c o l l e c t i o n rate i s a l s o low compared with the sample c o l l e c t i o n of the PKU screen which i s operating i n the province of B.C. and has a c o l l e c t i o n rate of approximately 95%. If the CF screen i s to be e f f e c t i v e , the 68.7% rate must be increased. A p o s s i b l e reason for the low c o l l e c t i o n rate could have been the concurrently running meconium screen. I t may have drawn a t t e n t i o n away and de-emphasized the importance of the f e c a l screen since both were screening for CF. There i s no doubt that the high percentage (49%) of under 3 day o l d in f a n t s present i n the VGH population tested increased the f a l s e p o s i t i v e r a t e . T r y p s i n a c t i v i t y increases s i g n i f i c a n t l y within the f i r s t few days 5 37 of l i f e , ' as in d i c a t e d by the values i n ug per g reported by 6 3 Mullmger : day 1 - 7 3 , day 2 -135, day 3 -332 and day 4 - 291 jig per g. Afte r day 3 the t r y p s i n a c t i v i t y reaches a f a i r l y stable l e v e l . The CF screen should therefore be performed on s t o o l specimens from i n f a n t s that are at l e a s t 4 days o l d . Since many in f a n t s are discharged from the h o s p i t a l by the 4th day, i t i s necessary to c o l l e c t the s t o o l specimen to be screened at home. The home c o l l e c t i o n rate was lower, at an unacceptable l e v e l of 45.6%. No data are a v a i l a b l e to determine the cause of t h i s poor compliance r a t e . I t i s assumed that a high percentage ( i f not a l l ) of mothers received the request l e t t e r and the c o l l e c t i o n card when - 99 -discharged. However the mothers may have been influenced s l i g h t l y by the a t t i t u d e of the VGH personnel towards yet another CF screen (the t h i r d on the same c h i l d ! ) . There was some i n d i c a t i o n that a few physicians, when asked by the parents about the screen, responded with a lack of enthusiasm and discouraged the s t o o l c o l l e c t i o n . As the high f a l s e p o s i t i v e rate became apparent to the physicians with time the c r e d i b i l i t y of the screen became questionable. H o s p i t a l and Home C o l l e c t e d Specimens The high f a l s e p o s i t i v e rate of the h o s p i t a l c o l l e c t e d specimens (5.0%) was due mostly to the young age of the i n f a n t at time of - c o l l e c t i o n and to a small degree to the error produced by the f e c a l pigment c o r r e c t i o n procedure. A s i g n i f i c a n t decrease i n the f a l s e p o s i t i v e rate was expected with the home c o l l e c t e d specimens since the l a t t e r .would be from c h i l d r e n over 4 days o l d . The decrease was however not obtained. Although a l l but 1 of the VGH c o l l e c t e d p o s i t i v e r e s u l t s became negative when tested on the same p a t i e n t s ' home c o l l e c t e d specimens, "new" p o s i t i v e r e s u l t s appeared. A l l but 1 of these p o s i t i v e s obtained were from infants who had previously tested negative. The one p a t i e n t that tested p o s i t i v e on both the VGH and home c o l l e c t e d specimens was not diagnosed as CF but was a normal healthy c h i l d . I t i s probably c o i n c i d e n t a l that the two p o s i t i v e r e s u l t s were obtained on the same p a t i e n t . - 100 -The f a l s e p o s i t i v e rate obtained for the home c o l l e c t e d specimens (3.4%) does not compare favourably to Crossley's rate of 0.1%* obtained on 2500 babies tested i n New Zealand and Forrest's rate of 0.02% obtained on 20,000 babies tested i n A u s t r a l i a . The f a l s e p o s i t i v e r e s u l t s obtained i n t h i s study on home c o l l e c t e d 31 specimens were due to f a c t o r s other than age. One d i f f e r e n c e apparent between the two populations, of h o s p i t a l and home c o l l e c t e d specimens was the s h i f t to the l e f t of the d i s t r i b u t i o n of net absorbance values from the home c o l l e c t e d specimens. These values were s i g n i f i c a n t l y lower than those of the VGH c o l l e c t e d specimens even though they were c o l l e c t e d during the same time period and analyzed at the same time with the same set of reagents. The decrease i n net absorbance values appears to be due to a thinner spread of s t o o l sample on the screening card prepared at home as compared to the VGH prepared card. V i s u a l examination i n d i c a t e d that the home cards d i d have a l i g h t e r spread of s t o o l . S t o o l samples from the s l i g h t l y older i n f a n t s at home tend to be more l i q u i d than the h o s p i t a l samples, e x p e c i a l l y i f the c h i l d i s breast fed. Other evidence that the low net absorbance values were caused by a thinner spread of s t o o l i s that the mean of the absorbances at 410 nm and the mean of the absorbances at 460 nm decreased by the same amount between h o s p i t a l and home specimens(see Figures II and I I I ) . The only ways i n which the equal reduction could occur are: * Crossley's f a l s e p o s i t i v e rate i s 0.4% a f t e r the i n i t i a l assay, reducing to 0.1% a f t e r re-assay of the same specimen using double the substrate concentration. - 101 -i) that the home c o l l e c t e d s t o o l smears are thinner i i ) that there i s a l o s s of t r y p s i n a c t i v i t y as we l l as a reduction i n the concentration of f e c a l pigment i n the home c o l l e c t e d specimens and that the two reductions are approximately equal. (See Appendix P) Since the p r o b a b i l i t y of the l a t t e r i s extremely small i t i s concluded that the s t o o l smears are thinner. The decrease i n net absorbance values d i d not appear to be caused by a l o s s of enzyme a c i t v i t y as a r e s u l t of mailing i n the s t o o l samples. There was no s i g n i f i c a n t d i f f e r e n c e between the r e s u l t s obtained on 25 samples before and a f t e r m a i l i n g . Nor was a s i g n i f i c a n t d i f f e r e n c e obtained when 20 samples were stored at room temperature for 3 to 7 days and compared to the same sample stored i n the f r e e z e r . Two other c o n t r i b u t i n g f a c t o r s were considered: that the lowered net absorbance values i n the home c o l l e c t e d specimens were due to a larger concentration of f e c a l pigment leading to overcompensation or that the home prepared s t o o l specimen was spread unevenly i n thickness on the cards leading to wider d i s p e r s i o n of r e s u l t s . Neither factor was found to have any s i g n i f i c a n t e f f e c t on the d i f f e r e n c e between the net absorbance values of the h o s p i t a l and home c o l l e c t e d specimens. Establishment of a New Cut-Off Point Since there were i n d i c a t i o n s that on the average l e s s p - n i t r o a n i l i n e was measured i n the t e s t s o l u t i o n s of the home c o l l e c t e d specimens, a lower c u t - o f f point could be applied to the CF screen which makes use of t h i s c o l l e c t i o n procedure. - 102 -Crossley's c u t - o f f point which was i n i t i a l l y adopted by us for the h o s p i t a l c o l l e c t e d specimens and by F o r r e s t i n A u s t r a l i a was an absorbance reading f o r the p - n i t r o a n i l i n e produced of 0.300 ( i . e . a net absorbance lower than 0.300 was considered p o s i t i v e ) . The mean net absorbance values for the population of VGH c o l l e c t e d specimens was 1.068 with an S.D. of 0.385. The c u t - o f f point was therefore approximately 2 standard deviations below the mean [1.068 - 2(0.385)=0.298]. If the same c r i t e r i o n i s used to e s t a b l i s h the c u t - o f f point for the home c o l l e c t e d specimens, the new c u t - o f f point w i l l be 0.94-2(0.377)=0.187 which could be rounded o f f to 0.200 for convenience. Using the new-cut-off p o i n t , the p o s i t i v e rate for the home c o l l e c t e d specimens i s lowered from 3.7 to 1.9%. This i s a s i g n i f i c a n t l y lower rate than the f a l s e p o s i t i v e rate obtained for the VGH c o l l e c t e d specimens but i s s t i l l not i d e a l and much higher than the rates obtained i n A u s t r a l i a 3 * and New Zealand. / f e The concern with lowering the c u t - o f f point i s that while i t increases the s p e c i f i c i t y to 98.1% i t w i l l l i k e l y a l s o decrease the s e n s i t i v i t y . Since CF p a t i e n t s were not detected with t h i s p i l o t p r o j e c t i t i s not p o s s i b l e to determine the e f f e c t s on the l a t t e r to see i f the s e n s i t i v i t y remains at an acceptable l e v e l . It i s p o s s i b l e however to determine the e f f e c t the new c u t - o f f point would have on the p o s i t i v e c o n t r o l specimens. A l l of the CF c o n t r o l specimens would have tested p o s i t i v e with the new c u t - o f f point of 0.20 0 including those that were mailed to the laboratory as part ,of the i n v e s t i g a t i o n i n t o the e f f e c t s of mailing. The c o n t r o l cards would have to be prepared spreading the s t o o l s of known t r y p s i n concentration the - 103 -same thickness as the home c o l l e c t e d cards. However, t h i s would only serve to lower the net absorbance values further and would not have a f f e c t e d the f i n a l outcome of the r e s u l t s . A f a l s e p o s i t i v e rate of 1.9% and a predicted incidence of 1:2000 of CF implies by Bayes theorem a l e s s than 2.6% p r o b a b i l i t y that a c h i l d with a p o s i t i v e r e s u l t does i n f a c t have CF (Appendix Q). A lower f a l s e p o s i t i v e rate i s therefore d e s i r e d i n order to increase the physicians' confidence i n the t e s t and to reduce unnecessary follow-up costs - both f i n a n c i a l and emotional. - 104 -FOLLOW-UP I. PROTOCOL A follow-up was i n i t i a t e d on a l l presumptive p o s i t i v e s for c y s t i c f i b r o s i s from two t e s t i n g procedures: e i t h e r an increased meconium albumin concentration as detected by the BMC t e s t on specimens c o l l e c t e d at the Vancouver General H o s p i t a l from A p r i l 1975 u n t i l June 1979, or a decreased f e c a l t r y p s i n concentration as detected by Crossley's BAPNA procedure which ran concurrently from November 1977 u n t i l June 1979. The two t e s t s were used i n p a r a l l e l . An i n f a n t was considered to be p o s i t i v e for CF i f the c h i l d tested p o s i t i v e i n eit h e r one of the tes t s and negative i f he tested negative i n both t e s t s . The follow-up protocol i s i l l u s t r a t e d i n Figure IV. The l e t t e r s to the physicians and the c l i n i c a l questionnaire used i n the follow-up procedure were prepared by the CF screening programme committee c o n s i s t i n g of Dr. A.G.F.Davidson, Dr. D.A. Applegarth and Dr. L.T.K. Wong. These l e t t e r s were i n i t i a l l y prepared for the meconium screen and modified l a t e r to include the s t o o l t r y p s i n screen when both were i n operation at the VGH simultaneously. FIGURE IV. CF SCREENING AND FOLLOW-UP PROTOCOL FLOW-CHART Newborns a VG t 1 Meconium Samples BMC S t r i p -t e s t for ALBUMIN \ Normal Results (negative for CF) Result recorded No further a c t i o n taken Infants at Home Stool Sample Cards Crossley BAPNA test for TRYPSIN Abnormal Results (positive for CF) Test repeated i n t r i p l i c a t e on the same sample Normal Abnormal Letter mailed to physician: request for s t o o l sample Quantitative Stool assay for Chymotrypsin Normal