Antibody Identification. Case Studies

Transcription

1 Antibody Identification Case Studies Karen Rodberg, MBA, MT (ASCP) SBB Director, Immunohematology Laboratory American Red Cross, Southern California Region The need is constant. The gratification is instant. Give blood ṬM

2 Commonly used problem-solving techniques: Proteolytic enzymes Reducing (thiol) reagents Titration and neutralization Adsorption Elution

3 Enzyme use for antibody i.d. 3 Antibody identification tool - Weak or equivocal reactions - Suspected multiple antibodies - Characterize unknown specificity Adsorption studies - Remove or separate antibodies

4 Proteolytic Enzymes 4 Proteases: cleave bonds in appropriate amino acid chains of membrane bound proteins or glycoproteins Ficin (figs) Papain (papayas) Bromelin (pineapples) Trypsin (bovine/porcine pancreas) -Chymotrypsin (pancreas) Pronase (Streptomyces griseus)

5 Effects of enzyme-treatment of RBCs 5 Proteases remove sialic acid-bearing glycoproteins reduction in: - Cell surface negative charge - Steric hindrance - Membrane-bound water Results in: RBCs closer together so IgG can span distance agglutinate

6 Other effects on RBCs 6 Effect on RBC antigens: Some antigens are denatured Some antigen-antibody reactivity is enhanced Therefore not used for routine antibody detection, but very useful in antibody identification

7 Example MNS System 7 - Cleave [ ] large portions of glycoproteins (e.g., GPA, GPB) from RBC - Site of action is enzyme-specific - Carbohydrates attached to the portion of protein affected will also be removed

8 Enzymes can denature or enhance: 8 Antigen denaturation (ficin/papain) M, N, S, En a TS, En a FS, Fy a, Fy b, Fy6, Ge2, Ge4, In b, Ch, Rg, JMH, Pr, Xg a, s*, Yt a * *variable Antigen-antibody reactivity enhancement P 1, I, i, Lewis, Rh, Kidd, Colton, Dombrock

9 Case Study #1 case history 9 33 y.o. female obstetrical patient Full term delivery No prenatal care 4 th pregnancy

10 Case Study #1 ABO group A 1 B Anti-A Anti-B RBCs RBCs Rh type Anti-D Cntl 0 0 Interpretation: Group O Interpretation: Rh negative D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT I II III

11 Case Study #1 Initial Panel D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT PT 0 0 Evidence of multiple alloantibodies some reactivity at RT and additional reactivity by indirect antiglobulin test. 11

12 Begin exclusion with cell #3 D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PT 0 0 PEG IAT 12

13 Examine RT reactivity Look first at Le a, Le b, P 1, M, N these antibodies most often react at RT D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PT 0 0 PEG IAT 13

14 Examine RT reactivity D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PT 0 0 Reactivity pattern matches anti-m, showing dosage, but need non-reactive RBCs for exclusion. PEG IAT 14

15 Test same panel ficin treated D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b IAT PEG PT 0 0 Ficin IAT 15

16 Test same panel ficin treated D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b IAT PEG PT 0 0 Ficin IAT (Some alloantibodies have already been excluded with cell #3) 16

17 Now exclude ficin resistant abys D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b IAT PEG PT 0 0 Ficin IAT 17

18 Also helpful to phenotype patient Anti-C Anti-E Anti-c Anti-e anti-s anti-s anti-k anti-fy a anti-fy b anti-jk a anti-jk b anti-m anti-n 0 4+ Patient can make alloanti-d, -C, -E, -S, -K, -Fy a, -Jk b, -M 18

19 Test selected RBC panel Focus on anti-d, -C, -E, -S, -Fy a, -M ( -K and -Jk b already excluded) D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b IAT PEG D C E S Fya M D C E S Fya M Ficin IAT 19

20 Exclude and confirm antibody i.d. D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b IAT PEG D C E S Fya M D C E S Fya M Anti-D and anti-m confirmed Ficin IAT 20

21 Case Study #1 conclusion Anti-D and anti-m confirmed Anti-D is likely an alloantibody, but cannot be distinguished serologically from passive anti-d (antenatal RhIg), although this patient had no prenatal care. Anti-M is likely naturally occurring and not clinically significant. * Transfusion recommendation: Group O Rh negative RBCs * Do cord blood studies on neonate (ABO/Rh, DAT) and watch baby s bilirubin, etc.

