Future Webinars. Continuing Education 8/29/2017. Transplant. Transfusion Service

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1 Immunohematology Case Studies Future Webinars September 29 October 31 December 6 RhD Molecular Testing HLA: Transfusion and Transplant Running a Remote Transfusion Service Link to register: All Content 2015 Immucor, Inc. Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is September 15, 2017 Certificates will be sent via only to those who have registered by September 29, 2017 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 1

2 Presentation Recording Session will be recorded and posted. Access information will be sent to each registrant when the recording becomes available CE credits will be issued to anyone who listens to the recording within one year of the original presentation date (today). All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. Immunohematology Case Studies Rebecca Coward, MT(ASCP)SBB CM WakeMed Health and Hospitals 2

3 Case 1 Patent DC: 26 year old Latin American female G5P2 at 37w4d Presents with contractions Forward Reverse Anti A Anti B Anti D1 Anti D2 Mono Ctrl A 1 B Patient DC R800 D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IAT R 1 R R 2 R rr POS Ctrl 4+ Echo CaptureR Extend II Rh MNS LU P Lewis Kell Duffy Kidd SP Lot DN096 D C E c e V M N S s Lua Lu b P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT 1 r r r r r r r r W rr rr rr rr rr rr rr rr rr R 2R Pos Ctrl / / / / / / / / / / Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 4+ / / / / / / / / / / / 0 3

4 DAT Echo DAT= 0 Ctrl= 3+ ABO Discrepancy Workup Repeat ABO/Rh in tube Patient DC Forward Reverse AntiA AntiB AntiD A 1 B AntiA 1 workup A 1 cells A 2 cells O cells IS Lot IS Lot IS Lot Cell # SC1 Cell # SC2 Cell # SC3 AntiA 1 lectin Lot IS Patient DC cells Positive Control Negative Control

5 Prewarm Prewarm C A 1 B Auto W *Use prewarm methods with caution! Note: no rouleaux noted with microscopic review Cold Screen a b a b D C E c e M N S s P1 K k Fy Fy Jk Jk IS RT R 1R R 2R rr Auto Ctrl C Cold Screen Rh MNS LU P Lewis Kell Duffy Kidd D C E c e V M N S s Lu a Lu b P 1 Le a Le b K k Fy a Fy b Jk a Jk b IS 1 R 1R W+ 2 R 1R R 1R W R 1R r r R 1R r r RT 15 4C 5

6 P1 P1P K blood group system Expression of antigen is variable Shows dosage based on zygosity Expression weakens with invitro storage Expressed more weakly on cord cells (than adult RBCs) P1 expression is weakened/inhibited In(Lu) phenotype Caucasians Blacks Cambodian/ Vietnamese P1 79% 94% 20% AntiP1 IgM (IgG is rare) Detected at RT or lower May be neutralized Hydatid cyst fluid Pigeon egg white Echinococcus cyst fluid Naturally occurring found in many P2 donors Not considered clinically significant (no HTR/HDFN) Case 1 Summary AntiD due to RhIg administration AntiP1 causing ABO discrepancy No phenotyping or titer performed 6

7 Case 2 Patient RP: 45 year old male; race unknown Trauma 1; head on motor vehicle collision Trauma 1 cooler issued: 4 O pos uncrossmatched RBCs 2 A plasma Forward Reverse AntiA AntiB AntiD1 AntiD2 Mono Ctrl A 1 B Patient RP R866 D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IAT R 1 R R 2 R rr POS Ctrl 4+ ECHO CaptureR ReadyID Rh MNS LU P Lewis Kell Duffy Kidd SP Lot ID339 D C E c e V M N S s Lua Lu b P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT 1 R zr R 1w R R 2R R or r r r r rr rr rr rr rr rr rr R 1R Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+ Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0 7

8 Antigen Typing Transfusion history is unknown AntiJk a Patient RP cells 0 Positive Control 3+ Negative Control 0 Antibody ID Review ECHO R866 D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IAT R 1 R R 2 R rr POS Ctrl 4+ Antibody ID Review Rh MNS LU P Lewis Kell Duffy Kidd SP Lot ID339 D C E c e V M N S s Lua Lu b P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT 1 R zr R 1w R R 2R R or r r r r rr ???? 8 rr rr rr rr rr rr R 1R Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+ Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0 8

9 PEG Screen PEG tube D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IS IAT CC R 1R R 2R rr AC Cold Screen Tube D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IS 15 RT A A SC SC SC AC C LISS Screen LISS tube D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IS 37C IAT CC R 1R R 2R rr AC

