Blood Grouping , its importance, method to asses

Describes how to do blood grouping in Lab

1. Grouping Dr. P K Maharana.

2. H–Antigen & Bombay Phenotype H-antigen is a precursor to A & B antigen, in the ABO blood group system . It is present over the surface of all RBCs in the ABO system ( A H , B H , AB H , O H ) As H-antigen acts as precursor, its absence means the absence of antigen A and B, hence these individuals identified as O blood group. Those O individuals, who do not express H-antigen ( also called substance H on their RBCs surface, which is normally present in O blood grou p ); produce isoantibodies to H-antigen as well as to antigens A and B. Hence a person with (hh) phenotype contain anti A, anti B & anti H . These individuals named as Bombay Phenotype. As all O group of cases ( both Rh + or Rh -), containing H antigen, when transfused to person who is not having H antigen, causes haemolysis.

3. Bombay Blood Group Bombay individuals lack all normal expression of the A, B, or O genes they inherited. The Bombay Phenotype Red Cells are devoid of normal A,B, H Ags. But in Bombay Phenotype Serum contains  anti-A, -anti-B ,and anti-H. In routine forward grouping, using anti-A, anti-B, and anti-AB. The Bombay would phenotype as an O blood group.

4. Testing with Anti Oh A ntibody As Bombay Phenotype dose not contains “h” antigen on the surface of the RBC it will show no reaction when treated with Anti Oh antibody, where as others will show positive reaction. Anti -D Anti -Oh Anti-Oh O+ O+ O+ Agglutination  O+ Agglutination  O+ No agglutination ( Bombay Type )

5. Testing with Anti Oh A ntibody As Bombay Phenotype dose not contains “h” antigen on the surface of the RBC it will show no reaction when treated with Anti Oh antibody, where as others will show positive reaction. Anti -D Anti -Oh Anti-Oh O+ O+ O+ Agglutination  O+ Agglutination  O+ No agglutination ( Bombay Type )

6. A2 Phenotype . It is observed that some of the A group of patients reacted( Showed haemolytic transfusion reaction ) to a group of similar A group of blood. This observation lead to the hypothesis that not all “A group” cases are similar in all aspects.

7. Anti A1 antibody ( Anti-A1 Lectin ) Anti- A1 Lectin is designed for use in agglutination tests for the detection of the A1 antigen on human red cells. Erythrocytes possessing the A antigen can be sub divided into A1 and A2 cells. Anti- A1 Lectin is designed for use in agglutination tests for the detection of the A1 antigen on human red cells. Group A red blood cells which are agglutinated with Anti- A1 Lectin are said to be of sub-group A1. Those which are not agglutinated by Anti-A1 Lectin fall into subgroups weaker than A1, the majority being classified as A2. Approximately 80 % of the population of blood group A is A1, while the remaining 20% are A2 or weaker subgroup.

8. Reverse Typing On reversed typing ( When plasma of the patient is treated with A & B type of cells, there is agglutination reaction to both the groups, means it is type O ). So when a blood group is diagnose as A group in forward typing & O group in reversed typing, in is thought to be case of A2 Blood Group.

9.   Persons with type A 2  blood group may possess anti-A 1  antibody . It is estimated that A2 type blood group is found in 1-8% of all A individuals and 22-35% of AB individuals)  . A2 cases will render the reverse typing discordant from forward typing. Such patients when grouped would display A in forward typing, but O in reverse typing. Antibodies to A 1  are present in the type A 2  patient. Forward( Cells) Reverse(Serum) Inference Anti -A1 A cells B -cells Positive Negative Positive A B A1 Forward Reverse Inference Anti A1 Serum A ( Clump) O ( No Clump) A2

10. Rh System The Rh system consists of over 50 antigens , of which 5 important antigens ( D, C&c , E,& e ) are of clinical implications and among them D being the major antigen expressed by Rh D protein . Molecular genetics has shown that there are two Rh genes, one encoding D, the other encoding the Cc and Ee antigens

11. Weak D represents Weak D represents a D phenotype where due to reduced D antigen expression on red cells, the antigen is not detected by routine techniques (spin tube method ). Weak D antigen positive cells have a weak expression of the D antigen and may be misclassified as D negative cells in routine Rh grouping procedures. However, the demonstration of this weakly expressed antigen can be undertaken by prolonged incubation and the use of anti-human globulin.

12. Procedure for Weak RhD antigen . ( Test Tube Method ) Equal volume of 5% red cells Suspension+ Anti D antisera  Incubated at 37C for 45 minutes  Centrifuge at 1000 rpm for 1 minute  Examined both macro & microscopically for agglutination. Agglutination Rh D positive No agglutination Rh D negative. Do not report Negative results. Perform Weak D antigen testing (Indirect antiglobulin test) by test tube or gel card methods using a commercial polyspecific Antihuman Globulin (AHG) reagent containing anti IgG and C 3 d.  Take the tested negative test samples of red cells, wash for 3 times with saline, decant extra saline add AHG ( IgG +C3d) 2 drops  Centrifuge for 1 minute at 1000 rpm  Re examine for agglutination both macroscopically/microscopically . Those samples that showed agglutination with addition of AHG serum were labeled as weak Rh D positive . Only blood sample that was negative macroscopically and microscopically in the weak D test was labeled as Rh D negative

13. Procedure Procedure: 1. Test Sample ( The sample which is D negative& to be evaluated for weak D positive) A 5% RBC saline suspension was made by washing the RBCs with isotonic saline in a test tube . 2. A control Saline in other test tube. 3. One drop of MEDICLONE D (IgG) and saline were added to the test tube and control tube, respectively. 4. After mixing, incubation was done for 30 min for sensitization at 37 C. 5.After washing the sensitized cells 3–4 times with normal saline and discarding the supernatant. 6. Two drops of Anti-human serum (Coomb's serum) were added and centrifuged for 1 min. 7. The sediment cells were gently dislodged and examined macroscopically as well as microscopically for agglutination . 1.Agglutination of sensitized RBC with Coomb's serum was considered as weak D antigen (Du) positive , 2. No agglutination noted as Rh D-negative blood .

