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Compatibility tests are performed in order to help prevent hemolytic transfusion reactions which may be caused by antibodies of the ABO blood group system or by antibodies to other blood group antigens. Compatibility testing includes verification of the ABO & Rh type of the donor blood and the following tests on recipient's blood:
Timing of Compatibility Testing A sample must be obtained from the patient within 3 days of the scheduled transfusion for compatibility testing if any of the following conditions exist:
ABO Typing ABO typing is accomplished by:
Interpretation of ABO Typing
Determination of ABO blood groups is the most important pretransfusion compatibility test. If tests are done to insure that donor and recipient belong to the same ABO blood group, then even if no other tests are done, the donor's red blood cells will be compatible with the recipient's plasma in about 97% of cases. Selection of RBC Units When ABO Specific Blood is not Available When ABO specific blood is not available, it is important to use RBCs that are still compatible with the recipient's serum; that is, crossmatch compatible. Donor RBCs must not contain A or B antigens that react with the anti-A or anti-B present in the recipient's serum. In this situation the large amount of anti-A or anti-B in the recipient's plasma would bind to transfused ABO incompatible RBCs and cause hemolysis. The reverse situation, in which the recipient's RBCs have an antigen that reacts with an antibody in the donor's plasma, is not as important. In this case, the small amount of antibody present in the 100 mL of plasma remaining in a RBC unit is rapidly diluted about 30 fold in the recipient's plasma before the antibody can injure enough RBCs to be clinically apparent. Whole blood should not be used when switching ABO blood groups since each unit contains 250 mL of plasma and therefore 2 to 3 times more anti-A or anti-B. The following table summarizes the proper selection of RBCs for Rh positive recipients when ABO group specific blood is unavailable. RBC Selection When Type-Specific Blood Is Unavailable
Rh Typing Rh typing is performed so that Rh positive red blood cells will not be given to an Rh negative recipient. This prevents Rh immunization in patients without pre-existing anti-D and prevents hemolytic transfusion reactions in patients who have already developed anti-D antibodies. The presence or absence of the D antigen in the Rh blood group system defines whether a person is Rh-positive or Rh-Negative. About 85% of the US population is Rh positive and 15% is Rh negative. In contrast to the ABO system, patients with D-negative red cells will not make anti-D unless they have been immunized previously by exposure to Rh positive red cells via fetomaternal transfer during pregnancy or prior transfusion. Rh-positive recipients can receive Rh positive or Rh negative RBCs, but Rh-negative recipients should only receive Rh-negative blood. For Rh negative recipients, the order of choices is the same as far as ABO groups are concerned.
Rh immune globulin should be administered to Rh negative female patients of childbearing potential who receive Rh positive units. Three hundred micrograms (1 vial) of RhIg is given for every 15mLof transfused Rh positive red blood cells, preferably within 72 h after transfusion. Intravenous Rh immune globulin (WinRho) is preferred over intramuscular RhIg when large volumes must be administered. Principles of Serologic Tests For Red Cell Antibody Detection When antibodies react with red cells, agglutination may occur particularly if the antibody is of the IgM class. Many IgG antibodies react with their corresponding antigen on the red cell but do not cause agglutination. For this reason, the antiglobulin test (Coombs Test) was developed. Antibodies formed in animals (usually rabbits or goats) against human IgG and complement can react with red cells that are coated with non-agglutinating antibodies and/or with complement components and cause visible agglutination. Sera containing anti-IgG and/or anti-complement are called antiglobulin reagents or Coombs sera. The antiglobulin test is either direct or indirect. Medical applications of the DAT and IAT are summarized in the following table. Medical Applications of the Direct & Indirect Antiglobulin Tests
The direct antiglobulin test (DAT) is performed to determine if a patient's red cells are coated in vivo with IgG or complement components. In the DAT, red cells are taken from the patient, washed to remove unbound IgG and then directly tested with antiiglobulin reagent (anti-IgG and/or anti-complement). If antibody is coating the patient's red cells, they are agglutinated by antiglobulin. The DAT is extremely sensitive; it can detect as few as 100 IgG and 400 C3d molecules per red cell. Approximately 1 in 9000 healthy persons has a positive direct antiglobulin test with no evidence of hemolysis. Some diseases may be associated with a positive DAT, even though the patient does not appear to be actively hemolyzing their red cells. Examples include chronic lymphocytic leukemia, multiple myeloma, systemic lupus erythematosis, infectious mononucleosis, mycoplasma infection, and AIDS. Different studies have reported that 0.3 to 1.5% of hospitalized patients have a positive DAT. Autoimmune hemolytic anemia has an incidence of one case per 80,000 and most often occurs in people older than 60 years. AIHA are divided into warm and cold autoantiobody types based on the temperatures at which the antibodies maximally react with red blood cells in vitro. Warm autoantibodies are more reactive at 37oC than at lower temperatures, whereas cold autoantibodies react optimally at 5oC and less strongly at higher temperatures. Characteristic Serological Findings in Autoimmune Hemolytic Anemias
