Hemolytic Disease of the Newborn

1. Hemolytic Disease of the Newborn Dr. Ayman Asfour, Director of Transfusion Medicine University of Mississippi Medical Center

2. Names for HDN Hydrops fetalis Icterus gravis neonatorum Kernicterus (outcome) Erythroblastosis fetalis

3. HDN Destruction of RBC Neonates and infants Intrinsic (membrane defect, enzymes) Acquired (infection, alloimmunization) Platelets and Granulocytes G-6-phosphate dehydrogenas, pyruvate kinase eliptocytosis, spherocytosis, , pyropoikilocytosis congenital syphilis, toxoplasmosis, CMV, rubella, coxackie and E-coli Rh, ABO maternal autoimmune hemolytic anemiaG-6-phosphate dehydrogenas, pyruvate kinase eliptocytosis, spherocytosis, , pyropoikilocytosis congenital syphilis, toxoplasmosis, CMV, rubella, coxackie and E-coli Rh, ABO maternal autoimmune hemolytic anemia

12. The DAT will detect antibodies coating the fetal cells�

15. History 1609 Diamond and colleagues Landsteiner and weiner Coomb, Mourant and Fischer levine discovered the anti(D) in anemic infants Mrs. Kell French midwife Hydrops fetalis, jaundice and anemia Guinea pigs injected with Rhesus monkey RBCs developed an antibody that reacted with 85% of human RBCsFrench midwife Hydrops fetalis, jaundice and anemia Guinea pigs injected with Rhesus monkey RBCs developed an antibody that reacted with 85% of human RBCs

16. Frequency ABO HDN 65% Rh HDN 33% Others 2%

17. Serology Maternal Antibody Fetal antigen Cross the placental barrier IgG1 and IgG3 High and low responders Primary and secondary response Anamnestic response

18. Basics� Mom lacks an antigen Baby has an antigen Mom produces Antibody (IgG)

19. Fetal-maternal hemorrhage Placental membrane rupture Trauma Amniocentesis Abortion Delivery

20. RBC antigens RBCs are formed by 2-3 weeks gestational age Most antigens are well developed by 10-12 weeks Except ABO, P1, Lewis and Cartwright

21. Clinical Erythroblastosis Extramedullary hematopoiesis Portal hypertension Hypoproteinemia and edema Hydrops Mother protects against high bilirubin Anemia leads to erythropoiesis expansion, extrameduullary hematopeiesis Hydrops, scalp edema, ascites, pleural effusion Portal hypertension, liver function deteriorates, hypoalbuminemia and hydrops indirect hemoglobinAnemia leads to erythropoiesis expansion, extrameduullary hematopeiesis Hydrops, scalp edema, ascites, pleural effusion Portal hypertension, liver function deteriorates, hypoalbuminemia and hydrops indirect hemoglobin

22. RES removes Ab labeled RBCs (Fc receptors on macrophages) Indirect bilirubin, maternal circulation Direct soluble, Indirect insoluble Mom protects baby

23. Anemia, more cells produced, erythroblastosis fetalis. Extramedullary erythropoiesis Hepatosplenomegaly Portal hypertension Edema

24. Basal ganglia and kernicterus

25. ABO Disease 31% Whites, 50% Asians Mild ABO antigens weak expression Anti-A, anti-B (IgM � doesn�t cross placenta) Anti-A,B in group O (IgG � crosses placenta) ABO substance in fetal plasma Only 10% require therapy Mother O ABO substance neutralize antibodies 10% with ABO incompatibility require therapyMother O ABO substance neutralize antibodies 10% with ABO incompatibility require therapy

26. ABO HDN Firstborn is at risk DAT positive in 20-40% Only 10% developed hemolysis Careful assessment of the newborn is the key Protects against Rh immunization Study by Dea-jardins 80% of those with hemolysis have positive DATStudy by Dea-jardins 80% of those with hemolysis have positive DAT

27. Non-ABO Disease Rh(D) most immunogenic 48-65% before RhIg prophylaxis 18.7% after RhIg History of previous transfusion K1, c, E

28. Prophylaxis 0.1 ml Rh(D) pos cells Expressed at 4 weeks of gestation Anti-D is formed by 6 months after the first pegnancy Rhogam (RhIg) 20 m g/ml RBCs Hypothetical mechanisms Steric changes in conformation