10 Case 2 Summary Solid phase platform may have increased sensitivity to Kidd antibodies Clinical significance of solidphase only antibodies has been debated Kay, et al. AntiJk a that are detected by solidphase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions. Transfusion 2016;56: Case 2 Summary CaptureR assays are designed primarily for the detection of IgG antibodies Cold antibodies detected by CaptureR May have an IgG component Indicator cells may carry antigen toward which the antibody is directed Antibody may link the Indicator cells to the immobilized RBC layer by binding to antigens on both Case 2 Summary Impact of ABID on transfusion recommendation and future transfusions Screening for antigen negative blood Extended crossmatches 10

11 Case 3 Patient AS: 18 year old African American female Past Medical History: sickle cell anemia (SSA) SS type Presents with pain Previous antibody ID: antis, warm autoantibody. Patient receives C, E, K, S RBCs per hospital protocol for SSA Forward Reverse AntiA AntiB AntiD1 AntiD2 Mono Ctrl A 1 B Patient AS ECHO R780 D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IAT R 1 R R 2 R rr POS Ctrl 4+ CaptureR ReadyID Rh MNS LU P Lewis Kell Duffy Kidd SP Lot ID310 D C E c e V M N S s Lua Lu b P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT 1 R zr R 1w R R 2R R or VS r r r r rr rr rr rr rr rr rr Di(a+) R 1R Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+ Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0 11

12 DAT Tube IgG = 0 CC = 3+ PEG tube Selected cell panel PEG tube D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IS IAT CC R 1 R NT R 1 R NT R 2 R NT r r NT rr NT AC CaptureR ReadyID Rh MNS LU P Lewis Kell Duffy Kidd SP Lot ID310 D C E c e V M N S s Lua Lu b P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT 1 R zr R 1w R R 2R R or VS r r r r rr rr rr rr rr rr rr Di(a+) R 1R Pos Ctrl / / / / / / / / / / / / / / / / / / / / / 4+ Neg Ctrl / / / / / / / / / / / / / / / / / / / / / 0 12

13 OSH Information TAS 2 weeks prior negative with S negative selected cell panel Received 1 unit XM compatible RBCs Phenotype shared: Group O; D+CEc+e+; K, Fy(ab), Jk(a+b+), M+N+Ss+; P1+ WARM identified 2007 AntiS identified 2012 Peg tube Selected cell panel PEG tube D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IS IAT CC rr VS Ror U R 1 R R 2 R Reference Lab Results AntiFy3 confirmed in current sample AntiSl a is ruled out Recommendation: transfuse phenosimilar cells Negative for C, E, K, Fy a, Fy b, S Transfusion Service where patient receives routine care was notified of ABID Clinicians at WM were made aware of ABID and potential delays in procuring blood for future transfusions 13

14 HEA BeadChip Analysis Fy3 Fy3 Occurrence Caucasians 100% Blacks 32% Asians 99.9% Absent from Fy(ab) RBCs Resistant to enzymes antify a and antify b sensitive to papain/ficin Expressed on cord cells and expression increases after birth (HDFN mild/rare) AntiFy3 IgG Clinically significant: HTR (mild/moderatedelayed); HDFN (mildrare) Formation of antify3 is usually preceded by antify a AntiFy3 produced by blacks does not react (or weakly reacts) with cord cells 14

15 AntiFy3 AntiFy3 vs antisl a Sl a frequency is 5060% in Blacks Sl a is expressed weakly on cord cells Most Fy(ab) cells are also likely Sl(a) Sl a is weakened/sensitive to papain/ficin AntiFy3 vs antify5 AntiFy3 agglutinates Rh null RBCs AntiFy5 does not agglutinate Rh null RBCs Case 3 Summary Even with previous antibody ID with panagglutinin, the possibility of a new antibody to HFA should always be considered There is power in the phenomatched reagent cell What to do with extra reactivity in SPRCA? Miller NM, et al. Patient factors associated with unidentified reactivity in solidphase and polyethylene glycol antibody detection methods. Transfusion 2017;57: % of patients studied with UID; had autoantibody or alloantibody identified on a subsequent screen or panel Patient AS developed warm autoantibody on subsequent sample Case 4 Patient DG: 19 year old African American female No pertinent past medical history Presents to the ED for generalized weakness, decreased appetite. Athome pregnancy test positive 15

16 TAS Echo Forward Reverse Anti A Anti B Anti D1 Anti D2 Mono Ctrl A 1 B Patient DG D C E c e M N S s P1 K k Fy a Fy b Jk a Jk b IAT R 1 R R 2 R rr POS Ctrl 4+ Echo ABO Reconfirm Tube Forward AntiA AntiB AntiD Patient DG Rh discrepancy workup Weak D tube AntiD CC Patient DG 3+ NT Control 0 3+ DAT Echo DG 0 16

