IMMUNOHEMATOLOGY LABORATORY LABORATORY METHODS FOR ABO BLOOD GROUP OUTLINE • The ABO Blood Group System o ABO Blood Group System o Nomenclature o ISBT • Introduction to ABO Forward Typing o Discussion o ABO Direct Blood Typing o Results & Post-Lab Discussion o Introduction to Blood Typing o Forward/Direct/Cell Grouping • Introduction to Reverse Blood Typing o Discussion o ABO Reverse Blood Typing o Results and Post-Lab Discussion o Reverse/Indirect/Backward or Serum Grouping • Determination of Secretor Status o Discussion o Secretor Status Determination o Interpretation of Results • Subgroup of A and AB Blood o Discussion o A & AB Subgroups o Interpretation of Results THE ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM • Most clinically significant blood group system in transfusion and transplantation therapy o Blood typing is required prior to transfusion & transplant; routinely tested o Small incompatibility in ABO blood group system may lead to immediate hemolytic transfusion reaction leading to severe rxn and eventual death of recipient • It is a unique blood group system because it is the only blood group system in which Ab is formed by the individual against Ag that is not found on the RBC. • The only system with reverse/backward typing GENETIC CONCEPT IN ABO INHERITANCE • ABO genes are located in long arm of chromosome 9 • Bernstein, 1924 o He explained the manner of inheritance of the ABO genes in the long are of chromosome 9 o 50% haplotype comes from the mother and the other 50% comes from the father. This manner of inheritance is called “codominance”. o Determine blood type of the offspring by ▪ Determining the parents’ blood type (homozygous or heterozygous) ▪ Use Punnett square ▪ Eg. Homozygous A & Heterozygous B A A B AB AB O AO AO • 50% probability of A; 50% probability of AB ▪ Eg. AB & O A B O AO BO O AO BO • 50% probability of A; 50% probability of B ▪ O is always expressed as homozygous o Mismatch in blood typing may be caused by infidelity & analytical errors LANDSTEINER’S LAW • Karl Landsteiner, 1901 • Established 3 laws for blood typing 1. The antigen on the RBC determines the blood type of the individual. (surface of RBC) 2. The corresponding antibody is never found on the individual’s serum. ▪ Ag + Ab = agglutination 3. The opposite antibody will always be present in the individual’s serum. • Blood type/Phenotype & Genotype Blood Type/Phenotype Genotype O OO A AA, AO B BB, BO AB AB Parent alleles A B O A AA “A” AB “AB” AO “A” B “AB” BB “B” BO “B” O AO “A” BO “B” OO “O” • Examples 1. Parent blood type OO x AO ▪ Possible blood type of children: 50% A; 50% O ▪ Exclusion: B, AB 2. Parent blood type: ▪ Possible blood type of children: ▪ Exclusion: NOMENCLATURE Landsteiner Jansky Moss O I IV A II II B III III AB IV I • Landsteiner - name is based on antigen present • Jansky & Moss - name is based roman numerals o Not routinely used INTERNATIONAL SOCIETY OF BLOOD TRANSFUSION (ISBT) • This system is created which aims to unify and standardize the manner of reporting of RBC Ag. Machine and IO readable system of reporting red cell Ag was used • ABO Blood Group system: 001:ABO o For the 4 major AG (New ISBT System) ▪ Blood type A = 001/ABO1 ▪ Blood type B = 002/ABO2 ▪ Blood type AB = 003/ABO3 ▪ Blood type O = 004/ABO4 • Numerical system of ISBT - composed of 6 digits o First 3 digits - ABO System; Last 3 digits - Ag ▪ A = 001001 ▪ B = 001002 ▪ AB = 001003 ▪ O = 001004
INTRODUCTION TO ABO FORWARD TYPING • Blood typing is a test that determines a person’s blood type. The test is essential if you need a blood transfusion or are planning to donate blood. Not all blood types are compatible, so it’s important to know your blood group. Receiving blood that’s incompatible with your blood type could trigger a dangerous immune response. DISCUSSION • Determination of blood groups is made with Anti-A, Anti-B, and Anti-1, b typing sera to demonstrate the presence of the antigen on the red cells. A check on this determination can be made by testing the serum for the presence of the specific antibodies with known red cells, B red cells, and O red cells. This test, referred to as reverse or serum grouping, is never used alone. • Blood grouping may be performed on the slide or in the tube using saline or serum suspensions of the red cells. However, the tube method is preferred over the slide method. Drying of the specimen is