29. Anti-idiotype feed-back inhibition Alloimmunization dropped from 14% to 2% Antepartum RhIg, 0.2% At 28-32 weeks gestation 1 m g/ml=5 IU IM 300 mg dose, IV available more expensive 1968 Rhig has been licenced in USA plasma pools , HCV non-lipid coated virus are not inactivated (parvo B19) nucleic acid testing and filteration1968 Rhig has been licenced in USA plasma pools , HCV non-lipid coated virus are not inactivated (parvo B19) nucleic acid testing and filteration

30. Indications for RhIg All Rh-D neg women at 28 weeks gestation Amnio, abortion, trauma, ITP and transfusion Partial D not Du Given within 72 hours after delivery 300 mg == 1500 IU for 15 ml RBCs Weak-D or Du no need for RhIg CAP and American college of ob and gyn 2 reports of fatal HDN due to partial D weak D express the complete antigen, but partial D with gene rearrangement (n 22)and point mutations (n 9) Studies showed that it is ok up to 13 days after exposure to Rh-D positive cellsWeak-D or Du no need for RhIg CAP and American college of ob and gyn 2 reports of fatal HDN due to partial D weak D express the complete antigen, but partial D with gene rearrangement (n 22)and point mutations (n 9) Studies showed that it is ok up to 13 days after exposure to Rh-D positive cells

31. Testing Conversion from neg to pos Rh typing Rosette test Weak-D antiglobulin-based phenotype study Kleihauer-Betke* (KNOW the calculations!) Adult cells (HbA) lose hemoglobin and look pale Fetal cells (HbF) retain hemoglobin during staining process and look bright pink Flow cytometry Rosette screening with Rh-D positive indicator cells coated with anti-D K B test, HbF and HbA differential solubility Tx of Rh pos blood;20 micro gram/ml of RBCs IM and 18 IV <12 weeks gestation 50 micro gram others 300 micro grams KB 5/1000 5/1000 X maternal blood volume (85 ml/kg)=25 vials=25/30=.83 doses round up to 1 + 1=2 vialsRosette screening with Rh-D positive indicator cells coated with anti-D K B test, HbF and HbA differential solubility Tx of Rh pos blood;20 micro gram/ml of RBCs IM and 18 IV <12 weeks gestation 50 micro gram others 300 micro grams KB 5/1000 5/1000 X maternal blood volume (85 ml/kg)=25 vials=25/30=.83 doses round up to 1 + 1=2 vials

32. Maternal Assays ABO, Rh including weak D(Du) IAT and titer A titer of 8-32 for anti-Rh-D 8 for anti-K1 Rhogam mimics anti-D Ultrasonography Age, hydrops, liver, blood velocity and placental thickness Area opaca vasculosa, blood islands of the yolk sac, hemangioblasts, peripheral angioblasts and central hemoblasts erythropoietin reaches the coelomic caviyt by decidualization of the endometrium. RhD antigen is is expressed as early as 38 dayintrauterine. K1 and Jkb 6-8 weeks K2, Fya, Fyb at 6-8 weeks of gestation Maternal serum + hetrozygous Rh-D positive cells + donor monocytes. Maternal IAT test replaced MMAArea opaca vasculosa, blood islands of the yolk sac, hemangioblasts, peripheral angioblasts and central hemoblasts erythropoietin reaches the coelomic caviyt by decidualization of the endometrium. RhD antigen is is expressed as early as 38 dayintrauterine. K1 and Jkb 6-8 weeks K2, Fya, Fyb at 6-8 weeks of gestation Maternal serum + hetrozygous Rh-D positive cells + donor monocytes. Maternal IAT test replaced MMA

33. Amniocentesis Bilirubin OD450 Spectral absorption curve Liley curve, useful after 27 weeks Queenan curve Transplacental puncture, can lead to fetomaterna hemorrhage, ultrasonographic guidance L/S ratio >2 lng maturity, surfactant. Lecithin/sphingomyelin Testing paternal blood with the same primers used to test fetal blood to determine the Rh-D status , PCR, to avoid the false positive results due to rearrangements in the RhD gene, if father not available a 4 fold increase in antibody titer is diagnostic Transplacental puncture, can lead to fetomaterna hemorrhage, ultrasonographic guidance L/S ratio >2 lng maturity, surfactant. Lecithin/sphingomyelin Testing paternal blood with the same primers used to test fetal blood to determine the Rh-D status , PCR, to avoid the false positive results due to rearrangements in the RhD gene, if father not available a 4 fold increase in antibody titer is diagnostic