In warm autoimmune hemolytic anemia, RBCs may be coated with IgG, IgG and complement, or complement alone. In warm AIHA, IgG is found alone in about 60% of cases and in association with complement in about 30% of cases. In contrast, cold autoimmune hemolytic anemia is caused by complement-fixing IgM antibodies that react more strongly in the cold than at higher temperatures. In these cases, the direct antiglobulin test detects only complement. Autoantibodies may appear to have specificity for a particular blood group antigen even though the patients' red cells express that antigen. Numerous drugs have been associated with a positive DAT including:
A DAT should be performed whenever there is:
In the indirect antiglobulin test (IAT), patient serum is incubated with commercially available normal red cells to allow in vitro coating of red cells. After incubation, the red cells are washed to remove unbound immunoglobulin and tested with anti-IgG. If antibody is present in the patient's serum, red cells become coated with antibody and are agglutinated by the antiglobulin reagent. Thus, the IAT detects the presence of antibody in serum. The major applications of the IAT are discussed in the following sections. Antibody Screen The antibody screen detects alloantibodies and autoantibodies in patient plasma, which have specificity for red blood cells. This test is performed by incubating patient plasma with two or three commercially available group O RBCs that have been extensively antigen typed. The FDA mandates that red cells for antibody detection possess the following antigens: C, D, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and JKb. Although not required, it is generally agreed that homozygosity for C, D, E, c, e, Fya and Jka is also preferable. The test is performed under conditions that detect clinically significant antibodies reactive at 37°C and the antiglobulin phase. It is designed to detect most unexpected antibodies to common red cell antigens other than anti-A or B. Screening cells cannot possibly be postive for all of the antigens that have a low frequency. Therefore, it is possible to get a falsely negative antibody screen. For example, if a patient has an antibody to Kpa and the screening cells are negative for Kpa antigen, the antibody will not be detected. Fortunately, the incidence of Kpa antigen is about 1%, so the likelihood of selecting a unit for immediate spin crossmatch that would be Kpa positive is very low. However, if a Kpa positive unit was selected, an immediate spin crossmatch would not detect any incompatibility and an incompatible unit would be transfused. Antibody binding to red blood cells can be enhanced by manipulation of environmental conditions or the red cell membrane. In general, physiological temperature and pH promote red blood cell antigen- antibody interactions. The negative charge of red cell membranes causes red cells to naturally repel each other. Enzyme modification of red blood cells reduces net surface charge and distance between cells, facilitating red cell agglutination by IgG molecules. The enzymes used in detection of blood group antibodies include ficin, papain, and bromelin. Suspension of red blood cells in low ionic strength solutions (LISS) decreases the ionic strength of the reaction medium and increases the attraction between positively charged IgG molecules and negatively charged red blood cells. Most LISS reagents contain 0.2% NaCl. LISS increases the rate of antibody binding to red blood cells and allows shorter incubation times. Polyethylene glycol (PEG) is a macromolecular additive within a LISS that brings antibody sensitized red blood cells closer together and promotes antibody cross-linking and enhancement of agglutination reactions. PEG is more effective than LISS alone in detecting weak antibodies. Crossmatch Tests If the antibody screen is negative and blood bank records show no previous history of antibody, the next step is to do an immediate spin crossmatch. The immediate spin crossmatch is sufficient to detect ABO incompatibility. However, if the antibody screen is positive, antibody identification and selection of antigen negative units must be completed prior to peforming an AHG crossmatch (see next section). A crossmatch consists of testing patient serum against a sample of red cells from the actual unit that has been selected for transfusion. There are two types of crossmatches:
