1. Non-Blood Group Antibodies A Nuisance in Immunohematology
Dr. Shamee Shastry
Professor and Head
Department of Immunohematology and Blood Transfusion

2. Overview Nonspecific reactions Causes and resolution Case discussion
Review of literature
Consensus and approach

3. Common Scenarios ABO discrepancy HDN workup AIHA
Alloimmunization – transfusion support
Transfusion reaction work up
Identify clinically significant antibodies
which could destroy incompatible donor red blood cells (RBCs) by intra- or extravascular hemolysis

4. Types of Antibodies in Immunohematology
Naturally occurring
Unexpected antibodies: Auto/Allo
Single antibody / multiple antibodies
Clinically significant / insignificant
Antibodies of undetermined specificity
Non blood group antibodies

5. Case 1 43 year/ Male Admitted for Urinary calculi removal
Request – 1 unit PRBC
Lab Parameters:
Hb: 11.2 gm/dL. PS – normal, RFT, LFT - normal
Coagulation screen – Normal
No past history of transfusion

6. Case 1 Immunohematology work up
Blood Grouping
Antibody Screening
Anti A
Anti B
Anti D
Anti H
A Cells
B Cells
O Cells AC
Interpretation 4+ 3+
O Rh D Positive
? Warm auto-antibody
Panel 1
Panel 2
Panel 3 AC
Interpretation
AHG 2+
Panreactivity with positive AC RT --

7. Case 1… Direct Antiglobulin Test : Negative
Cross-matching IAT method: Incompatible
Antibody Identification panel: Panreactive
Case of DAT negative, IAT and Auto control Positive

8. Case 1 D/D Resolution False negative DAT? Elution – negative
Antibody against high frequency antigen?
In vitro phenomenon?
Hemolysis of all the DAT positive cells?
Resolution
Elution – negative
Lab parameters – normal

9. Can It be In-vitro Reaction?
Testing with saline suspended pooled O cells
Testing with saline suspended donor RBCs
Testing with washed panel cells
Testing with saline suspended
Negative
Cross-match compatible
Diagnosis: In-vitro reaction due to warm antibodies against LISS

10. In-vitro Reactions Not Due to Blood Group Antibodies
Antibodies to chemicals present in RBC suspending medium
Antibodies to chemicals added to commercial antisera
Antibodies to chemicals added to commercial antibody potentiators
Miscellaneous Reactions

11. 1. Antibodies to Chemicals Present in RBC Suspending Medium
A. Antibodies to antibiotics
Neomycin, Chloramphinicol, Gentamycin
Do not bind covalently – Immune complex mechanism
Reacts in the presence of antibiotics
Resolution: washing

12. 1. Antibodies to Chemicals Present in RBC Suspending Medium
B. Antibodies to sugar:
Dextrose, Lactose, Glucose
Reacts with washed RBCs
Adsorption onto RBC membrane – similar to penicillin
C. Antibodies to hydrocortisone in reagent red cells
Hydrocortisone was added by the to prevent hemolysis of the reagent red cells.
The patient's antibodies were IgM, complement independent

13. 1. Antibodies to Chemicals Present in RBC Suspending Medium
D. Hemagglutination properties dependent on polycarboxyl group:
IgM agglutinins against EDTA (Ethylenediaminetetraacetic acid)
Do not react with washed cells
Reference:
Beck et al: antibodies react with polycarboxyl group
(citrate, acetate, succinate, butyrate)
Joshi et al: citrate dependent autoantibody causing ABO discrepancy
Zeigler et al: anti-A1 inhibited by EDTA

14. 2. Antibodies to Chemicals Added to Commercial Antisera
A. Antibodies to dyes
Acriflavin, yellow # tartrazine
ABO discrepancy seen with RBCs suspended in plasma
Resolution: by washing the cells
B. Antibodies to bacteriostatic agents :
Anti I cold agglutinin – enhanced in the presence of sodium azide
Washing resolved the problem

15. 3. Antibodies to Chemicals Added to Commercial Antibody Potentiators
A. Bovine albumin:
Golde et al: “albumin autoagglutinin” phenomenon
Due to antibodies to sodium caprylate - added as a stabilizer during the heating
B. LISS:
Antibodies to preservatives
- Methyl Paraben, Thimerosal
Paraben antibodies had anti Jka specificity and complement dependent

