1/31/2017. Advanced Transfusion Case Studies

Transcription

1 1/31/2017 Advanced Transfusion Case Studies

2 Case from NHSBT RCI When is anti-d+c not AntiD+C? 1/31/2017 Julie Molloy, MSc CSci FIBMS

3 AN patient with apparent Anti-C+D+E Booking sample, weeks gestation O neg, with positive antibody screen Previous pregnancy, delivered 18 months ago transfusion episode, at end of last pregnancy

4 NBS Reagents Liverpool Antibody Investigation Worksheet? anti-d + C Patient s Name Requestor Hematos Ref No. Tested by Date of Birth Hosp no Sample No. Date Tested Product Lot No. Product Lot No. Product Lot No. ID panel in Alsevers R ID panel Papainised in Alsevers R ID Panel in LISP R ID panel in CellStab R ID panel Papainised in CellStab R EXPIRY DATE : Rh M N S s P1 Lu a K k Kp a Le a Le b Fy a Fy b Jk a Jk b Other IAT ENZ 1 R1wR R1R R2R r r r r Co b rr rr rr rr rr Co b +Bg b Auto 0 0 Antibody Titre Dilution: Anti- Cell Id Group O D negative Conclusion Pheno D-C-c+E-e+K- Archive Anti- Archive DAT Batch No PS IgG IgA IgM C3c C3d Ctl

5 NBS Reagents Liverpool Antibody Investigation Worksheet? anti-d + C Patient s Name Requestor Hematos Ref No. Tested by Date of Birth Hosp no Sample No. Date Tested Product Lot No. Product Lot No. Product Lot No. ID panel in Alsevers R ID panel Papainised in Alsevers R ID Panel in LISP R ID panel in CellStab R ID panel Papainised in CellStab R EXPIRY DATE : Rh M N S s P1 Lu a K k Kp a Le a Le b Fy a Fy b Jk a Jk b Other IAT ENZ 1 R1wR R1R R2R r r r r Co b rr rr rr rr rr Co b +Bg b Auto 0 0 Antibody Titre Dilution: Cell Id Anti-C D-C+c+ Archive Group O D negative Conclusion Pheno D-C-c+E-e+K- Additional; anti-d quantified at 5.8 iu/ml Anti- Archive DAT Batch No PS IgG IgA IgM C3c C3d Ctl

6 Suspicious result Anti-D of 5.8 iu/ml Reactions with R2R2 cells should have been stronger Suggestions for further tests? Decided to perform parallel adsorptions of patient plasma. Vs. R2R2 cells Vs. r r cells. Resulting adsorbed plasmas were then panelled by IAT

7 Why do this? Any time we see anti-d+c in AN patient we should be considering if it might in fact be Anti-G (+anti-d, +anti-c?) R2R2 cells will remove anti-d and anti-g, leaving behind anti-c r r cells will remove anti-c and anti-g, leaving behind anti-d The combination of results will give us a clue as to what antibodies are really present

8 NBS Reagents Liverpool Antibody Investigation Worksheet? anti-d + C Ads vs R2R2 remove anti-d Patient s Name Requestor Hematos Ref No. Tested by and anti-g Date of Birth Hosp no Sample No. Date Tested Product Lot No. Product Lot No. Product Lot No. ID panel in Alsevers R ID panel Papainised in Alsevers R ID Panel in LISP R ID panel in CellStab R ID panel Papainised in CellStab R EXPIRY DATE : Rh M N S s P1 Lu a K k Kp a Le a Le b Fy a Fy b Jk a Jk b Other IAT ENZ R1wR R1R R2R Ads vs r r remove anti-c and anti-g r r r r Co b rr rr rr rr rr Co b +Bg b Auto Antibody Titre Dilution: Cell Id Anti-C D-C+c+ Archive Group O D negative Conclusion Pheno D-C-c+E-e+K- Additional; anti-d quantified at 5.8 iu/ml Anti- Archive DAT Batch No PS IgG IgA IgM C3c C3d Ctl

9 After a phone call... Previous pregnancy had delivered a group O D positive baby Sufficient prophylactic anti-d had been given to cover all recognised FMH One transfused unit was D-C-c+E-e+ One transfused unit was D-C+c+E-e+