Physician informed Well, no signs of CF Abnormal 1 Physician contacted for c l i n i c a l evaluation * I Questionable Symptoms ~1 Well, no signs of CF No further a c t i o n taken Sweat Analysis Abnormal CF l i k e l y I Normal CF not detected C h i l d r e f e r r e d to CLINIC Request for a 2nd s t o o l sample i Chymotrypsin abn. normal no further a c t i o n taken SCREENING DIAGNOSTIC FOLLOW-UP - 106 -A. Request for Stool Sample for Chymotrypsin The process s t a r t e d with a l e t t e r mailed to the p e d i a t r i c i a n or family physician informing him of the abnormal r e s u l t and asking for a s t o o l specimen for q u a n t i t a t i v e chymotrypsin a n a l y s i s . I n s t r u c t i o n s for sample c o l l e c t i o n were enclosed. The physician was also asked i n t h i s l e t t e r to contact the CF Assessment C l i n i c at Children's H o s p i t a l immediately i f the c h i l d had r e s p i r a t o r y or g a s t r o i n t e s t i n a l symptoms suggestive of CF. Response to t h i s i n i t i a l l e t t e r was very poor. An a d d i t i o n a l paragraph was therefore i n s e r t e d o f f e r i n g to contact the parents d i r e c t l y to arrange the s t o o l c o l l e c t i o n . This increased the response rate considerably. (The l e t t e r and s t o o l c o l l e c t i o n i n s t r u c t i o n s are enclosed i n Appendix D.) 1. Lack of Response I f a s t o o l specimen was not received from the c h i l d within two weeks a f t e r the l e t t e r was mailed, the doctor was contacted by phone to determine whether h i s o f f i c e had received the l e t t e r and whether the c h i l d i n question was h i s p a t i e n t . A verbal o f f e r to contact the parents d i r e c t l y was made and t h i s o f f e r was usu a l l y accepted. An inquiry was made at the same time as to the c h i l d ' s c l i n i c a l p i c t u r e . In several cases the doctor's o f f i c e neglected to n o t i f y the CF assessment c l i n i c laboratory at Children's H o s p i t a l that the family had - 107 -moved or that we had the i n c o r r e c t physician's name. This information was not obtained u n t i l the follow-up telephone c a l l . If the name of the family physician was not known, the parent's telephone number was obtained from Medical Records at VGH and the mother was contacted by telephone for t h i s information. This was accomplished without mentioning the CF screening programme or the p o s i t i v e r e s u l t . In a few cases where the family l i v e d outside the Greater Vancouver area or the family d i d not have a telephone, a l e t t e r was written to obtain the doctor's name. 2. D i r e c t Contact with Parents: The majority of physicians preferred that we contacted the parents d i r e c t l y . The sample c o l l e c t i o n and d e l i v e r y i n s t r u c t i o n s were given by telephone to one of the parents and the written i n s t r u c t i o n s were mailed to t h e i r home on the same day i n order to remove any doubts or confusion i n the parent's mind. If the s t o o l specimen was not received within two weeks the parents were contacted by telephone again and a f r i e n d l y reminder was given. This was repeated as many times as necessary u n t i l the specimen was received. B. Quantitative Chymotrypsin A n a l y s i s The follow-up s t o o l specimens were analyzed for chymotrypsin by the method of Smith et a l . Chymotrypsin was chosen because i t was reported - 108 -to show a c l e a r c o r r e l a t i o n with pancreatic exocrine i n s u f f i c i e n c y and i t s s e c r e t i o n was shown to be a f f e c t e d e a r l i e r and more severely than -28 t r y p s i n s e c r e t i o n . 1. Abnormally Low Chymotrypsin A c t i v i t y Since a spot s t o o l specimen rather than a 24 or 72 hour s t o o l specimen was analyzed, there was a low p r o b a b i l i t y that the abnormally low r e s u l t was due to normal day to day v a r i a t i o n . This p o s s i b i l i t y had to be kept i n mind, even though the c o r r e l a t i o n between spot s t o o l specimen r e s u l t s and 24 hour specimen r e s u l t s had been reported as very 412-good. As a r e s u l t , a re-assay on a new sample was requested by telephone i f an abnormally low chymotrypsin value was obtained. The physic i a n was also asked for a c l i n i c a l evaluation of the c h i l d at t h i s point i n time. I f i t was not p o s s i b l e to contact the physician d i r e c t l y , the request for a second s t o o l specimen was placed with the o f f i c e and the physician was asked to f i l l out a c l i n i c a l questionnaire (Appendix E) which was promptly mailed out to him on the same day. If the c l i n i c a l p i c t u r e was suggestive of CF, the second s t o o l • chymotrypsin a n a l y s i s was omitted and a sweat e l e c t r o l y t e determination was ordered immediately. I f the second s t o o l specimen a l s o displayed low chymotrypsin a c t i v i t y , sweat e l e c t r o l y t e s were determined. - 109 -2. Normal Chymotrypsin A c t i v i t y I f the q u a n t i t a t i v e chymotrypsin a n a l y s i s performed on the follow-up s t o o l specimen was normal the r e s u l t was mailed to the physician, along with the c l i n i c a l questionnaire. I f the c h i l d ' s c l i n i c a l p i c t u r e was not suggestive of CF no further a c t i o n was taken. In one instance, the c l i n i c a l p i c t u r e was questionable and the phy s i c i a n was encouraged to order a repeat chymotrypsin a n a l y s i s on a second s t o o l specimen and/or to order a sweat e l e c t r o l y t e determination despite the i n i t i a l normal chymotrypsin. C. Sweat E l e c t r o l y t e s The d etection of abnormally high l e v e l s of sodium and c h l o r i d e i n the sweat was used as the p r i n c i p a l d i a g n o s t i c c r i t e r i o n of CF. Since sweat c o l l e c t i o n can be a problem i n the f i r s t few weeks of l i f e , t h i s t e s t was u s u a l l y performed when the i n f a n t was at l e a s t two weeks of age. The p i l o c a r p i n e iontophoresis sweat te s t was performed at various l a b o r a t o r i e s and the r e s u l t s were mailed to the CF Screening Programme at Children's H o s p i t a l . 1. Abnormal Sweat E l e c t r o l y t e Concentrations If the concentration of the sweat s e l e c t r o y t e s was abnormally high the a n a l y s i s was repeated for confirmation. If the r e s u l t s from the - 110 -repeat a n a l y s i s were a l s o abnormally high then CF was l i k e l y and the c h i l d was r e f e r r e d to the CF c l i n i c for further assessment and confirmation. 2. Normal Sweat E l e c t r o l y t e Concentrations I f the sweat e l e c t r o l y t e concentrations were normal CF was considered u n l i k e l y . I t would be up to the p h y s i c i a n to decide on the bas i s of the sweat e l e c t r o l y t e r e s u l t , the c l i n i c a l p i c t u r e of the c h i l d and the family h i s t o r y whether or not CF was s t i l l a p o s s i b i l i t y and whether a repeat sweat a n a l y s i s was warranted. D. C l i n i c a l Follow-Up Only In some cases follow-up specimens for further laboratory a n a l y s i s were not obtained. With a few i n f a n t s , t h i s was because the physician f e l t that the request for the s t o o l specimen or sweat t e s t would r e s u l t i n severe anxiety i n the mother and that i t was more b e n e f i c i a l to the c h i l d and the family to j u s t monitor the c h i l d c a r e f u l l y from a c l i n i c a l standpoint. In these s i t u a t i o n s , the physician was contacted by telephone when the c h i l d was one year o l d and again at two years of age i n order to determine i f the c h i l d was "well with no c l i n i c a l symptoms of CF". L e t t e r s were mailed out to these physicians (Appendix F) along with the c l i n i c a l questionnaire. Stool specimens were sometimes obtained at t h i s l a t e r date when the physician f e l t that the mother was more able to cope - I l l -with the s i t u a t i o n or the c l i n i c a l p i c t u r e warranted a laboratory follow-up.' In a few cases, the c h i l d and family moved out of the greater Vancouver area. In these s i t u a t i o n s the family was contacted by mail to obtain the name of the new family p h y s i c i a n . The new physician was n o t i f i e d of the CF screening r e s u l t and asked to f i l l out a c l i n i c a l questionnaire. For a few i n f a n t s we received a s t o o l sample for chymotrypsin a n a l y s i s through the mail or the physician mailed us a sweat e l e c t r o l y t e r e s u l t but for most of these i n f a n t s a c l i n i c a l follow-up was a l l that was obtained. ' When poss i b l e the p h y s i c i a n was re-contacted by telephone for a second c l i n i c a l evaluation when the c h i l d was 2 years of age. E. Review of the F i l e s at Medical Records Another type of follow-up that was performed for the purpose of evaluating these two p i l o t p r o j e c t s was a comprehensive review of each baby's h o s p i t a l records f i l e d i n the Medical Records Department at VGH. The baby's c o n d i t i o n at b i r t h was noted and a search was made for p o s s i b l e reasons for f a l s e p o s i t i v e r e s u l t s . In the i n i t i a l follow-up procedure the s e c r e t a r i a l s t a f f mailed a l e t t e r informing the physician of a p o s i t i v e r e s u l t , and requesting a follow-up. From March to June, 1979 funds were a v a i l a b l e to h i r e a part-time person s p e c i f i c a l l y for the task of n o t i f y i n g the physicians of p o s i t i v e r e s u l t s and r e t r i e v i n g follow-up specimens by mail and telephone. The author became involved with the follow-up procedure i n - 112 -May 1979 and assumed f u l l r e s p o n s i b i l i t y i n J u l y 1979. She personally completed the follow-up on 57% of the meconium screen p o s i t i v e s and 70% of the f e c a l t r y p s i n screen p o s i t i v e s . . The review of the f i l e s at the Medical Records Department at the VGH was performed by the author on a l l p o s i t i v e s received i n both screening programmes. I I . RESULTS The number of babies tested and the number of presumptive p o s i t i v e s for CF i n each of the two p i l o t p r o j e c t s , the meconium albumin and the Crossley f e c a l t r y p s i n method, are l i s t e d i n Table XII. A. Meconium Albumin CF Screen 1. Diagnostic Follow-Up As in d i c a t e d i n Table XII, during a 4 year period 119 p o s i t i v e s (increased albumin concentration) were detected out of a t o t a l of 8891 infants tested. From t h i s number of p o s i t i v e s , 12 infants died s h o r t l y a f t e r b i r t h before further follow-up could be performed. The autopsy reports summarized i n Table XIII, implied that these infants d i d not have c y s t i c f i b r o s i s . I t was therefore necessary to follow up 107 infants for diagnostic studies and t h i s follow-up was completed on 103 of these i n f a n t s (96%) . Follow-up data were not obtained on 4 of the i n f a n t s : 3 of the c h i l d r e n were adopted and one family moved s h o r t l y a f t e r the b i r t h TABLE XII. CF SCREENING PILOT PROJECTS Specimen Time Pop- Number Abnormal Testing Number Period u l a t i o n * Tested Finding Procedure " P o s i t i v e " I Meconium April/75 - Albumin BMC June/79 10,091 8,891 Increase S t r i p - T e s t 119 II S t o o l : August/77 - Trypsin Crossley VGH June/79 4,675 3,210 Decrease BAPNA 160 Home+ September/78 - Trypsin Crossley June/79 