Karen Hodgins MLT ART GHEST Symposium September 29, 2012

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1 Antibody Case Studies The Good, the Bad and the Ugly Karen Hodgins MLT ART GHEST Symposium September 29, 2012

4 Case 1 ED from Saint John New Brunswick 77 yr old female with unstable angina Urgent cardiac surgery required A Rh Positive Previous Anti-E and anti-c R1R1, Kk, Jk(a+b+), Fy(a+b+), M-, N+, S- All panel cells reactive

5 Initial Panel (Immucor 10) D C c E e K k Fy a Fy b Jk a Jk b Le a Le y J J a b M N S s Gy Gel icin F

6 Enzyme treated cells Enzyme cleaves glycophorins A and and B which carry most of the negatively charged sialic acid Decreased zeta potential Cells can move closer together Removes some antigens completely and exposes other Ficin figs Papain Papayas Bromelin Pineapples Pronase Streptomyces griseus culture supernatant.

7 Case 1 DAT negative Destroyed by ficin Antigens destroyed by ficin M,N,S Fy Fy JMH Ch Ch Rg

8 Extra Cell Panel D C c E e K k Le a Le b M N S s Ch Rg Gel Ch/Rg Inhibition test t con nt 1/10 1/

9 Chido/Rodgers Inhibition Inhibition/neutralization technique Used to confirm antibody identity and to rule out other allo antibodies Source: Plasma (pool of 4 patients same blood group) Soluble antigen present in plasma will bind with antibody thus inhibiting antibody from attaching to red cells 6% albumin control must show positive results for test to be valid (1:2 dilution) High titre anti-rg may need additional dilutions to allow inhibition to work.

10 Chido/Rodgers Chido/Rodgers not true blood group antigens Located on the C4d of complement which are adsorbed on to the red cells from the plasma Rg antigen occurs in >98% population Absent or weak on cord RBCs Anti-Rg usually IgG and reacts best using IAT and does not bind complement Does not cause HDN or Transfusion Reaction The Blood Group Antigen Facts Book 2 nd Edition: Marion E. Reid and Christine Lomas-Francis

11 Case 1 Conclusion Anti-Rg Anti-E Anti-c Patient phenotype: Rg-

13 Case 2 DM from Windsor 80 yr old female pre-op Last transfused 6 months ago Antibody screen negative at that time O Positive

14 Initial Panel (Immucor 10) D C c E e K k Fy a Fy b Jk a Jk b Le a Le y J J a b M N S s Gy Gel icin F

15 Case 2 DAT positive Due to IgG Eluate non-specific R2R2, Jk(a-b+), Fy(a+b+), S-s+, K- Cells EGA treated to test for Fy and s antigen? Warm auto antibody, anti-e +? Allo absorption using R2R2 S-, K-, Jk(a-) cell WARM used to pre-treat cells before adsorption

16 Case 2 D C c E e K k Fy a Fy b Jk a Jk b Le a Le y J J a b M N S s Ay Allo

17 Case 2 Allo absorbed plasma indicates presence of other antibody(ies) Anti-e interfering e few e negative cells to perform exclusions Remove anti-e: Absorb onto rr, K-, S-, Jk(a-) After 9 absorptions R1R1 K- S- Jk(a-) non- After 9 absorptions R1R1 K-, S-, Jk(a-) nonreactive

18 Case 2 a b a b Le Le D C c E e K k Fy Fy Jk Jk a b M N S s Allo e abs

19 Case 2 Conclusion Conclusion Warm Autoantibody Anti-e Anti-Jk a Anti-K

21 Case 3 TL from Moncton NB 56 yr old male pre-op O Negative All Panel cells reacting

22 Case 3 D C c E e K k Fy a Fy b Jk a Jk b Le a Le y J J a b M N S s Gy Gel icin F

23 Case 3 DAT Negative Phenotype: rr, Fy(a-b+), Jk(a+b+), Ms, kk Decrease using ficin (variable results)?? Ch/Rg, Inhibition: antibody not inhibited. Other high incidence antigens tested Kn(a-), Yt(a-), Hy-, Lu(a-b-), Cs(a-), Sc: 1,2 All still reactive

24 Case 3 DTT treated cells: no change a-chymotrypsin treated cells: no change ZZAP treated cells: non-reactive Ficin treated cells: non-reactive (variable) could there be multiple antibodies? Where do we go from here.

