1. Alloantibodies causing Hemolytic Disease of the Newborn and Fetus. Nancy Benitez, BS, MHS (ASCP)SBB CM Reference Laboratory Director June 6, 2013

2. Case Saturday Night (SN) The referring hospital transfusion service does not have any previous history on this patient as samples were received from the outpatient clinic. 32 years old Caucasian Female. Second Pregnancy 2

3. SN..cont… They reported that the antibody screen was positive with all cells tested, but due to the limited resources no further testing was performed. Samples were sent to our IRL lab for antibody Identification. 3

4. Objectives Review the required serological techniques to determine the antibody specificity and clinical significance of alloantibodies causing Hemolytic disease of the fetus and Newborn. (HDFN) Review the critical role that the Reference laboratory plays in the procurement of uncommon blood to transfuse the newborn. Discuss the importance of the cooperation between patient, attending physician and the laboratory when alloantibodies are detected in the mother.

5. Routine Protocol- First Time Patients ABO-Rh DAT Screen (Liss and PEG) Panel Patient antigen testing.

6. ABO-Rh determination –O Pos FORWARD TYPE REVERSE TYPE ANTI-AANTI-BANTI-D A1 CELLSB CELLS 00444 6

7. SN Screen Liss Screen PEG Screen 7 RhKellDuffyKiddMNSsPLewisLu IS37⁰ CIAT DCcEeCwKkKp a Kp b Fy a Fy b Jk a Jk b MNSsP1P1 Le a Le b Lu a Lu b IR1R1++00+00+0++++0+++0++00+002+ IIR2R2+0++00++0++0++0+0++000+01+3+ IIIrr00+0+00+0+0+0++00+00+0+01+3+ IVAC 000 RhKellDuffyKiddMNSsPLewisLu ISIAT DCcEeCwKkKp a Kp b Fy a Fy b Jk a Jk b MNSsP1P1 Le a Le b Lu a Lu b IR1R1++00+00+0++++0+++0++00+02+ IIR2R2+0++00++0++0++0+0++000+03+ IIIrr00+0+00+0+0+0++00+00+0+03+

8. Patient phenotype CEceMNSsKk FyaFyb JkaJkb +00+0+++00++0

9. Initial Panel - LISS 9 RhMNSsPLewisKell Duffy Kidd DCEcefCwMNSsP1P1 Le a Le b KkFy a Fy b Jk a Jk b 37°CIATNo. 1rr000+++0+0+0++00++++0 2+ 1 2rr000+++00+0+ + vw 0+++++0+ 03+2 3r'r0+0+++0+0+++0+0+0++0 1+3+3 4r"r00++++0+0+++0+0++0++ 2+3+4 5rr000+++0++++ + vw 0+0++00+ 1+3+5 6RorRor+00+++00+0+++00+00++ 0 6 7R1R1R1R1 ++00+NT0+00+ + w +00++0+0 2+ 7 8R1R1R1R1 ++00+NT0++++00+++++0+ 02+8 9R1R1 w ++00+NT+++0+0000+++++ 1+2+9 10R2R2R2R2 +0++0NT0++++0+00+++0+ 2+3+10 11R2rR2r+0++++0++0+ + vw 0++00+++ 2+3+11

10. Problems to resolve Panel reacting with all cells tested. Reactions with different strengths. What do you think we should do next. 10

11. Let’s review the patient phenotype CEceMNSsKk FyaFyb JkaJkb +00+0+++00++0

12. FICIN Treated Panel 12 RhMNSsPLewisKell Duffy Kidd DCEcefCwMNSsP1P1 Le a Le b KkFy a Fy b Jk a Jk b IATNo. 1rr000+++0+0+0++00++++0 3+1 2rr000+++00+0+ + vw 0+++++0+ 4+2 3r'r0+0+++0+0+++0+0+0++0 3+3 4r"r00++++0+0+++0+0++0++ 4+4 5rr000+++0++++ + vw 0+0++00+ 3+5 6RorRor+00+++00+0+++00+00++ 6 7R1R1R1R1 ++00+NT0+00+ + w +00++0+0 07 8R1R1R1R1 ++00+NT0++++00+++++0+ 08 9R1R1 w ++00+NT+++0+0000+++++ 09 10R2R2R2R2 +0++0NT0++++0+00+++0+ 4+10 11R2rR2r+0++++0++0+ + vw 0++00+++ 3+11

13. Selected Cell Panel: FICIN Treated Cells 13 Donor/ Vial # RhMNSsPLewisLuKellDuffyKidd X Additional Antigens PEG DCEcef CwCw VMNSsP1P1 Le a Le b Lu a Lu b Kk Kp a Kp b Js a Js b Fy a Fy b Jk a Jk b Xg a IAT B6917 ++00+ 00+0+++0+0++00+0+++0++ 06/21/13 Do(a+) 0 9 550063 ++00+ 0 +00++0+0+++0+0+0++00 06/11/13 Co(a+) Co(b-) Di (a+) 0 9 M2975HM ++00+ 00+00+w+0++0+0 0 +00++5/25/20130 7

14. 0.01 DTT TREATED PLASMA Donor/ Vial # RhMNSsPLewisLuKellDuffyKidd X Additional Antigens PEG DCEcef CwCw VMNSsP1P1 Le a Le b Lu a Lu b Kk Kp a Kp b Js a Js b Fy a Fy b Jk a Jk b Xg a IAT B8392 ++00+ 00+0+0+0+0+0+0+0++++++Co(b+) 06/28/1302+ 3 B8390 ++00+ 00++0+00+++0+0+0++++++6/28/201301+ 20 B1051 ++00+ +0+0++++00+0+++0++00++ Co(b+) Bg(a+) 06/21/13 Do(a+) 02+ 2 14 0.01 DTT Treated plasma Control