1,920 875 Decrease BAPNA 30 * Number bf babies born at Vancouver General H o s p i t a l (VGH) plus number of babies t r a n s f e r r e d to VGH, s h o r t l y a f t e r b i r t h , from other h o s p i t a l s . + The mothers were given a CF screening card when discharged from VGH and were asked to mail i n a s t o o l specimen from home. T A B L E XIII SUMMARY OF AUTOPSY REPORTS MECONIUM SCREENING T E S T P O S I T I V E S NEWBORN • N O . F I N A L PATHOLOGICAL DIAGNOSIS 1 P u l m o n a r y I n t e r s t i t i a l emphysema, b i l a t e r a l p n e u m o t h o r a c e s a u b e p e n d y r a l p l a t e h e m o r r h a g e w i t h i n t r a v e n t r i c u l a r h e m o r r h a g e 2 M e n i n g i t i s , b l e e d i n g d i a t h e s i s w i t h i n t r a v e n t r i c u l a r a n d p u l m o n a r y h e m o r r h a g e , k e r n l c t u r u s 3 S u d d e n u n e x p e c t e d d e a t h s y n d r o m e , c y a n o s i s o f d i g i t s a n d mucous m e m b r a n e s , c e r e b r a l edema w i t h c o n g e s t e d c o r t i c a l v e i n s , p e t e c h i a e o n p l e u r a a n d c a p s u l e o f thymus 4 R e s p i r a t o r y d i s t r e s s ( c a u s e n o t d e t e r m i n e d ) , p e r f o r a t i o n o f s m a l l i n t e s t i n e 5 A s p i r a t i o n p n e u m o n i a , c o t t o n f i b r e e m b o l u s i n l u n g , p o s t n a t a l g r o w t h r e t a r d a t i c s u g g e s t i o n o f n e c r o t i z i n g e n t e r o c o l i t i s 6 P o s t m e n i n g i t i c h y d r o c e p h a l u s , p o s t o p e r a t i v e r i g h t v e n t r i c u l o p e r l t o n e a l s h u n t , s u b d u r a l hematorn • s e c o n d a r y t o s h u n t i 7 I n t r a u t e r i n e g r o w t h r e t a r d a t i o n , n e c r o t i z i n g e n t e r o p a t h y , p u l m o n a r y edema 8 S e v e r e p e r i n a t a l a s p h y x i a , n e c r o t i z i n g e n t e r o c o l i t i s w i t h p n e u m a t o s i s c y s t o i d e s i n t e s t i n a l i s a n d p e r f o r a t i o n , p u l m o n a r y c o n g e s t i o n w i t h i n t r a - a l u e o l a r h e m o r r h a g e 9 P e r i n a t a l a s p h y x i a , b r o n c h o p u l m o n a r y d y s p l a s i a , n e c r o t i z i n g e n t e r o p a t h y , s t o m a c h , i l e u m a n d p r o x i m a l c o l o n , a c u t e m y o c a r d i a l n e c r o s i s , h e p a t i c s t e a t o s i s a n d c h o l e s t a s i s 1 0 P e r i n a t a l a s p h y x i a , n e c r o t i z i n g e n t e r o c o l i t i s , s e p s i s , s u b e p e n d y m a l p l a t e h e m o r r h a g e w i t h i n t r a v e n t r i c u l a r e x t e n s i o n 1 1 D o w n ' s o y n d r o m o , c x a g g a r n t c d a l e l e c t a s i s i n b o t h l u n g o , m a s s i v e b i l a t e r a l o u b e p e n d y m a l c e l l p l a t e h e m o r r h a g e s 1 2 No a u t o p s y p e r f o r m e d . C a u s e o f d e a t h g i v e n an r e s p i r a t o r y f a i l u r e . * Common f i n a l d i a g n o s i s f o r a l l i n f a n t s e x c e p t N o . 3 i s p r e t e r m d e l i v e r y a n d n e o n a t a l d e a t h . N o . 3, a l s o a p r e m a t u r e b i r t h , d i e d a t 6 m o n t h s o f a g e . - 115 -of the c h i l d leaving no forwarding address and no other leads as to t h e i r new l o c a t i o n . Of the 103 remaining c h i l d r e n , a s t o o l was obtained for chymotrypsin for 67 i n f a n t s . Stool chymotrypsin was normal i n 45 of these. There were no c l i n i c a l suspicions of CF on the part of the attending physician and no further a c t i o n was taken. Twelve in f a n t s had abnormal chymotrypsin r e s u l t s . These were repeated i n 4, with normal r e s u l t s . In 2 there was no c l i n i c a l problem, and no further a c t i o n was taken. While another 2 had sweat assays performed, with normal r e s u l t s . The remaining 8 i n f a n t s who had a repeat chymotrypsin a n a l y s i s , again had abnormally low r e s u l t s and sweat e l e c t r o l y t e determinations were performed on a l l 8. Normal sweat r e s u l t s were obtained on 5 of these i n f a n t s with no c l i n i c a l s u s p i c i o n of CF on the part of the attending physician. Abnormal r e s u l t s were obtained on the remaining 3 i n f a n t s and these i n f a n t s were diagnosed as having c y s t i c f i b r o s i s . Fourteen of the i n f a n t s who had an i n i t i a l abnormal meconium screen had sweat a n a l y s i s performed without f i r s t assaying a s t o o l specimen for chymotrypsin. A l l 14 had normal r e s u l t s . I t was not p o s s i b l e to obtain a biochemical follow-up on 32 i n f a n t s . In these cases, e i t h e r the physician d i d not f e e l that any biochemical follow-up was warranted or the parents refused to co-operate. Follow-up i n the form of contacting the physician for a c l i n i c a l report has continued for at l e a s t 2 years on these infants and none of them have shown any need for further study. The r e s u l t s of the 103 follow-ups, categorized according to the i n i t i a l t e s t or evaluation that was performed, are presented i n Figure V. FIGURE V. RESULTS OF THE DIAGNOSTIC FOLLOW-UP ON POSITIVES FROM TKE MECONIUM SCREEN INITIAL CHYMOTRYPSIN J Normal 45 — n o further action taken Abnormal 12 _ repeat chymotrypsin Normal 4 no fu r t h e r a c t i o n taken 2 Abnormal 8 I^"^*--confirmation, sweat an a l y s i s Normal 2 1 Sweat analysis Normal 5 Abnormal 3 ^ C F Assessment C l i n i c T CF 3 INITIAL SWEAT ANALYSIS 1 . k Normal 14 >^  no further action taken CLINICAL EVALUATION T C h i l d "well, with no symptoms of CF, over 2 years o l d " 32 TOTAL 103 - 117 -2. Medical Records In v e s t i g a t i o n Since 3 out of the 119 p o s i t i v e s were true p o s i t i v e s , 116 f a l s e p o s i t i v e s were inve s t i g a t e d by reviewing the h o s p i t a l f i l e s on these i n f a n t s f o r p o s s i b l e causes of an increased albumin i n the meconium. a) Prematurity A high incidence of prematurity was found among the inf a n t s that gave f a l s e p o s i t i v e r e s u l t s (81%). As noted from the autopsy reports i n Table XIII, a l l of the 12 i n f a n t s that died s h o r t l y a f t e r b i r t h were born prematurely. Of the remaining 104 i n f a n t s that survived, 82 had also been born prematurely. » b) G.I. Disturbances and Necrotizing E n t e r o c o l i t i s Other s i m i l a r i t i e s noted were that 26 i n f a n t s out of the 104 had G.I. disturbances, 18 of these being n e c r o t i z i n g e n t e r o c o l i t i s . Among the autopsy reports another 5 l i s t e d n e c r o t i z i n g e n t e r o c o l i t i s as part of the f i n a l p a t h o l o g i c a l diagnosis, making a t o t a l of 23 nec r o t i z i n g e n t e r o c o l i t i s cases out of the 116 f a l s e p o s i t i v e s (20%). Other G.I. disturbances among the 82 premature l i v e i n fants were 3 adbominal d i s t e n t i o n , 2 i l e a l a t r e s i a , 1 intra-abdominal abscess, 1 meconium g a s t r i t i s and 1 Hirschsprung's disease. c) Diseases Related to Prematurity \ The other diseases that occurred i n several of the l i v e i n f a n t s were ones that were r e l a t e d to t h e i r prematurity such as hyaline membrane - 118 -disease (22) , and r e s p i r a t o r y d i s t r e s s syndrome (9). These same diseases were a l s o l i s t e d i n the autopsy reports. The r e s u l t s from the Medical Records i n v e s t i g a t i o n on the 104 c h i l d r e n that gave f a l s e p o s i t i v e s are presented i n Figure VI. B. Fecal Trypsin CF Screen 1. Diagnostic Follow-Up Testing the VGH and home c o l l e c t e d s t o o l screening cards with Crossley's t r y p s i n method, 190* p o s i t i v e s were detected out of a t o t a l number of 4085 samples tested (Table X I I ) . These represented 3393 in f a n t s since 692 i n f a n t s were tested by both methods. Diagnostic follow-up was completed on 175 inf a n t s (92%). The follow-up r e s u l t s , a d i s c u s s i o n of which follows, are summarized i n the accompanying flow chart (Figure VII) where they are categorized according to the i n i t i a l biochemical t e s t or evaluation that was performed. Of the 175 c h i l d r e n followed up, s t o o l samples for chymotrypsin a n a l y s i s were obtained from 152 of which 149 gave a completely normal r e s u l t and no further a c t i o n was taken. Three of the 152 chymotrypsin a n a l y s i s had border l i n e low chymotrypsin l e v e l s . Repeat chymotrypsin determinations were performed on 2 of these i n f a n t s both of which were normal and on the t h i r d i n f a n t a sweat t e s t was done which was al s o normal. Of the * 190 p o s i t i v e s were detected but these represented 189 infants since one c h i l d tested p o s i t i v e on both h o s p i t a l and home c o l l e c t e d specimens. FIGURE VI. RESULTS FROM AN INVESTIGATION FOR POSSIBLE CAUSES OF INCREASED MECONIUM ALBUMIN IN NON-CF INFANTS I. PREMATURE INFANTS 82 G.I. Disturbances 24 n e c r o t i z i n g e n t e r o c o l i t i s 16 abdominal d i s t e n t i o n 3 i l e a l a t r e s i a 2 intra-abdominal abscess 1 meconium g a s t r i t i s 1 Hirschsprung's disease 1 Ingestion of maternal blood 2 Intrauterine growth re t a r d a t i o n 1 Hemolytic disease of the newborn 3 Hyaline membrane disease 22 Respiratory d i s t r e s s syndrome 9 Down1s syndrome 1 Meconium a s p i r a t i o n 1 No s p e c i a l problems l i s t e d 19 i I I . FULL TERM INFANTS -f Hi M( L H< 22 | Necr o t i z i n g E n t e r o c o l i t i s 2 econium A s p i r a t i o n 2 Healthy 18 FIGURE VII. RESULTS OF THE DIAGNOSTIC FOLLOW-UP ON POSITIVES FROM THE FECAL TRYPSIN SCREEN INITIAL CHYMOTRYPSIN Normal 149 ^ no fur t h e r a c t i o n taken Abnormal 3 , ^ Sweat Analysis repeat chymotrypsin Normal 1 Normal 2 INITIAL SWEAT ANALYSIS Normal CLINICAL EVALUATION \ C h i l d "well with no symptoms of CF, over 2 years of age C h i l d "well with no symptoms of CF, under 2 years of age TOTAL - 121 -remaining 23 who were followed up 6 had normal sweat t e s t s and 17 were only evaluated c l i n i c a l l y . The l a t t e r 17 were f e l t by the family physician or p e d i a t r i c i a n to have no c l i n i c a l symptoms which would suggest c y s t i c f i b r o s i s and the physicians concerned f e l t that they d i d not warrant further follow-up. Twelve patients were l o s t to biochemical follow-up despite the request of the physician that a chymotrypsin a n a l y s i s be performed. The lack of further laboratory t e s t i n g on these 12 i n f a n t s was due to various reasons: 3 mothers were un w i l l i n g to co-operate, 6 mothers promised to d e l i v e r s t o o l specimens for chymotrypsin a n a l y s i s but had not done so at the time t h i s t h e s i s was being prepared and 1 p e d i a t r i c i a n would not give h i s permission for d i r e c t communication with the parents of three i n f a n t s , i n s i s t i n g on arranging for the s t o o l specimen c o l l e c t i o n himself. These specimens have not been received to date. These i n f a n t s are however completely healthy and w i l l be re-evaluated p e r i o d i c a l l y . Three i n f a n t s are l o s t to follow-up of any s o r t , for t h e i r f a m i l i e s had moved s h o r t l y a f t e r the b i r t h of these i n f a n t s and l e f t no forwarding address and we were unsuccessful i n our attempts to l o c a t e them. 2. Medical Records I n v e s t i g a t i o n A l l of the p o s i t i v e s from the VGH and home c o l l e c t e d specimens tested for t r y p s i n appear to be f a l s e p o s i t i v e s . A