Blood Bank Procedures

BLOOD BANK PROCEDURES ABO Blood Group System In 1900 karl landstainer discovered the blood groups ABO and classifieds human blood into A,B and O Groups. A fourth blood Group AB was discovered by landstainer’s associates. Von Decastello and sturli in 1902. The four groups are determined by the presence or absence of blood group antigens (agglutinations) on the red cells and accordingly an individual’s group A,B,AB, or O (O is donates the absence of A or B antigens). In addition, it has been shown that corresponding to antigens A and B, these are naturally occurring antibodies anti-A and B (agglutinations) in the plasma/serum of individually whose red cells lack the corresponding antigen. Group ‘A’ individually have anti-B, Group-B individually anti-A, Group ‘O’ Individually have both anti-A and anti-B and Group AB individually have no agglutination in the plasma/Serum. The A B O antigens and corresponding antibodies Antigen on RBC A B AB None

Antibody in Plasma/Serum anti-B anti-A Non anti-A and anti-B

Blood Group A B AB O

In 1991 von lungern Hirszfeid showed that Group ‘A’ could be divided into two principal sub Groups A1 and A2 on the basis of this the ABO system is classified into six main Groups A1,A2,B A1,B1, A2 B and O. ABH ANTIGENS: A, B and H antigens are present not only on the red cells but are also widely distributed through out the body tissues except in the central nervous system. A,B and H antigen city is determined by specific sugars linked to the terminal portion of oligosaccharides (short chain sugars) these are present on glycoproteins or glycolipids. In the red cell membrane both glycolipids and glycoprotein’s with ABH activity are present. In the plasma only glycolipids in soluble form are found. Cell membranes of endothelial and epithelial cells have both glycolipids and glycoproteins. Secretor Status A, B and H Substances are also found in the Secretions of 80 % of the population the ability to secrete B and H substances is determined by the presence of the secretor gene(se) in either the homozygous se se or heterozygous se se state, which is inherited independently of the ABO and Hh genes. Normally all secretors secrete H, in addition A and / or B sub stances.

Showing Sector Status Blood Group O A B AB Oh

Substance secreted H A&H B&H A,B&H Nil

SUB GROUPS OF A and AB: A and AB have divided into Subgroup A1, A2, A, B and A2 B depending up on the reaction with the extract of a lactin Dolichos biflorus seeds or human Anti-A1 serum. Anti-A sera very seldom differentiate between A1 and A2. both human anti-A1 and lactin anti-A1 agglutinate A1 and A1B cells but not A2 and A2 B cells. 20% of persons with A antigens in the A or AB group are A2 (or) A2 B. It is not necessary to classify group A patients or donors as A1 or A2 except when the individual serum contains anti-A1, anti-A1, occurs in the serum of 1-8% of A2 group person and 22-35% of A2 B groups person. Anti-A1 courses discrepancies between ABO cells and serum test and may also causes crossmatch incompatibilities but is considered clinically significant if it reacts at 37°C. Weak Subgroup of A: Sub group weaker than A2 occurs in frequently they are characterized by the declining number of A antigen sites or red cells and reciprocal increased in H reactivity. Weaker variants of A are mainly A3, Ag, Am and A. Intermediate. Classification of weak A subgroups based on. 1. Degree of agglutination by anti-A, Anti-A1 and Anti-AB 2. Degree of H reactivity on the red cells. 3. Presence or absence of anti-A1 in the serum. 4. Presence of A and H substance in saline of secretors. Serological Reactions of A and B phenotypes RBC Reactions of cells with Phenotype Know Anti Serum

A1 A2 A1 and 2 A3 Ag Am B B2 Bm

A +4 +4 +4 + 2mf -/w -/w -

Reactions of serum against RBC Saline of Secretors Contains

Saline of Secretors Contains

B -

AB +4 +4 +4 +2mg

H +,+2 +3 +3

A1 +4 +2 -

A1 -

+4 -/w -

+1/+2 -/w +4 -/+ -/w

+4 +4 +4 +4

-

+2 +4 +4 +4

-

+ to + 4 means = agglutination of increasing strength W means = weak agglutination - no agglutination Mf means = mixed field agglutination. Means = occurrence of anti-A1 is variable.

A2 -

B +4 +4 +4 +4

O -

A&H A&H A&H A&H

-/w +4 +4 +4

+4 +4 -

-

H A&H B&H H B&H

Weak Subgroups of B Sub groups of B are less common than subgroups of A and are only of theoretical value. They can be classified on the basis of the reactions shown in Table. Bombay Group or Oh Phenotype The phenotype (Oh) is characterized by the absence of A,B and H antigens on the red cells. The serum of these persons contains anti-A1, anti-B and anti-H, which reacts with all O blood groups, All these individuals are non-secretors of ABH. The red cells and secretions in Bombay group individuals track H as well as A and B substances, irrespective of ABO genotype, i.e. individual of Oh phenotype may have normal A/B genes but the corresponding antigens are not expressed on red cells. This type of blood was first found in Bombay hence its name. the frequency in India is around 1:7600. ANTIBODIES OF ABO SYSTEM Anti-A The antibody anti-A is found in group B and group O individuals and reacts well with A1 and A2 cells but not as well with weaker subgroups of A. Anti-B The antibody anti-B is found in group A and O individuals, and reacts almost with all B group cells but less effectively with weaker variants of B group. Anti-AB An antibody anti-AB is found in group O individuals. Serum from O group individuals is particularly useful in detecting some weak A and B antigens. Anti-A1 This is found in 1-8% of A2 and 22 to 35% of A2 B individuals. It is usually active at room temperature or below and is rarely clinically significant, except when it reacts at 37°C. Anti-H Anti-H very rarely occurs as cold reactive agglutinin in individuals with very low levels of H antigens on their cells and have little clinical significance. However, anti-H found in Bombay blood group (Oh) is an alloantibody and is clinically significant. It occurs as a heamolysis and agglutinates cells at 37 A B O Grouping: PRACTICAL ASPECTS OF ABO GROUPING (1) (2) (3) (4)

(5) (6) (7) (8) (9)

Routine ABO grouping must include both cells and serum testing as each test serves as check on the other. ABO grouping tests should be done at room temperature or lower; testing at 37°c weakens reaction. Anti-sera used in the ABO grouping must be used as per the instruction of manufacturer. Controls should always be run with respect to ABO grouping. Most laboratories have quality control of antisera once a day in order to eliminate the need to run individual controls every time the reagents are used. Tubes, slides or micro plates should be labeled properly. One should not rely on the colored dyes to identify the regent antiserum. Serum should always be added before adding cells and examine each tube after serum has been added to ensure that none has been missed. Tubes or slides should be clean and dry. Optical aid should be used to examine reactions that appear negative by the naked eye. Results should be recorded immediately after observation.

Techniques Blood Sample 1. Clearly labeled samples of clotted blood in sterile plain tubes are best for ABO and Rh tests. The sample can be stored at 4°Cand should be tested within 48 hours. No sign of heamolysis should be there. 2. If serum not completely separated or become clear, centrifuge the blood sample at 1000-3000 rpm for 3 min. 3. One to two milliliters of serum is pipetted into a pre-labeled tube (identification double checked) for serum grouping and other tests. 4. Take about 1 ml of cells into pre-labelled tube (identification double checked) and add 0.9% of normal saline and mix. Wash the cells thrice with normal saline and make 2-5% cell suspension in normal saline. ABO Typing Technique ABO typing should be performed using the manufacturer’s directions. There are three basic techniques: 1. Slide test 2. Tube sedimentation 3. Spin-tube. Slide testing It is used for emergency purpose ABO grouping for preliminary test. Slide test is not recommended as a routine test because it is not reliable for weakly reactive antigens on cells and for serum typing tests with low titre anti-A and anti-B. This is so because the slide test is less sensitive than the tube test. The other disadvantage is that drying of the reaction mixture can cause aggregation of cells that may be misinterpreted as agglutination. Method 1. The slide test may be performed on any non-porous clean surface, but it is usual to use glass or ceramic opaque tiles or microscopic slides. 2. Put one drop of anti-A serum and one drop of anti-B serum separately on the labeled plate or slide. 3. Add drop of about 20% red cell suspension to each drop of typing serum. 4. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide over an area of 15-mm-diameter. 5. Rock-rotate the slide or plate and leave the test for 2min at room temperature (20°-24°C). Then rock again and look for agglutination. 6. Record the results. Tube testing The tube technique is recommended because it is easy to perform and advantageous because the centrifugation involved enhances the reaction, allowing weaker antigens/antibodies to be detected and because the contents can be protected from drying and smaller amounts of reagents are required. Ideally cell typing and serum typing should be performed separately by different workers who check each other’s results by a ‘call-back procedure’. Tests in which discrepancies are identified, must be repeated. However, in smaller blood banks one trained worker is likely to perform all tests and discrepancies normally do not occur. Cell grouping Method 1. Prepare an appropriate 2-5% cell suspension in normal saline .It is advisable to wash the cells 23times in saline ,especially in case of cord blood or specimens in poor conditions