15. Titration Studies 15 TITER - ANTI-E PlasmaCellTesting phaseNeat(1:2)(1:4)(1:8)(1:16)(1:32)(1:64)(1:128)(1:256) TEST3144IAT3+ 2+ 1+00 TITER - ANTI- c PlasmaCellTesting phaseNeat(1:2)(1:4)(1:8)(1:16)(1:32)(1:64)(1:128)(1:256) TEST3342IAT3+ 2+1+000

16. Summary of mom’s results AntibodyTitrationImmunoglobulin Anti-c1:16IgG Anti-E1:32IgG Anti-MN/AIgM 16

17. Why Patient follow up…… Maternal IgG antibodies (anti-c, E) could be directed against the antigen(s) present on the fetal red blood cells. IgG antibodies cross the placenta to coat fetal antigens, cause decreased red blood cell survival and produce anemia and HDNF. Prevention for future pregnancies. 17

18. SECTION TWO

19. Case Saturday Night Baby (SNB) One of our local transfusion facilities requested testing for a baby sample with a possible Hemolytic Disease of the Newborn and Fetus. Mother’s records were immediately retrieve from the computer system 19

20. Case Saturday Night Baby (SNB) The hospital also confirmed that the mother has a history of anti-c, -E. RBCs fresh as possible, preferably <5 days old. CMV and Hemoglobin S negative Irradiated Requested O Neg, c, E negative (extremely difficult combination) 20

21. Laboratory Hospital Findings- Baby Positive Direct Antiglobulin Test ABO-Rh: O Neg Anemia Hyperbilirubinemia Reticulocytosis (6 to 40%) Abnormal Smear 21

22. Baby ABO-Rh & DATResults 22 Direct Antiglobulin Test (DAT) PolyIgGC3Interpretation 2+ 0Positive Forward Type Interpretation Anti-AAnti-BAnti-DRh Control O NEG 0000

23. SN Baby Elution RhKellDuffyKiddMNSsPLewisLu ElutionIAT DCcEeCwKkKp a Kp b Fy a Fy b Jk a Jk b MNSsP1P1 Le a Le b Lu a Lu b IR1R1++00+00+0++++00++0++00+ 0 IIR2R2+0++00++0++0++0+0++000+ 3+ IIIrr00+0+00+0+0+0++00+00+0+ 3+ 23

24. SN Baby Elution RhMNSsPLewisKell Duffy Kidd DCEcefCwMNSsP1P1 Le a Le b KkFy a Fy b Jk a Jk b IATNo. 1rr000+++0+0+0++00++++0 3+1 2rr000+++00+0+ + vw 0+++++0+ 4+2 3r'r0+0+++0+0+++0+0+0++0 3+3 4r"r00++++0+0+++0+0++0++ 4+4 5rr000+++0++++ + vw 0+0++00+ 3+5 6RorRor+00+++00+0+++00+00++ 6 7R1R1R1R1 ++00+NT0+00+ + w +00++0+0 07 8R1R1R1R1 ++00+NT0++++00+++++0+ 08 9R1R1 w ++00+NT+++0+0000+++++ 09 10R2R2R2R2 +0++0NT0++++0+00+++0+ 4+10 11R2rR2r+0++++0++0+ + vw 0++00+++ 3+11 24

25. Newborn- D testing EGA treated cell Immediate SpinAHGRh Control Anti-D (1)000 Anti-D (2)000 Anti-D (3)000 Interpretation D Negative Negative

26. Where could we find blood Where and how will we find a unit of blood that is Rh(D) negative and also c negative, as most of the units are dce/dce. Blood of this type was not available in the lab

27. The Rh complex The D antigen always travel with the C, c and E or e Antigens cannot be separated

28. Prevalence of Rh Haplotypes Fisher-Race HaplotypeModified Wiener Haplotype Prevalence (%) White Rh Positive DceR1R1 42 DcER2R2 14 DceR0R0 4 DCERzRz <0.01 Rh Negative Cer37 Cer'2 cEr''1 CEryry <0.01 28

29. Baby’s Rh Haplotype 29 Baby is dce / dCe Heterozygous for the Cc Antigen Mother R1R1 r' R1R1 R1R1R1R1 R 1 r' rR1R1 rr'

30. Prevalence Rh Haplotypes GENOTYPEDONOR FREQUENCY (%) dce/dcerr15.1020 dce/dCerr'0.7644 dCe/dCer'r'0.0097 30

31. Baby SN….Final outcome….. Baby’s total serum bilirubin level was at 16 mg/dL and did not increase after phototherapy. The Newborn was almost a Full-term an exchange transfusion was not necessary. Baby was not severely affected because he was heterozygous for the c antigen. 31

32. Be ready…… Think ahead, plan ahead, prepare ahead and work ahead. Communication among the medical team and blood provider is crucial to prevent additional complications. 32

33. References 1. Issitt PD, Anstee DJ. Applied Blood Group Serology. Montgomery Scientific Publications; Durham, NC; 1998. 2. AABB Technical Manual, 17 th edition. 3.Mollison, PL; Engelfriet CP and Contreras M (1997). Blood Transfusion in Clinical Medicine (10th ed.). Oxford, UK: Blackwell Science. 33

34. Questions and comments. 34