search of the Medical Records f i l e s on these i n f a n t s d i d not provide explanations for the f a l s e p o s i t i v e s . Most of the i n f a n t s were f u l l term healthy babies at b i r t h - 122 -(91.5%). Exceptions were: 7 premature birth, 1 Down's syndrome, 4 intrauterine growth retardation, 2 meconium aspiration, 2 hemolytic disease of the newborn and 1 infant that was small for gestational age. Serum b i l i r u b i n determinations had been performed on many of the infants involved in the CF screening programme during their hospital stay. Thirty-nine percent of the VGH collected CF screening positives and 40% of the home collected CF screening positives had a serum bi l i r u b i n above the normal level for their age at birth. A regression test indicated no linear relationship between the absorbance at 460 nm obtained with the Crossley method and the serum b i l i r u b i n concentration. III. Discussion A. Meconium Albumin CF Screen Diagnostic Follow-ups The follow-up which was completed on 96% of the infants who obtained a positive result with the meconium screen appears on the surface to have been thorough. In actual practice however the follow-up procedure was inadequate. In most cases too much time elapsed between the positive result, locating the patient and obtaining a sample for the i n i t i a t i o n of the follow-up investigation. Although 93% of the positives were detected before May 1979, only 43% had been followed up by that date. The delay was - 123 -i n i t i a l l y due mostly to inadequate manpower but a further delay r e s u l t e d when a l l e f f o r t s were d i r e c t e d a t s e t t i n g up the new f e c a l t r y p s i n screen. This delay i n follow-up r e s u l t e d i n s e v e r a l problems: 1. T h e o r e t i c a l l y e i t h e r true (CF diagnosed) or f a l s e p o s i t i v e r e s u l t s from the meconium screen could have been of b e n e f i t to the p h y s i c i a n because f a l s e p o s i t i v e r e s u l t s were associated with other problems such as G.I. disturbances (discussed l a t e r ) . Further biochemical t e s t i n g would have benefited the p a t i e n t under both of these circumstances i f i t had r e s u l t e d i n e a r l y diagnosis which then enabled the i n f a n t to obtain e a r l y medical treatment. The value of further d i a g n o s t i c t e s t i n g was questionable however when i t was delayed to the point where treatment for the symptoms was w e l l underway or completed when the p h y s i c i a n received the r e s u l t s . 2. The i n i t i a l contact with a report of the p o s i t i v e r e s u l t was u s u a l l y prompt but further follow-up was often delayed. The importance of the t e s t was g r e a t l y diminished i n the eyes of the physician when he was recontacted a f t e r some time had passed since the b i r t h of the c h i l d . Co-operation for further t e s t i n g was understandably more d i f f i c u l t to o b t a i n . 3. The longer the delay the more d i f f i c u l t i t became to trace the family. This was due to various reasons: e.g. the i n f a n t was no longer seeing the p e d i a t r i c i a n , the family doctor had been changed, the family had moved or i n s i t u a t i o n s where the i n f a n t had been r e f e r r e d to VGH from another h o s p i t a l the i n f a n t had, returned to h i s home outside of t h i s area. This hampered further biochemical t e s t i n g and i n some cases prevented i t . - 124 -4. Some parents were d i f f i c u l t to deal with when contacted at a l a t e r date. The delay seemed to increase t h e i r anxiety. The parents seemed to assume there was another serious problem with t h e i r c h i l d (most had been high r i s k premature). Unfortunately the manpower to deal with e x c e p t i o n a l l y anxious parents was a l s o not a v a i l a b l e . In a few cases, the parent was not s a t i s f i e d with the physicians explanation of the screen and the i n f a n t ' s r e s u l t s , and placed a c a l l to Children's H o s p i t a l but was unable to obtain peace of mind because a proper system had not been set up to deal with such c a l l s . 5. Other parents reasoned that i f a request f o r a s t o o l specimen was coming t h i s l a t e i t couldn't be very important and therefore they could not be bothered to c o l l e c t a s t o o l specimen. The d i f f i c u l t i e s i n obtaining s t o o l specimens f o r chymotrypsin a n a l y s i s i s r e f l e c t e d i n the high percentage (31%) of follow-ups that consisted only of a c l i n i c a l e valuation by the phys i c i a n . Medical Records I n v e s t i g a t i o n The i n v e s t i g a t i o n of the pa t i e n t s , d e t a i l e d records on f i l e i n the Medical Records department at the Vancouver General H o s p i t a l proved to be worthwhile. Out of a t o t a l of 104 i n f a n t s , 87 i n f a n t s records contained a p o s s i b l e explanation f o r the presence of the increased albumin concentration i n the meconium specimen. In the remaining 17, a c l i n i c a l reason f o r detecting an increased albumin concentration was not apparent. These i n f a n t s had a l l been c l a s s i f i e d as "healthy newborn i n f a n t s " with no complications. - 125 -1. Healthy Infants One i n f a n t out of the 18 had been given a g l y c e r i n e suppository 6 p r i o r to the meconium c o l l e c t i o n . Glycerine has been reported to produce the t y p i c a l blue colour with the BMC t e s t - s t r i p . On re-examination of the laboratory records no biochemical explanations were present f o r the f a l s e p o s i t i v e r e s u l t s on the remaining 17 t e s t s . None of the specimens had been reported as appearing bloody. Two of the specimens appeared to v i s u a l l y resemble s t o o l specimens rather than meconium but t h i s could lead to f a l s e negative r e s u l t s not f a l s e p o s i t i v e s . Whether these 17 p o s i t i v e s were due to the f a i l u r e of the BMC t e s t - s t r i p or whether these specimens a c t u a l l y contained an increased concentration of albumin due to some unknown cause i s not known. 2. Premature Infants Premature i n f a n t s o f t e n have lower than normal pancreatic a c t i v i t y due to the slow development of necessary enzyme systems. Albumin accumulates i n the meconium as a r e s u l t of a decrease or absence of these pancreatic enzymes and r e s u l t s i n a f a l s e p o s i t i v e i n the CF meconium screen. 3. G.I. Disturbances In the meconium specimens from i n f a n t s with G.I. disturbances, the specimens most l i k e l y d i d contain an increased concentration of p r o t e i n that was detected by the BMC t e s t - s t r i p . This increased p r o t e i n could - 126 -have been due to inflammatory p r o t e i n discharge, o c c u l t blood or a general c o n c e n t r a t i o n of the meconium specimen due to malabsorption. 4. N e c r o t i z i n g E n t e r o c o l i t i s There were re p o r t s of 50* cases of n e c r o t i z i n g e n t e r o c o l i t i s (NEC) i n the t o t a l p o p u l ation of 8,891 i n f a n t s tested with the meconium albumin assay using the BMC t e s t - s t r i p . This i s an incidence of 5.62 NEC per 1000 i n f a n t s a t VGH, both inborn and outborn. This incidence i s high compared with the incidence of NEC reported i n the l i t e r a t u r e : e.g. 2.02 cases per 1,000 b i r t h s during a two year period (1975 to 1977) with 13,860 b i r t h s and 3.61 per 1,000 b i r t h s i n 1978 as compiled by F i n t e r and M o r i a r t e y 3 ' i n A l b e r t a and an incidence of 3.95 per 1,000 b i r t h s during a 20 month p e r i o d (July 1977 to February 1979) with 8841 b i r t h s , inborn and Qi outborn, i n A t l a n t a , Georgia as reported by S t o l l e t a l . The higher VGH incidence rate f o r NEC i s p a r t i a l l y as a r e s u l t of VGH being a centre f o r high r i s k newborns but i s probably a l s o due to the manner i n which the s t a t i s t i c s were c o l l e c t e d . In both of the st u d i e s by Fine r and the one by S t o l l , the babies with c l i n i c a l symptoms which were suspect NEC but without radiographic evidence f o r NEC were excluded from the a n a l y s i s . T h i s procedure was not followed to compile the s t a t i s t i c s f o r t h i s meconium screen. A l l cases diagnosed by the p e d i a t r i c i a n or family p h y s i c i a n as NEC were included i n the data even though p o s i t i v e confirmation with x-ray a n a l y s i s had not been obtained f o r a l l of the cases. * According to an a n a l y s i s of the h o s p i t a l f i l e s on p a t i e n t s whose f i l e s were r e t r i e v e d by the Medical Records department through a diagnosis c l a s s i f i c a t i o n process. - 127 -According to the above c a l c u l a t e d incidence of NEC for VGH, 5.62 i n 1,000 b i r t h s , one would expect to f i n d 0.67 NEC cases among the 119 f a l s e p o s i t i v e s , whereas 23 were present. This i s a s i g n i f i c a n t increase. (See Appendix N, s e c t i o n 1) However, 82 of the 119 i n f a n t s , on which p o s i t i v e screening r e s u l t s were obtained were premature i n f a n t s . The incidence of NEC among premature babies increases d r a s t i c a l l y , the rate increasing with decreasing b i r t h w e i g h t . a 6 ' 3 / > 9 / A rough estimate of the incidence of NEC for premature babies, c a l c u l a t e d using the date presented i n S t o l l ' s at paper , would be 18 per 1,000 b i r t h s . According to t h i s rate, one would expect to f i n d 1.5 NEC cases among the 82 premature i n f a n t s that gave a f a l s e p o s i t i v e r e s u l t with the BMC t e s t - s t r i p . Again the a c t u a l number present was s i g n i f i c a n t l y higher, at 21 (See Appendix N, s e c t i o n 2). There was therefore a d e f i n i t e c o r r e l a t i o n between the p o s i t i v e r e s u l t s obtained i n the meconium screen and the presence of NEC. These s t a t i s t i c s point to the p o s s i b l e usefulness of the BMC t e s t - s t r i p (or other meconium albumin assay) for the e a r l y detection of NEC. The meconium albumin r e s u l t s were a v a i l a b l e w i t h i n 2 to 7 days of the b i r t h of the c h i l d . Since the specimen i s c o l l e c t e d within a day, the c o l l e c t i o n procedure could be re-organized so that the r e s u l t s are a v a i l a b l e w i t h i n 2 to 3 days. The age at diagnosis of NEC of the i n f a n t s at VGH ranged from 1 to 90 days, with most (62%) between 3 to 15 days. Most i n f a n t s who develop NEC do not develop the c l i n i c a l symptoms of NEC 35 f o r 2 to 5 days. At VGH, during the time period under study only 3 i n f a n t s were diagnosed under 3 days. As a r e s u l t the BMC t e s t - s t r i p appears to have p o t e n t i a l as an i n d i c a t o r of the p o s s i b l e development of - 128 -NEC and give the phys i c i a n an e a r l y warning of t h i s high m o r t a l i t y rate disease. There i s however one major draw back. The t e s t detected 23 NEC out of a t o t a l of 50 i n f a n t s diagnosed as having NEC. Twenty-seven i n f a n t s tested negative with the BMC t e s t - s t r i p . The negative r e s u l t s could not be c o r r e l a t e d with a suspicious and therefore p o s s i b l y i n c o r r e c t diagnosis of NEC. If the BMC t e s t - s t r i p were used as a di a g n o s t i c t e s t f o r NEC the physicians would have to be made aware that although a p o s i t i v e r e s u l t could be an e a r l y warning sign f o r NEC, a