An unpleasant visitor? The challenge of having a red cell antibody visit the lab Dr. Aseem Kumar Tiwari 1 1

Talk - Organization Expected Antibodies and ABO blood group system Other blood group systems and unexpected antibodies Detection of unexpected antibodies Antibody identification and Interpretation Rule of Three and Phenotyping Magnitude of thse problem Antibody Screen Negative – Change in algorithm Antibody Screen Positive – Approach to Patient 2

Expected Antibody

Laboratory Work-up – ABO system Expected Antibody (Regular Antibody/Typical Antibody) Forward(cell) Reverse(serum) Interpretation -A -B AC BC + - - + A - + + - B + + - - AB - - + + O Slide(1900)/Tube(1960)/Micro-titer plate(1980)/Solid phase adhesion/Column Agglutination Technique

Other Blood Group Systems Other blood group typing is not done routinely – ‘immunogenicity’ is considerably less than ABO and Rh

Unexpected Antibody – Unpleasant Visitor

Other Blood Group Systems and Unexpected Antibody Unexpected (Irregular Antibody/Atypical) Irregular Antibody – Develops either because of Transfusion/Pregnancy

Unexpected Antibody – Detection

Antibody Detection/Screening - Screening Cells Each vial (donor) has been phenotyped for each antigen 18 antigens are required on at least one of the vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb Single Vial (Reagent Pooled ‘O” cells) Two Cells (Selectogen) Three Cells (Surgiscreen) Protocol Reagent Pooled ‘O” cells for donors Three Cells (Surgiscreen) for patients

Antigram- An example An antigram (2 or 3 cells) will list the antigens present in each vial A reaction to one or more cells indicates the presence of an unexpected antibody AHG 37 IS P1 N M s S Leb Lea Jkb Jka Fyb Fya k K e c E C D Antigens 2+ - + I II Principle of testing ‘Unknown’ with ‘Known’

Antigram

If If an antibody is detected, it must be identified!

Antibody Panel - Identification An antibody panel usually includes at least 10 panel cells:

Antibody Panel - Identification

Antibody Panel - Identification

Antibody Panel - Identification Group O red blood cells

Antibody Panel - Identification Each of the panel cells has been antigen typed (shown on antigram) + refers to the presence of the antigen 0 refers to the absence of the antigen Example: Panel Cell #1 has 14 antigens present: C, c, e, f, M, N, S, s, P1, Lea, k, Fya,, Fyb and Jka

Antibody Panel - Identification The same phase (s) used in an antibody screen is used in a panel AHG

AHG Phase - Identification 3 3 3

Interpreting Antibody Panels Few basic steps to follow when interpreting panels “Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Consider antibody’s usual reactivity Look for a matching pattern

1. Ruling Out 2+    2+    2+   2+     Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage.

2. Circle antigens not crossed out 2+    2+    2+   2+    

3. Consider antibody’s usual reactivity 2+    2+    2+   2+     Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will usually react at warmer temperatures

4. Look for a matching pattern E doesn’t match and it’s a warmer rx Ab 2+    2+    2+   2+     …Yes, there is a matching pattern!

Interpretation anti-Lea

Rule of three! Patient serum MUST be: Rule of three must be met to confirm the presence of the antibody Patient serum MUST be: Positive with 3 cells with the antigen Negative with 3 cells without the antigen

Same 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

Screening and Identification only in AHG Phase (Customization !)

Antibody Screen Expected Antibody with 3 cell panel are (c, k, kpb, Jsb, Leb, N and s)

Antibody Identification In 11 cell panel identified anti-”c” in the patient. Confirmed patient red blood cell with rare antisera ”c”

Antibody Panels - Interpretation Few basic steps to follow when interpreting panels: “Ruling out” means crossing out antigens that did not react Circle the antigens that are not crossed out Look for a matching pattern

Another Example

Another Example

Phenotyping Antigen typing the patient red cells can also confirm an antibody If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why? Individuals DO NOT make allo-antibodies against antigens they have Patient has NOT been recently transfused (donor cells could react)

Magnitude of Problem

Scale of Problem 40/32560 (0.12%) or approximately 1 in 1000 15 out of 2026 (0.75%) 18/531 (3.4%) or approximately 34 in 1000 (multi-transfused patients) Rajendra Chaudhary, Nitin Agarwal.Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15 Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology. 2008 Oct;13(5):313-8.

Rh System 14 anti-D (35%) anti-D (5.6%), 6 anti-E (15%) anti-E (22.2%) 3 anti-c (7.5%) anti-c (38.8%) 2 anti-e (5%) 1 anti-C (2.5%) 3 anti-D with anti-C (7.5%) Kidd System 1 anti-Jk(b) (2.5%) anti-Jk(a) (5.6%) Lewis System 1 anti-Le(a) (2.5%) anti-Le(a) (11.1%) 1 anti-Le(b) (2.5%) anti-Le(b) (5.6%) MNS System 5 anti-M (12.5%) anti-M (11.1%) 1 anti-s (2.5%) INCONCLUSIVE 2 (5%) Total 40/32560 18/531 Thakral B, Saluja K, Sharma RR, Marwaha N. Red cell alloimmunization in a transfused patient population: a study from a tertiary care hospital in north India. Hematology. 2008 Oct;13(5):313-8.

