Pretransfusion Testing

Transcription

1 16 Pretransfusion Testing The purpose of pretransfusion testing is to select blood components that will have acceptable survival when transfused and will not cause harm to the recipient. If performed properly, pretransfusion tests will establish ABO compatibility between the component and the recipient and detect most clinically significant unexpected antibodies. AABB Standards for Blood Banks and Transfusion Services 1 requires the following procedures before components are issued for transfusion: 1. Positive identification of recipient and recipient s blood sample. 2. ABO and Rh typing of recipient s blood. 3. Red cell antibody detection tests using recipient s serum or plasma. 4. Comparison of current findings on the recipient s sample with records of previous results. 5. Tests on donor blood, as described in Section 8 of Methods. 6. Selection of components of ABO and Rh types appropriate for the recipient. 7. Performance of a serologic or computer crossmatch. 8. Labeling of components with the recipient s identifying information. Transfusion Requests Requests for transfusion must contain sufficient information for positive recipient identification. Standards 1 requires the first and last names of the potential blood recipient and an identification number unique to that individual. The name of the responsible physician should also appear, and additional information, such as gender and age of the recipient, diagnosis, and history of transfusion and pregnancy may be helpful. Blood request forms that lack the required information or are illegible are not acceptable. Requests may be transmitted by computer, provided the required information is complete. Telephoned requests are acceptable, but should be documented in a telephone log or verified by subsequent submission of a properly completed blood request

2 332 AABB Technical Manual form before the blood is issued for transfusion. Sample Collection Collection of a properly labeled blood sample from the intended recipient is critical to safe blood transfusion. Most hemolytic transfusion reactions result from errors in patient or sample identification. 2 The person drawing the blood sample must identify the intended recipient in a positive manner, most effectively done by comparing the information on the request form with the information on the patient s identification band. If a discrepancy is noted, the sample must not be collected. The phlebotomist must not rely on a bed tag or on charts or records placed nearby. In the absence of an identification band, the phlebotomist should await application of a suitable band before collecting the specimen. It is useful to remind clinical personnel that transfusions cannot be administered to a patient who lacks positive identification. When the patient s identity is unknown, an emergency identification number on a temporary band can be used. This number should be attached to the patient and blood samples and be cross-referenced with the patient s name and hospital identification number when they become known. Sample Labeling Before leaving the bedside, the phlebotomist must label the blood sample tubes with the patient s first and last names, identification number, and the date of collection. Imprinted labels may be used if the information on the label is identical to that on the wristband and request form. There must be a mechanism to identify the phlebotomist 1 ;this certification may go on the label of the tube or on the requisition. Confirming Sample Identity When a sample is received in the laboratory, a qualified member of the staff must confirm that the information on the label and on the transfusion request are identical. If there is any doubt about the identity of the patient, a new sample must be obtained. 1 It is unacceptable for anyone to correct an incorrectly labeled sample. Each laboratory should establish policies and procedures in this regard. Blood Sample Pretransfusion testing may be performed on either serum or plasma. 1 Plasma sometimes creates technical problems if small fibrin clots evolve and trap red cells into aggregates that resemble agglutinates. High levels of fibrinogen may induce rouleaux formation. Clotting may be incomplete in specimens not intended to be anticoagulated; this usually occurs in patients who have been treated with heparin. Adding thrombin or protamine sulphate to the sample usually corrects the problem or an anticoagulated specimen can be collected. It is permissible to collect blood from an infusion line. To avoid interference from residual intravenous fluid, the tubing should be flushed with saline and 5 ml or a volume of blood approximately twice the fluid volume in the line should be withdrawn and discarded before collecting the sample. 3 Appearance The appearance of the serum or plasma may create difficulties in detecting antibody-induced hemolysis. Whenever possible, a hemolyzed sample should be replaced with a new specimen. Test results observed with lipemic serum can also be

3 Chapter 16: Pretransfusion Testing 333 difficult to evaluate, but sometimes, hemoglobin-tinged or lipemic serum may need to be used. Each institution should have a procedure describing the indications for using hemolyzed and lipemic specimens. Age of Sample If the patient has been pregnant or transfused within the preceding 3 months or if the history is uncertain or unavailable, compatibility tests must be performed on blood samples collected within 3 days of RBC transfusions. 1 This is to ensure that the sample used for testing reflects the recipient s current immunologic status because recent transfusion or pregnancy may stimulate production of unexpected antibodies. Because it is not possible to predict whether or when such antibodies will appear, a 3-day time limit has been selected as an arbitrary interval expected to be both practical and safe. It is short enough to reflect acute changes in immunologic status but long enough to allow the results of preadmission testing completed on Friday (day 0) to be used for surgical cases performed on Monday (day 3). The 3-day requirement applies only to certain categories of patients, but many laboratories prefer to standardize their operations by setting a 3-day limit on all specimens used for pretransfusion testing. Exceptions may be made as needed if a patient has not been recently transfused or pregnant and the available specimen has been stored in a monitored refrigerator. Each institution should have a policy defining the length of time samples may be used. Retaining and Storing Blood Samples The recipient s blood specimen and a sample of the donor s red cells must be sealed or stoppered and kept at 1-6 C for at least 7 days after each transfusion. 1 Donor red cells may be from the remainder of the segment actually used in the crossmatch or a segment removed before issuing the blood. If the opened crossmatch segment is saved, it must be placed in a sealed or stoppered tube. Keeping the patient s and donor s samples makes it possible to do repeat or additional testing if the patient experiences adverse effects. Serologic Testing Except in emergencies, the red cells of the intended recipient must be typed for ABO and Rh before blood is issued for transfusion. If the patient is to receive whole blood or red blood cells, the serum must also be tested for unexpected antibodies. 1 ABO and Rh Typing To determine the ABO type of the recipient, red cells must be tested with anti-a and anti-b, and the serum or plasma with A 1 and B red cells. The techniques used and interpretation of results are described in Chapter 12. Any discrepant results should be resolved before blood is given. If transfusion is necessary before resolution, the patient should receive type O red cells. The patient s red cells must also be tested with anti-d, with suitable observations or controls to avoid a false-positive interpretation on D-negative cells. Chapter 13 discusses Rh typing reagents and appropriate control techniques. If problems in D typing arise, the patient should be given D-negative blood until the problem is resolved. Testing a recipient s red cells for weak D is not necessary, because giving D-negative cells causes no harm to recipients of the weak D phenotype. Omitting the test for weak D prevents misinterpretations arising from the presence of a positive direct