negative r e s u l t does not exclude the p o s s i b i l i t y of NEC developing. B-. F e c a l Trypsin CF Screen Diagnostic Follow-Up Out of a t o t a l of 190 p o s i t i v e s , 175 c h i l d r e n were followed up. A l l of the 175 c h i l d r e n were f e l t not to have c y s t i c f i b r o s i s and to-date information of a CF diagnosis has not been received. The completion of the diagn o s t i c follow-up on the p o s i t i v e s obtained with the f e c a l t r y p s i n CF screen was at a s l i g h t l y lower percentage (92%) than the meconium screen. Follow-up was not delayed to the same extent on these p o s i t i v e s as with the meconium screen, because some a d d i t i o n a l manpower had been made a v a i l a b l e . As a r e s u l t s t o o l specimens f o r chymotrypsin a n a l y s i s were obtained f o r a large proportion (81%) of the completed follow-ups. - 129 -Despite t h i s , there were some d i f f i c u l t i e s i n attempting to complete 8% of the follow-ups. The r e s i s t a n c e encountered i n obtaining the follow-up s t o o l specimens appeared to be due mainly to the lack of confidence i n the r e l i a b i l i t y of the Crossley method and i n the questionable s i g n i f i c a n c e of the low t r y p s i n r e s u l t . This view was presented by the physicians themselves. Many expressed the opinion that further t e s t i n g was not warranted although a few were kind enough to request a specimen "for the sake of the p r o j e c t " from mothers who they f e l t would not get anxious as a r e s u l t of the request. This view was a l s o forwarded by the mothers who were r e l u c t a n t to comply to the request of a s t o o l sample for chymotrypsin. According to these mothers they had been informed by t h e i r physicians that the s t o o l c o l l e c t i o n was not necessary and that i t was not important*. Because most of the p o s i t i v e s were from healthy c h i l d r e n who were a few weeks o l d at the time of the follow-up request, the physicians r e l i e d more on us to contact the parents d i r e c t l y . This d i d r e s u l t i n a more e f f i c i e n t r e t r i e v a l system. This agreed with studies i n Quebec i n 1972 which revealed that compliance rates for obtaining a follow-up sample improved g r e a t l y when the onus for c o l l e c t i n g and sending a t e s t was placed on the parents rather than on the p h y s i c i a n . This system appeared to arouse anxiety i n some of the parents (or at l e a s t made us aware of the anxiety that does r e s u l t ) . Lack of a system for handling anxious parents caused s i m i l a r problems i n the f e c a l t r y p s i n screen as * These i n s t r u c t i o n s may however have been given to reduce the anxiety that appeared i n some of the parents. - 130 -were present i n the meconium screen. Several telephone c a l l s , that we know o f , were placed to the Children's H o s p i t a l by anxious parents and these c a l l s were not channeled t o a p r o f e s s i o n a l person capable of dealing with such matters. Medical Records I n v e s t i g a t i o n A l l of the p o s i t i v e s detected i n the f e c a l t r y p s i n screen using the Crossley method appear to be f a l s e p o s i t i v e s to date. The few problem areas or disease s t a t e s that were present among the f a l s e p o s i t i v e p opulation such as premature b i r t h s , Downs syndrome and hemolytic disease of the newborn, were present i n percentages t h a t would be expected i n the normal i n f a n t p opulation. A l a r g e p r o p o r t i o n (40%) of the i n f a n t s that gave a p o s i t i v e r e s u l t had increased serum b i l i r u b i n l e v e l s a t b i r t h . Neonatal jaundice among the normal newborn population i s however found i n approximately 60% of newborns. 5* C. D i f f i c u l t i e s Encountered with the Follow-Up Procedure The form l e t t e r was not very s u c c e s s f u l i n obtaining a follow-up specimen i n e i t h e r screen. Many were f i l e d away and forgo t t e n * . It was therefore necessary to place personal phone c a l l s to the physicians i n * Probably, the i n t e n t i o n s were to review the l e t t e r with the mother when the i n f a n t and mother had the next appointment. - 131 -order t o complete the follow-up. I t was a l s o important to follow-up the o r i g i n a l request with a second phone c a l l (to the p h y s i c i a n or the parents) w i t h i n a reasonable time p e r i o d . This s t r e s s e d the importance of'the request ( e s p e c i a l l y to the parents) and u s u a l l y r e s u l t e d i n compliance. An a d d i t i o n a l problem, not a n t i c i p a t e d i n advance,* was the lack of resourcefulness of some parents f o r l o c a t i n g a container f o r the c o l l e c t i o n of the s t o o l specimen. There was a l s o h e s i t a t i o n i n mailing the specimen i n t h e i r containers and therefore the request for c o l l e c t i o n was conveniently ignored. Containers and mailing i n s t r u c t i o n s were made a v a i l a b l e f o r these parents when we were made aware of t h e i r problem. The follow-up procedure aroused anxiety i n some parents. This appeared to be s l i g h t l y worse i n the meconium screen and thought to be due mostly to the large delay i n forwarding the request. The anxiety associated with a screen should be reduced through d e l i c a t e treatment of the parents. Information should be provided about CF, the o b j e c t i v e of the t e s t and the follow-up procedure. Once the parents are t o l d of the screening r e s u l t , prompt completion of the follow-up t e s t i s necessary to minimize t h e i r a n x i e t i e s . Again because of minimum manpower the l a t t e r was not always accomplished. Anxiety could p o s s i b l y a l s o be reduced by not making the parents aware of the exact disease at such an e a r l y stage i n the follow-up procedure. The anxious state i n the parents seemed to be t r i g g e r e d by the term " C y s t i c F i b r o s i s " and a l l of i t s r a m i f i c a t i o n s . Towards the end * The assumption had been made that the doctor's o f f i c e would be looking a f t e r a l l the s t o o l c o l l e c t i o n s . - 132 -of the p i l o t p r o j e c t the use of the words C y s t i c F i b r o s i s was avoided with the f e c a l t r y p s i n screen. Parents were informed that t h e i r c h i l d had been screened by a neonatal screening programme and could p o s s i b l y have a d i g e s t i v e enzyme d e f i c i e n c y . The physicians of these i n f a n t s were completely i n favor of t h i s approach. Although the sample was small, there was a strong i n d i c a t i o n that t h i s approach reduced the amount of anxiety. This type of approach also has a negative side to i t , however, for i f the importance of the screen i s de-emphasized i n order to reduce anxiety, the importance of the follow-up i s a l s o de-emphasized and the parent i s more l i k e l y to respond with l e s s urgency or not at a l l . T h i s holding back of information i s probably not advisable for an e s t a b l i s h e d screening programme, but may be j u s t i f i a b l e for a p i l o t p r o j e c t that has npt-yet been proven r e l i a b l e . - 133 -SUMMARY AND CONCLUSIONS C y s t i c f i b r o s i s , which occurs most frequently among Caucasians, i s a co n d i t i o n whose e a r l y diagnosis and the r e s u l t a n t opportunity f o r e f f e c t i v e treatment could be missed. The incidence of CF as determined by a meconium albumin p i l o t screen i n operation i n Vancouver from 1976 to 1979 was 1 i n 2000. Thi s incidence i s s u f f i c i e n t l y high to warrant a CF screening programme for the province of B r i t i s h Columbia. Four methods were in v e s t i g a t e d to determine t h e i r s u i t a b i l i t y for Use i n a c y s t i c f i b r o s i s screening programme for the province of B r i t i s h Columbia. The e s s e n t i a l o b j e c t i v e s of a s a t i s f a c t o r y screening procedure for use i n the geographically dispersed areas of B.C. are that i t should provide the highest p o s s i b l e s p e c i f i c i t y and s e n s i t i v i t y and be adaptable to a m a i l - i n programme. Two of the procedures, the Robinson and E l l i o t t f e c a l t r y p s i n method and the determination of the f e c a l albumin:alpha-1 a n t i t r y p s i n r a t i o , were shown to be u n s a t i s f a c t o r y . The Robinson and E l l i o t t t r y p s i n method i n i t i a l l y appeared to have p o t e n t i a l because i t was performed, on dry s t o o l samples on f i l t e r paper cards which would be mailed to a c e n t r a l laboratory from the geographically dispersed areas of B.C. In our hands, however, the method f a i l e d to detect known CF specimens and to separate non-CF from c o n t r o l specimens. The method was shown to be non - s p e c i f i c because the colour - 134 -change of the assay was due not only to t r y p t i c a c t i v i t y but also to other unknown compound or compounds. Since t h i s compound was not i d e n t i f i e d , the s i g n i f i c a n c e of i t s presence i n s t o o l specimens from c y s t i c s i s not known. The interference of t h i s compound and the r e s u l t a n t lack of s p e c i f i c i t y makes the Robinson and E l l i o t t method unusable for the determination of f e c a l t r y p s i n a c t i v i t y . The albumin:alpha-1 a n t i t r y p s i n r a t i o has been used by Ryley to d i f f e r e n t i a t e between what appears to be pancreatic i n s u f f i c i e n c y i n healthy i n f a n t s from an a c t u a l pancreatic i n s u f f i c i e n c y . This concept was appealing because i t s use, i n combination with a f e c a l albumin screen, could p o t e n t i a l l y lower the number of f a l s e p o s i t i v e s that would t h e o r e t i c a l l y be obtained with that type of a screen. The i n v e s t i g a t i o n d i d confirm that the r a t i o could be used to d i f f e r e n t i a t e between f e c a l specimens with an abnormally high albumin concentration from c y s t i c s (with pancreatic i n s u f f i c i e n c y ) and from non-cystics. However the attempts made to f i n d a q u a l i t a t i v e albumin method for screening the s t o o l samples and to adopt the q u a n t i t a t i v e albumin and alpha-1 a n t i t r y p s i n Immunoelectrophoresis to the a n a l y s i s of dry s t o o l specimen on f i l t e r paper cards f a i l e d . Of the other two procedures i n v e s t i g a t e d , the meconium albumin assay (BMC t e s t s t r i p ) and Crossley's f e c a l t r y p s i n assay, the former had already been incorporated i n t o a p i l o t p r o j e c t at the Vancouver General H o s p i t a l . Since both methods seemed promising, the l a t t e r was incorporated i n t o a^second p i l o t p r o j e c t , and f a i r l y large scale i n v e s t i g a t i o n s were conducted. - 135 -The Boehringer-Mannheim Corporation t e s t - s t r i p , a s t r i p that detects an albumin content over 20 mg per g of meconium, proved to be a simple, r e l i a b l e and inexpensive t e s t . I t was used to assess 8,891 i n f a n t s y i e l d i n g 119 p o s i t i v e r e s u l t s . Of these 12 were from premature babies who had died s h o r t l y a f t e r b i r t h but who showed no signs of CF. Follow-up was completed on 96% of the remaining i n f a n t s . Of these, 3 in f a n t s were diagnosed as having c y s t i c f i b r o s i s . One f a l s e negative occurred, t o t a l l i n g 4 CF and g i v i n g