16. 4. Miscellaneous Reactions
A. Antibodies to formaldehyde:
3% of patients undergoing chronic hemodialysis had anti N-like Abs
Reacted against formaldehyde treated red cells

17. Case 2 32 year – posted for laparotomy - ruptured ectopic
Sample was sent for pre-transfusion testing
No previous h/o transfusion
H/o abortion +
Hb: 7.4gm/dL

18. Pre-transfusion Testing
Blood Grouping – no discrepancy noted
Antibody screening Antibody Identification
Anti A
Anti B
Anti D
Anti H
A Cells
B Cells
O Cells AC
Interpretation 4+ 3+
O Rh D Positive

19. Further work-up DAT: 2+ reaction Elution
Expected crossmatch report: Incompatible
Cross-Match
? Non blood group antibody

20. Repeat testing With cell panels of different manufacturer Negative
? warm autoantibody that is cross-reacting with suspension medium of panel cells

21. Washed cells
Mimicked antibody against high-frequency red cell antigens
Reacting in both saline phase as well as AHG phase
Reagent red cells without co-trimoxazole drug didn’t show the reaction

22. Reagent‑Dependent Reactivity: A Noise in the Immunohematology Laboratory

23. Nuisance antibodies in immunohematology
Commercial reagent red cells used are usually stored in buffered preservative suspension medium
Modified LISS buffer, co-trimaxazole & sodium azide
Pham et al. reported antibodies against co-trimoxazole
Garatty et al - reported antibodies against chemicals in suspension medium

24. Case 3 45 years / Female Diagnosis: Multiple myeloma on treatment
Sample was sent for pre-transfusion testing
Previous history of transfusion – 1 month back
Laboratory parameter
Hb: 8.4gm/dL, Hct:23%,

25. Pre-transfusion testing
Blood Grouping
Cross-matching: Incompatible
Antibody screening
DAT: Negative
Enzyme treatment –
resistant – no change in the strength of the reaction
Anti A
Anti B
Anti D
Anti H
A Cells
B Cells
O Cells AC
Interpretation 4+ 3+
wk+
Cold enhancement – Incubation at 4°C
O Rh D positive
Panel 1
Panel 2
Panel 3 AC
Interpretation
AHG 2+
Panreactive RT --

26. Further work-up
Antibody Identification: Pan reactivity; no variation in the strength of reaction (2 + with 11 cell panels)
Differential Diagnosis:
Allo-antibody, against high incidence antigen
Antibody against reagents/ chemicals
Ruled out- testing with washed panel cells
Patient received transfusion 1 month back – compatible unit
No signs of DHTR
DAT negative
No signs of hemolysis
Previous IAT – negative

27. Review of History Medication history Chemotherapy, antibiotics
DARZALEX ® (daratumumab)
Is a human monoclonal antibody for the treatment of multiple myeloma
 Recently approved by the FDA
Binds with high affinity to the CD38 molecule
Medication history
Chemotherapy, antibiotics
Daratumumab

28. Binds to CD38 a protein that is expressed on red blood cells (RBCs)
Interferes with blood bank compatibility tests, including the antibody screening and cross-matching

29. DARA – anti CD 38; Serologic interference
Cause positive reactions in indirect antiglobulin tests (IATs)
Agglutination may occur in all media (eg, saline, low ionic strength saline, polyethylene glycol)
Does NOT interfere with ABO/RhD typing or with immediate-spin crossmatches
Positive IATs can be observed for up to six months after anti-CD38 is discontinued
May cause a small decrease in hemoglobin in vivo (~1 g/dL), but severe hemolysis has not been observed among treated patients

30. How to Resolve? ABO/Rh D typing can be performed normally
Antibody detection (screening) and identification:
Dithiothreitol (DTT)-treated cells can be used to eliminate the interference
AHG crossmatch may be performed using DTT-treated donor cells
Alternative to DTT treated cells, antigen typed cord blood cells can be used

32. Case 4 43 years Male – Myelodysplastic syndrome
H/o Transfusion, Medication – Not available
Samples sent for Pre-transfusion testing
Blood grouping –
Cell Grouping: AB Rh D positive; Serum Grouping: Pan-agglutination
Antibody Screening: Pan-agglutination – at all phases
DAT - Negative

33. Case Enzyme treatment, DTT treated cells – Pan agglutination
Cord cells – 3+ agglutination (no change in the grade of reaction)
Antibody titration : >1024
? Potent alloantibody - to a high frequency antigen
Complete History: Patient had been in a clinical trial for a monoclonal antibody therapy: anti-CD47
Additional monoclonal therapies will continue to emerge. This case highlights the need for extensive communication between the Blood Bank and the clinical service regarding the use of novel monoclonal antibody treatment.

