LAB- ABO Grouping

Purpose: The purpose of this lab is to introduce the students to determining the ABO group of a patient by performing a forward and reverse type on the patients specimen. The student will interpret results and if there is a discrepancy, further testing shall be performed to determine the patients ABO blood type.

Principle: Forward typing- Using the antisera anti-A and anti-B, one can determine which of the ABO antigens is (are) present on the red cells. An antibody will bind to its specific antigen creating agglutination. Thus, anti-A will bind to the A antigen and anti-B will bind to the B antigen. A person who is type O will not have either A or B antigens and an AB patient will have both A and B antigens. Agglutination with antisera is a positive result indicating that specific antigen is present. Lack of agglutination is a negative result indicating that antigen is absent or not detectable. Reverse typing- Landsteiners Law states that a person can only make an antibody to an antigen he or she lacks. ABO antibodies are naturally occuring, meaning a person produces them shortly after birth with no known stimulus. Using this information, a blood bank technologist can use A1 cells and B cells with a patients plasma to determine if anti-A and/or anti-B is present.

Specimen Requirements: EDTA (purple tops) are preferred. Heparin, ACD, CPD, CPDA-1, CP2D or whole blood without anticoagulant are also acceptable. NO samples in gel separator tubes.

Supplies: Anti-A Anti-B A1 cells B cells Centrifuge agglutination viewer test tube rack test tubes transfer pipettes saline

Quality Control: Every reagent that is used must be QCd for each day of use. QC usually includes running a positive control and a negative control to determine the reagent(s) is(are) ready to use.

Directions: Record reagent lot numbers and expiration dates on worksheet. Tell whether each reagent is expired or in-date. Perform QC and check for reagent hemolysis/contamination. Record results on worksheet. Perform an ABO on all specimens. Ensure that the forward and reverse types agree for each specimen. Record results on daily blood bank worksheet. If a discrepency is present, try to resolve it. If you have done all you can procedure-wise, explain what further steps you would do in an actual blood bank lab.

Procedure: For QC: 1. Obtain 4 test tubes and label one with: a. Anti-A with A cells b. Anti-A with B cells c. Anti-B with A cells d. Anti-B with B cells 2. Add 2 drops of the appropriate antisera to its respective tubes. 3. Add 1 drop of the appropriate 2-4% red cell suspension to its respective tubes. 4. Mix well and spin for 30 seconds. 5. Read and record results. If reagents test correctly (pass QC), proceed to the rest of the procedure. If a reagent fails QC, repeat the QC procedure for that reagent. Sample Testing: 1. Obtain samples for ABO testing. 2. Label 2 test tubes for each sample. Both tubes should have sample information with one tube labeled plasma and the other labeled RBCs. 3. Spin the sample for 3 minutes to separate the RBCs from the plasma. 4. After centrifugation, separate the plasma and RBCs into their respective tubes. 5. Make a 3-5% red cell suspension of each sample. a. Add a small aliquot of packed RBCs to a newly labeled tube. b. Add enough saline to make a 3-5% suspension. c. Compare the suspension to a bottle of A or B cells. 2

6. For each sample, label 4 test tubes: a. Anti-A b. Anti-B c. A cells d. B cells 7. Add 2 drops of appropriate antisera/plasma to each tube. Plasma will be added to the A and B cell tubes. 8. Add 1 drop of appropriate RBC suspension to each tube. Patient RBCs will be added to the anti-A and anti-B tubes. 9. Mix all tubes well and spin for 30 seconds. 10. Read and record results. Interpret all results and make sure the forward and reverse typing agree. If they do not agree, that patients blood type CANNOT BE RELEASED until the discrepancy is resolved.

Worksheet
Record the reagent lot numbers and expiration dates below. Tell whether the reagent is expired or indate. Reagent Anti-A Anti-B A1cells B cells Lot number _ _____________ ______________ ______________ ______________ Expiration Date ____________ ____________ ____________ ____________ Expired or in-date _ ____________ ______________ ______________ ______________

Record your QC results below. Reagents in Tube Anti-A with A cells Anti-A with B cells Anti-B with A cells Anti-B with B cells Result Acceptable (Y/N)

Visible hemolysis and/or contamination? (Y/N) _________

Questions: 1. Explain the importance of daily QC.

2. Describe what each reagent is detecting in the patient. a. Anti-A

b. Anti-B 4

c. A1 cells

d. B cells 3. List 4 causes of error in ABO typing.

4. For each patient, list what RBCs (A, B, AB, O) would be compatible. Also list which plasma types would be compatible. Patient blood type AB B O A RBCs compatible Plasma compatible