22 Commonly used problem-solving techniques: Proteolytic enzymes Reducing (thiol) reagents Titration and neutralization Adsorption Elution

23 Reducing Antigen (thiol) Denaturation: reagents Effect of DTT (or AET) on RBCs Reduce disulfide bonds in structure of proteins denaturation of antigens Antigens destroyed: Kell antigens Knops JMH, Yt a, Gy, Hy Cromer (weakened) Lutheran (weakened) Vel (variable) 23

24 Usefulness of Enzymes and DTT: Ficin/ Possible Antibody Papain DTT or Antibody in System neg pos Fy a /Fy b ; Ch/Rg; Ge2, Ge4 neg neg Indian; JMH pos weak Cromer; Knops; Lutheran; Dombrock; AnWj; MER2 variable neg Yt a pos neg Kell; LW pos pos Rh; Jk3; Fy3; Diego; Colton; Ge3; Ok a ; I,i; P,LKE; At a ; Cs a ; Er a ; Jr a ; Lan; Vel; Sd a, Scianna

25 RBC antigens denatured by ZZAP ZZAP is a combination of enzyme and DTT (--frequently used for adsorptions) Antigens denatured: M, N, S, s*, Fy a, Fy b, Yt a *, Xg a, JMH, Ch, Rg, En a TS, En a FS, Ge2, Ge4, LW, Kell, Dombrock, Lutheran, and Scianna system antigens *variable

26 Commonly used problem-solving techniques: Proteolytic enzymes Reducing (thiol) reagents Titration and neutralization Adsorption Elution

27 Neutralization / inhibition Antigens in soluble form can be used to inhibit or neutralize reactivity to aid in antibody identification A, B, H, Le a, Le b, P 1 (blood group substance) Ch, Rg (pooled normal plasma) Sd a (urine)

28 Titration / neutralization Titrate to help classify HTLA-type reactivity ("high-titer, low avidity") HTLA clinically insignificant Neutralization or inhibition with plasma or other blood group substance

29 Examples of titration / neutralization 29 dilution plasma ± ± ± ± ± 0 albumin ± ± ± ± ± 0 plasma albumin ± ± ± ± ± 0

30 Neutralization with plasma 30 Neutralized anti-ch, -Rg Not neutralized anti-jmh, -Kn a, -McC a, -Sl a, -Yk a, -Cs a Possible other alloantibodies

31 Case Study #2 case history 59 y.o. woman with multiple myeloma, transfused 3½ months earlier No medication history given Antibody i.d. requested, no blood Patient discharged Group O Rh Positive Plasma: LISS-IgG = weak pos all RBCs autocontrol = neg

32 Case Study #2 ABO/Rh: A B IS IS Anti-A Anti-B RBCs RBCs Anti-D Cntl DAT: Anti- IgG Anti- C3 10% BSA Antibody screen: D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT I II III

33 Initial Antibody Panel D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT PT 0 0 ** Initial panel shows weak reactivity with 7 of 8 RBCs Autocontrol negative, so we assume this is alloantibody

34 Antibody Panel also tested with ficin and DTT treated RBCs D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Ficin IAT DTT IAT PT 0 0 ** IRL frequently tests ficin treated and DTT treated RBCs to characterize the antibody reactivity. This antibody appears to be DTT sensitive.

35 Exclusion of antibodies D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Ficin IAT DTT IAT PT 0 0 ** Using the DTT treated RBC panel, all common allos can be excluded, except anti K. Anti k can be excluded using cell #7, but is not a common alloantibody.

36 Titration/Neutralization Case # / Antibody tube # dilution diluent AB plasma 6% albumin RBC + AB pool control neat 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 Titer Interp w+ w+ w w+ w+ w ** Antibody appears to have HTLA characteristics, and is not neutralized.

37 Review: DTT-sensitive antigens associated with antibodies with HTLA characteristics Kell Knops LW JMH Indian Dombrock YT (variable) Lutheran (variable) Gerbich (variable) Scianna (variable) 37

38 Selected Rare RBCs D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Yt(a-) JMH Sc: Kp(b-) Ge: Kn(a-) K null LW(a-b-) Lu(a+b-) Lu(a-b-) Do(b-) Yk(a-) 0 1+ ** Selected cells focused on DTT sensitive antigens and antibodies that may have HTLA characteristics.

