Use of select cells in Immunohematology Dr. Aseem K Tiwari, Director, Department of Transfusion Medicine Medanta – The Medicity Hospital, Gurgaon

Acknowledgements Dr. Ravi Dara, Dr. Geet Aggarwal and my team

Outline Recapitulate – Ab identification Select cells – Definition Picking up “Select cells” conservatively Sources of Select cells (Not only extended panel cells!) Caution with “recently expired” panel cells Application of Select cells with case studies Case study 1 – cross-matched units Case study 2 – recently expired panel cells Case study 3 – extended and recently expired panel cells Case study 4 – identification panel cells and extended panel cells Case study 5 – blood donor units

Recap – Antibody Identification Exclusion by “cross out” technique (“rule-out”) where there is no agglutination [0] Rule out a specificity only if the RBC has double dose [homozygous] antigen expression This guards against eliminating an antibody that is showing dosage Look at what ‘is there’ [Pattern/Rule in]. Consider the Phase of reaction Variation in reaction strength Look for dosage

‘Rule of three’ “Three pos-three neg” rule - aid to assure that the results seen are due to a given antibody and not just random chance The rule of three must be met to confirm the presence of the antibody How is it demonstrated? Patient serum MUST be: Positive with 3 cells with the antigen Negative with 3 cells without the antigen

Example fulfills the “rule of three” 2+   3 Positive cells  2+    2+   3 Negative cells 2+     Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin

What if the “rule of three” is not fulfilled? If there are not enough cells in the panel to fulfill the rule, then additional cells can be used Select Cells Select cells are red cells that have been chosen because they express some specific antigens and lack others Select cells are chosen to confirm or eliminate the possible antibody Select cells are chosen depending on how many antibodies have to be identified

Select Cells For example: Let’s say you ran a panel and identified 3 different antibodies: anti-S, anti-Jka, and anti-P1 Selected cells could help… Selected cells S Jka P1 IS LISS 37° AHG #1 + 2+ #5 3+ #8 These results show that instead of 3 antibodies, there are actually 2: anti-S and anti-Jka

Picking ‘Select Cells’ Conservatively Choose cells that can help rule out more than one antibody at a time in order to help decrease supply usage and time Whenever possible, selected red cells should have a strong expression of the antigen being tested (i.e., from homozygous donors) Such red cells help ensure that non-reactivity with the selected red cell indicates the absence of the antibody

Example Ruling out C, Fyb, and M if you have a suspected Jka Instead of running 3 separate cells to rule out the antibodies, you can choose one that is homozygous positive for M, C, Fyb and negative for Jka Panel cell 9 works in this case. If the only antibody that is present is Jka, then your test results should be negative. If the results are positive then further ‘rule-outs’ will be needed to determine what is present

Sources of select cells Select cells can be chosen from 3-cell antibody screening panel Extended cell panels – Panel B, 22 cells panel Recently expired 11-cell panels Cross-matched units Phenotyped donor units Frozed rare RBC inventory

Recently expired panel cells – Caution! Duffy antigen are most labile antigens BCSH also recommends the use of controls, containing weak examples of antibodies (weak anti-D) and weak anti-Fya to assure the sensitivity of the test procedure and integrity of antigen expression of reagent red cells during storage British Committee for Standards in Haematology. Milkins C, Berryman J, Cantwell C, Elliott C, Haggas R, et al. Guidelines for pre-transfusion compatibility procedures in blood transfusion laboratories. British Committee for Standards in Haematology. Transfus Med. 2013;23:3–35

Application of select cells Antibody Identification – Multiple Identifying antibodies in patient with known antibody Titration – in case of multiple antibodies Differential adsorption

Case 1 (Mutiple Abs) - cross-matched units units) 49-year-old man diagnosed with ESLD scheduled for liver Tn needed 11 units of RBCs. On IH studies, anti-c and anti-E were identified. RBC units that were c– and E– were crossmatched with the patient’s serum using AHG. Some units were compatible and some were incompatible, suggesting presence of another unexpected Ab

Case 1 (Mutiple Abs) - cross-matched units units)

Case 2 (Mutiple Abs) - Recently Expired cells 33-year-old woman was admitted to the gynecology department with atypical adenomatous hyperplasia for D & C. She had a history of receiving two units of RBCs one year earlier for vaginal bleeding. Her hematocrit on admission was 21.3 percent (normal for females >36%). Two RBC units were ordered by her gynecologist

Case 2 (Mutiple Abs) - Recently Expired cells Selected cells were chosen from a recently expired panel for differentiation between the antibodies (Table 3). Anti-E was eliminated by selected cell number 7, and anti-Fya was eliminated by selected cell number 9. The positive reaction in selected cell number 3 suggested the presence of anti-Jkb in the sample. This case shows the use of selected cells in the confirmation and elimination of antibodies when multiple specificities were suspected. An expired panel (1-month past-date) was the source of selected cells. The reactivity of the expired RBCs was checked with appropriate controls before use as per our center’s SOP. Although low-prevalence antigens, Kpa and Jsa, could not be evaluated in this testing, they are rare in incidence, as the name suggests and should be considered as a last resort in specificity of exclusion.