Any blood component containing a significant number of RBCs needs to be crossmatched prior to transfusion including:
Antibody Screen & AHG Crossmatch Discrepancies The antibody screen can sometimes be positive when one or more of the crossmatches are negative. Some reasons are:
Antibody Identification If the antibody screen is positive, the specificity of the antibody is identified by testing the serum against a panel of 8 to 12 Group O RBCs of varying phenotypes. The pattern of positive and negative reactions with these cells identifies the antigen against which the antibody is directed. Antibody identification is accomplished by the "crossing out" method which consists of identifying each cell that is negative and crossing out all of the antigens present on that cell. The panel should also be observed to:
Antibody Panel Interpretation
If a clinically significant antibody is identified, only red cells negative for the relevant antigen will be selected for crossmatching and transfusion. For example, if the antibody is anti-K, RBC of the appropriate ABO and Rh type will be tested with anti-K anti-serum and only K-negative red cells will be selected for transfusion. For added safety, an AHG crossmatch is also performed. For clinically insignificant antibodies, it is permissable to crossmatch units that have not been antigen typed. When the antibody screen is positive, additional time is required to identify the antibody(ies), to find antigen-negative red cells, and to perform AHG crossmatches. This time can range from an hour to days if multiple antibodies, antibodies against high frequency antigens, or a mixture of autoantiobody and alloantibodies are present. If transfusion is medically necessary before compatible blood can be obtained, the attending physician and the transfusion medicine physician need to discuss the risk:benefit ratio of transfusing potentially incompatible blood. Autoantibodies An autoantibody is produced against a person's own red cells. When a patient has an autoantibody, the direct antiglobulin test and the autocontrol in an antibody panel will be positive. In addition, all cells in the panel will be reactive. If the antibody reactions are stronger at colder temperatures and weaker at warm temperatures, the patient probably has a cold autoantbody. If the antibody reactions are negative at colder temperatures and positive at warmer temperatures, the patient most likely has a warm autoantibody. Adsorption Procedures Sometimes, patients with autoantiobodies require red cell transfusion. The most important technical issue faced by the transfusion service is determining if the patient's serum contains alloantibodies in addition to the autoantibody. Between 15 and 40% of patients with autoimmune hemolytic anemia have alloantibodies. The most common alloantibodies detected in the sera of patients with AIHA in descending order are: anti-E, K, C, Fya, Jka, and c. The most frequently used method for detecting alloantibodies in the presence of a broadly reactive autoantibody is the warm autoadsorption procedure. Autoantibody is removed from autologous red cells by heat or chemical treatment and then the red cells are treated with an enzyme to enhance autoantibody adsorption. The most popular technique is to treat red cells with ZZAP, which is a combinination of dithiothreitol and papain. Autoantibody is then adsobed from plasma with these treated autologous red cells. Several adsorptions may be necessary to remove all of the autoantibody. If no antibody is detected, the adsorbed plasma can be used for crossmatching donor units. If alloantibody is present, it must be identified and antigen negative units selected for crossmatching. The warm autoadsorption procedure is not useful in patients who have been transfused within the past 30 days because even a small percentage of transfused cells may adsorb alloantibody, producing a falsely negative result. In this situation, allogeneic adsorptions may be required. By selecting two or three samples of red cells of varying phenotypes, almost all clinically significant alloantibodies can be detected. For example, adsorbing a serum containing an autoantibody and an anti-Jka alloantibody with Jka negative red cells will remove the autoantibody but not the anti-Jka . Once the autoantibody is removed, the remaining alloantibodies can be identified using a panel. This procedure is usually performed at a blood center reference laboratory instead of a hospital transfusion service.  