4 334 AABB Technical Manual antiglobulin test (DAT). Routine testing for other Rh antigens is not required. Detecting Antibodies to Red Cell Antigens Before deciding upon routine procedures for antibody detection, the blood bank director must decide which antibodies are considered clinically significant. In general, an antibody is considered clinically significant if antibodies of that specificity have been associated with hemolytic disease of the newborn, a hemolytic transfusion reaction, or decreased survival of transfused red cells. Antibodies reactive at 37 C and/or in the antiglobulin test are more likely to be clinically significant than those reactive only at room temperature or below. 4 Techniques for Antibody Detection Numerous serologic techniques have been developed that are suitable for detection of blood group antibodies (see Chapter 11). Goals in preparing compatible blood for a recipient are to: 1. Detect as many clinically significant antibodies as possible. 2. Detect as few clinically insignificant antibodies as possible. 3. Complete the procedure in a timely manner. Standards 1 requires that tests for unexpected antibodies must use unpooled reagent red cells, in a method that detects clinically significant antibodies, and includes an antiglobulin test preceded by incubation at 37 C. Each negative antiglobulin test must be followed by a control system of IgG-sensitized cells. If alternative procedures are used, there must be documentation of equivalent sensitivity, and the manufacturer s specified controls must be used. The method chosen should have sufficient sensitivity to detect very low levels of antibody in a recipient s serum. Transfusion of antigen-incompatible red cells to a recipient with a weakly reactive antibody may result in rapid anamnestic production of antibody with subsequent red cell destruction. The same antibody detection procedure may be used for all categories of specimens, including pretransfusion and prenatal tests on patients and screening of donor blood. Once a procedure has been adopted, the method must be described in the facility s standard operating procedures (SOP) manual and each member of the staff must know and follow the directions as written. Agglutination of Saline-Suspended Cells. The simplest serologic method is mixing serum with saline-suspended red cells. The tube may be centrifuged immediately, or incubated at either room temperature or 37 C before centrifugation. Antibodies reacting predominantly at temperatures below 37 C, such as anti- M, -N, -P 1,-Le a,and-le b, are most often detected at this phase. Many institutions omit incubation at room temperature to avoid finding antibodies that react only at low temperatures and have little clinical significance. When saline tests are included for detection of clinically significant alloantibodies, the tubes are usually incubated at 37 C for 30 minutes. Centrifugation and observation after incubation at this temperature may detect some antibodies (eg, potent anti-d, -K, or -E) that can cause direct agglutination of red cells. Also, some antibodies (eg, anti-le a,-jk a ) may be detected by their lysis of antigen-incompatible red cells during this phase. Most clinically significant antibodies bind to the cells during incubation, but do not cause agglutination and are not demonstrable until antiglobulin testing. If incubation at both room temperature and 37 C is desired, two sets of tubes are usually prepared. One set is incubated at room temperature and the other at 37 C, to avoid the positive reaction that poly-