an incidence of 1:2223 for the population t e s t e d . The negative r e s u l t was from a CF c h i l d who had low normal pancreatic function at b i r t h . The f a l s e p o s i t i v e rate was 1.3% which i s at an acceptable l e v e l . The f a l s e p o s i t i v e r e s u l t s were r e l a t e d to premature b i r t h and g a s t r o - i n t e s t i n a l disturbances. A s i g n i f i c a n t q o r r e l a t i o n was shown to be present with the p o s i t i v e BMC t e s t - s t r i p r e s u l t and n e c r o t i z i n g e n t e r o c o l i t i s (NEC) i n d i c a t i n g that the further i n v e s t i g a t i o n i n t o using the BMC t e s t - s t r i p as a d i a g n o s t i c t e s t f o r NEC would be worthwhile. The meconium screen d i d give s a t i s f a c t o r y r e s u l t s . The f a c t that the t e s t i s not s p e c i f i c for CF i s a disadvantage to a screening programme for CF but i t i s not a drawback i n i t s e l f since i t i s al s o important to recognize e a r l y the other c l i n i c a l conditions associated with a high concentration of albumin i n the meconium. The screen was reasonably s e n s i t i v e given i t s l i m i t a t i o n of i d e n t i f y i n g only patients with CF who have i n t r a u t e r i n e pancreatic i n s u f f i c i e n c y . The main disadvantages of the method are r e l a t e d to sample c o l l e c t i o n . Sample c o l l e c t i o n i s time constrained leading to a low c o l l e c t i o n r a t e . Good p r e c i s i o n with the BMC s t r i p i s only obtained i f a l l of the t e s t i n g i s - 136 -done in a central laboratory but this leads to d i f f i c u l t i e s in the organization of the sample collection away from larger c i t i e s since the meconium is unstable and needs to be transported frozen. These disadvantages make the method unsuitable for adoption as a CF screen for the whole province of B.C. A fecal trypsin method published by Crossley et a l provided a satisfactory solution to the sample collection for B.C. The trypsin a c t i v i t y in fecal samples spread on f i l t e r paper cards and mailed to a central laboratory was shown to be stable. The method was used to test dry stool specimens from 4085 infants. None of them to-date have cystic f i b r o s i s . The false positive rate was 4.7% which is an unacceptably high level financially and ethically and leads to d i f f i c u l t i e s in adequately following-up the patients. Two questionable areas of Crossley's method, the correction procedure for interfering fecal pigments and the visual evaluation of the test solutions were investigated and verified. Although the correction procedure for fecal pigments w i l l lead to a few false positive results due to overcompensation, the data indicate that the number w i l l be less than 0.6% as a conservative estimate. The visual evaluation was shown to be reliable, eliminating approximately 70% of the spectrophotometric work load. The use of a substrate solution twice the original concentration for re-analysing positive samples was shown to be unnecessary. This step in Crossley's procedure was therefore not adopted in this t r i a l screening programme. - 137 -The majority of the s t o o l specimens (3210) for the t r y p s i n screen were c o l l e c t e d i n the h o s p i t a l . The f a l s e p o s i t i v e rate for t h i s population was 5.0% and due mostly to the young age (under 3 days), of the infa n t s tested whose t r y p s i n enzymes are normally not present to the same l e v e l as 4 days or older i n f a n t s . The remainder of the specimens (875) were c o l l e c t e d by parents at-home and were therefore from i n f a n t s over 4 days o l d . These specimens were mailed i n to the laboratory. The f a l s e p o s i t i v e rate for t h i s population was 3.4%, a rate that was not s i g n i f i c a n t l y d i f f e r e n t from the h o s p i t a l c o l l e c t e d r a t e . The average concentration of p - n i t r o a n i l i n e i n the t e s t s o l u t i o n s of the home c o l l e c t e d specimens was however s i g n i f i c a n t l y l e s s than that present i n the t e s t s o l u t i o n s of h o s p i t a l c o l l e c t e d specimens. This was shown to be due to the thinner spread and therefore lower t r y p s i n a c t i v i t y of feces on the cards prepared at home. Since the o r i g i n a l c u t - o f f point chosen by Crossley was based on t h e i r data, our c u t - o f f point for the home c o l l e c t e d specimens could t h e o r e t i c a l l y be lowered even further based on our data i f i t s e f f e c t on s p e c i f i c i t y and s e n s i t i v i t y were c a r e f u l l y monitored. This could only be done however on a large scale evaluation of home c o l l e c t e d specimens. This was not f e a s i b l e economically and would cause further undue anxiety. For t h i s reason, and because of the a d d i t i o n a l concern that the f a l s e p o s i t i v e rate could not be lowered s u f f i c i e n t l y to enable the e f f i c i e n t completion of adequate follow-up, the t r y p s i n CF screen was al s o considered unsuitable for the province of B.C. - 138 -Many d i f f i c u l t i e s were encountered i n obtaining a follow-up on the presumptive p o s i t i v e s obtained i n each CF screening p i l o t p r o j e c t . The d i f f i c u l t i e s i n the meconium screen were caused by a delay i n i n i t i a t i n g the follow-up. As a r e s u l t of t h i s delay some anxiety was caused i n parents that could p o s s i b l y have been avoided, considerable more time and e f f o r t was required to c a r r y out the follow-up, and the t e s t r e s u l t s were of l e s s p o t e n t i a l value to the p h y s i c i a n . The d i f f i c u l t i e s i n the f e c a l screen were due to the high incidence of f a l s e p o s i t i v e s which d i s c r e d i t e d the programme. As a r e s u l t l e s s co-operation was obtained from both the physicians and the mothers i n c o l l e c t i n g the follow-up s t o o l sample for chymotrypsin a n a l y s i s . Unfortunately, both screening programmes were a f f e c t e d by budget c o n s t r a i n t s . At peak periods of laboratory work, and holiday time, l a c k of manpower r e s u l t e d i n an excessive delay time i n analysing and reporting the biochemical follow-up, unnecessarily lengthening the anxiety periods of some parents. Several phone c a l l s were placed to the family doctor or p e d i a t r i c i a n or to Childrens H o s p i t a l by parents who were anxiously waiting for the r e s u l t s of the follow-up s t o o l chymotrypsin t e s t s . The experiences gained i n following up the p o s i t i v e r e s u l t s from the meconium albumin and f e c a l t r y p s i n screen l e d to the following recommendations: i) Adequate information should be presented to parents before t h e i r i n f a n t i s screened. This may delay anxiety at the time of the t e s t and r e s u l t i n greater w i l l i n g n e s s to bring i n the sample (or infant) for the follow-up t e s t . - 139 -i i ) Prompt i n i t i a t i o n of the follow-up and prompt completion of the t e s t i s mandatory i n order to obtain a s u f f i c i e n t l y high compliance r a t e , provide the phy s i c i a n and i n f a n t with the f u l l b e n e f i t of the programme and minimize the parents' a n x i e t i e s . i i i ) The persons responsible for r e t r i e v i n g the follow-up specimen, requested to confirm a presumptive p o s i t i v e i n the screening programmej should be able to e x p l a i n both the screen and the follow-up t e s t i f c a l l e d upon to do so. They should be experienced i n dealing with anxious parents and should be r e a d i l y a v a i l a b l e to the parents. iv) I f the follow-up requires the r e t r i e v a l of a s t o o l or urine sample from the i n f a n t , convenience o f f i c i a l mailers should be provided to make the c o l l e c t i o n as easy as po s s i b l e for the parent. - 140 -ADDENDUM AN UPDATE In 1979, s h o r t l y a f t e r a d e c i s i o n had been reached at Children's -Ho s p i t a l to terminate the two e x i s t i n g CF screening p i l o t projects of meconium albumin and f e c a l t r y p s i n , C r o s s l e y ' 7 reported an e x c i t i n g new advance i n CF screeening. The New Zealand group had measured immunoreactive t r y p s i n (IRT) i n CF c h i l d r e n and found that i n the f i r s t few months of l i f e a l l CF c h i l d r e n had a r a i s e d serum IRT (including CF c h i l d r e n i n whom f e c a l t r y p s i n a c t i v i t y was not s i g n i f i c a n t l y decreased). IRT measurement appeared therefore to be capable of eli m i n a t i n g one of the strongest objections to both the meconium albumin and f e c a l t r y p s i n screens, the high f a l s e negative rate that r e s u l t s because they t e s t for pancreatic i n s u f f i c i e n c y . I t also appeared as i f the IRT method would eliminate or at l e a s t decrease i n number the f a l s e p o s i t i v e r e s u l t s obtain i n the f e c a l t r y p s i n screen because a l l 11 CF c h i l d r e n tested with the IRT method were c l e a r l y d i s t i n g u i s h a b l e not only from corresponding c o n t r o l s but al s o from non-CF babies with p e r s i s t e n t l y low s t o o l t r y p s i n . The IRT method appears to be capable of detecting CF c h i l d r e n who are e s s e n t i a l l y free of symptoms and these are the patients W so who are l i k e l y to b e n e f i t the most from the screen. ' Crossley had performed the IRT an a l y s i s on d r i e d blood spots. This i s a great advantage because drie d blood spots are c o l l e c t e d for s e v e r a l neonatal screening t e s t s i n many countries. I t i s advantageous from both - 141 -a p r a c t i c a l and economical standpoint to screen for s e v e r a l diseases from one sample. The cost of each a d d i t i o n a l new t e s t u s u a l l y adds l i t t l e to a screening programme c o s t . In B r i t i s h Columbia d r i e d bloot spots on f i l t e r paper are r o u t i n e l y c o l l e c t e d throughout the province and used to screen for phenylketonurea (PKU) and congenital hypothyroidism (T4). The quantity of blood c o l l e c t e d from each i n f a n t i s s u f f i c i e n t to perform three analyses, PKU, T4 and IRT and t e s t for a l l three genetic d i s o r d e r s . The IRT assay i s a r e l a t i v e l y simple radioimmunoassay s u i t a b l e for screening large numbers of specimens. Commercial k i t s are a v a i l a b l e which use the p r o t e i n binding p r i n c i p l e . The method requires f a i r l y expensive gamma counting equipment but since Children's H o s p i t a l had t h i s equipment on hand already for the T4 screen the cost of the IRT a n a l y s i s si i s reasonable and approximately equal to that of the T4 screen. When reports from other r e t r o s p e c t i v e studies confirmed Crossley's f i n d i n g that increased blood IRT was a c h a r a c t e r i s t i c of a l l newborn CF i n f a n t s whether or not they have r e s i d u a l exocrine pancreatic 3 f u n c t i o n . Children's H o s p i t a l i n i t i a t e d a p i l o t IRT screen. Only samples from newborn in f a n t s are tested since IRT value decreases i n CF c h i l d r e n , approaching the normal range or lower i n the older CF patient.