Welcome to today s webinar. Shedding Light on Bilirubin Presenter: Dennis J. Dietzen, Ph.D., DABCC. Sponsored by

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1 Welcome to today s webinar Shedding Light on Bilirubin Presenter: Dennis J. Dietzen, Ph.D., DABCC Sponsored by We will begin shortly. For technical assistance, call

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5 Today s Presenter Dennis J. Dietzen, Ph.D., DABCC Dennis Dietzen is Associate Professor of Pediatrics and Pathology at Washington University where he directs the Core Laboratory and Metabolic Genetics Laboratory at St. Louis Children s Hospital. Dr. Dietzen received a Ph.D. in Biochemistry from Indiana University in 1992 and completed a postdoctoral fellowship at Washington University in Prior to his return to St. Louis in 2002, he was a chemist at DuPont Diagnostics (now a part of Siemens Healthcare Diagnostics) and served as Director of Clinical Laboratories at the Memphis Veterans Affairs Hospital.

6 Neonatal Bilirubin: Yellow Means Caution Dennis J. Dietzen, Ph.D., DABCC Washington University School of Medicine St. Louis Children s Hospital

7 Bilirubin What is it? Where does it come from? Where does it go? How does it get there? Why is it a problem? How do we measure it? Diazo Spectral How much is too much? What is the best way to measure it? How can we do it better?

8 A B C D Which one of these is bilirubin?

9 A: Heme B: Bilirubin C: Folate D: THC Answer: B

10 A B C D Where does it come from?

11 A: Erythrocytes B: Muscle C: Brain D: Liver Answer:

12 A B C D Where does it go?

13 A: Erythrocytes B: Kidney C: Brain D: Liver Answer:

14 Where does it go?

15 How does it get there?

16 Infants have high hematocrit and immature livers bad combination. 60% of newborns become jaundiced Most of the time it s uncomplicated 1-2% exhibit bilirubin > 20 mg/dl Elevated unconjugated bilirubin may lead to kernicterus 5-40 infants/1000 receive phototherapy Exchange transfusions infrequent (<2/1000) Why is it a problem?

17 Exacerbating causes Rh incompatibility, erythroblastosis fetalis Hemolytic disorders (G6PD, PK deficiencies) Genetic disorders (e.g., Gilbert s) Late preterm/breastfed infants American Academy of Pediatrics Recommendations (2004) Total Serum Bilirubin Must be fast (phototherapy takes time) Must be accurate Must make certain that conjugated bilirbuin is not elevated. Conjugated hyperbilirubinemia suggests biliary disease. Why is it a problem (cont.)

18 Chemical Diazo Vanadate Bilirubin oxidase Optical Vitros Bu/Bc Transcutaneous Direct Absorbance Measurement of Bilirubin

19 Diazotized sulfanilic acid Ehrlich, 1883 Van den Burgh & Mueller, 1916 Evelyn & Malloy, 1937 Jendrassik & Grof, 1938 Doumas, 1983 Formation of purple pigment at 550 nm Chemical-1

20 Vanadate oxidation Wako Patent, 1996 Siemens Advia User-defined channel Bilirubin oxidase Abaxis Piccolo, 1990s Vanadate Bili Oxidase Chemical-2

21 Gastroenterolgoy 1978;74: Easy Right?...Not so fast! Internal hydrogen bonding Implications for measurement

22 Don t forget delta DMSO Caffeine Methanol Benzoate Theophylline Urea Chemical-3 What is Direct and Indirect Bili?

23 Direct = No accelerator Equals conjugated fraction plus ~1-10% of unconjugated. Not a problem in normal adults. (0-0.2 mg/dl) Large unconjugated fraction in kids can leave impression of conjugated hyperbilirubinemia (~ mg/dl). Total=Conjugated + unconjugated + delta Unconjugated = Total Direct Unless there is delta What are direct and indirect?