Antibody Screen Antibody Screen Screened Negative (999/1000) Screened Positive (1/1000)

Antibody Screen Negative 999/1000

Type & Screen Vs Cross-match 45373 patients for whom a total of 61668 units of packed red blood cells (PRBC) were cross-matched in the AHG phase using DiaMed; ID cards. An antibody screen was carried out in all the patients using the DiaMed; ID-DiaCell I+II+III. The AHG cross-match was also carried out for all recipients, irrespective of the result of the antibody screen. The results were compared to see if there were any cases where the antibody screening was negative but the AHG cross-match showed incompatibility. Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility. Type and screen policy can be safe, efficient, cost-effective, and beneficial to the transfusion service in India. Pathak S, Chandrashekhar M, Wankhede GR. Type and screen policy in the blood bank: Is AHG cross-match still required? A study at a multispecialty corporate hospital in India. Asian J Transfus Sci 2011;5:153-6

Type & Screen Vs Cross-match 11000 patients were cross-matched in the AHG phase Simultaneous Type and Screen. Not a single case was found where the antibody screen was negative and AHG cross-match showed incompatibility. Dr. Dolly Daniel – Personal Communication

Not as yet! Evaluated safety of T & S procedure for PTT in comparison with conventional test tube cross match. Study did not show the T and S to reach the expected safety level of 99%, it has achieved a reasonable level of safety (91.6%). Only one sample gave a negative antibody screen while conventional crossmatch was incompatible. This may be due to a rare antibody in the patient sera against which the corresponding antigen was not present on the screening cells but present on the donor red cell. A transfusion reaction would have occurred in this case since it was reacting at 37΀C in AHG. Since, the screening panel used in the present study was not from Indian subcontinent, therefore, it would be advisable to prepare screening cell panels that include RBC antigens which are prevalent within the local/regional population e.g, In, Mi a etc. Rajendra Chaudhary, Nitin Agarwal. Safety of type and screen method compared to conventional antiglobulin crossmatch procedures for compatibility testing in Indian setting. 2011:5 (2):157-15

Can Cross-match be Omitted? Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen were transfused a total of 10,899 red cell concentrates. Antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk. Heddle NM, O'Hoski P, Singer J, McBride JA, Ali MA, Kelton JG. A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing. Br J Haematol. 1992 Aug;81(4):579-84

Antibody Screen Positive 1/1000

Antigen negative compatible blood Coomb’s Cross-match

Antigen Negative Blood? Random Cross-match Type the stocked blood units Rare Donor Registry

Case study Case 1 Patient Name: Master S , (5 yr/M) Hospitsl- RML hospital, New Delhi Problem: Grouping discrepancy Blood group: forward- “A” Reverse- “O” Plan of Action Repeat grouping Screening with 3 cell panel Identification with 11 cell panel Finding ‘antigen negative blood using rare antisera, if red cell blood transfusion needed

Provisional diagnosis

Findings… Result of repeat blood grouping =Forward- “A”, Reverse- “O” Result of antibody detection(screening) =Probability of Anti-M and Anti- Fyb Result of antibody identification = Anti- “M” Confirmation of absence of M antigen on patients red cells = No agglutination with Antisera-”M”

Conclusion: Anti- “M” antibody present in the patient. Out of 21 unit got only one “M” negative unit, preserved for Master S.

Comparative study of frequency of Ag Antigen Pos. donor (%) Neg. donor (%) PGI Chandigarh (Pos %) (Neg %) D 85 93.4 6 6.59 93.39 6.61 C 77 84.60 14 15.38 84.76 15.24 c 57 62.63 34 37.36 52.82 47.18 E 23 25.27 68 74.79 17.9 82.1 e 88 96.79 3 3.29 98.3 1.7 K 4 4.39 87 95.6 5.56 94.44 k 91 100 Fya 79 86.81 12 13.18 86.75 13.25 Fyb 62 68.13 29 31.86 56.15 43.85 Jka 72 79.12 19 20.87 82.65 17.35 JKb 53 58.24 38 41.75 66.56 33.44 Lea 13 14.28 78 85.71 20.82 79.18 Leb 54 59.34 37 40.65 60.57 43.53 S 59 64.83 32 35.16 56.47 s 87.38 12.62 M 81 89.01 10 10.98 75.39 24.61 N 61.51 38.49 P1 71.92 28.08

Acknowledgements Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435; School of Health Related Professions; University of Mississippi Medical Center

Thank You

Antibody screen Crossmatch Cause Resolution Pos Neg Antibody directed against antigen on screening cell ID antibody, select antigen negative blood Antibody directed against antigen on donor cell which may not be on screening cell OR donor unit may have IgG previously attached ID antibody, select antigen negative blood OR perform DAT on donor unit Antibodies directed against both screening and donor cells Antibody ID, select antigen negative blood