BLOOD TYPING PROCEDURE

• The most commonly used immunohematology test is the ABO and Rh screen. ABO and Rh

screening determines the blood type of the patient and can also be used to decide if a pregnant

woman with Rh negative blood will need to receive Rh immune globulin (i.e., RhoGam). Rh

immune globulin injection helps prevent the mother from forming antibodies against the Rh

antigen, a condition that may lead to hemolytic disease of the newborn. The results of ABO/Rh

testing are critical to patient care. If a blood type is incorrectly interpreted, the patient may not

receive the proper treatment. The following information provides the essentials of ABO/Rh

testing and appropriate quality control (QC) procedures. However, it is not intended as a

substitute for adequate training in ABO/Rh testing.

• Specimen Collection

o ABO and Rh testing require both serum and red blood cells; therefore, the specimen

should be collected in a vacuum tube with no preservative (red topped tube). EDTA

tubes (lavender topped) may also be used.

o If using a red-topped tube, DO NOT USE GEL SEPARATOR TUBES. The gel

separator tube will put a layer of gel between the red blood cells and the serum when

centrifuged, making it very difficult to obtain the red blood cells. The gel may also

become mixed with the red blood cells, causing a negative reading to look positive.

Test the specimen as soon as possible. If there is a delay in testing, properly store the

specimen in a refrigerator.
• Specimen Preparation

o Test Tube Method

▪ For red-top tubes, after collection, allow the blood to stand until a clot forms,

then centrifuge the specimen to separate the serum from the red blood cells.

After centrifugation, store the serum in a separate test tube and retain the red

blood cells in the original tube. Be sure to label all tubes with proper patient

information to avoid confusing them with other patient samples. For purple-top

tubes, centrifuge the specimen to separate the plasma from the red blood cells.

The plasma may be stored in aseparate labeled tube like the red-topped

specimen, or kept in the original tube to reduce pre-analytical error.

▪ After separation, prepare a 2 to 5 percent red blood cell suspension for testing.

There are several methods that can be used for creating a cell suspension

preparation. Each laboratory should establish its own guidelines for the

preparation of cell suspensions. Some suggested procedures include:

1. Using a disposable pipette, transfer approximately 0.2 ml (two

drops) of the patients packed red blood cells to a 10 x 75 mm test

tube.

2. Fill the tube with saline. Cover and mix.

3. Centrifuge the tube at 3,400 rpm for 30 seconds, then decant the

saline.

4. Visually estimate a 2 to 5 percent suspension. Adding

approximately 1 ml of saline will make a 3 percent suspension,

suspension.

• Testing

o ABO/Rh testing entails putting a sample of the cell suspension in various reagents,

centrifuging at a specific speed for a specific period of time, and then gently shaking

the tube. If there is any clumping of blood cells, known as agglutination, the result for

that reagent is positive

o ABO Testing

▪ ABO testing should include both forward and reverse typing. Reverse typing is

a cross-check for forward typing. However, reverse typing is not recommended

when typing newborns and infants under the age of 4 months since they have

not developed the proper antibodies necessary for the test to be accurate.

▪ Forward typing uses the patient’s red blood cells. All red blood cells contain

antigens that are specific to the patient’s blood type. When antibody A (Anti-

A) or antibody B (Anti-B) reagent is added to the patient’s red blood cells the

antigens on the cells will cause the cells to react with the antibodies. For

example, if the patient has blood type A, the patient’s red blood cells will clump

with antibody A (Anti-A), but will not show any clumping with antibody B

(Anti-B). If the patient has blood type B, the patient’s red blood cells will clump

with antibody B (Anti-B), and will not clump with antibody A (Anti-A). The

forward typing results are considered the patient blood type.