34. Fetal Assays Blood type Hematocrit (30% indicates anemia) DAT Reticulocyte count Total bilirubin Middle cerebral artery blood velocity Cord insertion site should be avoided to avoid bradycardia, because vagal nerve distribution The vein should be sampled rather than the arteryCord insertion site should be avoided to avoid bradycardia, because vagal nerve distribution The vein should be sampled rather than the artery

35. Clinical Management IAT, antiglobulin phase Titer if positive every 2-4 weeks 32 critical value Amniocentesis, every 2 weeks Paternal typing If positive, fetal typing If positive, serial OD Hct ,30% IUT last OD at 37 weeks, then L/S ratio, induce labor if ok at 39 weeks.Hct ,30% IUT last OD at 37 weeks, then L/S ratio, induce labor if ok at 39 weeks.

36. 2nd pregnancy, titers are not helpful Queenan curve, doppler at 18 weeks Hematocrit <30% IUT

37. Marsh score Grade Score 4+ 12 3+ 10 2+ 8 1+ 5 +W 3

38. 1 2 4 8 16 32 64 3+ 3+ 3+ 2+ 2+ 2+ 1+ 10 10 10 8 8 8 5 Titer (64) Score 59

39. IUT 1963 intraperitoneal transfusion (IPT) 1981 intravenous transfusion (IVT) 1982 in Denmark, ultrasonographic guidance 1986 Yale and Mt.Sinai 1963 Liley 1981 Rodeck1963 Liley 1981 Rodeck

40. IVT less frequent transfusions shorter stay in ICU IPT (weeks - 20) X 10 = volume to transfuse

41. CMV negative Irradiated Fresh (to avoid ?Ca++) Maternal blood if possible Leukoreduced What type of blood to give fetus:

42. How much to transfuse Mandelbrot Vfetoplacental X (Hctfinal - Hctinitial) X Hcttransfused blood Giannina weight in grams X 0.04 will raise Hct by 20% Hct final 35-40% Hct initia <30 Hct unit 70-80% fetopalcental volume = (weight in grams + 1.046) X 0.14 Hct final 35-40% Hct initia <30 Hct unit 70-80% fetopalcental volume = (weight in grams + 1.046) X 0.14

43. IUT indications Hematocrit < 30% > 0.5 mg/dl/h bilirubin in Rh > 1.0 mg/dl/h bilirubin in ABO Serum bilirubin > 20 mg/dl Serum bilirubin > 15 for > 36 h kernicterus, basal ganglia, indirect bilirubin Survival 84% 92% in nonhydropic 70% in hydropic increased umbilical venous pressure and decreased heart rate acidic pH of transfused blood low fetal hemoglobin in transfused blood increased 2-3 DPG in transfused blood, increases release of O2 iron overload Kernicterus, basal ganglia, indirect bilirubin, deafness mental retardation, brain damage severe, extrapyramidal, gaze disturbance may appear after the first year of lifeSurvival 84% 92% in nonhydropic 70% in hydropic increased umbilical venous pressure and decreased heart rate acidic pH of transfused blood low fetal hemoglobin in transfused blood increased 2-3 DPG in transfused blood, increases release of O2 iron overload Kernicterus, basal ganglia, indirect bilirubin, deafness mental retardation, brain damage severe, extrapyramidal, gaze disturbance may appear after the first year of life

44. Phototherapy UV light, photooxidation 10-14 mg/dl bilirubin Exchange transfusion if failed 2 volumes Rh, ABO-compatible Rh-D negative ABO incompatibility, O negative washed reconstituted with AB plasma

45. Donor compatible with mother AHG phase Sickle negative blood Target hematocrit of 50% Fetal blood = 85 ml / kg Fetal blood volume 85 ml/kg 50% Hct of transfused bloodFetal blood volume 85 ml/kg 50% Hct of transfused blood

46. Conclusion IgG cross the placenta Rh-D, ABO, Kell RhIg, used as a prevention Can be fatal Maternal assays, fetal assays Amniocentesis, ultrasonography IPT, IVT, phototherapy and exchange Tx