25 Dithiothreitol Reducing agent Cleves disulphide bonds of IgM antibodies, 0.01M Antigen/antibody reaction cannot occur Cleves disulphide bonds within the structure of an antigen, 0.2M Kell antigens very susceptible to DTT Also Knops, Dombrock, Lutheran, Cartwright, Lw a, and JMH Helpful to categorize high incidence antibodies

26 α-chymotypsin treated cells Enzyme cleves proteins at specific amino acids (carboxyl terminus of leucine, phenylalanine to name a few Effects glycophorin B, Duffy glycophorin plus other glycophoirns gy Cromer antigen (Cr a )- sensitive to this enzyme, but not to Papain/ficin. In combination with DTT can narrow down the search for antigen specificity

27 Case 3 Absorption/elution technique Used unit that matched patient s phenotype. Ficin treated unit and tested against plasma Results: non-reactive Absorbed ficin treated unit with patient s plasma for 30 minutes at 37C DAT after 30 minutes: +/- Tested absorbed plasma and eluate made from Tested absorbed plasma and eluate made from absorbed cells against high incidence cells

28 Case 3 a No antibody eluted Absorbed plasma one negative cell?? Sc: 1,2 2 0 Did this work?? Ge: Set up pplasma against Abs plasma Hy- Wk 0 At(a-) 2 0 Cs(a-) 1 0 Co(a-) 2 0 Eluate Pt. Plasma negative cell: nonreactive!!! More Ge negative cells tested

29 Conclusion Ge- 0 Ge-1,-2,-3 0 Ge-1,-2,-3 0 Ge-1,-2,-3 0 Ge-2,-3,-4 0 Pt plasma a Appears to anti-ge These cells also ruled out all other common clinically significant antibodies Cells frozen for future testing

30 Gerbich Blood Group System Most Populations Melanesians Ge:2,3,4 (Ge+) > 99.9% 50-90% Ge-negative Ge:-2,3,4 (Yus type) Rare Not Found Ge:-2,-3,4(Gerbich type) Rare 10-50% Ge:-2,-3,-4 (Leach type) (Null) Rare Not Found The Blood Group Antigen Facts Book 2 nd Edition: Marion E. Reid and Christine Lomas-Francis

31 Gerbich Blood Group System The Blood Group Antigen Facts Book 2 nd Edition: Marion E. Reid and Christine Lomas-Francis

33 Case 4 RS chemo patient Metastatic colon cancer involving lung, liver and bone A Positive Last Transfusion February unit requested March 2012 Antibody screen negative

34 Case 4 Pre Electronic Post crossmatch Transfusion started Temperature Pulse Reaction 1400 Chills/rigors Dyspnea Tickle in throat BP 135/72 189/95 SpO2 99% Unable to obtain

35 Case 4 Post transfusion A Positive Ab Screen Negative DAT positive due to IgG Eluate non-reactive Transfused Unit gel crossmatch incompatible Antibody to low incidence id antigen Cell eluate Pre tx d Unit Wr(a+) 4 3 Wr(a+) 4 3 Wr(a+) (+) 4 3 Post tx d

36 Conclusion Acute Hemolytic Transfusion Reaction due to anti-wr a AHTR causes ABO incompatibility Other blood group incompatibility Rare cases of high titre antibody group O platelets transfused to a non-o recipient

37 Conclusions RBC alloantibodies Patient immunized from previous exposures Low levels of allo-ab in patient Error in screening Limitations in screening cells Uncrossed blood Outcome Less than 10% are fatal Over 50% result in no morbidity

38 AHTR Accelerated destruction of rbc in a transfused patient occurring during or within 24 hrs after a blood transfusion Characterized by sudden onset of fever, chills, facial flushing, chest pain or low back pain, hypotension and dyspnea May result in severe complications ie renal failure and DIC Leading cause of death associated ate with transfusion Estimated to occur in ~ 1 in 12,000 to 1 in 33,000 red cell units transfused Fatal AHTR occurs in 1 in 600,000 to 1 in 800,000 units transfused

39 The Wright Blood Group Identified in 1953 as cause of HDN Diego blood group system Antigens Wr a occurs in <0.01% of population Wr b occurs in 100% of population Anti- Wr a Resistant to ficin/papain Can be IgM or IgG React at RT or IAT Does not bind complement

40 The Wright Blood Group Anti- Wr a transfusion reactions: none to severe/immediate or delayed/hemolytic HDN: mild to severe Can be naturally occurring Found in 1% to 2% of blood donors Common specificity in patients with AIHA Francis The Blood Group Antigen Facts Book 2 nd Edition: Marion E. Reid and Christine Lomas-

42 Questions?

43 References Judd s Methods in Immunohematology, 3 rd Edition, 2008 Applied Blood Group Serology, ogy, Peter D. Issitt, David J Anstee, Fourth Edition, Apr 1998 The Blood Group Antigen Facts Book, Marion E Reid, Christine Lomas-Francis Francis, 2 nd edition, 2007

Antibodies Are Not Always What They Seem. Lindsay Peters MLS(ASCP)SBB Reference Laboratory Supervisor Mississippi Valley Regional Blood Center

Antibodies Are Not Always What They Seem Lindsay Peters MLS(ASCP)SBB Reference Laboratory Supervisor Mississippi Valley Regional Blood Center Case Study Patient: Mrs. Holmes is admitted to the ICU following