39 Selected Rare RBCs exclusion: D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Yt(a-) JMH Sc: Kp(b-) Ge: Kn(a-) K null LW(a-b-) Lu(a+b-) Lu(a-b-) Do(b-) Yk(a-) 0 1+ ** One example of Lu(a b ) RBCs was non reactive

40 Addi onal rare Lu(a b ) RBCs D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Lu(a-b-) Lu(a-b-) Lu(a-b-) Lu(a-b-) Lu(a-b-) Lu(a-b-) 0 0 ** Antibody specificity appears to be anti Lu3 but one example of Lu(a b ) RBCs reacted weakly Additional common alloantibodies could be excluded, including anti K.

41 RBC phenotyping Anti-C Anti-E Anti-c Anti-e anti-s anti-s anti-k anti-fy a anti-fy b anti-jk a anti-jk b anti-lu a anti-lu b anti-yt a

42 RBC phenotyping Anti-C Anti-E Anti-c Anti-e anti-s anti-s anti-k anti-fy a anti-fy b anti-jk a anti-jk b anti-lu a anti-lu b anti-yt a ** Individuals who make anti Lu3 would be expected to be Lu(a b ) This pa ent is Lu(a b+) so should not be able to make an Lu3.

43 Summary (note: this was August 2014) Plasma: DTT tt d RBCs = neg Reactive RBCs: Yt(a ), JMH, Kp(b ), K 0, LW(a b ), Kn(a ), McC, Yk(a ), Do(b ), Lu(a+b ) 6/7 Lu(a b ) = neg Anti Lu3 specificity? No, pa ent s RBCs type Lu(a b+) 43

44 Case #2 Preliminary conclusions Medications not listed on request form; called hospital: patient on daratumumab IRL Report: Antibody to Lutheran related high incidence antigen; unable to further identify the specificity All common alloantibodies excluded Reactivity in patient s plasma appearing to have Lutheran related specificity may be related to medication 44

45 2014 AABB Meeting (October) Hannon JL, et al. Transfusion 2014;54Suppl: 162A (abstr) [Transfusion 2015;55:2770] 3/6 myeloma patients with positive IATs after DARA (PEG 1+, solid phase 1 4+) Chapuy CI, et al. Transfusion 2014;54Suppl: 157A (abstr) [Transfusion 2015;55: ] 5/5 DARA treated myeloma patients with positive IATs (weak 1+, tube & solid phase) DTT pretreatment of reagent RBCs a robust method to negate DARA interference 45

46 Hindsight is 20/20 46

47 Daratumumab Daratumumab (DARA) is an IgG1κ human monoclonal antibody to CD38 CD38 type II transmembrane glycoprotein Expressed on immune cells, e.g., T lymphocytes; also widely distributed on non immune cells, e.g., RBCs, platelets, neurons.. Functions include: Receptor that mediates adhesion & signaling Ectoenzyme that contributes to intracellular calcium mobilization 47

48 Daratumumab FDA approved Nov. 16, 2015 (Darzalex, Janssen) indicated for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory agent or who are doublerefractory to a PI and an immunomodulatory agent. accelerated approval based on response rate 16 mg/kg; weekly (weeks 1 8), every 2 weeks (weeks 9 24), every 4 weeks (week 25 on) 48

49 Daratumumab Product Insert Interference with Serological Testing: DARA binds to CD38 on RBCs, resulting in positive indirect antiglobulin tests (IATs), i.e., antibody screens & crossmatches DARA mediated positive IATs may persist for up to 6 months after the last DARA infusion* DARA bound to RBCs masks detection of antibodies to minor antigens ABO and Rh blood type determinations are not impacted 49

50 DTT treated reagent RBCs Chapuy CI, et al. Transfusion 2015;55: Chapuy CI, et al. Blood 2015;126:3567 (abstr) DTT more efficient than trypsin Advantage: DTT is inexpensive & already used by blood banks Disadvantage: some antigens are disrupted by DTT treatment (CROM, DO, IN, JMH, KEL, KN, LW, LU, RAPH, YT) Provide K blood to DARA pa ents Rarely a potentially clinically significant antibody could be missed (e.g., anti k, Do a, Do b ) 50