5 Chapter 16: Pretransfusion Testing 335 specific antihuman globulin (AHG) serum may elicit if cold-reactive antibodies have bound complement to the cells. Antiglobulin Testing. For the antiglobulin phase of antibody detection and crossmatching, it is permissible to use either anti-igg or a polyspecific antiglobulin reagent that contains anti- IgG and anti-c3d,g. The anti-c3d,g activity in polyspecific reagents may detect some rare antibodies demonstrable only because they activate complement, especially those with Kidd blood group specificity, 5 or enhance reaction strength for some other antibodies. The disadvantage of using antiglobulin reagents that contain anticomplement activity is that they enhance detection of antibodies that are clinically insignificant, such as cold-reactive autoanti-i or -IH. The director of each transfusion service should decide which reagent is best suited for that laboratory, and establish a procedure for routine use. A variety of procedures and reagents can be individually applied to special investigations. Reagent Volumes. Thevolumeofserum and red cells used in routine test systems should be standardized. This is particularly important for tests using low-ionic-strength saline (LISS) that require optimal proportions of serum and low-ionic reagent. Tremendous variability in serum-to-red cell ratios can result from differences in the internal diameters of transfer pipets and reagent droppers. 6 Drops of serum dispensed from some disposable transfer pipettes tend to be small and highly variable, while droppers in reagent vials tend to deliver large drops. Many serologic procedures specify 2 drops of serum, but this volume may be too small for detection of some weak alloantibodies. 7 Increasing the serumto-red cell ratio will increase the amount of antibody uptake per cell and increase test sensitivity. If a weak antibody is suspected, the volume of serum can be increased to 3 or 4 drops, unless LISS or polyethylene glycol (PEG) is being used. Reagent Red Cells Group O red cells suitable for antibody screening are commercially available and are offered as sets of either two or three vials of single-donor red cells, or as a pool of cells from two donors. The pooled cells are for use in testing serum samples from donors and may not be used for recipients specimens, 1 because a weak antibody against an antigen present on only one of the cells might not produce a detectable reaction. Antigen Expression. The decision to use two or three cells in an antibody screening procedure should be based on circumstances in each individual laboratory. The reagent red cells are selected to express the antigens associated with most clinically relevant antibodies. Reagent cells licensed by the Food and Drug Administration (FDA) for this purpose must express the following antigens: D, C, E, c, e, M, N, S, s, P 1,Le a,le b, K, k, Fy a,fy b,jk a,andjk b. 8 There are no requirements for other antigens, such as Lu a,v,orc w. Some weakly reactive antibodies react only with screening red cells from donors who are homozygous for the genes controlling expression of these antigens, a serologic phenomenon called dosage. Antibodies in the Rh, Duffy, and Kidd systems most commonly manifest dosage. Some workers recommend that Jk(a+b ) red cells be used to provide adequate detection of anti-jk a. Preparation of Cells. Reagent red cells are suspended in a preservative solution. Aliquots may be washed and resuspended in saline or LISS solution to their original concentration, because test sensitivity diminishes with a cell suspension that is too heavy. Cells should not be stored in these wash solutions more than 24 hours because the

6 336 AABB Technical Manual solutions may not be sterile and some antigens, Fy a in particular, deteriorate rapidly in a low-ionic environment. 9 At their expiration date, reagent red cells may be as old as 9 weeks postphlebotomy. They should not be used for required tests beyond their expiration date because the strength of some antigens decreases during storage. Antigen loss may, unpredictably, be more pronounced on the red cells of some donors than of others. When not in use, reagent red cells should be refrigerated. Reading and Interpretating Reactions In serologic testing, the hemolysis or agglutination that constitutes the visible endpoint of a red cell antigen-antibody interaction must be observed accurately and consistently. Using a light source and an illuminated concave mirror enhances the sensitivity and consistency. Because a single test may result in both hemolysis and agglutination, the supernatant fluid should be observed for free hemoglobin immediately after centrifugation and, after that, the cells should be gently dispersed to observe for agglutination. The manner in which cells are dislodged from the bottom of the tube affects detection of agglutination. The tube should be held at an angle so that the fluid cuts across the cell button as the tube is gently tilted. The reaction strength (or grade) should be determined when all cells have been resuspended. Shaking tubes too vigorously may break up large agglutinates or disperse weakly cohesive agglutinates. The strength of agglutination or degree of hemolysis observed with each cell sample should be recorded immediately after the test is read. All personnel in a laboratory should use the same interpretations and notations and be consistent in grading reactions. Consistency in grading is especially important in antibody identification. Some laboratories prefer to use a numerical scoring system to indicate reaction strength and others use notations of ± to 4+. Microscopic observation is not routinely recommended, but an optical aid such as a convex mirror enhances discrimination. A microscope can be useful in distinguishing rouleaux from true agglutination and detecting specific patterns of agglutination characteristic of some antibodies, such as the mixed-field pattern seen with anti-sd a. Autologous Control Standards 1 does not require an autologous control or DAT as part of pretransfusion testing. Some workers find that routine observation of an autologous control provides useful information. Others 10 consider it of limited value, even for patients who have recently been transfused. Practical Considerations Antibody detection tests may be performed in advance of, or together with, a crossmatch between the patient s serum and donor red cells. Performing antibody detection tests before crossmatching permits early recognition and identification of clinically significant antibodies and makes the decision about immediate-spin crossmatching easier. Precrossmatch detection of antibodies allows more time to screen for donor units that lack the relevant antigen, facilitating the timely provision of blood for transfusion. Comparison with Previous Records Results of tests on a current specimen must be compared with previous transfusion service records if there has been prior testing, and the comparison must be documented. 1 Concurrence between previous and current ABO and Rh find-