2. Set up three rows of clean test tubes and label them. Add two volumes (drops) of anti-A in the tube labeled A two volumes (drops) of anti-B in tube labeled B and two volumes (drops) of antiAB in tube labeled AB. 3. Add one volume (drop) of test cell suspension in each tube. 4. Mix the contents of each tube by gentle shaking and leave at room temperature (20°-24°C) for 30-60 min. in urgent cases, centrifuge the tubes at 1000 rpm for one minute after 5-10 min incubation at R.T. (spin tube method). 5. Observe the supernatant fluid for the presence of heamolysis against a well-lighted background. 6. Gently disperse the cell button and check for agglutination against a well-lighted background. 7. Where no agglutination is seen macroscopically examine the contents under microscope. 8. Record the results immediately. Table 4.5 Serum grouping Use a similar tube technique to test patients’/donors’ serum against saline suspensions of pooled 2-3 samples of group A cells, B cells and O cells. Method 1. Label three tubes A cell, B cell and O cell. 2. Place two volumes (drops) of the test serum in each tube. 3. Add one volume (drop) of A cells to tube labeled A, one volume (drop) of B cells to tube labeled B and one volume (drop) of O cells to tube labeled O. 4. Mix the contents of each tube by gentle shaking and leave at R.T. for 30-60 min. in urgent cases, centrifuge after 5-10 min incubation at R.T. (spin tube method). 5. Observe the supernatant fluid for the presence of heamolysis against well lighted background. 6. Gently disperse cell button and see for agglutination. 7. All negative results must be examined under microscope. 8. Record the results immediately. (Table 4.5) Table 4.5: Record of the results (ABO Grouping) Reaction of test red cells with Anti-A + + -

Anti-B + + -

Anti-AB + + + -

Reaction of serum with pooled cells A +/H +/H +/H

B +/H +/H +/H

O +

Interpretation (blood group of test cells) AB A B O Oh

+ = Agglutination; - = No agglutination H = Heamolysis Controls It is generally unnecessary to run controls each time ABO grouping is performed; however, reagent antisera and cells used should be controlled on a daily basis as a part of routine quality control programme. GRADING OF AGGLUTINATION REACTION + 4 Single clump of agglutination with no free cells. + 3 Three or four individual clumps with few free cells. + 2 Many fairly large clumps with many free cells. + 1 Fine granular appearance visually, but definite small clumps (10-15 cells) per low power field. W 2 to 3 cells sticking to gather per low power field, uneven distribution. - All cells are free.

+ H Heamolysis (partial or total) must be interpreted as positive. ABO sub grouping There are several sub groups of A, as already described but the main division is into A1, A2, A1 B and A2 B. Eighty per cent of group A and AB blood will be agglutinated by anti-A1 serum and are classified as A1 and A1B respectively. Those negative with anti-A1 are classified as A2, A2 B. Anti-A1 reagents can be obtained from three main sources: 1. Human anti-A1 (prepared by absorption of group B anti-A serum with A2 cells). 2. The lactin (plant agglutinin) Dolichos biflorus which is specific for A1 antigen. 3. Anti-A1 found as a naturally occurring antibody in the serum of A2 and A2 B individuals. Method 1. Place 1 volume each of Anti-A1 reagent into three clean test tubes. 2. To the first add 1 volume of 2% saline suspension of the donor’s / patient’s red cells. To the second tube add 1 volume of known A cells. To the third tube, add 1 volume of known A2 cells (A1 and A2 cells as positive and negative controls should always be included). 3. Mix the contents of each tube by gentle shaking and leave at room temperature for 30-60 min. In case of emergency centrifuge the tube after 5-10 min. at 1000 rpm for 1 min.] Read results and record. Testing for A1 and A2 Reaction of red cells with anti - sera Anti-A Anti-B Anti-AB Anti-A1 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 +4 -

Reaction of serum with cells A B +/H - or +/H* +/H - or +/H* -/H

interpretation O -

A1 A2* A1 B A2B*

+ to +4 = agglutination of increasing strength H = Heamolysis, - = No agglutination *Occasionally A2 and A2B serum contains anti-A1 and thus gives reaction with pooled A cells. Reading Gently shake the tube and examine. Those showing agglutination are labeled as A1 and A1B and those not showing agglutination as A2 and A2B. TITRATION OF ANTIBODIES-DOUBLE DILUTION STEP-I 1. 2. 3. 4.

Label a row of test tubes, according to the serum dilution, usually 1:1 through 1:152. Deliver 0.1 ml or 1 volume of saline into all tubes except the first tube. Add 0.1 ml or 1 volume of serum to tubes 1 and 2 (dilution 1:1 and 1:2). Mix the contents of tube 2 with a clean pipette and then transfer 0.1 ml or 1 volume from the mixture to tube 3 (1:4 dilution). 5. Continue the same technique, through all dilutions and remove 0.1 ml or 1 volume from the dilution tube with dilution of 1:152 and discard or save for further dilution if required. DILUTION METHOD 1. Arrange two sets of test tubes (10) in a test tube rack. 2. In the tubes add 0.1 ml of saline in to all (two sets) test tubes.

3. Add 0.1 ml of serum to test tube 1 and 2 (dilution 1:1 and 1:2). 4. Mix the content of tube 2 with a clean pipette and then transfer 0.1 ml or (1 volume) OD the mixture to tube 3 (1:4 dilution). 5. Continue the same technique through all dilutions and remove 0.1 ml from the dilution tube will dilution of 1:512 and discord or save for further dilution if required. STEP-II 6. Add 0.1 ml or 1 volume of 2-5% saline suspension of appropriate red cells to each tube. 7. Incubate in the appropriate manner according to the antibody being tested. In case of anti-A and anti-B, incubate at room temperature for 60 min. 8. Gently dislodge the red cells and observe macroscopically for agglutination. The agglutination titre is recorded as the reciprocal of the highest serum dilution in which there is (W) agglutination. Thus a serum which gives weak (W) agglutination at 1:256 is said to have a titre of 256. in describing the titre of the serum, it is usual to ignore the diluting effect of the cells suspension. AVIDITY Speed and strength of agglutination is termed as avidity. The test is done by mixing one drop of antiserum with one drop of 10% cell suspension on a slide or title and rocking gently at room temperature (RT). The time for a clearly visible reaction (+1) and then for strong (+4) reaction to occur is recorded with the help of a stopwatch. GROUPING OF CORD OR INFANT BLOOD Special precautions should be taken while testing cord or new-born infant blood since ABO antigens are not fully developed and allo-agglutinins are usually absent. Cord red blood cells should be washed 3-4 times with saline to minimize error due to wharton’s jelly. Serum grouping is not recommended in newborns. SOLVING PROBLEMS OF DISCREPANCIES Once a discrepancy is detected in ABO cells and serum grouping repeat the test before additional investigation as carried out. Quality assurances of reagents correct techniques. Care full observations and interpretation of results resolve many problems. Repeat Preliminary Procedures 1. Obtain a fresh blood sample from the donor unit of patient to rule out discrepancy due to contamination or un identification samples. 2. Wash the cells 3-4 times in normal saline to rule out rouleax formation and prepare 2-5% cell suspension. 3. Perform direct agglutination test on the cells are coated with antibody as in HDN and AIHA. 4. Rest the cells with fresh and potent anti-A, anti-B, anti-AB and anti-A, or anti-H as appropriate for individual problem. 5. Test the serum against appropriate A1, A2 and B cells. Group ‘O’ cells and autologous cells should be used as controls to detect allo-agglutinins and auto-agglutinins. Preferably pool the different cells sample, of A1, A2, B and O-group cells. This helps to confirm antibody specificity and levels little to chance. 6. Use group O cord cells if anti-1 is suspected 7. keep the tests at room temperature and at 4°C for 30-45 min and then interpret the results. Weak and negative reaction should be read microscopically.

Rh BLOOD GROUP SYSTEM REAGENTS FOR Rh GROUPING The different types of anti-Rh (D) sera available are: 1. Polyclonal human anti-Rh (D) serum. Potentiating substances such as albumin, enzyme, AHG serum are used to bring about agglutination with human IgG anti-D. 2. Anti-Rh (D) serum of slide test or rapid tube test (High protein anti sera). This contains macromolecular additives and gives rapid reliable results when used in accordance with manufactures directions. 3. Anti-D sera for saline tube test. The two kinds of saline active anti-Rh (D) sera available are: i) Traditional saline reagents are made from raw material containing predominantly IgM antibodies which agglutinate antigen positive cells suspended in saline. These are relatively scarce due to nonavailability of raw material. ii) Saline-active anti-Rh (D) sera prepared form IgG antibodies that have been chemically modified to convert them to agglutinate in saline medium. 4. Monoclonal antibodies The first blood group specific human monoclonal antibodies were produced from culture of lymphoblastoid cells produced by transformation of human B lymphocytes with Epstein Barr Virus (EBV). By taking lymphocytes from Rh-immunized donors both iGM and IgG antibodies have been obtained The method was not successful because many of cell lines die after a month Another approach has been made by fusing lymoblastoid cells (produced by EBV-transformed B lymphocytes) with mouse myeloma producing hetero-hybridoma. The heterohybridoma cells. In tissue culture, produce both IgM and IgG, approximately 20-50μg/l. for a period of 1 year. These antibodies are highly specific and react equally well at 20°C as well as at 37°C and are reliable for slide for rapid test tube technique. The same method has been used to produce anti-A, anti-B, anti-AB, etc. The types of monoclonal anti-D reagents are: 1. IgM anti-D monoclonal reagent 2. IgM and IgG anti-D monoclonal reagent 3. Blend of IgM anti-D monoclonal and IgG anti-D polyclonal (human anti-D) reagent. Rh Grouping Procedure Rh (D) typing should be performed according to the instructions given by the manufacturers of the anti-sera or as described below, There are three methods: 1. Slide test 2. Tube sedimentation 3. Spin tube The blood sample should be collected with or without anticoagulant in a sterile tube and stored at 4°C. it should be tested within 48 hours. No sign of heamolysis should be there. Slide Testing This may be used for emergency Rh (D) typing if centrifuge is not available. The slide test is not recommended for routine tests because is it not reliable especially for weakly reactive cells and it also has the disadvantage that driving of the reaction mixture can cause aggregation of the cells that may be misinterpreted as agglutination.