51 2015 AABB Meeting DARA can be Mistaken for Lutheran or Knops Antibody Aye T, et al. Transfusion 2015;55 Suppl:28A 5/6 pts nonreac ve with most Lu(a b ) RBCs Using flow cytometry, showed nonreactive Lu(a b ) RBCs had low levels of CD38 RBCs from one in house donor, with weak expression of Lu b and very low levels of CD38, were nonreactive with all 6 patients plasma Velliquette RW, et al. Transfusion 2015; 55 Suppl:26A DARA can also be mistaken for anti Kn 51

52 Flow Cytometry Untreated RBCs % Pos = 62% RBC Background (Autofluorescence) 0.2M DTT tt d RBCs % Pos = 13% RBCs + PE anti CD38

53 Flow Cytometry Results CD38 Expression on Selected RBCs Flow cytometry (% positive) #2 10 #1 0 Fy(a+) Fy(b+) Fy(a b ) Lu(a b ) Cord DTT tt'd DARA Pts

54 AABB Association Bulletin #16 02 Jan. 15, 2016 Positive IATs may occur in all media & by all methods (gel, tube, solid phase); usually weak (1+) but stronger in solid phase (up to 4+) Adsorptions with untreated or ZZAP treated RBCs don t eliminate interference Anti CD38 doesn t interfere with IS crossmatch; variably interferes with DATs & autocontrols Anti CD38 may cause small Hb decrease in vivo ( 1 g/dl) but severe hemolysis not observed 54

55 AABB Association Bulletin #16 02 If patient s history of anti CD38 unknown: ABO/RhD typing = no issues Antibody detection (screen) test = all cells pos Antibody identification panel = all cells pos, autocontrol may be neg DAT = pos or neg AHG crossmatches = all units pos Post adsorptions = all cells still pos Thus, 1) delays in issuing blood, & 2) clinically significant alloantibodies could be masked 55

56 AABB Association Bulletin #16 02 BEFORE patient starts anti CD38: Perform baseline type & screen Baseline phenotype or genotype recommended 56

57 AABB Association Bulletin #16 02 AFTER patient starts anti CD38: DTT treated RBCs can be used for Ab screen/id Provide K units, unless pa ent known to be K+ Abs to other DTT sensitive agns can be missed, but are infrequent If DTT treated Ab screen neg, may use electronic or IS crossmatch (ABO/D compat, K matched) For patients with known alloabs, phenotypically or genotypically matched units may be provided; AHG xmatches will still be incompatible; some clinically sig abs may be missed, but infrequently AHG crossmatch with DTT tt d donor cells may be performed 57

58 Communication is Critical Patients should be advised to inform healthcare providers that they are taking anti CD38 prior to receiving blood transfusions Hospital Transfusion Services & Immunohematology Reference Labs need to be informed that patients have received anti CD38 Patients should have type and screen performed prior to starting anti CD38 58

59 Selecting Blood for Transfusion Anti CD38 not removed by adsorptions Proposed solutions: 1. Treat reagent RBCs with DTT or trypsin to denature/remove cell surface CD38 2. Use results of phenotyping & genotyping to select antigen matched units 3. Inhibit anti CD38 using anti idiotype or soluble CD38 4. Test a panel of antigen typed group O cord RBCs 59

60 Panel of CD38-depressed RBCs Patients on DARA tend to have depression of CD38 on their RBCs 1 NYBC published abstract in 2016 suggesting the use of a panel of DARA RBCs 2 Phenotype DARA RBCs if DAT and constuct a selected cell panel 1 Sullivan HC, et al. Transfusion 2016; 56 Suppl:25A 2 Velliquette RW, et al. Transfusion 2016; 56 Suppl:26A 60

61 Current SoCal IRL Approach Hope for a good medication history or accurate diagnosis on request form Review hospital s panel if submitted If hospital tests by solid phase or gel, reactions will probably be stronger than by tube If the serology is suggestive of DARA (i.e. weak to moderate reactivity by PEG and DTT sensitive) then test cord RBCs and CD38 depressed RBCs (e.g., from DARA patients) Exclude or identify alloantibodies 61

62 Example of current serology D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PT 0 0 PEG IAT

63 Example of current serology D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Ficin IAT DTT IAT PT 0 0

64 Example of current serology D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Ficin IAT DTT IAT PT 0 0 ** Using the DTT treated panel RBCs, all common allos can be excluded, with the exception of anti K (and anti k).