7 Chapter 16: Pretransfusion Testing 337 ings gives assurance, but not proof, that no identification errors have occurred and that tests have been correctly performed and interpreted. Records are reviewed for presence of clinically significant red cell antibodies, for difficulties in testing, and for occurrence of severe adverse reactions. 1 Clinically significant red cell alloantibodies may become undetectable in a recipient s serum over time. Between 30 and 35% of antibodies become undetectable within 1 year and nearly 50% become undetectable after 10 or more years. 11 Even if the current antibody detection test is negative, the antiglobulin phase of the crossmatch is required for patients with a history of any clinically significant alloantibody. 1 Blood lacking the relevant antigens should be selected for transfusion even though red cells possessing those antigens are presently compatible in vitro. Crossmatching Tests Unless there is urgent need for blood, the recipient s serum or plasma must be crossmatched with the donor s red cells before transfusion of any red cell component. The major significance of the crossmatch is as the final check on ABO compatibility between donor and recipient. The methods used for crossmatching may be the same as those used for red cell antibody detection or they may be different. When the recipient has no clinically significant red cell antibodies and no history of such antibodies, the antiglobulin phase of the crossmatch can be omitted. 1 In this situation, only a test to detect ABO incompatibility, such as an immediate-spin or computer crossmatch, is required. It is very rare for the antiglobulin phase of the crossmatch to detect a clinically significant unexpected antibody if the patient s antibody detection test is negative. 12,13 The potential benefits of omitting a routine antiglobulin crossmatch include decreased workload, reduced reagent costs, and more effective use of blood inventory. The policy to omit the antiglobulin phase of the crossmatch for patients who meet these criteria must be made by the medical director. For patients who currently have, or have previously had, a clinically significant antibody, the crossmatch method must include antiglobulin testing as well as testing to detect ABO incompatibility. 1 Suggested Procedures for Routine Crossmatching The simplest serologic crossmatch method is the immediate-spin saline technique, in which serum is mixed with saline-suspended red cells at room temperature and the tube is centrifuged immediately (Procedure A in Table 16-1). This method is designed to detect ABO incompatibilities due to anti-a, anti-b, or anti-a,b. It can be used only if the patient has no present or previous clinically significant antibodies. Because the testing is performed at room temperature, antibodies, such as anti-m, -N, -P 1, -Le a,and-le b, may be detected that were not observed if antibody detection tests omitted room-temperature testing. Procedure B in Table 16-1 illustrates an antiglobulin crossmatch that meets the requirements of Standards 1 for all routine situations. The red cells used for crossmatching should be obtained from a sealed segment of tubing originally attached to the blood container. The cells should be washed once and resuspended to a 2-4% concentration in saline or LISS. Washing the donor s red cells removes plasma and can prevent the formation of fibrin clots. Because the ratio of serum to cells

8 338 AABB Technical Manual Table Two Acceptable Crossmatch Procedures Procedure A: * 1. Label 1 tube for each donor sample to be tested. 2. Add 2 drops of patient s serum to each tube. 3. Add 1 drop of 2-4% saline- or EDTA-saline-suspended donor red cells to each tube. 4. Centrifuge immediately, read, grade and record results. Procedure B: 1. Label 1 tube for each donor sample to be tested. 2. Add 2 drops of patient s serum to each tube. 3. Add 2 drops of 2% LISS-suspended donor red cells to each tube. 4. Centrifuge immediately, read, grade and record results. 5. Incubate at 37 C for minutes. 6. Centrifuge, read, grade and record results. 7. Perform the antiglobulin test. 8. Confirm validity of negative tests with IgG-coated red cells. *Acceptable method only if antibody detection tests are negative and the patient has no history of clinically significant antibodies. See Method 1.7. Immediate-spin tests are acceptable. However, some workers incubate the tubes at room temperature for 5 minutes to improve the sensitivity of the test. Optional step in testing. Trudeau et al 14 have demonstrated that an immediate-spin crossmatch is not necessary for detection of ABO errors when LISS-suspended red cells are used for 37 C and antiglobulin testing. markedly affects the sensitivity of agglutination tests, it is best to use the weakest cell suspension that can easily be observed for agglutination. If too many red cells are present, weak antibodies may be missed because too few antibody molecules bind to each cell. Many workers find that a 2% concentration yields the best results. Compatibility Testing for Infants Requirements for compatibility testing for infants are discussed in Chapter 22. An initial pretransfusion specimen must be obtained from the infant to determine ABO and Rh type. For ABO typing, testing only the cells with anti-a and anti-b is required. Serum or plasma from either the infant or the mother may be used to detect unexpected red cell antibodies; the infant s serum need not be tested for ABO antibodies unless non-group O cells will be transfused. If no clinically significant unexpected antibodies are present, it is unnecessary to crossmatch donor red cells for the initial or subsequent transfusions. Repeat testing may be omitted for an infant less than 4 months of age during any one hospital admission. Computer Crossmatch When it is permissible to omit the antiglobulin phase of the crossmatch and perform only a procedure to detect ABO incompatibility, computerized selection