Method 1. Place one drop of reagent anti-Rh (D) on a labeled slide. 2. Place one drop of control medium or 22% albumin on another labeled slide. 3. Put one drop of 40-50% red cells suspended in plasma or serum on both the slides. 4. Mix the cell suspension and reagent, using a clean stick for each slide and spread the mixture evenly on the slide over an area of 15 mm diameter. 5. Place both slides on a view box surface (lighted), tilt gently and continuously for two minutes. Observe for agglutination. Interpretation A positive test has agglutination with anti-Rh (D) in the ‘test’ and smooth suspension of the cells in the control. A negative test has a smooth suspension of cells in both the ‘test’ and control. If there is agglutination in the control, the test results must be considered invalid and the test with saline-reacting anti-D must be performed. The interpreted result may be false positive in the following cases: i) Drying of reaction mixture may be confused with agglutination. ii) Small fibrin clots may give appearance of agglutination. iii) Incomplete Anticoagulated blood may clot on the heated slide. The interpreted results may be false negative in the following cases: i) Saline suspended cells react poorly or not at all. ii) A weak suspension of cells may agglutinate poorly. 40-50% cell suspension must be used. Whole blood from a severely anemic patient may be concentrated by centrifugation or by removing plasma or serum. iii) Weakly active cells, which normally take two full minutes to agglutinate, are read before time. iv) Reagents may be identified incorrectly resulting in the wrong reagent being used in place of anti-Rh (D). Tube testing Method 1. Place 1 drop of anti-Rh (D) serum in a tube labeled ‘test’. 2. Place 1 drop of control diluting reagent or 22% albumin in a tube labeled control. 3. Add 1 drop of 2-5% cell suspension in plasma or serum in each tube. 4. Mix well and keep at 37°C for one hour (sedimentation method). 5. Gently re-suspend the cell button and observe for agglutination. All negative results must be confirmed under microscope. Interpretation A) The interpreted result may be false positive in the following causes: i) The anti-Rh (D) used may contain antibodies of other specificities in addition to anti-Rh (D). Some workers routinely perform the test in duplicate using antisera from two different sources. ii) Immunological coating of the patient’s cells of factors in patient’s serum causing cellular aggregation can cause false agglutination in the control tube containing immunologic inert reagent. Serum factors can be washed by washing the cells in normal saline. If after washing the agglutination persists in control test, then it is most likely the cells are coated with immunoglobulin. In such cases the cells are tested with saline reactive antiserum. iii) Antisera and regents may be contaminated with bacteria, foreign substance or another antiserum. iv) Poly-agglutinable red cells may cause false agglutination with any reagent containing human serum because the antibodies anti-T and anti-Tn which agglutinate these surface altered cells are present in most adult human sera.

B) The interpreted results may be false negative in the following cases: i) ii) iii) iv)

Inadvertent failure to add the antiserum. Failure to properly identify reagents resulting in the wrong reagent being used in place of anti-Rh (D). Too heavy cell suspension in the tube test may result in poor agglutination. Cells possessing weak expression of the D antigen may not react well in immediate spin-tube method.

Anti-Rh (D) sera for saline test tube technique Saline agglutinating anti-Rh sera should be used when the control of high protein anti-D gives a positive test that persists even when washed red cells are used. Cells giving a positive direct antiglobulin test can usually be tested with saline-reacting anti-D serum. These sera are not suitable for the slide test. They are also not suitable for D* test as IgM antibody generally reacts poorly in the indirect antiglobulin test. Method 1. Place one drop of saline reactive anti-D in a properly labeled ‘test’ tube. 2. Add one drop 2-5% cell suspension of well-washed red cells with normal saline. 3. Mix gently and incubate at 37°C usually for 15-30 min. 4. Centrifuge usually 1000 rpm for 1 min. 5. Gently re-suspend the cell button and observe for agglutination. Negative result must be confirmed under microscope. Note: A saline suspension of known Rh (D) positive (O R1 R2) and Rh (D) negative (Orr) cells should be run parallel with the test as controls. Ensure that the concentration of cells in the controls is comparable to that of the test cells. Observe the controls before reading the test. Interpretation Agglutination in the test indicates that the red cells are Rh (D) positive. The result is valid only if the results of controls are satisfactory. MONOCLONAL ANTI-Rh (D) REAGENT These reagents work equally well at 20°-37°C and are reliable for emergency Rh (D) typing by slide or immediate spin tube technique besides the routine sedimentation tube technique. Methods Methods for Rh (D) typing with monoclonal regents are the same as described for anti-Rh (D) sera for slide or rapid tube test, or saline-reacting anti-D tube method. Controls i) ii)

A negative control if supplied with the test reagent. The controls for known Rh (D) positive cells (OR1 R2) and O Rh (D) negative cells (Orr) should be put in parallel with the test cells as in saline-reacting anti-D controls. Controls should be put at the end and read first to ensure that they work in the shortest incubation period compared to the test sample.]

Testing for D Cells of low-grade D possess the D antigen but expressed so weakly that they are not directly agglutinated by most anti-D sera D can be detected by antiglobulin test. (IAT)

All anti-D sera, especially anti-D sera for saline tube test and IgM monoclonal sera are not suitable for testing as IgM react poorly in the IAT. The manufacturer’s package insert will tell if the reagent can be used for D testing. Polyclonal anti-D (human IgG anti-D); IgM and IgG anti-D monoclonal reagent, and IgM anti-D (human IgG anti-D) can be used to detect D by IAT. Method 1. 2. 3. 4. 5. 6.

Take one drop of anti-Rh (D) serum in a clean labeled ‘test’ tube. Take one drop of appropriate control reagent in labeled tube. Add 2-5% of cell suspension to be tested to both the tubes. Mix and incubate both the tubes at 37°C for 15-30 min. Centrifuge at 1000 rpm for 1 min. Gently re suspend the cell button and examine for agglutination. If there is strong agglutination of cells in ‘test’ tube, then sample is Rh (D) positive and there is no need to proceed with antiglobulin phase of test. 7. If no agglutination or doubtful reaction observed, wash the cells 3-4 times with saline can decant the last washing. 8. Add 1-2 drops of anti-globulin reagents (Coombs serum). Mix gently and centrifuge at 1000 rpm for 1 min. 9. Resuspend the cell button gently and examine for agglutination and record the test result. 10. If the test is negative, the reaction may be confirmed by adding known IgG sensitized cells, re centrifuge and re-examine for agglutination. The presence of agglutination confirms the test result. Interpretation Agglutination in the ‘test’ tube and none in ‘negative control’ tube constitutes a positive test result and the blood is accordingly labeled D”. if the negative control test is positive no valid interpretation of D test is made. ANTIGLOBULIN (COOMBS) TEST & DIRECT ANTIGLOBULIN TEST (DAT/DCT) This test is used to demonstrate the coating (sensitization) of red cells in vitro with immune antibody (IgG) or the complement component (generally C3) in. 1. Diagnosis of haemolytic disease of new born (HDN) 2. Diagnosis of auto immune haemolytic anaemias (AIHA) 3. Investigation of drug induced red cell sensitization. 4. Investigation of haemolytic transfusion reaction. INDIRECT ANTIGLOBULIN TEST (IAT/ICT) This test is used to detect the presence of incomplete antibodies and complement binding antibodies in the serum after coating on red cells in vitro in. 1. Compatibility testing 2. Screening and detection of unexpected antibodies in serum. 3. Detection of red cell antigens not detected by other techniques. Control cells for AHG Tests Positive Control: Sensitized O Rh (D) Positive cells. Negative Control: Sensitized O Rh (D) Negative cells Un sensitized O Rh (D) Positive cells. A best general control for the DAT / DCT and IAT / ICT is the addition of IgG sensitized OR h (D) Positive cells to any AHG test that are non-reactive. A positive result indicates the following. 1. The AHG reagents has remained active and not been neutralized. 2. The red cells have been adequately washed. 3. The final volume of saline did not exclusively dilute the AHG serum. 4. AHG reagent was added to the test tubes.