▪ Reverse typing uses the patient’s serum. The serum contains antibodies that

react with the reagent red blood cells which are coated with antigen A (A cells)
 or antigen B (B cells). The type of antibodies present in the patient’s serum will

determine which reagent red blood cells will cause agglutination, and will

therefore confirm the blood type. For example, if the reagent A cells are

negative (no agglutination), the patient does not have the antibody A in their

serum, so the blood type is A. The results from the reverse typing will be the

opposite of the forward typing. Reverse typing results are not considered to be

the blood type, but a confirmation of the forward typing results

o Rh Testing

▪ All patients are either Rh D positive or Rh D negative. Testing for the Rh D

factor is done by using reagent Anti-D and patient red blood cells. If the

patient’s red blood cells contain the D antigen (Rh D factor), there will be

agglutination when tested with Antibody D (Anti-D) and the patient is

considered Rh D positive. If there is no agglutination of cells, then the patient

is usually Rh D negative. See next section on Weak D (Du) testing.

▪ With each patient test, a patient control (Rh control) may be indicated

depending on the recommendation of the reagent manufacturer. The patient Rh

control consists of patient red blood cells and a control substance. This control

substance could be albumin, saline or other material defined by the

manufacturer. If both the patient control and patient test are positive, the result

is considered invalid and should be repeated with washed patient red blood

cells. A positive patient control demonstrating agglutination indicates that

another substance is present and reacting with the control portion of the Anti-D

reagent and not the Anti-D.

▪ Weak D (Du) is a weaker expression of antigen D. If a patient is antigen D

negative they may be Weak D (Du) positive. Since Weak D (Du) is a weaker

expression of antigen D, it will require an incubation period at a higher

temperature to cause agglutination. After performing Rh testing, all negative

tubes should be incubated at 37ºC for 15 minutes. After incubation, antihuman

globulin is added to help enhance any weak reactions, and the tubes are then

centrifuged and read. If the Weak D (Du) test and patient control are negative,

it is recommended that Coombs check cells be added.

▪ Coombscheck cells are reagent red blood cells coated with antibody and used

to determine that the antihuman globulin (AHG) was not inactivated by

substances in the patient's cell suspension. Agglutination should be noted after

the addition of Coombs check cells. If agglutination does not occur, the AHG

has been inactivated and the test is not valid. Repeat the test on a well-washed

cell suspension.

▪ It is important to run a patient control (Rh control) with the Weak D (Du) test.

If the patient control (Rh control) is positive, the typing is not valid and the

patient cells may need to be washed and retested, or checked for a positive direct

antiglobulin test (DAT). If the Weak D (Du) test results are negative, the patient

is considered Rh D negative. If the Weak D (Du) test results are positive the

patient is considered Rh D positive.

o When recording results, a plus sign (+) is considered positive, a zero (0) is considered

negative, and all reactions are graded on a scale of zero (negative) to most positively

reactive (4+). For example, if the patient is blood type A, then the forward results will

have a 3 to 4 plus positive result with the Anti-A and a negative result with the Anti-B.

The reverse typing should be the opposite of the forward typing. A cells will be negative

and the B cells will be 2 to 4 plus positive.

BLOOD GROUP CHART

Forward Typing Reverse Typing

Anti-A Anti-B A Cells B Cells

Group A 3–4+ 0 0 2–4+

Group B 0 3–4+ 2–4+ 0

Group AB 3–4+ 3–4+ 0 0

Group O 0 0 2–4+ 2–4+

• The crossmatch test has traditionally meant the testing of the patient’s serum with the donor

RBCs, including an antiglobulin phase or simply an immediate spin phase to confirm ABO

compatibility.

• Purpose

o The purposes of crossmatching are to:

▪ It is a final check of ABO compatibility between donor and patient.