9 Chapter 16: Pretransfusion Testing 339 of blood can be used to fulfill the requirement, provided that the following conditions have been met 1 : 1. The computer system has been validated, on site, to prevent the release of ABO-incompatible components. 2. The recipient s ABO group has been determined at least twice, with complete testing of both serum and cells. One determination must be made on a current sample. The second determination may be made on the same sample, on a second current sample, or by comparison with previous records. If previous computer records are used, the system must have been validated to accept only the results of testing on specimens known to have been accurately identified. 3. The computer system contains the identification number and the component name of the donor unit, the ABO and Rh types of the component, the interpretation of the blood group confirmatory test, and the identification and ABO and Rh type of the recipient. 4. A method exists to verify correct entry of data prior to release of blood or components. 5. The system contains logic to alert the user to discrepancies between information on the donor unit label and the interpretation of the blood group confirmatory test, and to ABO incompatibilities between recipient and donor unit. Butch et al 15,16 have described in detail the components of a model computer crossmatch system. Potential advantages of a computer crossmatch include: decreased workload, reduced volume of patients blood samples, and reduced exposure of personnel to blood specimens. Use of a computer crossmatch requires FDA approval of a request for a variance to regulations on compatibility testing, found in 21 CFR The regulations regarding use of alternate procedures are found in 21 CFR Routine Surgical Blood Orders Blood ordering levels for common elective procedures can be developed from previous records of blood use. Because surgical requirements vary among institutions, routine blood orders should be based on local transfusion utilization patterns. The surgeons, anesthesiologists, and the medical director of the blood bank should agree on the number of units required for each procedure. See Chapter 3 for more detailed discussion. Routine blood order schedules are successful only when there is cooperation and confidence among the professionals involved in setting and using the guidelines. Once a surgical blood ordering schedule is established, the transfusion service routinely crossmatches the predetermined number of units for each patient undergoing the designated procedures. Routine orders may need to be modified for patients with anemia, bleeding disorders, or other conditions in which increased blood use is anticipated. As with other circumstances that require rapid provision of blood, the transfusion service staff must be prepared to provide additional blood if an unexpected problem requires additional blood. Type and Screen Type and screen is a policy in which crossmatched blood is not labeled and reserved for patients undergoing surgical procedures that rarely require transfusion. Instead, the patient s blood sample is tested for ABO, Rh, and unexpected antibodies, and then stored in the blood bank for immediate crossmatching, should this prove necessary. The blood bank must have enough donor blood available to meet unexpected needs of

10 340 AABB Technical Manual patients undergoing operations on a type and screen basis. If transfusion becomes necessary, ABO- and Rh-compatible blood can be safely released, for patients with no clinically significant antibodies, after an immediate-spin or computer crossmatch. If the antibody screen is positive, the antibody(ies) must be identified and antigen-negative units identified for use if needed. Interpretation of Antibody Screening and Crossmatch Results Negative Antibody Screen, Compatible Crossmatch The vast majority of samples tested have a negative antibody screen and are crossmatch-compatible with units selected. A negative antibody screen does not guarantee that the serum is free of clinically significant red cell antibodies, only that it contains no antibodies that react with the screening cells by the techniques employed. A compatible crossmatch does not guarantee normal red cell survival. Negative Antibody Screen, Incompatible Immediate-Spin Crossmatch In the following situations the antibody screen may be negative but one or more immediate-spin crossmatches are incompatible: 1. The donor red cells are ABO-incompatible. When agglutination is noted at immediate spin, the first thing to do is determine the donor s ABO group. Labeling errors are more likely to cause this problem than technical errors. Most ABO antibodies in a recipient s serum react strongly with most mislabeled A, B, or AB cells at room temperature or 37 C, and in the antiglobulin test. Rarely, donor red cells with weak expression of A or B antigens (eg, A x ) have been incorrectly labeled as group O, but the serum of a group O recipient is unlikely to agglutinate such cells on immediate spin. 2. Anti-A 1 is present in the serum of an A 2 or A 2 B individual. If an immediate-spin crossmatch is performed, anti-a 1 will cause some, but not all, group A donor units to be incompatible. If the antibody reacts only at room temperature, it is unlikely to shorten the survival of transfused cells. 4 If it is reactive at 37 C, crossmatch-compatible blood should be given. 3. Other alloantibodies, reactive at room temperature, are present. If the antibody detection protocol does not include a room temperature phase, antibodies such as anti-m may not be detected until an immediate-spin crossmatch with cells that express the antigen. To identify the responsible antibody, identification procedures should include testing at room temperature. 4. The donor cells are polyagglutinable. All normal adult sera contain varying levels of non-red-cellstimulated antibodies (anti-t, -Tn, -Tk, -Cad, etc) that react with red cells of abnormal surface characteristics. These antibodies are rarely apparent because reagent red cells and red cells from most donors do not manifest the corresponding antigens. The recipient s serum is nonreactive with antibody screening cells, panel cells, and cells from all other donors, but as these antibodies are direct agglutinins that react best at room temperature, the immediate-spin or room temperature phase of the crossmatch would identify the donor s abnormality.