24 Clin Chem 1984;30: Vitros Plasma (no hemoglobin mostly) Serendipitous Separation of Bu and Bc spectra Does not detect delta Delta = Total (Bu + Bc) Optical Methods Whole blood Use λ ~500 Hematocrit Correction Total Only Speed, blood volume Radiometer, ~10 years Siemens and IL recently

25 Diazo Vanadate Bu/Bc Who is doing what? CAP neonatal bilirubin 2010 NB-B

26 HPLC Peak α β γ δ Bilirubin Species Unconjugated Singly Conjugated Doubly Conjugated Conjugated to Albumin Traditional Methods Indirect (Total Direct) Total? Direct Total Vitros Methods Unconjugated (Bu) Conjugated (Bc) Neonatal (Bu+Bc) Delta (Total [Bu+Bc]) Direct (Total Bu) Complements of Dr. Phil Bach, University of Utah. Summary

27 Shed a little light

28 H H Biochem Biophys Res Comm 1979;90: Shed a little light

29 H H C-4,5 H H C-15,16 Configurational Structural 4Z,15Z 4E,15Z 4Z,15E 4E,15E Cyclo Shed a little light

30 EE,EZ Total Direct Diazo Cyclo Bu Bc AAP recommendations include following only TOTAL. Formation in vivo or in vitro? Shed a little light- Effect on diazo methods

31 Absorptivity of native circulating bilirubin forms is roughly equal. Absorptivity of configurational isomers is reduced, but not absent. Absorptivity of structural isomers is minimal Effect of light- Impact on Optical Methods J Chromatography 1986;383:

33 Central Laboratory Direct & Indirect Diazo (Adult) Vitros BuBc (Pediatric) Transport, spin, analyze (blood volume, light) Implementation/Validation Fresh specimens-no photoisomers Know direct & indirect component Focus on simple unconjugated Relative Imprecision (WB & TC ) Nursery to NICU specimens F/U inconsistencies-tip of iceberg Phototherapy specimens Check adults for HbF influence Lipemic for turbidity correction Check vs. total or BuBc (neonatal) Lab confirmation limits Periodic accuracy comparison Near-Patient Options No plasma/serum Eyeball method Transcutaneous Whole Blood Now that we know everything, what should practice be?

34 Hyperbilirubinemia is common and dangerous Bilirubin assays are not created equal. For uncomplicated, unconjugated hyperbili: Find one. Know it. Stick with it. Tell your physicians to do the same. AAP recommendations based on Total Serum. Hard to manage equivalence of multiple assays. Direct assays overestimate the presence of conjugated bilirubin in infants. This is bad. Take home message.

35 Wet technique specific for conjugated Harmonizing standard? Matrix effects. Reference technique (e.g., LC/MS/MS) Simultaneous enumeration of multiple bilirubin metabolites.bilirubinomics Improve diagnosis of cholestatic disorders Newborn screening? (biliary atresia). Can we do it better?

37 Continuing Education Credit After today s webinar: Land on Evaluation form with link to eval Forward to colleagues who attended with you CRCE Contact Hour by the American Association for Respiratory Care (Available for Live session only) Florida CE (Available for Live session only) PACE (California approved) Credit Certificate of Attendance Must complete Eval to receive CEU and Certificate Receive certificate via

38 Questions

39 Continuing Education Credit After today s webinar: Land on Evaluation form with link to eval Forward to colleagues who attended with you CRCE Contact Hour by the American Association for Respiratory Care (Available for Live session only) Florida CE (Available for Live session only) PACE (California approved) Credit Certificate of Attendance Must complete Eval to receive CEU and Certificate Receive certificate via

40 Thank you for attending today s session. For a complete listing of upcoming and recorded Radiometer sponsored educational webinars and resources, visit:

UNIT: Total and Direct Bilirubin

UNIT: Total and Direct Bilirubin 13bili.wpd Task Determination of total and direct bilirubin. Objectives Upon completion of this exercise, the student will be able to: 1. Explain formation, excretion,