faster if slide method is used, and may be mistaken for false positive reaction. In addition, hemolysis is not detected in slide method. If the methods described below differ from the recommended procedures given by the manufacturer of the antiserum in use, the manufacturer’s instructions should be used. RESULTS AND POST LAB DISCUSSION RESULTS • How do you grade the agglutination reaction? Anti-A Antisera Anti-B Antisera Anti-AB Antisera Blood type “A” + - + Blood type “B” - + + Blood type “AB” + + + Blood type “O” - - - INTRODUCTION TO BLOOD TYPING • Blood typing o It is the process of testing of blood sample to determine an individual's blood group. o It uses commercially prepared antisera (forward) or known red cell suspension (reverse) ▪ Only ABO blood group system has reverse typing ▪ RCS is used for reverse typing • Rules in Blood Typing o The rules of specificity are applied o Rules of Specificity ▪ If the antigen is specific to the antibody, there is always a + reaction ▪ If the antigen is non-specific to the antibody, there is no reaction. • Methods of Blood Typing o Slide Method ▪ Used for rapid blood typing (tissue fluid affect agglutination rxn due to finger prick) ▪ Used for bed-side typing ▪ Only forward/direct blood typing is performed o Tube Method ▪ Routine blood typing ▪ Requires cell washing (3x) ▪ Both forward and reverse typing are performed ▪ Involves centrifugation FORWARD/DIRECT BLOOD TYPING /CELL GROUPING • The screening procedure • Principle : To detect unknown antigen using commercially prepared typing sera (contains antibody). • Reagent : Commercially prepared typing sera that has 1:256 potency level • Potency Level - relative strength of ab present in reagent. REAGENTS IN FORWARD BLOOD TYPING • Anti-A typing serum – detects A antigen o Components ▪ Blood type B serum - is the source of monoclonal anti-A antibody ▪ Coloring agent/dye – BLUE dye • Examples of blue coloring agents: Thymol blue, bromphenol blue, phenol blue patent blue, trypan blue ▪ 0.1 Sodium azide - used as a preservative in typing sera. It has antimicrobial properties that prevents the growth of molds and microorganisms in the typing serum. • Anti-B typing serum – detects B antigen o Components ▪ Blood type A serum - is the source of monoclonal anti-B antibody ▪ Coloring agent/dye – YELLOW dye • Examples of yellow coloring agents: Acriflavin yellow, tartrazine yellow ▪ 0.1 Sodium azide - used as a preservative in typing sera. It has antimicrobial properties that prevents the growth of molds and microorganisms in the typing serum. • Anti-AB typing serum – detects A and B antigens o 3 utilities ▪ Detects A and B antigens ▪ Also useful in ABO subgroupings. ▪ Used as control in forward typing o If used as a control: ▪ If Anti A and/or Anti B is positive= Anti AB should be positive ▪ If Anti A and/or Anti B is negative= Anti AB should be negative o Components ▪ “O” Serum - source of polyclonal anti-AB antibody ▪ 0.1 Sodium azide - used as a preservative in typing sera.
MATERIALS • 10 or 12 x 75 mm test tubes • Slides • Droppers • Serofuge • Applicator sticks • Anti-A typing sera • Anti-B typing sera • Anti-A,B typing sera • Saline solution • Expired blood (washed RBC) PROCEDURE • Test tube method : use of 10 or 12 x 75 mm tubes is recommended. 1. Prepare a 2 to 5 percent saline suspension of red cells to be tested. 2. Place 1 drop of Anti-A typing serum in a properly indentified tube. Place 1 drop of Anti-B typing sera in a properly identified second tube. Place 1 drop of anti-A,B typing sera in properly identified third tube.* Always label the test tubes 3. Add 1 drop of the cell suspension to each tube. 4. Mix by shaking gently. The tube may be left at the room temperature for five minutes before centrifugation or may be spun at once 1000-2000 r.p.m. for 1 minute. As an alternative to centrifugation, sedimented cells may be read after standing one hour at room temperature, but this is less sensitive. Tube must be observed in proper lighting and white bg. 5. Gently dislodge cell button and inspect macroscopically using an optical aid or place a drop of the solution on a slide and read under the low power of the microscope. o *This is used to confirm group O blood and to identify rare subgroups of A. This antiserum will agglutinate cells of groups A, B and AB, but not group O. It will also detect the weakly reacting subgroups of A. • Slide Method . Use a flat slide and red cells from clotted or anti coagulated blood. 