11 Chapter 16: Pretransfusion Testing 341 Negative Antibody Screen, Incompatible Antiglobulin Crossmatch If an antiglobulin test is part of the crossmatch procedure, some of the following situations may be encountered. 1. Donor red cells have a positive DAT. Approximately 1 in 10,000 healthy donors have red cells that are coated with IgG and/or complement and cause incompatibility in the antiglobulin phase of the crossmatch. 4 These units are not identified during processing because the only donor units subjected to antiglobulin testing are D-negative units being tested for weak D. The problem can be quickly and easily identified by performing a DAT on the cells. The significance of a positive DAT in a normal donor is unknown. Because all crossmatches are positive if carried to the antiglobulin phase, the unit should not be transfused. Transfusion services should develop a procedure to ensure that these units are disposed of properly. 2. Antibody reacts only with cells having strong expression of a particular antigen. A random donor s red cells may have stronger expression of a particular antigen than screening cells, either because of dosage (eg, Rh, Kidd, Duffy, and MN antigens) or because of intrinsic variation in antigen strength (eg, P 1 ). If the potential recipient has a very weakly reactive antibody of that specificity, the antibody screen may be negative but the crossmatch is incompatible. The antibody can usually be identified by testing a full panel of red cells, because some of the panel cells are likely to have a double dose of the antigen in question. More sensitive techniques may be necessary for identification. 3. Antibody reacts with a low-incidence antigen. As many as 1% of unselected sera may contain antibodies to some of the low-incidence antigens. Low-incidence antigens are generally absent from cells used in screening tests, but may be present on cells of a single donor, resulting in a negative antibody screen and an incompatible crossmatch. 4. Passively transferred antibody is present. It is very rare for transfused blood or components to contain clinically significant unexpected antibodies, but significant circulating levels of anti-a or -B may be present after infusion of group O plasma. Recent transfusion of group O platelets to a non-group O recipient is the most frequent causative event. Positive Antibody Screen, Compatible Crossmatches If serum reacts with all reagent red cells but many or all of the crossmatches are compatible, antibodies of one of the following specificities may be responsible: 1. Auto-anti-H (-IH). Group O cells have large amounts of H antigen while A 1 and A 1 B cells have very little. Serum containing auto-anti-h may agglutinate all group O reagent cells, but not A 1 and A 1 B donor cells. Most examples of apparent autoanti-h are actually anti-ih. These antibodies usually react only at room temperature and are not clinically significant. Reactivity in the antiglobulin phase can usually be circumvented by the use of a prewarming technique and/or a monospecific anti-igg antiglobulin reagent. 2. Anti-Le bh. This antibody reacts with Le(a b+) red cells that are group O, but not with A 1 or A 1 B cells that are Le(a b+). It is most often made by group A 1 or A 1 B Le(a b ) individuals. Anti-Le bh is often reactive at room temperature but may be de-

12 342 AABB Technical Manual tected at the antiglobulin phase. See Chapter 12. Positive Antibody Screen, Incompatible Crossmatches Alloantibodies, autoantibodies, untoward interactions with reagents, and rouleaux formation may cause the antibody screen, the crossmatch, or both to be positive. The problem should be identified before issuing blood for transfusion unless the need for transfusion is urgent. 1. Alloantibody is present and autologous control is negative. When unexpected alloantibodies are present, the antibody screening test will usually be positive, but the frequency of the antigen influences how many crossmatches will be incompatible. When the antibody screen is positive, the specificity of the antibody(ies) should be identified and reagent antibody used to confirm that red cells from crossmatch-compatible units lack the corresponding antigen. Alternatively, donor units can first be screened with reagent antibody and negative units be selected for crossmatching. It is not necessary to confirm the absence of antigen if the patient s antibody is anti-m, -N, -P 1,-Le a, and/or -Le b. 17 If multiple antibodies or an antibody reacting with a high-incidence antigen is present, or if the antibody is present in very low concentration, it may be impossible to identify the antibody(ies) with available resources. If time permits, a sample can be sent to a reference laboratory for resolution. For the patient with an antibody to a high-incidence antigen, siblings are the most promising source of compatible blood. The rare donor file can be contacted, if necessary. 2. Alloantibody is present and autologous control is positive. An autologous control is ordinarily included in the procedures used to identify alloantibody detected in a screening test. 18 In several circumstances, an alloantibody may cause a positive reaction with autologous cells in patients who have received blood or components within the preceding 2-3 months. Alloantibody in the patient s serum may be reacting with transfused donor cells. Mixed-field agglutination is usually noted because only the subpopulation of red cells positive for the antigen react with antibody. These results are seen in patients experiencing delayed serologic or hemolytic transfusion reactions. Antibody may be present in such low concentration that it is demonstrable only in an eluate prepared from the antigen-positive red cells. Enhancement techniques are often needed to demonstrate antibody in serum or eluates. An alloantibody reacting with circulating donor cells can be easily misinterpreted as autoantibody. The distinction is important because selection of blood for transfusion differs in the two situations. In most circumstances it is not necessary to find donor blood compatible with an autoantibody, whereas alloantibodies should be identified and, if clinically significant, subsequently transfused cells should lack the corresponding antigen. The patient s diagnosis and transfusion history may help in making the distinction. Anti-Fy a eluted from red cells would very likely be alloantibody, but apparent anti-e in an eluate could be either alloantibody or autoantibody. Alloantibody reactive with the recipient s cells has been passively transferred in transfused plasma or