Preparation of O Rh (D) Positive Sensitized cells Procedure 1. Select a dilution of anti-Rh (D) sera that costs the O Rh (D) Positive cells at 37°C in vitro but does not agglutinate them one has to determine by experience to what extent anti-D serum should be diluted to give sensitized cells (No agglutination). 2. Add 5% washed cell suspension of O Rh (D) positive cells equal to the volume of diluted anti-D serum. 3. Mix, incubate at 37°C for 30 minutes. 4. Look for the agglutination (if there is agglutination the procedure is repeated by taking more diluted anti-D serum). 5. If there is no agglutination wash the cells three times with a large volume of saline. Decant the supernatant saline completely every time. Make 5% suspension of sensitized cells in saline. 6. Add 1 drop AHG serum to 1 drop of the 5% washed sensitized cells. 7. Mix, spin immediately at 100 rpm for 1 min. 8. Cells should show +2 agglutination (if there is no agglutination the whole procedure in repeated by taking less diluted anti-D serum). DIRECT ANTIGLOBULIN TEST (DAT /DCT) Procedure 1. Place one drop of 2-5% cell suspension to be tested in a clean labeled tube. Note: The clotted sample should be as fresh as possible (not more than 24 hold) other wise the sample may be taken in EDTA to prevent the uptake of complement in vitro). 2. Wash the red cells 3-4 times in a large volume of saline care should be taken for adequate removal of the supernatant after each wash. Completely decant the final supernatant wash. 3. Add 1-2 drop of poly specific AHG serum immediately. 4. Mix centrifuge at 1000 rpm for 1 min. 5. Gently shake the tube to dislodge the cell button and examine for agglutination using an optical aid and record the result. 6. Leave an apparently non-reactive test tube at room temperature for 5 min. Centrifuge and read again. Though this step is optional all manufacturers recommend it when maximum sensitivity for complement or IgG is desired. The additional step should be never be substituted for the immediate reading because reaction due to IgG coating may become weaker after incubation. 7. Add one drop of IgG coated red cells to any test that is non-reactive mix, centrifuge at 1000 rpm for 1 min. look for agglutination if a negative result (no age) is obtained the test result is invalid and whole test should be repeated. INTERPRETATION DAT is positive when agglutination is observed either after immediate spin or after spin following RT incubation. DAT is negative when no agglutination is seen at either phase provided IgG coated red cells are added in step 7 of test. A negative DAT does not necessarily mean absence of coating globulin poly specific reagents detect approximately 500 molecules of IgG per red cell but auto-immune haemolytic anaemia has been reported with an IgA coating below this level. INDIRECT AGGLUTINATION TEST or INDIRECT COOMBS TEST Procedure 1. Place two or four drops of the test serum in a tube (sample should be fresh for detecting complement-binding antibodies otherwise fresh AB serum should be added to it).

2. Add one drop of 4-5% suspension of washed cells (i.e. donors / patients red cells or screening red cells etc) 3. Mix and incubate at 37°C for 30-60 min. 4. Centrifuge at 1000 rpm for 1 min 5. Examine for haemolysis or agglutination using an optical aid; record results. Agglutination at this stage indicates the presence of saline reacting antibodies. 6. If no agglutination is seen wash three or four times in large amount of saline Decant supernatant in each wash as completely as possible. 7. Add 1 – 2 drops of AHG serum. 8. Centrifuge at 1000 rpm for 1 min. 9. Gently shake the tube to dislodge the bottom and examine for agglutination using an optical aid. 10. Leave an apparently non-reactive test at RT for 5 min centrifuge and read again as in DAT. 11. Add 1 drop of IgG coated red cells to any test that is non-reactive. Mix, centrifuge at 1000 rpm for one minute. Look for agglutination if a negative result (no agglutination) is obtained the test result is in valid and the whole test should be repeated. Note: AUTO CONTROL SHOULD BE KEPT WITH THE IAT. Interpretation IAT is positive when agglutination is observed either after immediate spin or after spin following RT incubation. IAT is negative who no agglutination is seen at either phase, provided IgG coated cells are added in step 2 of IAT test. FACTORS AFFECTING THE SENSITY OF IAT Temperature Most IgG antibody and red cell reaction occurs optimally at 37°C. incubation at lower temperature decreases the rate of association between antigen and antibody, while incubation at higher temperature may damage red cells or antibody molecules Incubation time For saline or albumin techniques 30 min incubation at 37°C is adequate to detect most of the clinically significant coating antibodies. For some weak reactive in LISS medium incubation time is 10-15 min. SERUM RED CELL RATIO Increasing the ratio of serum to red cells increases the degree of antibody coating on red cells. A commonly used ratio is 2 drops of serum to 1 drop of 2-5% cell suspension. Equal volume of serum and 2% suspension of red cells in LISS should be used. Suspending Medium The sensitivity of IAT can be increased with the addition of 22% BOVINE ALBUMIN or ENZYME or LISS. BOVINE ALBUMIN (22%)-IAT / ICT Of increase the rate of the antigen-antibody reaction and reduces the incubation time with out effecting sensitivity for description of the action of bovine-albumin sec chapter on special method. Procedure A) One stage method-Additive method 1. Two drops of albumin (22%) are added in step 2 of saline-IAT 2. Mix and incubate for 20 min at 37°C 3. Proceed further as in saline-IAT

B) Two stage method-Layering method 1. First two steps are the same as in saline-IAT 2. Mix and incubate at 37°C for 30 min 3. Add one drop of 22% bovine albumin along the side of the tube 4. Incubate further for 15-20 min 5. Spin at 1000 rpm for minute and examine for agglutination / haemolysis 6. Proceed further as in saline-IAT step-6 This procedure should be attached in “9th page” ENZYME-IAT Enzymes after the configuration of erythrocyte surface and increase the mobility of antigen and clustering of the antigenic site, which increase the chance of effective antigen antibody collisions. Besides they reduce the negative charge on the red cell surface. For details see chapter on screening and identification for antibodies. Procedure Mostly one stage method with papain cystein is conducted for determining antibodies by enzyme-IAT. 1. Add one drop of papin cystein solution in step 2 of saline-IAT 2. Incubation at 37°C for 30 min 3. Proceed further as step 4 in saline-IAT Note: For the preparation of papain-cystein solution sec chapter on special methods. BOVINE ALBUMIN SERUM This used in blood group serology as (i) 22% concentration as enhancer at agglutination; and (ii) Concentration of 1.7% as a stabilizer in other reagents specially those to be stored at 4°C Methods A) One stage method (Albumin used as an additive) 1. Add 2-3 drops serum to a labeled tube 2. Add 1 drop of 2-4% red cell suspension in the tube 3. Add 2 drops of bovine albumin 22% 4. Mix and incubate at 37°C for 15-20 min 5. Centrifuge at 1000 rpm for 1-2 min 6. Gently re suspend the cell button and observe for agglutination / haemolysis. Note: 1. Manufacturers instructions should be followed. 2. Agglutination or haemolysis is a positive result B) Two stage method (Albumin layering) 1. Add 2-3 drops of serum to a labeled tube 2. Add 1 drop of 2-4% red cell suspension in the tube 3. Mix and incubate at 37°C for minutes 4. Centrifuge at 1000 rpm for 1-2 minutes. 5. With out disturbing the cell button allow 2 drops of 22% albumin to run down the in side of the tube albumin will form a layer on the top of top of the cells “Do not mix”. 6. Incubate of 37°C for 10-20 minutes 7. Gently re suspend the cell button and observe for agglutination / haemolysis. 8. Confirm all negative results under microscope. Note: 1. Manufacturers instructions should be followed. 2. Agglutination or haemolysis is a positive result.

Bovine Albumin Serum Stage-I Serum Cell suspension 2 – 4 % Bovine albumin 22%

1 – 2 drops 1 drop 2 drops

Mix and incubation 37°C 15-20 minutes and centrifuge at 1000 rpm for 1-2 min. Gently re suspends the cell button and observes agglutination. Control all negative result under m/s Stage-II Serum 2 – 3 drops 2 – 4 cell suspension 1 drop Mix at 37°C incubate for 30 min Centrifuge at 1000 rpm at 1 – 2 min Without disturbing allow two drops of 22% albumin bovine inside of the test tube. Incubate at 37°C for 10 – 20 min. Gently re suspend the cell button and observe agglutination / haemolysis. V. SCREENING AND IDENTIFICATION OF ANTIBODIES UNEXPECTED ANTIBODIES These antibodies are screened and detected in pre-transfusion testing of patient’s blood and during antenatal care in mother’s blood. If the unexpected antibodies are detected during screening test or compability tests the technologist has two choices; 1. To identify the antibody (antibodies) and select blood that lacks the corresponding antigens (s) for cross-matching. 2. To perform cross-matching using several other units an attempt to find units that fail to react with the recipient’s serum. These units are regarded as compatible and issued for transfusion. 3. These second course of action is much less desirable than the first and should be taken in emergency only. Screening of Antibodies The candidates of antibody screening are i) Patients requiring a transfusion. ii) Donors blood iii) Antenatal patients Patients requiring a transfusion Routing antibody screening in recipient blood sample will enable identifications of unexpected antibodies (ies) and to find compatible blood. Donors Blood Every donor’s blood should be screened for antibodies. The presence of antibodies in donor’s blood may cause a mild transfusion reaction of decrease of patient red cells. Antenatal patients All antenatal patients should be screened for a antibodies besides anti Rh (D), other antibodies also which could be problem for both the mother and the child. Early detection of an antibody will give time to the pediatrician and the blood bank to prepare for intrauterine or exchange transfusion in the baby or for an emergency transfusion to the mother.

All the screening cells are group ‘O’ it should be remembered that ABO in compabiilty will not be detected unless a cross-match is also carried out. Thus cross matching and antibody screening both are every necessary. SELECTION OF SCREENING CELLS For screening of antibodies a set of two specially selected group OR1 R1 red cells are used. These cells must carry the main antigens of Rh, kell, duffy, kidd, MNSs, P and Lewis (and Lutherian is possible) blood group systems. Many antibodies can be detected more casily if the antigens are homozygous on the red cells, particularly of common group. The presence of rare antigen should also be determined and noted. TYPICAL PANEL SCREENING CELLS Donor No 1 2

Geno Type R1 R1 R2 R2

Rh ( c DE e +-+-+ -+-+-

MNSS MNS s +++-+-

P P1 + +

Kell Kk ++ -+

Duffy Fya Fyb +++

Lewis Lea Leb -+ +-

Kidd jka jkb -+ +-

Lutheran Lua Lub -+ -+

To maintain the optimum strength of the antigens the two cells OR1 R1 and OR2 R2 should be used separately (and, of course, in conjunction with each other) and not pooled. This will increase the chance of detecting the weak antibodies. Cell panels are commercially available in developed countries; how ever they can also be prepared by the individual laboratory institutions that prefer to prepare their own panel, should determine the full phenotype of individuals on staff and bleed them regularly, or freeze large donations from these individuals in glycerol. For freezing or red cells in glycerol see chapter on storage and preservation of blood. Methods for Screening of antibodies Three techniques are commonly used: 1. Saline test at room temperature 2. Enzyme test – papain cystein technique 3. Indirect antiglobulin test. 1. Saline test 1. Set up 2 tubes of (size 10x75 mm) 2. Add 2 drops of patients serum to each tube 3. Add 1 drop of screening cells to each appropriate tube 4. Mix and incubate at RT (20-25°C) for 1 hour. 5. Shake the tubes gently and read microscopically negative readings should be checked microscopically. 6. Result should be scored and recorded. 7. Any haemolysis must be noted as this indicates and positive result. 2. Enzyme test Procedure 1. Set up 2 tubes. 2. Place 2 drops of patient’s serum in each tube. 3. Add one drop of papin cystein to each tube. 4. Add one drop of screening cells to each tube. 5. Mix well and incubate at 37°C for 1 hour. 6. Shake the tubes gently and read microscopically negative readings should be scored and recorded. 7. Result should be scored and recorded.