10 Conclusion Antibody specificity present anti-g+c Against standard panels anti-g (with or without anti-c) will appear to be anti-d+c Clue that anti-d might not be present stronger reactivity with R1R1 (and often r r) cells than with R2R2 cells If lab had known anti-d wasn t present, would not have performed anti-d quantification

11 What advice was given to hospital? There is a risk of HDFN as the titre is 32 or greater Continue with anti-d prophylaxis Send samples for re-assessment at 28 weeks gestation Take the samples before prophylactic anti-d is given

12 Next Sample taken between 31 & 32 weeks gestation 500 iu of prophylactic anti-d had been administered (routinely, at 28 weeks) Same work-up as before, including absorptions with R2R2 and r r cells

13 NBS Reagents Liverpool Antibody Investigation Worksheet? anti-d + C Ads vs R2R2 remove anti-d Patient s Name Requestor Hematos Ref No. Tested by and anti-g Date of Birth Hosp no Sample No. Date Tested Product Lot No. Product Lot No. Product Lot No. ID panel in Alsevers R ID panel Papainised in Alsevers R ID Panel in LISP R ID panel in CellStab R ID panel Papainised in CellStab R EXPIRY DATE : Rh M N S s P1 Lu a K k Kp a Le a Le b Fy a Fy b Jk a Jk b Other IAT ENZ R1wR R1R R2R Ads vs r r remove anti-c and anti-g r r r r Co b rr rr rr rr rr Co b +Bg b Auto Antibody Titre Dilution: Anti Cell Id Group O D negative Pheno D-C-c+E-e+K- Archive Anti- Archive DAT Batch No PS IgG IgA IgM C3c C3d Ctl

14 Interpretation? Anti-D and anti-c are present From these results you can t tell if anti-g is present As anti-g previously ident, it seemed reasonable to assume it was still present Titration results suggest level of anti-g+c was unchanged Was the anti-d immune or prophylactic?

15 Next steps? Performed anti-d quantification. If immune anti-d was present the level might be higher than it was before. After all, the level of anti-g+c had not changed 17.1 iu, so suggested immune anti-d is present? In addition quantified the antibody in the plasma adsorped with r r cells (contains only anti-d) 0.11 iu, so only prophylactic anti-d is present?

16 What does this mean? Anti-D quantification results can be affected by the presence other antibodies, e.g., anti-c The results from the first sample show this. There was no anti-d but we recorded a level of 5.8 iu Must have been due to the presence of anti-g+c Titres against r r cells for the first and second samples were the same, so the level of anti-g+c was unchanged Why had the quant result risen to 17.1 when the concentration of anti-d was actually 0.11iu/mL?

17 Advice given Anti-G+C present, no significant change in antibody level Anti-D present likely, but not certain, of prophylactic origin Refer patient to a Fetal Medicine Centre Send more maternal samples Send a paternal sample If RCI Lab hadn t detected the anti-d, would not have asked for more samples

18 What came next Sample taken between 34 & 35 weeks gestation No anti-d given, since last investigation Same work-up as before, including absorptions with R2R2 and r r cells and quantification against R1R1 cells No paternal sample received

19 What did we find? Anti-D and anti-c still present after absorptions Anti-G presumed present Titred to 128 against r r cells Raw plasma (anti-d) quantified at 15.6 Pure anti-d quantified at 0.08 Nothing else done at this point ran out of ideas as to how to proceed!

20 Advice given Anti-G+C present, no significant change in antibody level Anti-D present likely, but not certain, of prophylactic origin Refer patient to a Fetal Medicine Centre Send more maternal samples Send a paternal sample De-ja vu

21 Next Sample taken between 36 & 37 weeks gestation No anti-d given, since last investigation No paternal sample was going to be received This time RCI Lab used a little more imagination

22 What was done? Absorptions with r r and R2R2 cells leaving behind pure anti-d and pure anti-c Perform an elution on the r r cells elution contained a mixture of anti-c and anti-g adsorb this elution with R2R2 cells then perform an elution on these cells elution contained pure anti-g Quantified anti-d from r r adsorption (0.17) and raw plasma (16.4)

23 Something to think about R1R1 cells For every anti-d binding site one site for anti-c binding two sites for anti-g binding R2R2 cells For every anti-d binding site one site for anti-g binding no anti-c binding site r r cells For every anti-c binding site one site for anti-g binding no anti-d binding site