13 Chapter 16: Pretransfusion Testing 343 plasma derivatives. If antibody is present in the serum, the antibody screen and crossmatches will probably be positive, and antigen-negative blood can be selected until the antibody is no longer demonstrable. 3. Cold-reactive autoantibody is causing spurious positive results. Potent cold-reactive autoantibodies may cause problems with ABO and Rh typing, antibody detection, antibody identification, and/or crossmatching. The most common specificity of cold autoantibody is anti-i, but transfusion of i adult blood is unnecessary. It is, however, important to determine the patient s correct ABO and Rh types and determine whether any clinically significant alloantibodies are present. 4. Warm-reactive autoantibody is causing spurious positive results. Warmreactive autoantibodies rarely cause discrepancies in ABO testing, but commonly interfere with Rh tests that use reagents with a high concentration of potentiators. See Chapter 13 for a more detailed discussion. If autoantibody is present in the serum, antibody screening tests will be positive and crossmatches incompatible. Before selecting blood for a patient with warm-reactive autoantibodies, the serum must be evaluated for the simultaneous presence of clinically significant alloantibodies. 5. Rouleaux formation is causing spurious positive results. Rouleaux formation results from serum characteristics that cause all cells tested to appear agglutinated, both at room temperature and at 37 C. Rouleaux formation is usually identified microscopically as clumps of cells said to resemble stacked coins; however, rouleaux can be difficult to identify when the cells adhere in irregular clumps that resemble agglutination. Rouleaux and agglutination can be distinguished by adding 1-3 drops of saline to the tube; rouleaux will disperse but antibody-mediated agglutination remains unchanged. See Method 3.4. Rouleaux formation does not usually affect the antiglobulin test, because serum is removed by washing before addition of antiglobulin serum. 6. Reagent-related problems. Antibodies to a variety of drugs and additives can cause positive results in antibody screening and/or compatibility tests. See Chapter 17. Labeling and Release of Crossmatched Blood A blood transfusion record indicating the recipient s name, identification number and ABO and Rh types must be completed for each unit of donor blood or component. 1 The record must also include the identification number and the ABO and Rh type of the donor unit; the interpretation of crossmatch tests, if performed; and the date of transfusion. It is good practice to include identification of the person(s) performing the test and, if blood is issued before resolution of compatibility problems, the status of the serologic findings. Before issuing a unit of blood, blood bank personnel must: 1. Securely attach to the container a label or tag that contains the recipient s first and last names and identification number; the identification of the donor unit; and the interpretation of compatibility testing, if performed. If compatibility testing is not complete at the time of issue, this must be conspicuously indicated. 2. Check the expiration date of the blood to avoid issuing an outdated component.

14 344 AABB Technical Manual 3. Inspect the unit to make certain it does not have an abnormal color or appearance and document the inspection. 4. Record in a permanent manner the identity of the person issuing the blood; the identity of the person to whom the blood was issued or the destination of the unit; and the date and time of issue. Final identification of the recipient and the blood container rests with the transfusionist, who must identify the patient and donor unit and certify that identifying forms, tags, and labels are in agreement. See Chapter 20. Selection of Units ABO Compatibility Whenever possible, patients should receive ABO-identical blood components; however, it may be necessary to make alternative selections. If the component to be transfused contains 5 ml or more of red cells, the donor s red cells must be ABO-compatible with the recipient s plasma. Because plasma-containing products can affect the recipient s red cells, the ABO antibodies in transfused plasma should be compatible with the recipient s red cells. Requirements for components and acceptable alternative choices are summarized in Table Rh Type Rh-positive blood components should routinely be selected for D-positive recipients. Rh-negative units are permissible in special circumstances, but should be reserved for D-negative recipients. D- negative patients should receive redcell-containing components that are D- negative to avoid immunization to the D antigen. Occasionally, ABO compatible Rh-negative components may not be available. In this situation, the blood bank physician and the patient s physician should weigh alternative courses of action. The clinician may elect to postpone transfusion, or may decide that the 80% risk of immunization is less significant than the risk of delaying transfusion. Depending on the child-bearing potential of the patient and the volume of red cells transfused, it may be desirable to administer Rh Immune Globulin to a D-negative patient given D-positive blood components. 19 Other Blood Groups Antigens other than ABO and D are not routinely considered in the selection of units of blood. However, if the recipient Table Selection of Components When ABO-Identical Donors Are Not Available ABO Requirements Whole Blood Must be identical to that of recipient. * Red Blood Cells Must be compatible with the recipient s plasma. Granulocytes, Pheresis Must be compatible with the recipient s plasma. Fresh Frozen Plasma Should be compatible with the recipient s red cells. Platelets, Pheresis * All ABO groups acceptable; components compatible with recipient s red cells preferred. Cryoprecipitated AHF All ABO groups acceptable. *Since a large volume of plasma is administered, components compatible with the recipient s red cells are preferred.

