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

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

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

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

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

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 --

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reagent‑Dependent Reactivity: A Noise in the Immunohematology Laboratory

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

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%,

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 --

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

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

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

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

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

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

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.

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.

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

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

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

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

AUS: Natural history

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

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

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..

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.

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.

Thank you Team IHBT – Kasturba Medical College, Manipal