Ordering Blood for Surgery Type and Crossmatch If it is likely that a patient will require transfusion, then the physician should order a type and crossmatch of an appropriate number of units. This order will insure that blood will be available at the time of surgery. Blood crossmatched for surgery and not transfused is released the morning after surgery. Type And Screen A type and screen should be ordered when there is a reasonable possibility that a surgical patient will require blood but the likelihood of transfusion is too low to justify reserving crossmatched units. As a general guideline, if a surgical procedure requires transfusion in less than 10% of cases, a type and screen rather than type and crossmatch is appropriate. When a type and screen is ordered, ABO and Rh typing and antibody screen are performed. This allows the Blood Bank to identify patients with unusual blood types or complex antibodies ahead of time so that problems can be resolved prior to surgery. If a patient requires blood during surgery, and the antibody screen is negative, units are issued after an immediate spin crossmatch. This process takes 5 to 10 minutes. If clinically significant antibodies are detected during the antibody screen, antigen-negative units are identified and crossmatched using AHG. Maximum Surgical Blood Order Schedule A "Maximum Surgical Blood Order Schedule" (MSBOS) is a listing of surgical procedures performed at a given institution with recommended maximum blood orders for each procedure These blood orders are based on actual intraoperative blood use at the institution and are typically set to cover 90% of patient needs. The MSBOS is a useful guideline for surgical blood ordering. Blood orders may be periodically monitored by the blood bank staff to assure conformity with these guidelines. Emergency Transfusion In an emergency, the patient's physician must weigh the risk of transfusing uncrossmatched blood against the hazard of waiting for a completed crossmatch. If the physician believes the urgency of the situation warrants the use of uncrossmatched blood, they must sign an "Emergency Blood Release Form" to document the reason for the urgent need and to acknowledge that units are not crossmatched at the time of transfusion. Direct consultation with Blood Bank personnel is highly recommended to expedite requests. The following options are available for emergency transfusion. Uncrossmatched Group O, Rh-Negative Red Cells O-negative red cells are available for immediate transfusion to any patient, but should be used only when the patient's blood type is not known and there is insufficient time for typing. This situation will sometimes occur in the trauma setting and should only apply to the first few units given. Uncrossmatched Type Specific Blood The preferred medical practice is to transfuse ABO type-specific blood rather than O-negative red cells whenever possible in order to conserve the O-negative blood supply and to minimize transfusion of potentially incompatible group O plasma. Blood typing can be completed and type-specific blood made available within 5 minutes after receipt of a patient sample. Blood type cards or dog tags from other facilities are not acceptable documentation of blood type. In all emergency situations, standard compatibility testing for units issued uncrossmatched is initiated and completed without delay and additional crossmatched blood is made available as soon as possible. If any compatibility problem is detected during follow-up testing, the physician is notified immediately. Circumstances in Which It Is Difficult or Impossible To Find Compatible Blood Some patients have multiple red cell alloantibodies. For example, when a patient has anti-Fya, anti-Jkb, anti-E and anti-S, blood bank technologists will test units of the appropriate ABO type until they find units that are negative for all four antigens and then must perform an AHG crossmatch. In the example given, the probability of a given unit of red cells lacking all four antigens is 3% (1 in 33 units). If transfusion is required on an emergency basis or if large numbers of units are required urgently, it may be impossible to supply antigen-negative blood. Patients with alloantibody to an antigen that is on an extremely high percentage of homologous red cells (ie, a high frequency antigen) may be incompatible with all red cells tested. Some of these antibodies are clinically insignificant but others may cause shortened red cell survival. In these cases, the regional blood center may have to search for suitable donors among family members or rare donor files. Some patients have red cell autoantibodies that react with all homologous red cells tested. In this instance, it is almost impossible to provide compatible blood for transfusion. For extreme life-threatening emergencies, utilization of blood that one expects may be hemolyzed rapidly is nevertheless warranted, particularly since such dire expectations are not always realized. In Vivo Compatibility Test The presence of an antibody in a patient's plasma does not necessarily indicate that transfusion of incompatible blood will result in shortened survival of transfused red cells. When no compatible red blood cells can be found by in vitro compatibility testing and the need for transfusion is urgent, then an in vivo crossmatch might be helpful in assessing the possible outcome of a transfusion. The principle underlying this test is that intravascular hemoIysis of as little as 5 mL of red cells will raise the plasma hemoglobin concentration of an adult by about 50 mg/dL, which is an amount that can be easily visualized. The medical director of the hospital transfusion service in consultation with the patient's physician must approve an in vivo crossmatch before it is undertaken. The most practical method is to:
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