17 Other troubleshooting Instrument Tech Reagent Call Immucor! Immucor Investigation Testing by Echo ECHO Confirm Assay ECHO Weak D Assay ECHO DAT Testing by tube D hemagglutination assay Weak D Immucor DX Reference Laboratory RHD and RHCE BeadChip Analysis ECHO Testing Testing by Echo ECHO Confirm Assay Group B, Dnegative ECHO Weak D Assay 4+ ECHO DAT negative 17

18 Tube Testing Testing by tube D hemagglutination assay Weak D AntiD1 AntiD2 Monoclonal Control IS Weak D RHD BeadChip assay Weak D type 4.0 or 4.3 (hemizygous or homozygous) partial D AntiD has been observed (allo/auto?) Consider patient D negative for the purposes of transfusion and/or RhIg administration As a donor, consider patient D positive Further classification could be accomplished by sequencing RHCE BeadChip assay Predicted phenotype: C c+ E e+ Alleles detected: ce(48c)/ce(48c, 733G) ce(48c) encodes a normal e that may react weakly with some monoclonals ce(48c, 733G) encodes partial c, partial e, V+, VS+, hr B 18

19 Case 4 Summary Weak reactivity detected by tube (manual) tests may not always be reproducible using automated hemagglutination assays D typing discrepancies often point to altered D expression Variant RHD alleles are often inherited with variant RHCE alleles Workup and management of patients with Rh variants is not standardized References The Blood Group Antigen FactsBook. 3 rd ed. Reid, LomasFrancis, Olsson Human Blood Groups. 3 rd ed. Daniels Technical Manual. 18 th ed. Fung, et al. Rhesus Base last accessed 8/21/17 Thank you! 19

20 We like you! Like us on social media! All Content 2015 Immucor, Inc. Thierry PEYRARD PharmD, PhD, European Specialist in Clinical Chemistry and Laboratory Medicine Director, National Immunohematology Reference Laboratory National Institute of Blood Transfusion Paris France Immunohematology Case Studies Presented as a Webinar for the Immucor Global UserGroup August 30 th, 2017 PRESENTATION OUTLINE Short presentation of the French National Immunohematology Reference Laboratory Case studies Discussion 20

21 CNRGS: THE FRENCH NATIONAL IMMUNOHEMATOLOGY REFERENCE LABORATORY (IRL) Centre National de Référence pour les Groupes Sanguins Department of the French National Institute of Blood Transfusion (INTS) Only official national IRL in France Staff: 40 people CNRGS INTS headquarter MAJOR MISSIONS Complex case solving: referrals from continental France, French overseas territories and foreign countries (serology, molecular testing) National Rare Blood Program Manufacturing of the National Reference Identification Panel of ReagentRBCs Contribution to the control of performance of immunohematology reagents according to the European "CEmarking" scheme Scientific research Continuing education and university 21

22 CASE STUDIES CASE STUDY 1 37 year old pregnant patient, 30 th week of gestation RBC antibody screen positive => Antibody identification RH KEL FY JK LE MNS P LU DO YT CO XG IATpapain IAT Autocontrols 22

23 37 year old pregnant patient, 30 th week of gestation RBC antibody screen positive => Antibody identification AntiM, with dosage effect Negative autocontrols => probable alloantim but M/N typing required to conclude MNS phenotype MN+ => alloantibody RH KEL FY JK LE MNS P LU DO YT CO XG IATpapain IAT Autocontrols RH KEL FY JK LE MNS P LU DO YT CO XG IATpapain IAT Autocontrols New blood sample investigated a few weeks later in a second laboratory: antim confirmed But the phenotype this time was found to be M+N+! Strong reaction for M (4+), equivalent to the M+N control RBCs => Blood samples referred to our reference laboratory 23

24 MN+ type found in our laboratory, with two different sources of reagents Control with the antim of laboratory #2 who found a 4+ reactivity => confirmation of the M+ type! Patient M+ or M? Auto or alloantim? Typical issue of crossreactivity interference. Some reagents may more or less react with another antigen than the test antigen. Some widely used antim clones (e.g. 2514E6 and M11H2) strongly crossreact with the lowprevalence He antigen (Henshaw, MNS6) 710% of people of African descent are The antim clones that crossreact with He are, however, considered the most peformant antim Of note, antim clone BS57 does not crossreact with He Some antim clones (E3, E6, 425/2B) also crossreact with the lowprevalence M g antigen (MNS11) => less problematic because this antigen is very rare in all populations (except in Switzerland but prevalence still <1%) 24

25 CONCLUSION Beware of possible crossreactions with some monoclonal antibodies, responsible for false positive results Always carefully read the package insert of the manufacturer and limitations of the reagent May explain discrepancies between phenotype and genotype May also explain apparent parentage exclusion (mother and father previously typed as MN+ with a noninvolved reagent, and child typed as M+N+ with a ti t ith H ) CASE STUDY 2 25

