Routine Pretransfusion Testing

Click on image to see transfusion service laboratory.

Pretransfusion testing has several components. An ABO group and Rh(D) typing are done to determine the type of donor red cells to be crossmatched. An antibody screen is done to detect any possible unexpected clinically significant antibodies in the patient. An immediate spin (IS) crossmatch at RT with donor cells --or an electronic crossmatch-- are done to confirm ABO compatibility between donor and patient. Controls may (or may not) include an autocontrol and IAT positive and negative controls. Each of these components is discussed below.

ABO Group

A front group (using anti-A and anti-B) and a reverse group (using A₁ cells and B cells) are done. The ABO group is read after an IS at RT, macroscopically only, and again upon completion of the test.

Rh(D) type

Commercial anti-D is used for routine D typing. If the anti-D reagent is high protein, an Rh control is set up that must be negative for the Rh(D) test to be valid. The manufacturer's supplied Rh control has the same protein concentration as the commercial antiserum being used. If a low protein reagent is used (e.g., monoclonal/polyclonal blend anti-D), no Rh control is required unless the patient types as AB Rh(D) positive, i.e., all tests using the patient's red cells and low protein antisera are positive. In this case, a suitable Rh control would contain the same protein concentration as the commercial anti-D (typically 6- 10% albumin).

Antibody Screen for Irregular Alloantibodies

In this test the patient's serum is tested against two or three separate antibody screen cells. AABB Standards requires that screen cell be single-donor cells, not pooled red cells.

Characteristics of screen cells:

• These cells are group O because we want to detect unexpected antibodies (not anti-A and anti-B).

• Their antigenic content is known and is provided on antigrams supplied by the manufacturer.

• These cells should be positive for all common blood group antigens, usually those with a frequency of about 8% or more.

• The cells should also come from donors who are homozygous for genes that produce antigens showing dosage, i.e., antigens in the Rh, Kidd, Duffy and MNSs systems.

• Some labs use a three cell screen: usually screen cell 1 is R₁R₁ (CDe/CDe), screen cell 2 is R₂R₂ (cDE/cDE), and screen cell 3 is rr (cde/cde). Normally only one of the three cells is positive for the K antigen. If two screen cells are used, both are Rh(D) positive (usually R₁R₁ and R₂R₂) and often only one is K-positive. A potential advantage of a three cell antibody screen is that more antigens can be homozygous than with a two cell screen.

Advantages of screen cells: Screen cells can detect antibodies more effectively than can donor cells because they come from donors who are homozygous. Donor cells, if positive for the corresponding antigen to a patient's antibody, could be from either homozygous or heterozygous donors. Donor antigens (except for ABO and D) are unknown. If the patient had a very weak antibody whose corresponding antigen showed dosage, the serum might be falsely negative with heterozygous donor cells but should be detected by the homozygous screen cells.

Note:It is incorrect to refer to cells as being homozygous or heterozygous; it is the donors whose genes are homozygous or heterozygous. However, for the sake of brevity, throughout this module the terms homozygous cells and heterozygous cells will be used occasionally.

Phases of the Antibody Screen

Depending on the method used, one, two, or three phases may be done, often all in the same test tube:

• RT Saline (or LISS) IS:

  Today most labs omit this phase with antibody screen cells, because it will detect IgM antibodies that are capable of reacting at RT, but not at body temperature. These include IgM cold-reactive antibodies that are usually clinically insignificant such as anti- M, -N, -Le^a, -Le^b, and -I. (A saline IS at RT may be done with donor cells to confirm ABO compatibility.)

• Saline (or LISS) 37⁰ C Phase:

  This phase detects IgM antibodies that have a higher thermal range and are considered to be clinically significant. Some of the IgM antibodies listed above for the saline IS phase are capable of reacting at body temperature and causing in vivo red cell destruction. This phase is usually read only macroscopically for agglutination; if clotted patient specimens are used, the phase is also read for hemolysis (C9 binding).

• IAT (37⁰ C Phase):

  The antiglobulin phase can detect IgG antibodies in four main systems (Rh, Kell, Kidd and Duffy) as well as anti-S, anti-s and sometimes anti-M (if it is IgG). If clotted specimens and polyspecific antiglobulin serum (AHG) are used, the IAT can also detect IgM antibodies that can bind C3 (due to the anti- C3 in the AHG serum), e.g., some anti-Le^a, -Le^b, and -I.

  This phase is read macroscopically for agglutination, either using the naked eye or against a lighted magnifying mirror. Some labs (particularly those in Canada), if the IAT is negative, also read it microscopically. IgG sensitized cells are added to all negative tests and must produce a positive result for the test to be valid.

• Autocontrol:

  If done, the autocontrol consists of testing patient serum with the patient red cells in the same phases as the antibody screen cells. Alternatively, the autocontrol may be a direct antiglobulin test (DAT) on the patient's red cells. Many labs do an autocontrol only if the patient has an unexpected antibody. Otherwise time and money is spent investigating clinically insignificant positive DATs.

• IAT Positive/Negative Controls:

  Depending on lab policy, these may be run only upon receipt of a new lot of AHG serum, run once daily as part of the morning controls, or may be run in parallel with each antibody screen. A weak antibody (for example, 1/250 dilution of commercial IgG anti-D) is tested with antigen-positive and antigen-negative cells (such as O R₁r cells and A rr cells) to control the reactivity and specificity, respectively, of the AHG serum. Note that the addition of IgG sensitized cells to negative tests serves the same purpose as the positive IAT control (tests the sensitivity of AHG serum); also, if the AHG serum has lost its specifity, this should be apparent by the occurence of false positives.

Regulation

Routine Pretransfusion Testing

Possible Results