Note: Any haemolysis must be noted as this indicates agglutinations for preparation of papain cystein solution, see chapter on special methods. INDIRECT ANTIGLOBULIN TEST Procedure 1. Setup 2 tubes 2. Place 2-4 drops of patient’s serum in each tube. 3. Add 1 drop of screening cells to each tube. 4. Mix and incubate at 37°C for 30-60 min. 5. Examine for agglutination and for haemolysis and record the result. 6. If there is no agglutination or haemolysis. Wash the contents of both tubes 3 – 4 times in large volume of saline Decand each wash the completely as possible. 7. Add 1 – 2 drops of antiglobulin serum to each tube. 8. Mix well and centrifuge at 1000 rpm for 1 min. 9. Gently shake the tube to dislodge the cell button and examine for agglutination / haemolysis macroscopically negative readings should be checked microscopically. 10. Add 1 drop of IgG anti-D coated cells in non-reactive tests. Mix and centrifuge at 1000 rpm for 1 min and examine for agglutination. This acts as control for AGT. If a negative result no agglutination) is obtained the result is in valied and whole test should be repeated. For more details sec chapter on agglutination (Coombs) test ANTIBODY IDENTIFICATION Before beginning the antibody (ies) identification, it is use full to review the following. 1. Medical history-diagnosis. 2. History of transfusion or pregnancy. 3. Drug therapy (including Rh immunoglobulin) 4. Result of previous testing. Repeat Preliminary Test 1. ABO cells and serum grouping should be carried out using anti-A, anti-B, anti-AB; A, B, O red cells and A2 cells (identified). 2. If patients is group A perform A sub typing using anti-A (icctin) 3. Perform Rh (D) typing 4. direct AHG test (DAT) Selection of panel of cells These panel cells are commercially available though they can be prepared in the individual laboratory. These are usually8 – 10 groups ‘O’ cells from different donors. These cells must carry the maximum antigens of Rh, kell, Duffy, kidd, MNSs, Lewis, P and Lutheran blood group system namely. C, D, E, c, e, K, k, Fya Fyb, jka jkb, M,N,S, s, P, Lea Leb, Lua Lub ,

Method of antibody detection The serum should be tested against panel cells (8 – 10) patient’s own cell (auto control) and two cord cells. Three techniques are commonly used. 1. Saline test at room temperature

2. Enzyme test. 3. Indirect antiglobulin test. 1. Saline test Purpose: The saline test at RT is used to identify IgM agglutinations, namely anti-M, anti-N, anti-Lea, anti Leb, anti-I, anti-H, anti-P, etc. Procedure: 1. Setup the appropriate number of (10x75) test tube for panel cell, auto control, two cord cell) 2. Place 2 drops of patient’s serum in each tube. 3. Add 1 drop of Panel cell, patient’s cell suspension for auto control, two group O cord cell suspension [one Rh (D) + one Rh (D) - ] to each appropriate tubes. 4. Mix and incubate at RT (20-25°) for 1 hour 5. Shake the tube gently and read macroscopically negative results should be checked microscopically. 6. Result should be a cored and recorded. 7. Any haemolysis must be noticed as this indicates agglutinations many lewis antibodies produce haemolysis in saline when fresh serum is used. Note: The above procedure can be performed at a lower temperature (4°C) which tends to enhance the reaction of many cold antibodies. It auto agglutination is due to cold agglutinings, reaction will commonly be negative at 37°C. if the serum fails to react against the cord cells. There may be anti-I. ENZYME TEST Purpose: Enzyme tests are useful for the identification of warm reacting (IgG or complement binding) allo-and auto-antibodies. This technique serves to enhance the reaction of Rah Lewis and kidd antibodies. Enzymes may weaken or inactivate certain antigens like M.N.S. Fya and Fyb. Method 1. Set up appropriate number of 10x75 mm tubes. 2. Place 2 drops of patient’s serum in each tube. 3. Add 1 drop of papain cystein solution to each tube. 4. Add 1 drop of panel cell, patient’s cell suspension for the auto control and 2 group ‘O’ cord cell suspension (ORh (D) + ORh (D)-) to each appropriate tubes. 5. Mix well and incubate at 37°C for one hour. 6. Shake the tube gently and read macroscopically. Negative reading should be checked microscopically. 7. Result should be scored and recorded. Note: Any haemolysis must be noted as this indicates agglutination. INDIRECT ANTIGLOBULIN TEST Purpose: The indirect AGT is done to detect warm reacting IgG and complement binding allo-and autoantibodies. Method: Saline-AGT Procedure 1. Set up appropriate number of tubes. (for pond cells, auto control, 2 group ‘O’ cord cells) 2. Place 2 – 4 drops of patient’s serum in each tube. 3. Add one drop of panel cell, patient’s washed cell suspension for the auto-control and 2 group O cord washed cell suspension {O Rh (D) +, O Rh (D) -} to each appropriate tubes. 4. Mix and incubate at 37°C for 30-60 min. 5. Examine for agglutination and haemolysis record the result.

6. If there is no agglutination or haemolysis, wash the contents of each tube 3 to 4 times in large volume of normal saline; decant wash as completely as possible. 7. Add 1 – 2 drops of antiglobulin serum to each tube. 8. Mix well. 9. Centrifuge at 1000 rpm for 1 min. 10. Gently shake the tube to discord the cell button and examine for agglutination and / or haemolysis macroscopically negative reading should be checked microscopically. 11. Add 1 drop of IgG anti-D coated cells in non-reactive tests: Mix and centrifuge at 1000 rpm for 1 min and examine for agglutination. This acts as control for AGT. If negative result (no agglutination) is obtained the result is invalid and whole tests should be repeated. Note: For more defails see chapter on antigloblulin (Coomb’s) test. Interpretation of result To define the antibody (ies) that has been found. The result of the three techniques must be looked at individually and then as whole. By what techniques do reactions occur? For identification cross out all the antigenic determinates occurring on the panel cells that did not react with test serum beginning with the first cell and proceeding to the end of the panel see examples 9.1 and 9.2 Example – 9.1 Table.

Example 9.2 Table

VI COMPABILITY TESTING (CROSS MATCHING) Compability tests are done to ensure that particular unit of blood may be safety transfused to a patient. Normally group specific blood (ABO and Rh (D) group) as that of the patient is selected. However in certain situation because of the non-availability of group specific blood group O blood is selected for an A or B patient or A/B blood for an AB patient. SALINE TECHNIQUE FOR IgM COMPATIBILITY The recipient serum or plasma must be cross-matched with donor’s red cells before whole blood or red cells are used for transfusion this is major cross match must be done except when the demand is very urgent and there is no time for cross-matching. The donor’s red cells must be taken from the tube attached to the bag. The methods used must include those that will demonstrate IgM and IgG antibodies compatibility. Saline technique is designed to detect IgM antibody (ies) that react optimally RT (22°C) or lower in recipient’s serum against donor’s cells. This technique also serves to detect major ABO grouping error. Saline technique is in adequate as a compability test because clinically significant IgG antibodies are not detected by this method. Saline method demonstrate ABO compability

Method 1. Label 1 tube for each donor sample to be tested. 2. Put 2 drops of patient’s serum in labeled tube. 3. Add 1 drop of 2-4 drops saline suspended red cells of the donor to the tube. 4. Mix and incubate for 5-10 min (spin method) or incubate for 30-60 min (sedimentation method) at RT. 5. Centrifuge at 1000 rpm for 1 min in spin method centrifugation is optimal in sedimentation method. 6. Read the result observe for haemolysis and agglutination. Interpretation Agglutination or haemolysis indicates a positive result (incompatible) Note: Immediate spin test is acceptable but the incubation improves sensitivity of the test if the recipient has weakly reactive anti-A or anti-B or if the donor’s red cells have weak expressions of an antigen eg A2 B red cells. COMPATIBILITY TEST FOR IgG ANTIBODIES Anti-Human Globulin Test Indirect anti-human globulin test (IAT) is the most important and widely serologically procedure in modern blood banking to test the compatibility between recipients serum and donor’s cells. The majority of incomplete antibodies is IgG and is detected by AHG test. It a consistency high standard of performance of indirect antiglobulin test is not available in any laboratory, another technique (enzyme/albumin) capable of detecting antibodies reactive at 37°C may be in reported as an alternative.