Case 3 (Additional Ab) – Extended/Expired 16-year-old boy with thalassemia intermedia; received multiple transfusions during 15 years from tertiary center, relocated to new city/PHC for Tx. At this center, he had adverse reactions to Tx. Record mentioned that he was alloimmunized with anti-e. Two units of RBCs were prescribed by the hematologist because his hematocrit was low (18.9%, normal for males >41%). His blood type was group O, D+ with an unexpected antibody identified as anti-e (Table 4). Typically, e is present on most panel cells (10 out of 11 cells were e+)

Case 3 (Additional Ab) – Extended/Expired 3 e– selected cells (R2R2) were chosen from extended-cell antibody identification panel for testing. Presence of an additional antibody(ies); after the rule-in/rule-out, anti-N was suspected (Table 5). To confirm this antibody specificity one more e–, N+ cell was required to fulfill the two antigen-positive/two antigen-negative rule. One more selected cell (cell number 3: e–, N+) from an expired panel was chosen and found to be positive (Table 6). Anti-K and anti-Fya could not be excluded. RBCs (e–N–) were provided with no adverse reaction

Case 4 (Ab Titration) – Identification & Extended Titration is important in prenatal testing and change in titer helps decide the need for intervention (i.e. IUTx). Commonly performed for anti-D in Rh isoimmunized mothers, but many other unexpected antibodies are also responsible for severe HDFN & need monitoring. D– pregnant woman with only one living child (gravida 4, para 2) was alloimmunized with 3 antibodies: anti-D, anti-S, and anti-Jkb. Specific antibody titration monitoring was advised. To perform the titration of each specific antibody, selected cells were needed that were positive for one antigen and negative for the other two antigens

Case 4 (Ab Titration) – Identification & Extended For titration of anti-D, selected cell was chosen from an antibody identification panel that was D+, S–, Jk(b–), cell number 4: R0r (Table 7). For titration of anti-S, a selected cell was chosen from the same antibody identification panel that was S+, D–, Jk(b–), cell number 9: rr. For titration of anti-Jkb, selected cell number 21 from an extended-cell antibody identification panel (Table 8) was used that was Jk(b+), D–, S–. Titrations were performed and found to be 64 for anti-D, 128 for anti-S, and 16 for anti-Jkb

Case 5 (Allo-adsorption) – blood donor units patient with warm AIHA admitted for symptomatic anemia had pan-agglutination with all 11 cells on antibody identification panel. Autoadsorption was not possible as there was not enough blood sample available; patient’s hemoglobin was 6 g/dL (normal for males >13 g/dL, females >12 g/dL) and there was history of recent transfusion (15 days earlier). An allo-adsorption was planned.

Case 5 (Allo-adsorption) – blood donor units

Case 5 (Allo-adsorption) – blood donor units

Case 5 (Allo-adsorption) – blood donor units R1R1, R2R2, and rr cells were identified. Among these three cells, R1R1 was negative for Jka and K, and rr was negative for Jkb. Finding R1R1 and rr RBCs was easy, but finding R2R2 RBCs was difficult. R1R1 RBCs were obtained from a freshly phenotyped unit of blood, and R2R2 and rr RBCs were obtained from institutional rare donor inventory. After alloadsorption, anti-E was identified as an unexpected antibody, which was masked by the autoantibody

Summary Select Cells play important role in IH (serological testing) The select cells can be sourced from conventional sources Extended cell panel 3-cell Screening panel And several unconventional sources Cross-matched blood units Phenotyped blood units (phenotyped donors from registry) Recently expired cells (after adequate validation/QC) Select Cells can have various applications: Ab confirmation or exclusion Titration Differential adsorption

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Case 5 (Allo-adsorption) – blood donor units

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Cross outs - Exceptions EXCEPTION 1: K, Kpa, Jsa and Lua can be excluded when the antigen is present in a heterozygous manner (both antigens of a pair are present on the RBC) a) Antibodies of the Kell system (K, Kpa and Jsa) do not typically exhibit dosage b) These antigens are all low prevalence. It is difficult to find a cell that expresses these antigens, especially in a homozygous manner. EXCEPTION 2: If anti-D is present in the patient’s plasma, it may be difficult to find a cell that is D - C + c - (r’r’) in order to exclude C In this situation only it is acceptable to exclude C using a cell with heterozygous antigen expression D- C+ c+ (r’r) This exception is true for the E antigen also (rule out using r”r cell). Source- CLS 422 Clinical Immunohematology I Student Lab