14. Grouping by Tube Method with Albumin : Prepare a washed RBC 3-5% suspension of RBCs. Add 50ul Anti D & 1-2 drops Albumin in a tube containing 50ul 3-5% Red cell suspension. Incubate the tube at 37°C for 45 minutes . After 45 minutes, suspend the tube & examine agglutination, if agglutination occurs it means Rh is positive if no agglutination present it confirmed that Rh is negative.

15. Gel Card Method Gel card system used was Diamed ID Microtyping System containing polyspecific AHG. 1. A 1% red cell suspension of blood sample was prepared in Low Ionic Strength Solution (LISS). Fifty microlitre of 1% RBC suspension was taken in microtube of IgG gel card followed by Addition of 50 μL of monoclonal anti IgG (ID Diaclon Anti-D). 2.This was followed by incubation at 37°C for 15 min and fixed centrifugation for 10 minute then Examined . Look for Agglutination .

16. Principle of Matrix Gel Card Method PRINCIPLE IgG antibodies are also known as incomplete antibodies. 1. These antibodies can sensitize red cells when allowed to incubate at TM 37°C in presence of corresponding antigen. 2. Matrix Anti-D IgG contains monoclonal Anti-D IgG in a standardized TM concentration. 3.Red blood cells when allowed to incubate with Matrix Anti-D IgG, red blood cells will get sensitized if Rho (D) TM antigen or weaker expression is present. 4.This reaction can be detected when Matrix gel card containing Coombs Anti-IgG is centrifuged under controlled conditions. 5.Cells sensitized with Anti-D IgG will be trapped within the gel matrix in presence of Coombs Anti-IgG. 6.The red blood cells, which do not react are not trapped in the gel column and get settled at the bottom of the microtube

17. Anti-D IgG for Weak D testing on Matrix Gel System SAMPLE COLLECTION: ( No special preparation of patient is required prior to sample collection by approved techniques ). For optimal results, freshly collected samples should be used. Anticoagulants like EDTA, CPD-A and Citrate can be used. Samples should be centrifuged at 1500g for 10 minutes to avoid fibrin residue which may interfere with results. SAMPLE PREPARATION for TM : ( Prepare 0.8% red blood cell suspension in Matrix Diluent - 2 LISS as follows ): 1. Bring the Matrix Diluent -2 LISS to room temperature before use. 2. Dispense 1.0 ml of Matrix Diluent -2 LISS into a clean test tube. 3. Add 10µl of packed red cells to Matrix Diluent -2 LISS collected in test tube and mix gently. 4. Red blood cell suspension so obtained should be used for testing. REAGENT: Matrix Anti-D IgG contains monoclonal Anti-D IgG (Clone- MCAD6).

18. Tulip Matrix Gel card Method TEST PROCEDURE TM : 1 . Label the appropriate microtube of the Matrix Coombs Anti-IgG card with patient's / donor’s name or identification number. Remove the aluminium foil of required number of microtubes carefully by pulling it backwards. 2. Pipette 50 ìl of 0.8% patient's / donor’s red blood cell suspension to the microtube, taking care to ensure that the micropipette tip does not touches the microtube. 3. Add 25ìl of Matrix Anti-D IgG to the above microtube. 4. Incubate the Matrix gel card for 15 minutes at 37°C in an incubator. 5. After incubation, centrifuge the Matrix gel card for 10 minutes in the gel card centrifuge. 6. Retrieve the card from centrifuge, read and record the results. INTERPRETATION OF RESULTS: Positive reaction : Agglutinated red blood cells forming a clear line at the top of the gel column or agglutinates dispersed in the gel column. Negative reaction : Non-agglutinated red blood cells settle at the bottom of the microtube forming a compact button.

19. Discrepancies Some other things to keep in mind when performing a weak D test include: A positive DAT result will invalidate the Du test.  Low red blood cell concentrations can make it difficult to visualize red blood cells, which can lead to weak positive results being missed.  Aged or hemolyzed blood may produce weaker reactions than fresh red blood cells.  Rouleaux caused by high concentrations of protein in serum or plasma can cause difficulties in interpreting the results.

20. Interpretation of Results 4+  Agglutinated red blood cells form a line at the top of the gel microtube. 3+  Most agglutinated red blood cells remain in the upper half of the microtube. 2+  Agglutinated red blood cells are observed throughout the length of the microtube. A small button of red blood cells may also be visible at the bottom of the gel microtube. 1+  Most agglutinated red blood cells remain in the lower half of the microtube. A button of cells may also be visible at the bottom of the gel microtube. ±  Most agglutinated red blood cells are in the lower third part of the gel microtube . Negative: All the red blood cells pass through and form a compact button at the bottom of the gel microtube. Mixed field agglutination : Agglutinated red blood cells form a line at the top of the gel and non-agglutinated red blood cells form a compact button at the bottom of the gel microtube. H  Hemolysis of red blood cells

21. ADDITIONAL REAGENT MATERIALS REQUIRED for Tulip Matrix Diluent-2 LISS for preparation of red cell suspension. Matrix Coombs Anti-IgG Card (Refer package insert before use). Gel card centrifuge (85g), Incubator (37°C), Work station, Micropipette capable of delivering 5-50µl of specimen and Bottle top dispenser.