Indirect anti-human globulin test (IAT) for IgG compabtibility Method 1. Put 2 drops of patient’s serum in a labeled tube 2. Add 1 drop of 2-4% saline suspended red cells of donor. 3. Incubate for 30 – 60 min at 37°C. 4. Centrifuge at 1000 rpm for 1 min. check for haemolysis/agglutination. 5. Wash the cells three times with normal saline. 6. Perform AHG test. 7. Add IgG coated red cells to negative AHG test. 8. Centrifuge and check for agglutination-if there is no agglutination tests in valid. For details see chapter on AHG test. Interpretation: Haemolysis or agglutination at any stage indicates in compability. ENZYME TECHNIQUE For method see chapter special methods ALBUMIN TECHNIQUE For method see chapter on special method. PROFORMA FOR CROSS MATCH 1. Patient’s ABO and Rh (D) Testing

Cell Grouping -A - A1 - B

- AB

Rh (D) test -D

Serum Grouping A cells B cells

ABO Group & Rh (D) O cells

2. Antibody screening on patient cell. 3. DAT in patient’s cells 4. Cross matching Routine test for cross match 1. Normal saline technique for IgM 2. IAT or enzyme / albumin technique for IgG. Donor’s Number

Saline test at RT

IAT or enzyme albumin test at 37°C

Compability Yes/No

Note: A positive result (agglutination and or haemolysis indicates in compatibility. LOW IONIC STRENGTH (Liss) for cross-match (15 min test) 1. Normal saline technique for IgM 2. Liss-IAT Donor’s No

Saline test at RT

Liss-IAT test at 37°C

Compatible (Yes/No)

Note: A positive result (agglutination and or haemolysis indicates in compability. Issue of blood 1. Cross match result should be sent along with the blood to be issued. The cross match report form must include. i) ii) iii) iv) v) vi) vii) viii)

Donor’s blood identification number Donor ABO and Rh (D) group Expiry date Patients name and identifications number etc. Patients ABO and Rh (D) Group Interpretation of cross matching test Date and time of issue Identification of the person performing the tests.

2. Label or tag is securely attached to the unit of blood it should contain: i) ii)

Donor’s blood identification number. Donor’s ABO and Rh (D) group

iii) iv) v)

Patients name and identification number Patients ABO and Rh (D) groups, and Interpretation of cross-matching test.

3. At the time of issue of blood observe the following: i) ii) iii)

Recheck the identification of the patient and donor blood. Check the blood expiry date to avoid issuing out dated blood. In spect the unit to make certain it does not have abnormal colour or appearance.

Note: If blood issued before compability test are completed label should clearly state uncross matched blood. VII. ADVERSE EFFECT OF BLOOD TRANSFUSION Adverse effects of Transfusion Immunological effects Immediate Effects Haemolysis with symptoms Febric non haemolytic reaction Anaphylaxis Urticaria Non cardiac pulmonary oedema

Usual Etiology Red cells incompability Antibody to leucocyte antigens Antibody to IgA Antibody to leucocytes or complement activation Antibody to leucocytes or complement activation

Delayed Effects Alloimmunisation of RBC, WBC. Platelets antigens or plasma proteins. Haemolysis

Usual Etiology Primary alloimmunisation due to exposure to antigens of donor origin. Anaemestic or secondary response of antibody to red cell antigens. Engraftment of transfused functional leucocytes. Development of antiplatelet antibody usually PL

Graft-vs-host discat Post transfusion purpura

NON IMMUNOLOGICAL EFFECTS Immediate Effects Fever with shock Congestive heart failure Haemolysis with symptoms

Delayed Effects Iron Hepatitis AIDS Protozoal infection Syphilis Mono nucleosis like effects

Usual Etiology Bacterial contamination Volume over load Physical destruction of blood due to freezing or overheating mixing non-isotonic solutions with red cells.

Usual Etiology Multiple transfusion Hepatitis B NANB rarely Hepatitis-A Retrovirus in donors blood (HIV) Staiamal parasites Treponoma polida Cytomegalo virus Epstein Barr virus (EBV) Immune complications of blood transfusion may be caused by red cell leucocyte or platelet incompability or allergic reactions to plasma components.

RED CELLS INCOMPATIBILITY The manifestations of red cell in compability include acute intravascular haemolysis, which is usuall, associated with in compability in ABO system and extravascular haemolysis, nearly always due to Rh incompability and sometimes due to K, K, S, Fya systems. In some cases extravascular haemolysis delayed for Up to two weeks or more after transfusion. Causes of haemolytic transfusion reactions 1. Clarical Errors i) in adequate or incorrect labeling of blood (pilot tube, blood container, recipient sample) ii) Confusion in the identity of patients at the time of collection of sample or at the time of transfusion. 2. Technical Errors i) Error in blood grouping and cross-matching. ii) Incompatibility not detected in cross-matching due to improper method. iii) Weak antibodies not detected by routine tests. iv) Destruction of recipient red cells. By the donor antibodies. It occurs when donor blood has antibodies against antigen (S) in recipient. It is not serious as donor antibodies are diluted. It occurs in the transfusion of group ‘O’ blood to group A1 group B (or) group AB recipients. This usually results from the indiscriminate use of group ‘O’ blood which has to been screened to ascertain the anti-A and anti-B titre and haemolysis. Signs and symptoms of haemolytic transfusion reaction There are 4 phases of reactions. All phases may not occur in patients. 1. SHOCK PHASE Fever, chill Byring sensation at the site of transfusion Pain in chest, lumbar region of back Haemorrhage Shock 2. POST SHOCK PHASE It the reaction is not checked there may be haemoglobinaemia Hyper bilirubinaemia Haemoglobinurea Disseminated intravascular co agglutination 3. ANURIC PHASE Renal failure Oliguria Anuria Uraemia 4. RECOVERY PHASE Patient phases large amount of urine. In the beginning urine is glomerular filtrate with a low concentration of urea. After some days the tubular functions return and diuresis may have effect on blood urea and creatinine. There may be excessive loss of potassium. In case of suspected transfusion reaction-the transfusional must take the following actions 1. Stop transfusion and inform the patient’s physicians. 2. Keep the intravenous line open with infusion of normal saline or other suitable intravenous infusion. 3. Recheck all labels, forms and identity of the patient to determine if the patient received the correct blood or component.

4. Send the fresh blood sample of the patient, care fully drawn to avoid mechanical haemolysis, to the having remaining blood or component, the administrative set attached and all related labels and form. 5. Preserve the urine passed after transfusion to check the presence of haemoglobin produced by the lysis of red cells. Laboratory investigations 1. Check identity of the patient donor blood and all relative and all relevant papers to ensure that there was no clerical error. 2. Compare the patients are and transfusion specimen for the colour of serum of plasma. i) Pink or red discolouration in post-transfusion sample indicates the presence of free haemoglobin due to the destruction of red cells. ii) Yellow or brown discolouration in samples drawn 4-10 hours after the transfusion indicates increased bilirubin. 3. Repeat ABO Rh (D) testing in the patients pre-and post transfusion blood sample, blood from the bag or from a segment of the table still attached to the unit, to check any error in ABO and Rh (D). 4. Direct antiglobulin test i) If direct AGT is negative the red cells are not coated with IgG antibody there is no in compability. ii) If antibody coated donor-in compatible cells are not immediately destroyed the direct AGT on the post reaction sample will be positive. iii) If the patients blood sample is drawn after several hours of the suspected reaction the antibody coated donor-in compatible cells are destroyed. The direct AGT will be negative. iv) In non-immunologic reactions the direct AGT will be negative. Interpretation of Laboratory Finding 1. If nothing abnormal is found in the above findings, it indicates that there has not been an acute haemolytic reaction. 2. If any findings is positive or doubtful or the patients clinical condition strongly suggest a haemolytic reaction the following investigations are warranted. i) Repeat the cross-match testing both pre and post transfusion samples of the patients against the sample of blood from the bag or segment of tube still attached to the unit by saline, enzymes albumin and indirect antiglobulin test techniques. ii) Repeat antibody screedning in patients pre and post transfusion samples and in blood from the bag or segment of tube still attached to the unit with OR1R1 and OR2 R2 screening cells by saline, enzyme and indirect AGT technique. iii) If irregular antibodies is detected on screening a) identify antibodies by panel of cells by saline enzymes and indirect AGT technique Autocontrol and two cord cells (one D Rh +, one D Rh - ) are also included. b) Identify the antigen (S) on donors red cells corresponding to the implicated antibody(ies) iv) In case of the multiple transfusion donors – donor compability is done. 3. IgA is tested in recipient blood in case of anaphylactic compability is done. 4. Bacteriological smear and culture of donor’s blood is done. VIII SCREENING FOR SUITABILITY OF TRANSFUSION The main diseases transmitted through blood are hepatitis, syphilis, malaria and AIDS and infrequently Brucellosis, Toxoplasmaosis and some other viral infections like CMV, EBV, Pollio, Herpes etc.