34. Anti CD47 - Overview
CAMELLIA (anti-CD47) is a monoclonal antibody used to treat AML and myelodysplastic syndrome.
CD47 is widely expressed on human tissues and red cells.
CD47 acts as a marker of self, a "Do not eat me" signal for healthy tissue.
Blocking of CD47 on the surface of Red blood cell (RBC) with the use of targeted monoclonal antibodies decreases the protective signal and increases the phagocytosis of circulating RBCs by macrophages in the spleen.
Clinically manifest with signs of extravascular hemolysis.

35. Anti CD47
Treatment with anti-CD47 is likely to cause anomalous results in serology
After treatment, if the ABO group cannot be concluded, group O red cells may be required for transfusion
Antibody testing was successfully completed using Gamma clone anti-IgG sera
A clone without anti-IgG4 is used
A Novel Use of Human Platelet Concentrate to Resolve Interference of Anti-CD47 in Serologic Testing
HPC – Used for adsorption of anti CD47

36. Antibody of Undetermined Specificity (AUS)
Definition: Unexplained reactions following ruling out the antibody against FDA specified red cell antigens
Not a universally used term (Liu et al . Washington University School of Medicine)
AUS was reported in 18% of cases with antibodies – Majority gave 1+ reaction
Causes: Non BG antibodies, anti HLA, antibody to low-prevalence antigen

37. AUS: Potential Etiologies
New antibodies experience affinity maturation
Antibodies with low titer/affinity to RBC antigens
Antibodies to low frequency antigens
Antibodies against non-RBC antigens
Agglutination artifact

38. Should we perform additional work up of AUS ?
Advantages
Additional clinically significant antibodies found
Potentially decrease frequency of HTR
Disadvantages
Delays identifying and releasing compatible RBCs
Increase resources needed
? Clinical significance

40. AUS: Natural history

41. Observed an 11-fold increased odds of detecting a new UID using SPRCA
Common in females, obstetric patients, Surgery or trauma patients, cancer, chronic or autoimmune disease, and obstetric patients had the majority of UID reactivity
Conclusion: UID results are of mixed significance, and future work is needed to both definitively characterize and eliminate excess solid-phase reactivity from substances unrelated to RBC antibodies

42. Laboratory Techniques - Immunohematology
Tube Technique, Column Agglutination Technique
Microplate Technique, Solid phase assay
Which is better?

43. Trade off between sensitivity and specificity
Can alternative antibody screening modalities offer the sensitivity of gel and solid phase with improved specificity?
What actions can be taken to help evaluate the possibility that an AUS represents a low titer alloantibody?
What are reasonable approaches to transfusion therapy in a patient with AUS?
As mentioned in the Transfusion editorial..

44. Consensus & Approach Clinical details and history Blood grouping
No discrepancy
Discrepancy
Resolution
Antibody screening and Identification and AC/DAT
Auto Antibody
Alloantibody
DTT/Enz/Adsorption/Elution
AUS
Panagglutination with Pos AC
Panagglutination with
Neg AC and crossmatch
Antibody to red cell suspension medium
Antibody to Sugar, Monoclonal Abs
Antibody to low frequency antigen
Follow-up
Antibody to preservative
Antibiotics
Washed cells/ DTT treatment
Washed cells / use reagent of different manuf.

45. Summary
Haemagglutination in serology can be due to non-blood group antibodies
Review the history, clinical signs & symptoms
Step-wise approach, Perform specific tests
Review the literature, results of other lab tests
Determine the clinical significance, review the clinical condition of the patient.
Provide timely transfusion support
The sensitivity of an antibody detection method may come with caveat of detecting unwanted antibodies of little clinical significance. These unwanted findings rarely provide additional clinical benefit. Instead they may significantly increase the time and effort of work up and delay urgent transfusions.
AUS is by definition dependent on ruling out all known clinically significant specificities covered by FDA-approved panel RBCs. AUS remains an interpretation of exclusion
Even though most AUS may represent clinically insignificant antibodies, a fraction of AUS may be important alloantibodies in development or clinically significant antibodies to low-prevalence antigens.

46. Thank you
Team IHBT – Kasturba Medical College, Manipal