1. Prepare a 10% saline suspension of RBCs to be tested. 2. Add 1 drop of anti – A typing sera to left side of a slide; add 1 drop of anti-B to the right side half of slide. 3. Add 2 drops of the cell suspension to each half of the slide. Mix cells and antiserum with an applicator stick over an area of about 1 inch in diameter. A separate clean toothpick or wood applicator stick must be used for mixing the contents of each area. 4. Rotate or tilt the slides and observe for macroscopic agglutination. Agglutination will begin within a few seconds. The maximum time for completion of agglutination is about two minutes. 5. Do not place slide on a heated viewing stage or box for observation, since agglutination may be weakened at temperatures above 22°C. RESULTS Anti-A Antisera Anti-B Antisera Anti-AB Antisera Blood type “A” + - + Blood type “B” - + + Blood type “AB” + + + Blood type “O” - - - INTRODUCTION TO REVERSE BLOOD TYPING DISCUSSION • This method is frequently used as a check on the accuracy of the blood grouping performed on the red cells. The test is performed by examining the serum of the blood under test against known A ₁ red cells, B red cells, and O red cells, for the presence of anti-A and or anti-B. The principle follows that whatever a blood group antigen is present on the red cell, the opposite antibody is present on the serum. • This method suffers from certain deficiencies: the anti-A and the anti-B antibodies may be so weak as to be virtually undetected; certain typical antibodies may be present capable of reacting with antigens other that A or B, hence confusing the issue; also, some A ₂ or weaker subgroups of A have anti A ₁ in the serum and thus may react as group O in both direct and reverse grouping. • Reverse grouping should be performed in the test tube only. Group A ₁ (or a pool of five unselected group A) B cells and O cells should be washed and prepared fresh daily as 2-5% suspension in saline. ABO REVERSE BLOOD TYPING: SERUM GROUPING PRINCIPLE • To detect unknown antibodies in the patient’s serum using laboratory prepared known red cell suspension MATERIALS • 10 or 12 x 75 mm test tubes • Droppers • Serofuge • Applicator sticks or Toothpicks • O cells • A ₁ cells • B cells • Saline solution • Expired blood
• Patient Sample : Serum (It is preferred over plasma because plasma contains fibrinogen that may cause spontaneous in vitro agglutination.) • Known Reagent : Laboratory Prepared Red Cell Suspension RESULTS AND POST-LAB DISCUSSION INTERPRETATION OF RESULTS A 1 Cells Unknown Serum Tested Against ABO Group Antibodies in Serum B cells O cells + + - O Anti-A, B - + - A Anti-B + - - B Anti-A - - - AB None REVERSE/INDIRECT/BACKWARD OR SERUM GROUPING • Confirmatory Procedure • Principle : To detect unknown antibodies in the patient’s serum using laboratory prepared known red cell suspension REAGENTS/SAMPLES • Sample : Patient’s serum o Preferred over plasma fibrinogen of plasma can cause spontaneous in vitro agglutination (false positive) • Reagent : Laboratory prepared known RCS (2-5%) volume by volume concentration of RCS o In practice, RCS prepared can only be stored for up to 24 hours (Refrigerator temperature) o Prepared daily • Known A RCS - detects anti-A • Known B RCS - detects anti B • Known O - Detects anti-H o Also used as control in reverse typing o Known O should always having negative reaction o Positive Known O denotes Bombay phenotype • • *Known AB is not essential/prepared in the lab o AB - universal recipient PROCEDURE • Test Tube Method 1. Prepare 2% suspensions of A ₁ red cells, B red cells and O red cells in saline. 2. Place 2 drops of serum to be tested into each of three tubes identified by specimen and labeled “A”,”B” & ”O”. 3. Add 1 drop of the suspension of A cells to tube “A”, 1 drop of the suspension of B cells to tube “B” and 1 drop of suspension of O cells to tube “O”. 4. Mix by shaking gently. The tubes may be left at room temperature for five minutes before centrifugation, or spin at once at 1000-2000 r.p.m. for 1 minute. 5. Observe the supernate for hemolysis* against a white background. 