IX. SPECIAL METHODS INSTRUCTION FOR PREPARATION OF SOLUTION The definitions, calculations and methods given below are based upon elementary chemistry. DEFINITION • Mole, gram-molecular weight: weight in grams of a substance is equal to the molecular weight of a substance. • Molar solution: a one molar (IM) solution contains one mole of solute in a titre of solution. The solvent is generally distilled water unless other wise indicated. • Normal solution: a one normal (IM) solution contains one gram-equivalent weight of solute in a liter of solution. • Gram equivalent weight: weight in gram of substance which will produce or react with / more of hydrogen ion. • Percentage solution: the percentage of a solution gives the weight or volume of solute in 100 units of total solution. Weight/weight (w/w) giving gram of solute in 100 gm of solution Volume/volume (v/v) giving milliliters of solute in 100 ml of solution Weight/volume (w/v) giving grams of solution in 100 ml of solution Examples: Atomic weights (rounded to whole numbers) H=1; O=16; Na=23; P=31; S=32; Cl=35; K=39 Molecular weight 1) Na H2 Po4 = 23+2+31+64=120 2) KH2 PO4 = 39+2+31+64 = 136 Molar Solution: 1 M KH2 PO4 Molecular weight of KH2 PO4 = 39+2+31+64=136g Up to 100 ml of distilled water 2. 0.15m Na H2 PO4 2H2O Molecular weight of Na H2 PO4 2H2O = 23+2+31+64+36 = 156 g 0.15 Na H2 PO4 2H2O requires 0.15x156 = 23.4 g Of solution Na H2PO4 2H2O made up to 1000 ml of D/W. Normal Solution 1N NaoH Molecular weight of NaoH = 23+1+16 + 40 g 1N NaoH requires 1x40 = 40 g of solution (NaoH) Made up to 1000 ml of D/W. one mole of NaoH dissociates with one mole H; so grame equivalent weight and gram molecular weight are same. Percent Solution 0.9% of Nacl required 0.9 gm solute made up to 100 ml of D/W. Normal Saline Saline for use in blood group serology should have Nacl 9 gms/L D/W. Nacl-9 gms D/W mark to 1 liter pH 5.5 – 8.0 Phosphate buffer ISO osmatic Prepare two stock solutions

A) 0.15M NaH2PO4 2H2O – B) 0.15M Na2 HPo4

23.4 g/L 21.3 g/L

Prepare working buffer solutions of the desired pH by mixing appropriate volumes of the two solutions. A few examples are. pH 7.0 7.2 7.4 7.6 7.7

Solution-A 32 ml 24 ml 18 ml 13 ml 9.5 ml

Solution-B 68 ml 76 ml 82 ml 87 ml 90.5 ml

CYANMETHEMOGLOBIN (HICN) METHOD The basis of the method is dilution of blood in a solution containing potassium cyanide and potassium ferricyanide, Hb, Hi and Hbco are all converted to HICN (cyanmethaemoglobin). The absorbance of the solution is them measured in photoelectric calorimeter at a wave length of 540 nm ± 15 nm (or with a light green filter) Blood Sample 1. Fresh blood or free flowing capillary blood added to any solid anticoagulant (1 mg EDTA 1 ml) can be used. Measurement can be carried on blood which has been stored at 4°C. 2. Fresh capillary blood can also be used if added immediately to reagent solution. Reagent (Diluent) Modified Drabkin’s reagent Potassium ferricyanide – 200 mg Potassium cyanide – 50 mg Potassium dihydrogen phosphate – 140 mg Nomidet P40 (shell chemical co) 1 ml D/W – 1 liter Other non ionic detergents which can be used in place of nomidet include sterox SE (Harleco) 0.5 ml Triton X-100 (Rohm and Haas) 1ml and saponic 218 (alcoac Inc) 1 ml pH should be 7.0 – 7.4. The reagent should be clear and pale yellow in colour when measured as blank in a photometric calorimeter at a wave length of 540 nm, the absorbance must read zero. CYNAMETHAEMOGLOBIN (HICN) STANDARD SOLUTION Standard solutions are available commercially. The unused solution should be discarded at the end of the day on which the ampule is opened to avoid contamination. The HICN standard solution is used for direct comparison with blood which is also converted to HICN Method 1. Switch on the photoelectric calorimeter and wait for 15-20 min to warm up before use. 2. Add 20 µl of blood to 5 ml of diluent stopper the tube containing the solution and invert it several times. Allow to stand at RT for 5-10 min to ensure the completion of the reaction. This solution of HICN is ready to be compared with standard. 3. Select filter of wavelength 540 nm. 4. Set the colorimeter at zero against blank 5. Measure the absorbance value of the test solution prepared as in step 2. Interpretation

1. Record the absorbance value directly in the calorimeter calibrated for direct reading of Hb% (or mg/dl) 2. If the calorimeter is not meant for taking direct reading of haemoglobin g/dl record the absorbance reading and haemoglobin can be calculated from the following formula. g/dl of Hb = OD of the test / OD of the std x conc. of std g/dl Precautions 1. Blood should not be clotted. 2. The reagent should be discarded, if it becomes turbid. 3. The mixture of blood and reagent should be clear turbidity is due to contamination and give false result. 4. Pipette should be accurate to take 20 µl of blood. 5. Standard solution should be discarded at the end of day on which ampoule in opened. Bovine Albumin Serum This is used in blood group serology as i) 22% conc. as enhancer of agglutinations and ii) Conc. of 1-7 % as a stabilizer in other reagents. Especially those to be stored at 4° Use of Albumin in Blood Group Serology 1. Albumin increases the dielectric constant of the medium and thus reduces to zeta potential 2. Due to this effect the electrical repulsion between the red blood cells is less and cells agglutinate. 3. Now it is believed that the effect of albumin in enhancing the agglutination is actually due to the potentiating effect of the low ionic strength saline (LISS) in which albumin is dissolve (mostly 22% albumin is used in serology as 30 % albumin has the tendency to cause rouleax). 4. Albumin is used as an additive to the serum cell mixture or can be layered on the cell button. 5. The addition technique takes less time and is more frequently used but the layering method is more sensitive for detecting IgG antibodies. Methods A) One-Stage method (Albumin used as an additive) 1. Add 2-3 drops serum to a labeled tube 2. Add 1 drop of 2-4% red cell suspension in the tube 3. Add 2 drops of bovine albumin 22% 4. Mix and incubate at 37°C for 15-20 min 5. Centrifuge at 1000 rpm for 1-2 min 6. Gently re-suspend the cell button and observe for agglutination (or) haemolysis. 7. Confirm all negative results under microscope. Note: 1. Agglutination or haemolysis is a positive result 2. Manufactures instruction should be followed. B) Two stage method (Albumin layering) 1. Add 2-3 drops serum to a labeled tube. 2. Add 1 drop of 2-4 % red cells suspension in the tube 3. Mix and incubate at 37°C for 30 min. 4. Centrifuge at 1000 rpm for 1-2 min. 5. With out disturbing the cell button allow 2 drop of 22% albumin to down the inside of the tube. Albumin will form a layer on the top of the cells. Do not mix. 6. Incubate at 37°C for 10-20 min. 7. Gently re-suspend the cell button and observe for agglutination/haemolysis. 8. Confirm all negative results under microscope. Note: 1. Agglutination (or) haemolysis is positive result 2. Manufactures instructions should be followed.

ENZYMES The action of enzymes on red cells potentiates agglutination in at least 2 ways. First it removes. Sialic acid from the red cell surface and thus reduces the surface charge. (zeta potential) and allowing the cells. To cone close to one another second, enzyme may potentional the agglutination by removing structure which sterically interfere with the access of antibody molecule. The increased agglutinability of enzyme treated red cells is much more. With IgG antibodies than with IgM antibodies enzymes enhance, the activity at red cells with antibodies. Viz: Rh-=hr, kidd, lewis and P system certain antigen a re-destroyed or modified by enzymes viz M; N, Sa, and Duffy. Methods The 2 stage technique is used for detection and the one stage technique issued for detection and identification of IgG antibodies and for cross matching also the two stage techniques is more sensitive than the one stage technique. In one stage technique protealytic effect of enzymes inhibited by the serum the method in which one volume of serum is placed in a test tube one volume of enzyme solution (low) is layered carefully on top of the serum mixing between enzymes and serum. The tube one incubated for 1 hour and the cells then examined for agglutination. Two stage enzyme technique Preparation of enzyme solution a) Preparation of stock solution i) Papain is dissolve in 0.85% Normal saline. ii) Trypsin is dissolve in N/20 Hcl. iii) Ficin is dissolve in phosphate buffer saline pH 55. iv) Bromaline is dissolve in phosphate buffer saline pH 55 All stock solutions are prepared as 1% strength ie 1 gram is dissolved in 100 ml diluent. Preparation of working solution 1. Before use 1 volume of stock solution is diluted with 9 voloumes of diluent which in case of papain is sorensens phosphate buffer pH 7.3. Procedure (2-stage test using papain) 1. Prepare a papain solution as follows. a) Suspend 1 g of papain (BDH/merck 1:350) in 100 ml of 0.85% Nacl (this solution may be kept for several months at 4°C although this solution is best kept at-20°C). it is a stock solution. b) If desired this suspension can be centrifuge dafter storage for 24 hours at 4°C with occasional agglutination. The clear supernatant is slightly less active than the original suspension. c) For use 100 ml of the stock solution is added 109 volumes of M/15 phosphate buffer, pH 7.3 prepared by adding 3 volumes of M/15 Na2 HPO4 (9.46 g/l) to 1 volume of M/15 – M – KH2 PO4 (9.07 g/l) 2. a) b) c) d)

Pre treatment of red blood cells. Take 0.1 ml of 3 times washed cells in a test tube. Add 0.2 ml of papain working solution. Incubate at 37°C for 15-30 min. Make 2-4 % cells suspension in normal saline.

3. a) Take 2 volumes of serum in a test tube b) Add 1 volume of papainsed red cells c) Incubate at 37°C for 30 min. d) see for agglutination (Read microscopically). Note: Auto control should be kept.