▪ It may detect the presence of an antibody in the patient’s serum that will react

with antigens on the donor RBCs but that was not detected in antibody

screening because the corresponding antigen was lacking from the screening

cells.

o The crossmatch procedure attempts to fulfill these goals in the following ways:

▪ The crossmatch serves as a double check of ABO errors caused by patient

misidentification or donor unit mislabeling.

o If the recipient possesses a clinically significant antibody or a history of one, the

crossmatch provides a second means of antibody detection and checks the results of the

antibody screen.

o The crossmatch, designed to detect donor units unlikely to survive normally once

transfused, must be rapid and simple enough to be practical. Actual measurement of

the survival rates of transfused red cells may be considered to constitute the best

crossmatch. This technique can be performed directly through the use of radioisotope

labeling, but this is impractical for regular use. Posttransfusion hematocrit or

hemoglobin values are often determined to provide a working measure of successful

hematocrit by 3% and the hemoglobin by 1 g/dL.

• Principle

o Cross-matching will detect incompatibilities between the donor and recipient that will

not be evident on blood typing. There are two types of cross-matches: Major cross-

match and Minor cross-match.

▪ The major crossmatch involves testing the patient’s serum with donor cells to

determine whether the patient has an antibody which may cause a hemolytic

transfusion reaction or decreased cell survival of donor cells. This is the most

important cross-match.

▪ The minor crossmatch involves testing the patients cells with donor plasma to

determine whether there is an antibody in the donor’s plasma directed against

an antigen on the patient’s cells.

• Methods

o Serologic Crossmatch

▪ The serologic crossmatch test consists of mixing the recipient’s serum with

donor RBCs. Several procedures can be used for serologic crossmatch testing,

including the immediate spin and antiglobulin crossmatch. The objective of

testing is to select donor units that can provide maximal benefit to the patient,

which should be kept in mind when developing the test protocol. Crossmatch

methods can generally be categorized by the test phase in which the procedure

ends.
 o Immediate Spin Crossmatch

▪ The purpose of this test is to detect ABO incompatibility. Equal volumes of 2%

saline suspension of red cells of donor and recipient’s serum are mixed,

incubated at room temperature for 5 minutes, and centrifuged. Agglutination or

hemolysis indicates incompatibility.

o Antiglobulin Crossmatch

▪ The antiglobulin crossmatch procedure begins in the same manner as the

immediate spin crossmatch, continues to a 37°C incubation, and finishes with

an antiglobulin test. Several enhancement media may be applied to boost

antigen-antibody reactions. These may include albumin, low ionic strength

solution (LISS), polyethylene glycol, and polybrene. For greatest sensitivity, an

antihuman globulin (AHG) reagent containing both anti-IgG and

anticomplement may be selected for the final phase of this crossmatch method.

However, many laboratories routinely use monospecific anti-IgG AHG

reagents.

• Procedure

1. Prepare donor and recipient blood samples:

- For Major crossmatch : Donor’s red cell and recipient serum or plasma

- For Minor crossmatch : Recipient red cells and donor’s serum or plasma

2. Prepare 3 – 5% cell suspensions of red cells.

3. Major Crossmatch:

- Label a test tube. Add two drops of the patient serum and one drop of the appropriate

donor cell suspension.

- Label a test tube. Add two drops of the appropriate donor serum and one drop of the

patient cell suspension.

5. Mix the tubes and incubate at 37°C for about 45 minutes.

6. Add two drops of AHG (Antihuman globulin) and mix well.

7. Centrifuge for 1 minute at 1500 rpm

8. Read macroscopically and microscopically and record the results

• Interpretation

o Tubes (gel cards, etc.) should be carefully labeled so that the contents can be identified

at any stage of the procedure. After centrifugation of tubes, the supernatant should be

examined for hemolysis, which, if present, must be interpreted as a positive result.

Results should be read against a white or lighted background, and a magnifying mirror

or hand lens can be used to facilitate reading. The button of RBCs should be gently

resuspended. A “tilt and wiggle” method of resuspension is ideal. The initial tilt, when

the clear supernatant sweeps over the button of RBCs, immediately indicates a positive

or negative reaction. A jagged or firm button edge is indicative of a positive

agglutination reaction, whereas a smooth swirling of free cells off the RBC button

indicates a negative reaction.

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