15 Chapter 16: Pretransfusion Testing 345 has a clinically significant unexpected antibody, antigen-negative blood should be selected for crossmatching. If the antibody is weakly reactive or no longer demonstrable, reagent antibody should be used to screen donor units before a crossmatch is performed. If, on the other hand, there is an adequate quantity of patient s serum and the antibody reacts well with antigen-positive red cells, a more economical approach is to use the serum to screen units and use the reagent antibody to confirm that the compatible units are antigen-negative. When commercial reagents are not available stored serum specimens from patients or donors can be used, especially for low-incidence or uncommon antigens. Release of Blood in Urgent Situations When blood is urgently needed, the patient s physician must weigh the risk of transfusing uncrossmatched or partially crossmatched blood against the risk of delaying transfusion until compatibility testing is complete. Ideally, a transfusion service physician should provide consultation. The risk that the transfused unit might be incompatible may be judged to be less than the risk of depriving the patient of oxygen-carrying capacity. Required Procedures When blood is released before pretransfusion testing is complete, the records must contain a signed statement of the requesting physician indicating that the clinical situation was sufficiently urgent to require release of blood. 1 Such a statement does not absolve blood bank personnel from their responsibility to issue properly labeled donor blood that is ABO-compatible with the patient. When urgent release is requested, blood bank personnel should: 1. Issue uncrossmatched blood, which should be: a. Group O Red Blood Cells if the patient s ABO group is unknown. It is preferable to give Rh-negative blood if the recipient s Rh type is unknown, especially if the patient is female with the potential to bear children. b. ABO and Rh compatible, if there has been time to test a current specimen. Previous records must not be used, nor should information be taken from other records such as cards, dog tags, or driver s license. 2. Indicate in a conspicuous fashion on the attached tag or label that compatibility testing was not complete atthetimeofissue. 3. Begin compatibility testing and complete crossmatches promptly. If incompatibility is detected at any stage of testing, the patient s physician and the transfusion service physician should be notified immediately. Standard compatibility tests should be completed promptly for those units issued for initial replacement of the patient s blood volume. Massive Transfusion Massive transfusion is defined as infusion, within a 24-hour period, of a volume of blood approximating the recipient s total blood volume. Exchange transfusion of an infant is considered a massive transfusion. Following massive transfusion, the pretransfusion sample no longer represents the blood currently in the patient s circulation and its use for crossmatching has limited benefit. It is only important to confirm ABO compatibility of units administered subsequently. The blood bank director should decide what procedures to use in these situations, put them in writing, and ensure their

16 346 AABB Technical Manual consistent application by all laboratory personnel. Blood Administered After Non-Group-Specific Transfusion When patients have been transfused with blood of an ABO group other than their own and additional transfusions are needed, it may be desirable to use ABOidentical units. The safety of this conversion depends on the status of anti-a and/or anti-b in a current sample from the intended recipient. When red cells of the patient s original ABO group are compatible with serum from a freshly drawn specimen, they may be issued for transfusion. If the crossmatch is incompatible because of ABO antibodies, transfusion with red cells of the alternative group should be continued. Transfusion services sometimes release units for transfusion during emergencies before they receive a sample for blood typing. When it arrives, the sample is a pretransfusion specimen. In most cases, the sample is tested and units of that ABO group are issued for transfusion without concern for anti-a and/or anti-b remaining from the initial emergency-release units. Because most donor units are Red Blood Cells with comparatively little supernatant plasma, or red cells in additive solutions with even less residual plasma, the risks in this practice are minimal. If the change in blood type involves only the Rh system, return to type-specific blood is simple, since antibodies are unlikely to be present in the plasma of either recipient or donor. If a patient has received blood of an Rh type other than his or her own before a specimen is collected for testing, it may be difficult to determine the correct Rh type. If there is any question about the recipient s D type, D-negative blood should be transfused. References 1. Klein HG, ed. Standards for blood banks and transfusion services, 17th ed. Bethesda, MD: American Association of Blood Banks, Sazama K. Reports of 355 transfusion associated deaths: 1976 through Transfusion 1990;30: Procedures for the collection of diagnostic blood specimens by venipuncture. 3rd ed. NCCLS document H3-A2, approved standard. Villanova, PA: National Committee for Clinical Laboratory Standards, Mollison PL, Engelfriet CP, Contreras M. Blood transfusion in clinical medicine, 9th ed. Oxford: Blackwell Scientific Publications, Howard JE, Winn LC, Gottlieb CE, et al. Clinical significance of the anti-complement component of antiglobulin antisera. Transfusion 1982;22: Beattie KM. Control of the antigen-antibody ratio in antibody detection/compatibility tests. Transfusion 1980;20: Brown PJ. Antibody identification. In: Rudmann SV, ed. Textbook of blood banking and transfusion medicine. Philadelphia, PA: WB Saunders, 1995: Code of federal regulations. Title 21, part 660, subpart D Washington, DC: US Government Printing Office, 1994 (revised annually). 9. Lizza C, Myers J, Gindy L. Blood Groups. In: Petz LD, Swisher SN, Kleinman S, et al, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill Livingstone, 1996: Judd WJ, Barnes BA, Steiner EA, et al. The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited. Transfusion 1986;26: Ramsey G, Smietana SJ. Long term follow-up testing of red cell alloantibodies. Transfusion 1994; 34: Oberman HA. The present and future crossmatch. Transfusion 1992;32: Meyer EA, Shulman IA. The sensitivity and specificity of the immediate-spin crossmatch. Transfusion 1989;29: Trudeau LR, Judd WJ, Butch SH, Oberman HA. Is a room-temperature crossmatch necessary for the detection of ABO errors? Transfusion 1983;23: Butch SH, Judd WJ, Steiner EA, et al. Electronic verification of donor-recipient compatibility: the computer crossmatch. Transfusion 1994;34: Butch SH, Judd WJ. Requirements for the computer crossmatch. Transfusion 1994; 34:187.