* 0-'' 8 5'* 9 The present theory i s that IRT i s r a i s e d i n the newborn as a r e s u l t of s p i l l a g e from the pancreas i n t o the c i r c u l a t o r y system because of blocked pancreatic ducts. In the older c h i l d the IRT l e v e l i s thought to f a l l as a r e s u l t of the d e t e r i o r a t i o n of pancreatic f unct i o n . a 7 > s 9 In 1980, Crossley's second p u b l i c a t i o n on the IRT method included a prospective study t e s t i n g over 5,040 bewborns^ 9 The f a l s e p o s i t i v e rate was 0.67% which i s at an acceptable l e v e l for a routine screening \ - 142 -programme. At Children's H o s p i t a l 5,949 in f a n t s have been analysed at the time of w r i t i n g t h i s addendum and a f a l s e p o s i t i v e r a t e of 2.2% has been obtained i f a c u t - o f f point of 45 u n i t s i s used, or 0.35% i f a c u t - o f f point of 70 u n i t s i s used. The c u t - o f f point has yet to be e s t a b l i s h e d . False p o s i t i v e s are found i n the newly born i n f a n t with j e j u n a l a t r e s i a , b i l a r y a r t r e s i a and pancreatic a c h y l i a . Follow-Up Procedure Because of the d i f f i c u l t i e s encountered i n the follow-up on both the meconium albumin and f e c a l t r y p s i n screening p i l o t p rojects i n Vancouver, emphasis was placed on incorporating i n t o t h i s new screening programme the recommendations that were made as a r e s u l t of the previous experience. In order to guarantee success i n the follow-up an i n i t i a l request goes out to p e d i a t r i c i a n s and family physicians throughout the province for permission to t e s t an i n f a n t (or infants) under h i s care. Confidence i n the IRT assay i s conveyed to the ph y s i c i a n . By asking h i s p a r t i c i p a t i o n i n the programme, the l i k e l i h o o d of the physician's f u l l co-operation following-up p o s i t i v e r e s u l t s , both chemically and c l i n i c a l l y , i s g r e a t l y increased. A r e g i s t e r e d nurse was hi r e d as the co-ordinator for the IRT programme. This means the programme has the manpower to follow-up a l l r e s u l t s immediately and to pursue the i n i t i a l request within f a i r l y narrow time l i m i t s . A separate telephone l i n e was es t a b l i s h e d for the CF screening programme and an answering s e r v i c e was incorporated i n order to ensure that a l l enquiries regarding the programme get answered. - 143 -A request for a second d r i e d blood specimen i s made on a l l p o s i t i v e IRT l e v e l s found on the o r i g i n a l screening card. Contact i s made with the p h y s i c i a n d i r e c t l y by phone requesting the c o l l e c t i o n of the second specimen at the in f a n t s "6-week check". The parents' anxiety i s therefore not aroused by making a s p e c i a l v i s i t to the doctor's o f f i c e for the sole purpose of c o l l e c t i n g a blood specimen. The phone c a l l to the doctor i s immediately followed with a l e t t e r and c o l l e c t i o n i n s t r u c t i o n s . The physi c i a n decides whether or not the parents should be t o l d at t h i s stage that the blood specimen i s for CF. D i r e c t contact with the parents i s not necessary because the specimen i s i n most cases c o l l e c t e d a t the doctor's o f f i c e . At the physician's request, however, the sample c o l l e c t i o n could be made at Children's H o s p i t a l as an outpatient. The second blood specimen i s analysed immediately and the r e s u l t , whether negative or p o s i t i v e , i s phoned to the physi c i a n , followed by a confirmation l e t t e r . I f the second specimen a l s o has an increased IRT l e v e l a request for a s t o o l chymotrypsin or sweat c h l o r i d e i s placed with the p h y s i c i a n by one of the C l i n i c i a n s from the CF C l i n i c a l Assessment C l i n i c . A pamphlet e n t i t l e d "IRT Screening for C y s t i c F i b r o s i s . What i s i t ? " i s provided for the parents at t h i s stage. In many cases t h i s i s the f i r s t time the parents make contact with the CF screening programme co-ordinator. I t was f e l t that an R.N. would better serve the needs of the anxious parents both i n person and i n answering telephone queries. The implementation of the recommendations have r e s u l t e d i n increasing the number of completed follow-ups and reducing the anxiety of the parents involved. I t i s too e a r l y to e s t a b l i s h whether the f a l s e - 144 -p o s i t i v e rate i s at an acceptable l e v e l and the f a l s e negative rate won't be known for some time. To date the IRT neonatal screen for CF looks very promising. - 145 -APPENDIX A SCREENING CARD FOR COLLECTION OF STOOL SAMPLE FECAL TRYPSIN CF SCREEN - 146 -C.F. SCREENING PROGRAMME CHILDREN'S HOSPITAL DATE OF SPECIMEN CHILD'S NAME. BIRTH DATE _ .HOSPITAL PARENT'S NAME ADDRESS _ BABY'S DOCTOR ADDRESS Tel. « S o .± c o O 1) L to • o c c5 o • - o o ° c ° ^ C 0) 0) OJ > c E ° | v r H Q. O « v> g> 73 "U > CO S C QJ .S * Q. II « c a > Q. W v - 147 -APPENDIX B •— LETTER TO PARENTS: REQUEST FOR COLLECTON OF INFANT'S STOOL SAMPLE FOR CF SCREENING PROGRAMME - 149 -APPENDIX C CALCULATION OF % ERROR RESULTING FROM USE OF CROSSLEY'S FECAL PIGMENT CORRECTION PROCEDURE - 150 -1. C a l c u l a t i o n of True Net Absorbance I t i s not p o s s i b l e to subtract the absorbance reading at 410 nm for the f e c a l pigment (column b i n Table III) d i r e c t l y from the TEST s o l u t i o n absorbance set 410 nm because each reading was obtained using a d i f f e r e n t sample d i s c with varying amounts of feces. The f e c a l pigment Abs 410/Abs 460 r a t i o should, however remain constant despite the use of a separate sample d i s c . As a r e s u l t the true net absorbance value can be c a l c u l a t e d using the f e c a l pigment r a t i o e s t a b l i s h e d f o r each sample and the f a c t that the p - n i t r o a n i l i n e absorbance at 460 nm i s 4.4% of i t s absorbance at 410 nm i n the fo l l o w i n g system of simultaneous equations. / Absorbance readings reported i n Table I I I f o r the f i r s t s t o o l sample are used i n the example c a l c u l a t i o n . f e c a l pigment + p - n i t r o a n i l i n e = 1.675 1/1.61 f.p. + .044 p - n i t r o a n i l i n e = 0.164 . [1.675 - 1 . 6 K . 1 6 4 ) ] T [1-1.61(0.044)] = p - n i t r o a n i l i n e Abs 410 = 1.519 Therefore, p - n i t r o a n i l i n e absorbance at 410 nm for the f i r s t s t o o l smaple i n Table I I I should have been 1.519 2. C a l c u l a t i o n of % Error The c a l c u l a t i o n performed above r e s u l t s i n the true net absorbance reading of p - n i t r o a n i l i n e at 410 nm. When Crossley's c o r r e c t i o n procedure i s applied however the net absorbance represents only 91.2% of the p - n i t r o a n i l i n e (as discussed on page ). Therefore 91.2% of the net absorbance c a l c u l a t e d above was compared, with the r e s u l t obtained using Crossley's c o r r e c t i o n procedure i n order to c a l c u l a t e the % e r r o r . ^ X 1.519 = 1.385 - 151 -APPENDIX D LETTER TO PHYSICIAN: REQUEST FOR STOOL SAMPLE FOR QUANTITATIVE CHYMOTRYPSIN ANALYSIS INSTRUCTIONS FOR STOOL SAMPLE COLLECTION STOOL COLLECTION FOR CHYMOTRYPSIN 1. Collect a few grains (walnut-sized sample) of a random stool. 2. Place stool in a closed container. Label container with: ^ NAME: DATE OF BIRTH: DATE OF SAMPLE: DOCTOR'S NAME: 3. For specimens from Vancouver, send fresh or fresh frozen to: C.F.Screening Programme Children's Hospital 250 West 59th Avenue Vancouver, B.C. V5Z 1X2 *•* *** *** *** BOARD OF DIRECTORS: G. H. Tullidge. president; J. S. McKendy. first vice president; 0. M. Clark, O.C.. second vice president; Mrs. M. S. Duffus. recording secretary. Also on the Board: C. W. Bawlf; M. Belkin; B. S. Brown: Mrs. W. Clark; J. H. Green; H J. Grey, Q.C.; Mrs. R. S. Hager; D. M. Howard: W. S. McQuaid: Mrs. G. L. Claman. government representative. ADMINISTRATION: J. W. Short, administrator; D. R. McAmmond, assistant administrator; J. L. Greenan. treasurer. - 154 -APPENDIX E CLINICAL QUESTIONNAIRE - 155 -DATE: RETURN TO: C.F. SCREENING, CHILDREN'S HOSPITAL, 250 WEST 59TH AVENUE, VANCOUVER, B.C. V5X 1X2 NAME: B.D: (1) CLINICAL SUMMARY (2) TESTS OR RECENT WEIGHT DATE: Well - no problems Chest i n f e c t i o n Diarrhoea F a i l u r e to t h r i v e Other S t o o l chymotrypsin Sweat e l e c t r o l y t e s R e s u l t s : I w i l l arrange above t e s t s Other suggestions DOCTOR N.B. Specimen f o r s t o o l chymotrypsin should be walnut-sized, weighing approximately 3-5 grams, l a b e l l e d and forwarded to Chi l d r e n ' s H o s p i t a l . - 156 -APPENDIX F LETTER TO PHYSICIAN: REQUEST FOR CLINICAL FOLLOW-UP TO PHYSICIAN WHO FELT LABORATORY FOLLOW-UP WAS NOT WARRANTED. - 158 -APPENDIX G CHI-SQUARE TEST FOR SIGNIFICANCE OF DIFFERENCE BETWEEN FALSE POSITIVE RATES FOR HOSPITAL AND HOME COLLECTED SPECIMENS IN FECAL TRYPSIN CF SCREEN - 159 -Ho s p i t a l c o l l e c t e d samples n = 3,210 no. of p o s i t i v e s = 160 Home c o l l e c t e d samples n = 875 no. of p o s i t i v e s = 30 Ho s p i t a l Home Neg. 3050 845 3895 Pos. 160 30 190 3210 875 4085 The chi-square value with one degree of freedom i s 4085113050 (30) - 845(160)1 - 4085/2 1 2 (3895) (190) (3210) (875) = 3.41 This i s l e s s than the c r i t i c a l value 3.84 of chi-square at the l e v e l . Therefore the d i f f e r e n c e i n f a l s e p o s i t i v e s rates between h o s p i t a l and home c o l l e c t e d samples i s not s i g n i f i c a n t . - 160 -APPENDIX H CHI-SQUARE TEST FOR SIGNIFICANCE OF DIFFERENCE BETWEEN FALSE POSITIVE RATES FOR LESS THAN 3-DAY OLD INFANTS AND AT LEAST 3-DAY OLD INFANTS - 161 -H o s p i t a l c o l l e c t e d samples were checked for age of i n f a n t at time of c o l l e c t i o n number of p o s i t i v e s checked 108 n under 3 days 84 n 3 days or older 24 number of negative r e s u l t s checked 300 n under 3 days 117 n 3 days or older 183 3 days 3 days Pos. 84 24 108 Neg. 117 183 300 201 207 408 The chi-square value with one degree of freedom i s : 408 [|184 (183) - 24(117 ) 1 - 408/2 ] 2 (108) (300) (210) (207) = 46.23 This i s more than the c r i t i c a l value 10.83 of chi-square at the 0.1% l e v e l . Therefore the d i f f e r e n c e i n f a l s e p o s i t i v e r a t e between the under 3 day o l d i n f a n t and the 3 day or older i n f a n t i s highly s i g n i f i c a n t . - 162 -APPENDIX I CHI-SQUARE TEST FOR SIGNIFICANCE OF CHANGES IN RESULTS FOR HOSPITAL AND HOME COLLECTED SPECIMENS ON THE SAME INFANT - 163 -Both a h o s p i t a l and a home c o l l e c t e d specimen were c o l l e c t e d on 692 i n f a n t s . One i n f a n t gave a p o s i t i v e r e s u l t on both specimens, 34 inf a n t s were postive on the h o s p i t a l c o l l e c t e d specimen only Using McNemar t e s t for s i g n i f i c a n c e of changes*, corrected for c o n t i n u i t y the chi-square value with one degree of freedom i s : ( 21-34 - l ) 2 21 + 34 = 2.62 This i s l e s s than the c r i t i c a l value of 2.71 of chi-square at the 10% l e v e l . Therefore the changes i n r e s u l t s between the two c o l l e c t i o n methods are not s i g n i f i c a n t . * Sidney S i e g e l , Nonparametric S t a t i s t i c s , New York, McGraw-Hill, 1956, pp. 63-64. - 164 -APPENDIX J 1. CHI-SQUARE TEST FOR SIGNIFICANCE OF DIFFERENCE BETWEEN DISTRIBUTIONS OF NET ABSORBANCE READINGS OF HOSPITAL AND HOME COLLECTED SPECIMENS 2. TEST FOR SIGNIFICANCE OF DIFFERENCE BETWEEN MEANS OF HOSPITAL AND HOME COLLECTED SPECIMENS NET ABSORBANCE VALUES - 165 -1. D i s t r i b u t i o n s of Net Absorbance Values of H o s p i t a l and Home C o l l e c t i v e Specimens Net Absorbance Value HOSPITAL COLLECTED n = 485 ac t u a l