24 Theoretical ratio of total antibody binding capacity (assuming equal antigen density) R1R1 R2R2 r r anti-g+c 6 : 2 : 2 anti-g+c+d 8 : 4 : 2 anti-g 4 : 2 : 1 anti-c 2 : 0 : 1 anti-d 2 : 2 : 0 If the different cells are able to bind differing amounts of Ab, then interpreting titres quite difficult 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

25 Ab Titres compared Raw Anti-D* Anti-C R1R R2R r r * NB Anti-D expressed as reciprocal of diltution NOT IU/mL 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

26 IgG Subclasses determined Various Ab/dilutions incubated Vs R1R1 cells by IAT Anti-IgG1 Anti-IgG3 Anti-IgG Ctrl 1:1 1:100 1:1 1:100 1:10 G+C G C D /31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

27 This is of concern IgG1 is more efficiently transported across the placenta Close to term IgG1 concentrations are often, relatively, higher in the fetus than in the mother Results in this case indicate a lytic anti-g

28 What was advised Anti-G+C present, no significant change in antibody level Anti-D present likely, but not certain, of prophylactic origin Refer patient to a Fetal Medicine Centre Refer maternal samples for molecular typing of fetal DNA Send maternal samples every 2 weeks

29 What came next Sample from baby boy, delivered at about 39 weeks (on a Sunday) No request form received Request form later faxed stating preexchange samples requesting DAT+ Nothing from mother

30 Next steps? Visual examination of the sample showed lysis Rh phenotype on baby C-c+D+E+e+ Elution of baby s cells looked like anti-d+c, however as baby C-, must be either anti-g or anti-g+d adsorbed eluate with r r cells Anti-D left behind Elution of r r cells (used in adsorption) Anti-G eluted (i.e. looks like anti-d+c)

31 Baby DAT Anti-IgG1 Anti-IgG3 Anti-IgG Ctrl 1:1 1:100 1:1 1:100 1: /31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

32 Confirming the culprit Anti-IgG1 Anti-IgG3 Anti-IgG Ctrl 1:1 1:100 1:1 1:100 1:10 G+D G D The various specificities incubated with R2R2 cells and tested with AHG 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

33 What about the baby? At delivery 4.07 kilos Hb 15.5! bilirubin 349 umol/l Given double dose phototherapy Hb derived from colorimetric method, so don t know what it actually was Bilirubin level is sufficient to cause kernicterus

34 Following on... Next day Hb 18.1 bilirubin 339 triple exchange transfusion performed Next day Hb 19.1 bilirubin 301 single dose phototherapy performed Four days after delivery the bilirubin had fallen to 232 Eight days after delivery the FBC derived Hb was 19 Ten days after delivery the baby was thriving

35 Could the investigation have proceeded differently? If Ab had been mistaken this for anti-d+c would the pregnancy have been managed differently? Did the need to ensure the patient would be given prophylactic anti-d lead to underestimation of the seriousness of the case? Later became apparent that the woman had suffered a number sensitising events, after Ab had been identified as anti-g+c. If prophylactic anti-d hadn t been given the outcome might have been much worse!

36 Cold Hemagglutinin Disease (CHAD) Case 1/31/2017 Julie Molloy, MSc CSci FIBMS

37 Patient PW Male, DOB 1938 CHAD Requested investigation - Antibody ID Previous Tx 11 days Manual ABO/D using x 6 warm washed cells Gp O D Positive Known case - unable to group on Bio-Rad IH-1000 so manual groups needed - pre-warm/warm washed tube done 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

38 CHAD Group O Rh D Positive DAT Positive IgM 2+. No alloantibodies detected using prewarmed and warm washed anti-igg LISP tube IAT 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

39 High risk patient Known case, Minimal investigation due to risk of patient 1/31/2017 Julie Molloy, MSc CSci FIBMS

40 High Risk Patient ST Male, DOB 1944 Aplastic Anaemia Requested investigation - XM 2 units RC Previous Tx 4/52 Previous results - Gp A D Positive, C-c+E+e-K+k+Kpa-, Anti-C+e+Kpa+Auto-pan 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

41 HR Pt A Positive R 2 R 2 K+k+Kp(a-) Anti-Kp a detected by IAT. Previous anti-c not apparent. Known anti-e 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