26 25 year old female sickle cell patient No history of recent transfusion Group O, D+CEc+e+, K Positive antibody screen and identification Autocontrols IAT Papain Pattern of reactivity consistent with an antin (dosage effect) Negative autocontrols alloantin? Exceptional antibody! Is this really an alloantin? N is papain and trypsin sensitive 26

27 rongly positive reactions on trypsintreated RBC ++ Autocontrols IAT Pap Tryp Extended phenotype of the patient Fy(ab), Jk(a+b), M+NSs Rare Ss phenotype (12% in Africans, up to 35% in Equatorial Africa) 50% are U (no glycophorin B) 50% are U+ var (altered glycophorin B, weak/partial U) Possible antiu alloimmunization, either in U or U+ var GPA M/N Trypsin sensitive GPB S/s/U Trypsin resistant 27

28 Example of a M+N+S+s+ individual M N GPA 10 6 molecules/cel l S s GPB molecules/c ell N N GPA(N) GPB Same 26 amino acids than those which define N on GPA(N) => N or Nlike Whyisit possible to type for N on GPA? Number of GPA molecules >> GPB => antin diluted enough to react only with N on GPA GPA GPB Same 26 amino acids than those which define N on GPA(N) => N or Nlike 28

29 But N typing quite often shows weak or unclear reactions Number of GPA molecules >> GPB => antin diluted enough to react only with N on GPA GPA GPB Possible detection of N on GPB if antin too concentrated or Number of GPB molecules above the average, e.g. in S+s (express 50% more GPB than Ss+) => highest risk in M+NS+s (6% Caucasians, 2% Africans) Patient M+NSs U M M GPA No GPB => No N => "True" N May form an antibody to a highprevalence antigen named anti N or antinlike (antimns30), that ressembles antin when starting to develop (first reacts with N on GPA because GPA expression >> GPB) Patient M+NSsU+ var M M GPA Altered GPB => No N in >90% of cases => "True" N GPB Presence of the He (MNS6) antigen => abolishes N expression Rare blood NSsU+ var or NSsU required! 29

30 Patient M+NSsU+ var M M GPA Altered GPB => No N in >90% of cases => "True" N GPB (one Presence of the dose) He (MNS6) antigen => abolishes N expression Rare blood NSsU+ var or NSsU required! CONCLUSION AlloantiN is exceptional in Caucasians as they are all virtually N positive (N on GPB) => most antin are autoantibodies directed to glycophorin A/B and correspond in N patients (28%) to the socalled «mimicking alloantibodies» In addition to the M/N type, the discovery of an antin must systematically lead to a Ss typing, CONCLUSION Beware of the presence of any antin in a N patient of African descent If Ss => rare MNS:30 type! If not Ss (S+s for example), can also be a rare MNS:30 type because some rare African GPB variants may also loose N! => A MNS genotype must be performed in any case of antin discovered in an African patient, 30

31 CASE STUDY 3 Pregnant woman, group O, DCEc+e+, K. 4 th pregnancy. 26 th week of gestation IAT Negative autocontrols What do you think? Pregnant woman, group O, DCEc+e+, K. 4 th pregnancy. 26 th week of gestation IAT IAT Papai n IAT Papai n AntiD + AntiC pattern of reactivity 31

32 HYPOTHESES AntiD immunoprophylaxis. Batches of antid often contain a small amount of antic => But antic too reactive here After investigation, no antid immunoprophylaxis performed here: consistent with a real antid+c alloimmunization => logically no need to inject antid between 28 th 32 nd week of gestation because antid alloimmunization considered being HYPOTHESES But may also be an antig! G is a common epitope between D and C antigens => antig ressembles anti D+C! Several possible configurations AntiG AntiC+G AntiD+G AntiC+D+G Essential in pregnancy to know if HOW TO RULE OUT THE PRESENCE OF ANTID Adsorption of plasma on a DC+Ec+e+ (r r) RBC Only antic and antig will adsorb onto r r RBCs and antid, if present, will remain free in the adsorbate => antibody identification in the adsorbate If no antid found in the r r adsorbate, antid immunoprohylaxis is required! 32

33 CONCLUSION Any antid+c pattern of reactivity in a pregnant woman must lead to the systematic search for the presence of a real alloantid (risk of the D reactivity being osberved due to anti G!) If not considered, no antid immunoprophylaxis will be carried out, whereas the patient is not yet alloimmunized to the D antigen => i l ff t b t t i l f t THANK YOU FOR YOUR ATTENTION Future Webinars September 29 October 31 December 6 RhD Molecular Testing HLA: Transfusion and Transplant Running a Remote Transfusion Service Link to register: All Content 2015 Immucor, Inc. 33

34 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is September 15, 2017 Certificates will be sent via only to those who have registered by September 29, 2017 All Content 2015 Immucor, Inc. Thank you! All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 34