65 Selected cell panel D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT KT RBCs cord RBCs cord RBCs CD38 RBCs CD38 RBCs CD38 RBCs CD38 RBCs CD38 RBCs 0 0

66 Selected cell panel DARA RBCs D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT KT RBCs cord RBCs cord RBCs CD38 RBCs CD38 RBCs CD38 RBCs CD38 RBCs CD38 RBCs 0 0 ** Using the cord RBCs, KT s RBCs, and CD38 depressed RBCs we can also exclude anti K and anti k.

67 Transfusion recommendations If no alloantibody, random ABO/Rh compatible units may be transfused; select least reactive (agn neg, if appropriate). Recommend K if an K cannot be excluded If hospital has a phenotype/genotype, antigennegative units may be transfused without repeated serological investigations. (Disadvantage: may cost more than the workup, depending on phenotype.) 67

68 Commonly used problem-solving techniques: Proteolytic enzymes Reducing (thiol) reagents Titration and neutralization Adsorption Elution

69 Adsorption options 69 Remove auto-antibody to detect/rule out alloantibodies Types Autologous only if pt not recently tx d Allogeneic - differential Methods ZZAP Enzyme PEG Temperature (warm and/or cold)

70 Example of Adsorption Empty 7ml tube Add 1 vol of adsorbing RBCs Add 1 vol of pt plasma Mix 70

71 Example of Adsorption Procedure, continued 37C Incubate at 37C for 30 min Centrifuge Harvest ads plasma to fresh tube & discard ads RBCs Test ads plasma 71

72 Pre prepared ZZAP treated RBCs Our laboratory does so many adsorptions on behalf of our local hospitals that it is more efficient for us to prepare these adsorbing cells ahead of need, and have them available for use. 72

73 Differential adsorption Allogeneic adsorbing RBC selection Differential adsorption Do not need to know pt RBC phenotype RBCs from 3 donors whose RBC phenotypes collectively lack all common clinically significant antigens D, C, E, c, e, S, s, K, Fy a, Fy b, Jk a, Jk b

74 Example of adsorbing cells Allogeneic adsorbing RBC selection Differential Example: Donor #1: E c S K Fy(a ) Donor #2: C e s K Jk(b ) Donor #3: D C E Fy(b )Jk(a ) Note: treatment of the adsorbing RBCs with enzymes or ZZAP destroys certain antigens which changes the adsorbing RBC phenotype making selection easier

75 Untreated vs treated RBCs Adsorption treatment comparison D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb #1 UT #1 Ficin #1 ZZAP

76 Selection of adsorbing RBCs for PEG Untreated RBCs for PEG Adsorption D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb A B C

77 Ficin-treated adsorbing RBCs Ficin RBCs for Enzyme Adsorption Before treatment D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb A B C After treatment D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb A B C

78 ZZAP-treated adsorbing RBCs RBCs for ZZAP Adsorption Before treatment D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb A B C After treatment D C E c e M N S s K k Lea Leb Fya Fyb Jka Jkb A B C

79 Case study #3 case history 60 year old Caucasian female Dx: lymphoma Multiple transfusions Jan April 2010 History of Anti-K Last transfusion 2 months ago, 2 units of K RBCs when only anti-k id d Hb 7.3 g/dl

80 Case #3 initial testing 80 Cell Typing: Reverse Typing: Anti- A Anti- B anti-d control A 1 B D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT I II III

81 Case #3 initial panel 81 anti-igg anti-c3 control IS 4+ 0 RT 4+ 0 Chloroquine treated RBCs: anti-igg IS RT 0 D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT Ficin IAT LISS IAT PT CDPttd 0 4+

82 ZZAP treated RBCs for differential adsorptions adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr ZZAP adsorbed x2 double 37C for 30 minutes 82

83 ZZAP denatures MNSs, Kk, Fy adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr ZZAP treatment: combination of enzyme + DTT, so affects antigens on adsorbing cells 83

84 R1 adsorbed serum adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr R1 adsorbed serum: would contain anti E, -c, -S, -s, -K, -k, -Fy a, -Fy b, Jk b 84

85 R2 adsorbed serum adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr R2 adsorbed serum: would contain C, -e, -S, -s, -K, -k, -Fy a, -Fy b, -Jk b 85

86 rr adsorbed serum adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr rr adsorbed serum: would contain D, -C, -E, -S, -s, -K, -k, -Fy a, -Fy b, -Jk a 86

87 Additional selected RBCs Selected cell panel: adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr

88 R1 column Selected cell panel: adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr

89 R2 column Selected cell panel: adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr

90 rr column Selected cell panel: adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr

91 Panel interpretation Selected cell panel: adsorbed sera D C E c e S s K k Fy a Fy b Jk a Jk b R1 R2 rr R R rr E E+ K K E

92 Case #3 conclusions The patient has anti E in addition to anti K, plus a warm autoantibody Transfusion recommendations give E K units compatible with adsorbed sera, or least incompatible with unadsorbed serum 92

93 Commonly used problem-solving techniques: Proteolytic enzymes Reducing (thiol) reagents Titration and neutralization Adsorption Elution

94 Purpose of elution 94 Cause dissociation of antigen and antibody from antigen-antibody complexes. The objective is to: Recover antibody in a usable form or Recover intact RBCs free of antibody (Ig removal)

95 Uses for elution Investigation of + DAT Autoimmune hemolytic anemia Hemolytic transfusion reaction Hemolytic disease of fetus/newborn Drug-induced immune hemolytic anemia Antibody identification Adsorption/elution, antibody separation Preparation of antibody-free intact RBCs (eg, for phenotyping, autoadsorption) 95

96 Elution methods to recover antibody 96 Heat (56C) Lui freezethaw Acid Chemical/ organic solvents ABO HDFN, IgM agglutinating antibodies ABO HDFN only Warm auto- & alloantibodies Warm auto- & alloantibodies Easy, poor recovery for IgG Quick, small vol RBCs Easy, kits available Chemical hazards

97 Case Study #4 case history 97 Pt is a 65 year old male who had cardiac bypass surgery about 2 ½ weeks ago. During surgery he was transfused 2 units of RBCs and has received 1 unit per week since then. His hemoglobin and hematocrit are still gradually dropping, so 2 more units of RBCs are ordered for transfusion today. His antibody screen was previously negative, but now it is weakly positive with 2 of the 3 screening cells. Both of the units being crossmatched are weakly incompatible. Should you just crossmatch a couple more units (the floor keeps bugging you), or first identify the antibody?

98 Case Study #4 initial testing ABO/Rh typing Anti-A Anti-B A RBCs B RBCs IS Anti-D IS Cntl DAT Anti- IgG Anti- C3 10% BSA Rh phenotyping Anti-C Anti-E Anti-c Anti-e 1+ mf 1+ mf What is ABO/Rh? What is DAT? What is Rh probable genotype? What does the mixed-field (mf) reactivity indicate? 98

99 ABO/Rh typing Anti-A Anti-B A RBCs B RBCs IS Anti-D IS Cntl DAT Anti- IgG Anti- C3 10% BSA Rh phenotyping Anti-C Anti-E Anti-c Anti-e 1+ mf 1+ mf What is ABO/Rh? O Positive What is DAT? DAT + with complement only What is Rh probable genotype? Might be R o r, but recently transfused, so can t say for sure. Mixed-field reactivity is evidence of two or more RBC populations. 99

100 Case Study #4 antibody screen and crossmatches D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT I II (+) III Crossmatches: Unit #1 Unit #2 0 (+) 0 (+) (+) microscopic positive 100

101 Case #4 initial panel D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT (+) (+) (+) (+) s (+) (+) PT

102 Exclusion D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT (+) (+) (+) (+) s (+) (+) PT

103 D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT (+) (+) (+) (+) s (+) (+) PT

104 D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT (+) (+) (+) (+) s (+) (+) PT

105 Antibody identified: anti-jk a showing dosage D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b RT PEG IAT (+) (+) (+) (+) s (+) (+) PT

106 Case #4 eluate D C E c e Le a Le b P 1 M N S s K k Fy a Fy b Jk a Jk b Eluate Last Wash s PT 1+ mf 0 Anti-Jk a also present in eluate Why? 106

107 Next steps Patient s RBC phenotype: anti-s anti-s anti-k anti-fy a anti-fy b anti-jk a anti-jk b 1+ mf mf 3+ mf 1+ mf 3+ mf Unit # 1 Unit # Both units are Jk(a+b+) Patient needs Jk(a ) RBCs 107

108 Case Study #4 - conclusions 108 The patient has made alloanti-jka which is present in both serum and eluate. Transfusion recommendations: Provide Jk(a ) units Alert patient s physician that his hemoglobin may continue to drop slowly as Jk(a+) RBCs are cleared from circulation. It may not be evidence that the patient is bleeding.

109 Questions? 109