42 Pan Reactive Ab 1/31/2017 Julie Molloy, MSc CSci FIBMS

43 Pre-op Patient KM Male, DOB 1945 Pre-op splenectomy Requested investigation - XM 4 units RC Ab Screen Positive, Pan reactive No previous Tx 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

44 Pre-op PAN A D Positive C+c-E-e+K- Solution dependant antibody, reacting to preservative. NAD in preservative free cells by IAT 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk

45 Pre-op PAN 1/31/2017 Julie Molloy, MSc CSci FIBMS

46 Neonate 1/31/2017 Julie Molloy, MSc CSci FIBMS

47 Neonate Patient MJ Male, DOB 3 days ago! Mother has anti-c+e Investigation requested Group DAT 3+ No record of IUT Group O D Positive, no reverse Gp C+c+E-e+K- 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

48 Neonate Anti-c in eluate 1/31/2017 Julie Molloy, MSc CSci FIBMS

49 Pt with Myelodysplastic Syndrome (MDS) 1/31/2017 Julie Molloy, MSc CSci FIBMS

50 Patient NT Male, DOB 1935 Myelodysplastic Syndrome (MDS) Investigation requested Antibody ID, Urgent XM 4 units RC DAT + IgG Positive Ab Sc (capture) Ab NOT removed by x 5 Auto Adsorption! No history of Tx, pt not seen before by RCI 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

51 MDS Auto Pan by gel, Enz and tube IAT. Auto anti-d by gel IAT in plasma AA x 4 Vs rr NAD by gel IAT in plasma AA x 4 Vs R1R1 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

52 Following discussion with NHSBT MO Gp O Pos C-K- issued as suitable Summary of results Pt Gp O R2r K- DAT 5+ IgG Hb 64 g/l Strong Pan Agg AA x 4, auto anti-d detected. Units 3+ (gel IAT) Vs. rr ads plasma, NAD/1+ Vs. R1R1 ads plasma 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

53 SCD Patient Antibody ID & XM 1/31/2017 Julie Molloy, MSc CSci FIBMS

54 Patient MB Male, DOB 2000 Sickle Cell disease Ab ID & XM 12 units RC (Ro or rr, K- HbS neg, CMV neg) Known pt Knopps/McCoy with known -C and K Investigated using inhibition agent 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

55 SCD O Rh D??, Phenotype Ro but recorded as?? Due to recent Tx with Orr Anti-C, anti-k, anti-kna/mcca & Pan previously detected by IAT. 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

56 Able to supply Gp O (rr) D-C-E- K-CMV neg HbS neg units following XM Vs. inhibited plasma 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

57 Iron Def Patient Antibody ID & XM 1/31/2017 Julie Molloy, MSc CSci FIBMS

58 Patient LF Female, DOB 1934 Iron Deficiency, Hb 53 g/l Ab ID & XM 2 unit RC Referring lab Ab Sc pos PAN Ab Capture & IAT

59 IDA A Rh D Pos R1rK- DAT 5+ Insufficient sample for auto adsorptions, strong pan reactive autoantibody removed following 3 x allo adsorption 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

60 Low Hb Ab ID 1/31/2017 Julie Molloy, MSc CSci FIBMS

61 Patient LR Female, DOB 1954 Low Hb, level not stated Requested investigation Ab ID DAT 3+ IgG

62 LR Low Hb O Rh D Neg C-c+E-e+K-M-N+S-s+Jk(a+b+) DAT 3+ IgG Previously detected anti-d+k+pan 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

63 LR Low Hb O Rh D Neg C-c+E-e+K-M-N+S-s+Jk(a+b+) DAT Additionally 3+ IgG this time anti-fya detected Previously detected anti-d+k+pan Additionally on this occasion anti-fya detected 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk

64 Summary of results Historical Results Gp O Rh D Negative C-c+E-e+K-M-N+S-s+Jk(a+b+) Allo Anti-D+K+ Anti-Lua, anti-fya not detected Auto anti-pan reactive Ab Current sample Gp O Rh D Negative C-c+E-e+K-M-N+S-s+Jk(a+b+) Allo Anti-D+K+Fya detected by IAT Tube Auto anti-pan reactive Ab 1/31/2017 Julie Molloy, MSc CSci FIBMS julie.molloy@uwe.ac.uk; Julie.molloy@nhsbt.nhs.uk