6. Gently dislodge the cell button and inspect for agglutination with the aid of an optical device or place a drop of the solution on a slide and read under the low power of the microscope. o Weak reactions will be enhanced if the tubes are left for more than five minutes room temperature before centrifugation. Avoid excessive shaking which may disperse weak agglutination. o *Hemolysis should be considered a positive test. Certain group O donors may have anti-A hemolysis, which is usually evidence of “immune” anti-A. The serum should NEVER be inactivated to circumvent this problem, unless only that portion to be used in this procedure is removed and heated at 56°C. RESULTS A RCS B RCS O RCS “A” - + - “B” + - - “AB” - - - “O” + + - DETERMINATION OF SECRETOR STATUS DISCUSSION • Blood type isn’t just in blood, it’s also in saliva, sweat, tears, seme, vaginal secretions, mucus, digestion, breast milk o Regulator genes ▪ Sese - regulates formation of ABH genes in other bodily fluids (except CSF due to BBB) ▪ Zz genes - regulates formation ABH genes in RBC • To determine the secretor status through testing the saliva and to identify the soluble substance/s that is/are present in the saliva by applying the principle of neutralization and hemagglutination-inhibition reaction (+) • Secretors are defined as individuals with blood group soluble substances namely A,B, and H in secretions such as saliva. • For a person to be considered a secretor, he must inherit the Secretor gene (SeSE or Sese) and H gene (HH or Hh). • The water-soluble ABH substances (glycoproteins in nature) has the capacity to neutralize or prevent their corresponding agglutinins to react or agglutinate to its corresponding erythrocyte antigens. This reaction is termed as neutralization or hemagglutination-inhibition reaction. SECRETOR STATUS DETERMINATION MATERIALS • 10 or 12 x 75 mm test tubes • Droppers • Serofuge • Saline solution • Test tube rack • Hot plate • Beaker • Nescofilm REAGENTS/SAMPLES • 5% red cell suspension of known A,B and O cells • Unknown saliva samples • Anti-A serum ( 1:4 dilution ) 1 mL anti A + 3 mL NSS • Anti-B serum ( 1:4 dilution ) 1 mL anti B + 3 mL NSS • Anti-H lectin from Ulex europaeus ( 1:4 dilution ) 1 mL anti-H + 3 mL NSS • Normal Saline Solution (0.85-0.9% NaCl) in wash bottle
PROCEDURE • Collection and Preparation of Saliva 1. Collect approximately 2mL of saliva in a small beaker and transfer it into a test tube. Be sure to cover it with nescofilm. 2. Place tube with saliva in a boiling water for 10 mins. 3. Centrifuge at 3,400 rpm for 10 minutes. 4. Transfer the clear or slightly opalescent supernatant into a clean test tube. Discard any opaque or semi-solid material. Make a 1:2 dilution ( 1mL clear supernatant + 1mL NSS ) of the collected supernatant with normal saline solution. • Test for Secretor Status 1. Prepare 1:4 dilution of Anti-A, Anti-B and Anti-H antisera. 2. Prepare 5% suspension of A,B and O cells. O cells are used because it has the greatest amount of H antigen. 3. Prepare 3 test tubes and label as A,B, and H. 4. Add 1 drop of diluted saliva to the 3 test tubes. A 1 drop of diluted Anti-A to tube A, 1 drop of diluted Anti-B to tube B, and 1 drop of diluetd Anti-H to tube H. 5. Mix and cover with nescofilm. Incubate all tubes at room temperature for 10 minutes. 6. Add 1 drop of 5% Known A cells to tube A, 1 drop of 5% Known B cells to tube B and 1 drop of 5% Known O cells to tube H. 7. Mix and cover with nescofilm. Incubate at room temperature for 30-60 minutes or for 15 minutes in a 37oC water bath. 8. Centrifuge for 15 seconds at 3,400 rpm. 9. Gently dislodge the cell button and examine for agglutination. 10. Grade each reaction and record the results. INTERPRETATION OF RESULTS POSSIBLE RESULTS Blood Group A-Tube B-Tube H-Tube Soluble substances detected A 0 + 0 A, H B + 0 0 B, H AB 0 0 0 A, B, H O + + 0 H • Blood group - secretor • A-Tube - known A-cells • B-Tube - known B-cells • H-Tube - known O-cells INTERPRETATION • Non-secretor : agglutination of red cells by antiserum- saliva mixture • Secretor : No agglutination of red cells by antiserum and saliva mixture. The antiserum has been neutralized by the soluble blood group substances or antigens in the saliva, which react with their corresponding antibody. Therefore, no antibody sites in the antisera are free to react with the antigens on the reagent red cells used in the testing. This negative reaction is a positive test for the presence of ABH soluble antigens and indicates that the individual is a secretor. • TEST FOR ABH SOLUBLE SUSBTANCES SALIVA NEUTRALIZATION TEST • Principle: hemagglutination inhibition o (+) = absence of agglutination o (-) = presence of agglutination • 2-Step Procedure o Materials ▪ Saliva ▪ Known RCS ▪ Antisera / Lectin o Step 1 : Saliva + Source of Antibody (antisera/lectin) o Step 2 : Step 1 Mixture + Known RCS o In step 1, if there is neutralization, agglutination will be inhibited in step 2 • Protocols in Saliva Neutralization Test o 2mL of saliva ▪ Check saliva for presence of blood • Presence of blood may affect results ▪ Filter saliva before use to remove impurities ▪ Saliva must be heated at 56C for 10 mins (to inactivate enzymes - salivary amylase) • Clear sample: complete heating • Hazy: incomplete heating; extend heating o Saliva must be diluted using NSS to reduce non-ABH glycoproteins present in the sample which may cause postzone phenomenon (excess antigen) ▪ Add 1 mL of NSS to 1mL of saliva o Antisera must also be diluted using NSS • Results Step 1 Step 2 Reaction Soluble Substance Present Saliva + Anti A Known A RCS + Step 1 Mixture No agglutination A substance in saliva Saliva + Anti B Known B RCS + Step 1 Mixture No agglutination B substance in saliva Saliva + Anti H Known O RCS + Step 1 Mixture No agglutination H substance in saliva o A substance in saliva - no agglutination occurred due to neutralization of anti A by saliva in step 1 o E.g. Saliva + Antisera A cell B cell O cell Anti A 4+ 0 0 Anti B 0 0 0 Anti H SUBGROUP OF A AND AB BLOOD DISCUSSION • A distinction can be made between A ₁ cells and those of the weaker subgroups of A by the use of an absorbed anti-A (anti-A ₁ ) serum. Anti-A found in group B sera consists of two antibodies, designed as anti-A and anti-A ₁ which are separable in the laboratory by the addition of appropriate A cells. For example, when A ₁ cells are added, both anti-A and anti-A ₁ are removed from the serum; however, when A ₂ cells are added, only anti-A is removed and the specific anti-A ₁ remains. It is in this manner that the commercial reagent, Absorbed anti-A (anti-A ₁ ) Serum is prepared, which detects only those cells of subgroups A ₁ or A ₁ B but will not react with cells of subgroup A ₂ , A ₂ B, or weaker.
• Two anti-A ₁ reagents are available: (1) anti-A (from a group B subject) absorbed with A ₂ cells so that the absorbed serum has anti-A ₁ specify; or (2) an extract (“Lectin”) made from the seeds of dolichos biflorus prepared and diluted so that it agglutinates A ₁ or A ₁ B red cells, but not A ₂ , A ₂ B or other weaker subgroups. The commercially obtained Dolichos extract is ready to use as anti-A ₁ and needs no further dilution. • Since some anti-A ₁ reagents are standardized for use in slide tests and others for tube tests, substitution of methods is not always possible, and the manufacturer’s direction must be followed. Samples of A ₁ and A ₂ red cells are advisable as controls. Recommended test times should not be exceeded, since many anti-A ₁ reagents will weakly agglutinate A ₂ cells if the tests are left long enough. • Dolichos biflorus lectin / Anti-A lectin - used to differentiate A1 & A2 subgroup of A A & AB BLOOD SUBGROUPS MATERIALS • 10 or 12 x 75 mm test tubes • Droppers • Serofuge • Saline solution • Absorbed anti-A (anti-A ₁ ) serum • Expired blood (type A or type AB) PROCEDURES • Test Tube Method 1. Prepare a 2% suspension of red cells to be tested in saline. 2. Add 1 drop of absorbed anti-A (anti-A ₁ ) serum to small test tube (10 x 75 mm). 3. Add 2 drops of the 2% cell suspension. 4. Incubate at room temperature no longer than 15 minutes, or centrifuge immediately at 1000-2000 rpm. for one minute. 5. Examine agglutination. • Slide Test Method 1. Prepare a 10% suspension of red cells to be tested in saline. 2. Add 1 drop of absorbed anti-A serum to slide. 3. Add 1 drop of the 10% cell suspension. 4. Mix well and tilt slide back and forth. Do not place slide directly in viewbox, since agglutination may be weakened at temperatures above 22°C. 5. Examine for agglutination. Do not observe longer than two minutes. o NOTE: Absorbed anti-A (anti-A ₁ ) serum is prepared by absorption so that specific anti-A ₁ remains. Thus, the serum will react with A ₁ but not A ₂ cells or weaker. INTERPRETATION OF RESULTS Cells Anti-A Serum Absorbed anti-A (anti-A 1 ) serum (Dolichos biflorus) A 1 + + A intermediate + + / - (variable) A 2 +* - • * weaker than A 1 • Confirm A subgroups by using Anti-A/Dolichos biflorus lectin