One-stage Enzyme technique Preparation of Papain Cystein reagents Preparation of phosphate buffer (pH 6.2) i) Phosphate buffer stock solution M/15 Na2 HPO4, 2H2O 11.866 gm + 1000 ml D.W M/15 KH2 PO4 9.066 gm + 1000 ml D.W Filter and keep them separately in sterile condition in refrigeration. ii) Preparation of working solution (pH 6.2) at the time of preparing papain cystein reagent. Na2HPO4 KH2PO4 1 Volume 4 Volumes for pH 6.2 4 volumes 1 volume for pH 7.4 0.4 volume 9.6 volumes for pH 5.4 Preparation of papain cystein reagent 1. 10 grams of papain (BDH/Merck 1:350) is dissolving in 250 ml of phosphate. (pH 6.2) 2. Mix, grind if necessary. 3. Centrifuge the solution after 15 min. separate he supernatant and discard. Deposit or filter through whatman’s filter No.1 4. 4.85 g of Cystein Hcl (german) is added in 25 ml of phosphate buffer (pH 6.2). 5. Mix papin solution and cystein solution and add phosphate buffer (pH) to make the volume 1000 ml. 6. Incubate at 37°C for 1 hour. 7. Adjust pH to 6.2 if pH is less it can be increased. By adding 5 N NaoH (10 grams of NaoH in 50 ml H2O) if pH is more it is discarded. 8. Store in small aliquot – at 20°C. Methods 1. Take 1 volume serum in a test tube 2. Add 1 volume of papain cystein reagent. 3. Add 1 volume of 2% cell suspension 4. Incubate at 37°C for 1 hour 5. See for agglutination (read microscopically) Note: Auto control should be put. LOW IONIC STRENGTH SALT SOLUTION (LISS) In normal saline Na+ and cl- ions form ionic clouds around oppositely charged sites on antigen and antibody molecules respectively and partially neutrialized them. When the ionic strength of the reaction mixture is reduced the number of ions available to form ionic clouds around cells or protein molecules is decreated and they unit more effectively it result in an in-created at + reaction between the oppositely charged antibody molecules and red cells. Thus the rate and degree of antibody uptake is increased 2 to 4 folds in comparison with normal saline. However all antibodies are not equally response to LISS solution (anti-A and anti-B usually remain un effected. The effect of LISS solution 1. Reduces electro static carrier surrounding red cells and antibody molecules. 2. Increases the rate of antibody uptake 2 so 4 folds compared with normal saline and also increases the total amount of antibody taken up.

3. 4. 5. 6.

Titre of antibody increases. Detection of antibody increases. Detection of antibody with low equilibroium constant Incubation period is reduced.

Preparation of LISS Solution LISS Solution described by low and messeter consists of 0.17 M Saline - 180 ml 0.15 M Phosphate buffer pH 6.7 - 20 ml 0.3 M Sodium glycinate pH 6.7 - 800 ml The procedure of preparation of 1 liter of 0.03M.LISS solution is as follows. 1. 18 g of glycine is dissolve in about 500 ml of distilled water (sodium glycinate is not available commercially). 2. The pH is adjusted to 6.7 by dropwise addition of 1 N NaoH. 3. 20 ml of phosphate buffer (0.15) pH 6.7 as added to the glycine solution. 4. 1.79 g of Nacl dissolve in 100 ml of distilled water is added to the solution. 5. The solution is made up to 1 liter with distilled water, mix thoroughly 6. Adjust pH to 6.7 with 1N NaoH 7. Dispense into 100 ml amounts. It is sterilized by seitz filtration or autoclaving for storage at 4°C stored at 20°C to avoid pacterial growth. Alternatively a low conc. of sodium azide-0.1 g/l may be added. Quality Control A) Non-Serological 1. pH should be within the range 6.65-6.85 2. conductivity should be 3.6-3.7 mmol/cm at 23°C 3. Osmolarity 270-285 mmol. Serological A weak IgG anti-D (0.25 iu/ml) should give a +/++ reaction with R1 red cells by the routine LISS-ANG test. This examination should be carried out in paralled test using the previous batch of LISS. Method (LISS-IAT) 1. Wash the red cells twice in normal saline 2. Wash there cells one in LISS 3. Make 2-3% cell suspension LISS. 4. Take equal volume of serum and LISS suspended cell in a tube. 5. Incubate at 37°C for 15 min in routine and 5 min in emergency. 6. Centrifuge examines the supernatant for haemolysis resuspend the cells and observe for agglutination. Grade and record results. 7. Wash the cells 3 times in large amount of normal saline decant supernatant the each wash as completely as possible. 8. Add 1-2 drops of AHG serum 9. Centrifuge at 1000 rpm for 1 min (500 g for 1.5s) 10. Gently shake the tube to dislodge the solution N and examine for agglutination using optical aid and record the result. 11. Leave atapparently non-reactive test at room temp for 5 min centrifuge and read again as in DAT. 12. Add 1 drop of IgG coated red cells to any test that is non-reactive mix centrifuge at 1000 rpm for 1 min. look for agglutination if a negative result (no agglutination) is obtained the test result is invalid and few hole test should be repeated.

Interpretation It is same as in DAT or IAT Haemolysis or agglutination is a positive result. Precautions 1. For routine work LISS should be used at ambient temp as could LISS straight from 4°C storage increases un wanted cold antibody reaction. 2. Red cell should be washed 2 in normal saline to free there from serum before adding LISS to them traces of residual serum will results in non-specific uptake of auto logous serum complement 3. Equal volume of serum and LISS suspend 2-3% cell should be used. 4. The to be should be shake gently to see agglutination 5. Bottle of LISS in used should be discarded after 48 hours. Uses of LISS 1. Screening identification and quantitation of antibody. 2. It is useful in emergency cross-matching due to sort incubation time. 3. useful in electine surgery patient serum is kept after doing blood group 4. Antibody screen and cross matching is done on request at the time of operation. ALL-ANTIBODY ABSORPTION TECHNIQUE Application To remove an unwanted antibody from serum for the purpose of antibody identification or to prepare reagent antisera Method 1. Wash a large volume of red cells to be used in the absorption, 3 times in normal saline these cells must possess the antigen corresponding to the antibody that is to be absorbed. And shall lack the antigen corresponding to another antibody if any that is not to be absorbed shall lack the antigen corresponding to another antibody if any that is not to be absorbed i.e remain in the serum. 2. After the final wash centrifuge the cell so that they are tightly pached and removed as much of saline as possible. 3. Divided the cells into two aliquots. 4. Add serum equal to the volume to packed cells in one aliquot. 5. Incubate at the optional temp at which antibody to be absorbed will react to 30 – 60 mm mixing occasionally. 6. Centrifuge and recover the serum. 7. Test the serum to see that absorption is complete. If not repeat the procedure using another aliquot of packed red cells always test to ensure that the antibody that is to remain in the serum is sufficiently reach before continuing with repeat absorption. ABSORPTION AND ELUTION [for weak sub groups of A or B] 1. Wash 1 ml of cells to be tested at least 3 times with saline Discard the supernatant after last wash. 2. Add 1 ml of anti-A1 to red cells is weak variant of a is suspected or 1 ml of anti-B if weak variant of B is suspected. 3. Mix the cells with antisera and incubate at room temperature for one hour. 4. Centrifuge the mixture and discard the supernatant antisera. 5. Wash the remaining red cells for a minimum of 5 times with a large volume of salin (10 ml or more) save the supernatant of the fifth was to test for free antibody. 6. Add an equal volume of saline to the washed and packed cells and mix

7. Elute the absorbed antibody by placing the tube at 56°C water bath for ten min and mix the red cell saline mixture at least one during this period. 8. Centrifuge and remove the cherry colored elute and discard the cells. Testing of ELUTE 1. If anti-A was used, test the elute against three different samples A1 cells and 3 group O cells at room temp at 37°C and with antiglobulin serum. 2. If anti-B was used test the elute against 3 sample of B cells and 3 group fo O cells at room temp at 37°C and with antiglobulins serum. 3. Test the fifth saline was in the same manner to show that washing has removed all antibody not bound to the cells. Interpretation If the elute agglutinates or reacts with antiglobulin testing with specific A or B cells and does not react with O cells tested have active A or B antigen or their surface capable of binding with specific antibody. If the elute also reacts with O cells it indicates non-specific reactivity in elute and the results are not valid. If the fifth saline wash material is reactive with A and B cells the results of the test made on elute are not valid because it indicates that active antibody was present in the medium un attached to the cells being tested. Haemolysin test The haemolysin test detects incomplete IgG, ABO antibodies by their ability to haemolyse appropriate red cells in the presence of complement, it is not describe. To include substantial amount of such antibodies in grouping sera because they can complete with the complete (IgM) antibodies and may inhibit agglutination (prozon effect) Haemolysin test issued to determine the ability of anti-A and anti-B jin group O subjects to cause the haemoysin of cells and therefore to determine their safety when group O is transfused out cross matching or when group is give to an individual of an other blood group. Method 1. Place 2 drop of fresh serum not more than 24 hold in each of he tubes labeled A and B. if seru is un avoid by more than 24 hour old add an equal volume of fresh. Human AB serum free from lysine as a source of complement which is important to reaction. 2. Add 2 drops of fresh 2-5% saline suspension of washed A cells to tube A and 2 drops of B cells suspension to tube B. 3. Mix gently and incubate of 37°C for 2 hours 4. Centrifuge and examine the supernatant against a well lighted white back ground to detect haemolysis. 5. Score the degree of lysis according to the intensity of the supernant colour (pink ro red) and cell button size washed packed red cells. Note: use of weaker cell suspension or large amount of serum will increase incidence of haemolytic activity. Alternative Method 1 volume of 50% suspension of washed A1 or B cells in suspended in 9 volumes of fresh test anti-A or anti-AB sera the mixture is incubated at 37°C for 2 hours then centrifuge and the supernatant is inspected for haemolysis.