expected1* HOME COLLECTED n = 470 ac t u a l expected 0 - .299 31 32.5 33 31.5 .300 - .599 46 57.9 68 56.1 .600 - .899 49 73.1 95 70.9 .900 - 1.199 104 116.8 126 113.2 1.200 - 1.799 255 204.7 148 198.3 The Chi--square value with 4 degrees of freedom i s z i 2 (Obs. - Exp.) 1 1 = 49.22 Expi This i s more than the c r i t i c a l values of 18.46 of chi-square at the 0.1% l e v e l . Therefore the d i f f e r e n c e between d i s t r i b u t i o n s of net absorbance readings of h o s p i t a l and home c o l l e c t e d specimens i s highly s i g n i f i c a n t . * I f population d i s t r i b u t i o n s are the same - 166 -2. Means of H o s p i t a l and Home Co l l e c t e d Specimen Net Absorbance Values H o s p i t a l c o l l e c t e d specimens mean 1.068 Home c o l l e c t e d specimens mean 0.941 d i f f e r e n c e 0.127 The standard e r r o r of h o s p i t a l c o l l e c t e d specimens i s 0.0175 The standard e r r o r o> of home c o l l e c t e d specimens i s 0.0174 The standard e r r o r of the d i f f e r e n c e i s V (.0175) 2 + (.0174) 2 = 0.0247 Therefore the d i f f e r e n c e 0.127 between means, which due to sample s i z e i s normally d i s t r i b u t e d , i s 0.127 = 5.14 standard deviations 0.0247 from zero and i s therefore highly s i g n i f i c a n t . - 167 -APPENDIX K -1. COMPARISON OF SCREENING RESULTS FROM FREEZER AND ROOM TEMPERATURE STORED SPECIMENS 2. COMPARISON OF SPECIMEN RESULTS BEFORE AND AFTER MAILING 3. COMBINED DATA FOR ROOM TEMPERATURE STORED SPECIMENS - 168 -1. COMPARISON OF RESULTS FROM FREEZER AND ROOM TEMPERATURE STORED SPECIMENS. Using a normal approximation to the binomial d i s t r i b u t i o n , with c o r r e c t i o n for c o n t i n u i t y , the p r o b a b i l i t y of g e t t i n g a decrease i n 12 or more specimens out of 20 due to random error alone can be c a l c u l a t e d as follows: (12-0.5) -0.5(20) 0.5 V20 = 0.6708 The p r o b a b i l i t y of obtaining a Z value as high as 0.6708 i s 25.1% which i s not s i g n i f i c a n t . 2. COMPARISON OF RESULTS BEFORE AND AFTER MAILING Again using a normal approximation to the binomial d i s t r i b u t i o n , with c o r r e c t i o n for c o n t i n u i t y , the p r o b a b i l i t y of g e t t i n g a decrease i n 14 or more specimens out o f 25 due to random erro r alone (ignoring the s i n g l e t i e ) , can be c a l c u l a t e d as follows: = (14-0.5) - 0.5(24) 0.5 V24 = 0.6124 The p r o b a b i l i t y of obtaining a Z value as high as 0.6124 i s 27.0% which i s not s i g n i f i c a n t . 3. COMBINED DATA OF SECTIONS 1 AND 2 ABOVE Using a normal approximation to the binomial d i s t r i b u t i o n with c o r r e c t i o n for c o n t i n u i t y , the p r o b a b i l i t y of get t i n g a decrease i n 26 or more specimens out of 45 due to random error alone (again ignoring the si n g l e t i e ) can be c a l c u l a t e d as follows: = (26-0.5) - 0.5(44) 0.5 V44 = 1.0553 The p r o b a b i l i t y of obtaining a Z value as high as 1.0553 i s 14.6% which i s not s i g n i f i c a n t . - 169 -APPENDIX L CHI-SQUARE TEST FOR SIGNIFICANCE OF DIFFERENCE BETWEEN DISTRIBUTIONS OF ABSORBANCE VALUES AT 460 NM OF HOSPITAL AND HOME COLLECTED SPECIMENS - 170 -D i s t r i b u t i o n of Abs 460 Readings of H o s p i t a l and Home C o l l e c t e d Specimens Net HOSPITAL COLLECTED HOME COLLECTED Absorbance n = 682 n = 633 Value a c t u a l expected a c t u a l expected 0 - .049 79 135.9 183 126.1 .050 - .099 370 339.7 285 315.3 .100 - .149 137 111.0 77 103.0 .150 - 1.199 40 38.4 34 35.6 .200 - 1.249 24 24.9 24 23.1 .250 - .249 10 9.9 9 9.1 .300 - .349 5 8.3 11 7.7 .350 - 1.000 17 14.0 10 13.0 The Chi-square value with 7 degrees of freedom i s : 2 (Obs. - Exp.) = 72.04 L ExPi This i s more than the c r i t i c a l values of 24.32 of chi-square at the 0.1% l e v e l . Therefore the d i f f e r e n c e between d i s t r i b u t i o n s of Abs 460 readings of h o s p i t a l and home c o l l e c t e d specimens i s hig h l y s i g n i f i c a n t . * I f population d i s t r i b u t i o n s are the same - 171 -APPENDIX M COMPARISON OF THE PRECISION OF THE RESULTS OF HOSPITAL AND HOME COLLECTED SPECIMENS - 172 -SPECIMENS HOSPITAL COLLECTED HOME COLLECTED 10 0.1242 0.1469 0.0465 Difference in x = 0.1242 - 0.1065 = 0.0177 S.D. of difference = A/(0 .0465)2 + (0.0140)2 = 0.0486 0.0177 - 0.364 S.D. which i s not significant. 0.0486 (i.e. no significant difference in precision between hospital and home collected repeat analysis) n S« D-S.D. S.D.j 37 0.1065 0.0850 0.0140 - 173 -APPENDIX N CORRELATION BETWEEN POSITIVE MECONIUM SCREEN RESULTS AND PRESENCE OF NECROTIZING ENTEROCOLITIS o - 174 -1. Out of 119 p o s i t i v e CF meconium r e s u l t s 23 i n f a n t s were diagnosed as having n e c r o t i z i n g e n t e r o c o l i t i s . Using the Poisson d i s t r i b u t i o n as an approximation to the hypergeometric d i s t r i b u t i o n , with the expected NEC being 0.669 cases i n 119 p o s i t i v e s (based on 50 occurences of NEC i n the 8891 c h i l d r e n tested at VGH), the p r o b a b i l i t y of f i n d i n g 23 or more NEC i n 119 p o s i t i v e s i s 22 ,,n.x -0.669 (0.669) e  x=0 which i s i n s i g n i f i c a n t . 2. Using the same procedure for the number of NEC among premature i n f a n t s , where the expected number of NEC's i n 82 i s 1.48, (based on an incidence of 18 NEC cases per 1000 b i r t h s as estimated from S t o l l ^ ) , the p r o b a b i l i t y of f i n d i n g 22 or more NEC i n 82 premature p o s i t i v e s i s --— 21 ,, , Q.x -1.48 1 - £ (1»48) e  x=0 which i s i n s i g n i f i c a n t . - 175 -APPENDIX 0 1. RANK TEST FOR SIGNIFICANCE OF THE EFFECT OF DOUBLING THE BAPNA SUBSTRATE CONCENTRATION 2. TEST FOR CORRELATION BETWEEN % INCREASE IN ABSORBANCE DUE TO DOUBLING SUBSTRATE CONCENTRATION AND THE ORIGINAL ABSORBANCE VALUE - 176 -1. The 30 samples were tested 4 times with the 0.25 mg BAPNA substrate and once with the 0.50 mg BAPNA substrate. In each case the rank of the l a t t e r among the f i v e r e s u l t s was determined. In 14 out of the 30 samples, the double substrate r e s u l t ranked highest of the f i v e . The p r o b a b i l i t y of obtaining t h i s r e s u l t by chance alone, assuming equal p r o b a b i l i t y for each of the 5 pos s i b l e ranks, i s : P (14/30 are ranked highes't) = ( 1 / 5 ) 1 4 ( 4 / 5 ) 1 6 = 0.00067 Therefore the e f f e c t of doubling the substrate concentration i s s i g n i f i c a n t at the 0.1% l e v e l . 2. The l i n e a r regression of the % increase i n absorbance value due to doubling the substrate concentration on the mean absorbance value of the 0.25 mg BAPNA r s u l t s for each sample l e d to the following equation: Y = 0.2047 - 0.000991X with a c o e f f i c i e n t of determination of 0.000129 and a standard e r r o r of the slope c o e f f i c i e n t of 0.016 i n d i c a t i n g no s i g n i f i c a n t c o r r e l a t i o n . - 177 -APPENDIX P CONDITIONS UNDER WHICH AN EQUAL PERCENT REDUCTION IN ABSORBANCE AT 410 AND 460 NANOMETERS FROM HOSPITAL TO HOME COLLECTED SPECIMENS CAN OCCUR - 178 -By the p r i n c i p l e of a d d i t i v i t y , the absorbance of h o s p i t a l and home t e s t s o l u t i o n s can be expressed as the sum of absorbances due to p - n i t r o a n i l i n e and f e c a l pigment as follows: PMA fp h4W = A 4 1 0 + 410 _ « PNA . - fp A460 _ A 460 + A460 where e.g. * 4 & ) - *4f60 * b x c f P and where e.g. a460 = m o ^ - a r a b s o r p t i v i t y of f e c a l pigment at 460 nm c P M = concentration of p - n i t r o a n i l i n e (PNA) i n the t e s t s o l u t i o n (as a r e s u l t of the typ s i n a c t i v i t y i n eit h e r the h o s p i t a l or home c o l l e c t e d specimens). b = c e l l depth The mean absorbance values at both 410 and 460 nanometers of t e s t s o l u t i o n s prepared with home c o l l e c t e d samples were 12% lower than the h o s p i t a l c o l l e c t e d samples.* (Sample s i z e was 500.) This decrease i n absorance values at both wavelengths could be as a r e s u l t of one or more of the following: 1. The home c o l l e c t e d s t o o l smears were thinner than the h o s p i t a l c o l l e c t e d s t o o l smears. * For complete d i s t r i b u t i o n s of net absorbances and absorbances at 460 nm for home and h o s p i t a l c o l l e c t e d samples see f i g u r e s II and I I I r e s p e c t i v e l y . Note that since net absorbance A = h4lD ~ 2 A 4 6 0 as discussed on page 65, mean net absorbance A = A 4 l 0 - 2 A 4 6 Q , and a simultaneous reduction of 12% i n A 4 ( c, and A 4 6 0 r e s u l t s i n a 12% reduction i n net absorbance, A, as indicated i n f i g u r e I I . - 179 -2. There was a decrease i n concentration of f e c a l pigment i n home c o l l e c t e d feces. 3. There was a decrease i n t r y p s i n concentration i n home c o l l e c t e d feces r e s u l t i n g i n a decrease i n p - n i t r o a n i l i n e i n the t e s t s o l u t i o n s . An average 12% reduction i n s t o o l smear thickness from h o s p i t a l to home (item 1 above) would have the e f f e c t of decreasing both C P h A and C^ p by the same amount and therefore c l e a r l y reduce A 4 1 0 and &4&Q by the same amount from h o s p i t a l to home. A decrease i n f e c a l pigment concentration i n the home c o l l e c t e d feces (item 2) would reduce A^, and A | £ 0 equally (proportionally to the reduction i n C-p) but since A*£ 0 i s not the same f r a c t i o n of the A^jo as i s the A ^ o of A 4 6 o , the percentage reductions i n A 4 i 0 and A^ 6 0 would not be the same. The same argument holds for item 3 above. Of course an extraordinary coincidence of simultaneous equal changes i n t r y p s i n concentration and f e c a l pigment concentration could r e s u l t i n a 12% decrease i n both. - 180 -APPENDIX Q CALCULATION OF CONFIDENCE THAT CHILD WITH POSITIVE FECAL TRYPSIN SCREEN RESULT HAS CF - 181 -The p r o b a b i l i t y that a c h i l d with a p o s i t i v e r e s u l t from the f e c a l t r y p s i n screen does i n f a c t have CF i s c a l c u l a t e d as follows: Let P(CF) = incidence of CF i n population = 0.0005 P(Normal) = 1 - P(CF) = 0.9995 P( + /Normal) = f a l s e p o s i t i v e rate =0.019 P( + /CF) = true p o s i t i v e rate (unknown) We want to c a l c u l a t e : P(CF/+) = p r o b a b i l i t y that c h i l d with p o s i t i v e r e s u l t has CF p , c p / + l = P(+/CF) P(CF)  v ' ' P(+/CF) P(CF) + P(+/Normal)P(Normal) An upper bound for the value of P(CF/+) occurs when P(+/CF) = 1.0 i . e . the true p o s i t i v e r a t e i s p e r f e c t . This would r e s u l t i n : P ( C F / + ) = (1.0X0.0005) 1 ' ' (1.00) (0.0005) + (0.019) (0.9995) = 2.6% Thus the p r o b a b i l i t y that a c h i l d with a p o s i t i v e r e s u l t does have CF i s l e s s than 2.6%. - 182 -BIBLIOGRAPHY 1. Adriaenssens, K.H., H. Janssens and H. van Soom: Two t i e r screen for c y s t i c f i b r o s i s . Lancet i : 833, 1981. 2. Applegarth, Derek A.: Biochemical screening for p a e d i a t r i c d i s o r d e r s . Talk given to Royal College of Physicians and Surgeons of Canada, Montreal, 1979. 3. Applegarth, Derek A., A.G.F. Davidson, L.T. Kirby, M. Bridges, P. Sorensen, L.T.K. Wong and D.F. Hardwick: Dried blood spot screening for c y s t i c f i b r o s i s . Lancet i i : 1236, 1979. 4. Armstrong, D. and J.C. Kramer: Studies i n c y s t i c f i b r o s i s . Determination of sweat e l e c t r o l y t e s i n s i t u with d i r e c t reading e l e c t r o d e s . Pediatr 43_: 794, 1969. 5. Barbero, G i u l i o J . , Maarten S. 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