Red cell alloantibodies

Transcription

1 17 Identification of Alloantibodies to Red Cell Antigens Red cell alloantibodies other than non-red-cell-stimulated anti-a or -B are called unexpected red cell alloantibodies and may be found in % of the population, depending upon the group of patients or donors studied and the sensitivity of the test methods used. 1,2 In contrast to red cell autoantibodies, which may also react with red cells from other individuals (see Chapter 18), alloantibodies react only with antigens present on red blood cells from persons other than the self. Non-self immunization to red cell antigens may result from pregnancy or transfusion, or from injection with immunogenic material. In some instances no immunizing event can be identified. Significance of Alloantibodies Alloantibodies to red cell antigens may be initially detected in any test that uses a serum [including ABO testing, the antibody detection test (antibody screen), or the crossmatch] or eluate. Ordinarily, once an antibody is detected, its specificity should be determined and its clinical significance assessed. A clinically significant red cell antibody is one that shortens the survival of transfused red cells or has been associated with hemolytic disease of the newborn (HDN). The degree of clinical significance may vary, however, even among antibodies with the same specificity. Some antibodies cause destruction of incompatible red cells within hours or even minutes, others decrease the survival by only a few days, and some cause no discernible cell destruction. Antibodies of some specificities are known to cause HDN; some may cause a positive direct antiglobulin test (DAT) in the fetus without clinical evidence of HDN; and others do not cause HDN. Reported experience with other examples of antibody with the same specificity can be used in assessing clinical significance. Table 14-2 summarizes the expected reactivity and clinical significance of commonly encountered alloantibodies, and Marsh et al 3 have published

2 350 AABB Technical Manual a review of these and other specificities. For some antibodies, few or no data exist, and decisions must be based on the premise that clinically significant antibodies are usually those active at 37 C and/or by the indirect antiglobulin test (IAT). It is not necessarily true, however, that all antibodies active in vitro at 37 C and/or by the IAT are clinically significant; not all antibodies serologically reactive only below 37 C are benign. Antibodies encountered in pretransfusion testing should be identified, for assessment of the need to select antigennegative blood for transfusion. Patients with clinically significant antibodies should, whenever practical, receive red cells that have been tested and found to lack the corresponding antigen. In prenatal testing, the specificity and immunoglobulin class of an antibody influence the likelihood of HDN. While identification of unexpected antibodies in donor blood is not required, results of such testing are useful for characterizing the units for transfusion use and for procuring blood grouping reagents or teaching samples. General Procedures Specimen Requirements Either serum or plasma may be used for antibody identification; serum is used more widely, and that is the only term that will be used in this chapter. Plasma is not suitable for detection of complement activation. When autologous red cells are studied, the use of a sample anticoagulated with EDTA avoids problems associated with the in-vitro uptake of complement components by red cells that may occur with a clotted sample. A 10 ml aliquot of whole blood usually contains enough serum for identifying simple antibody specificities; more may be required for more complex studies. Medical History It is useful to know a patient s clinical diagnosis, history of transfusions or pregnancies, and recent drug therapy. In patients who have had recent red cell transfusions, the circulating blood may be so admixed with donor cells that special procedures are needed to separate the autologous red cells for typing (see Method 2.15). Other procedures will be necessary for patients known to have autoantibodies, which may be diseaseassociated or induced by drugs (see Chapter 18). Reagents Red Cell Panels Identification of an antibody to red cell antigens requires testing the serum against a panel of selected red cell specimens, usually eight or more. They are usually obtained from commercial suppliers, but institutions may assemble their own by using red cells from local sources. Panel cells are (except in special circumstances) group O, allowing serum of any ABO group to be tested, and the phenotypes for antigens of the major blood groups are recorded. Each cell of the panel is from a different individual. The cells are selected so that, taking all the cells into account, a distinctive pattern of positives and negatives exists for each of many antigens. To be functional, a reagent red cell panel must make it possible to identify with confidence those clinically significant alloantibodies that are most frequently encountered, such as anti-d, -E, -K, and -Fy a. The phenotypes of the reagent red cells should be distributed such that each of the common alloantibodies, if it is the only one in a serum, can be clearly identified and most others at least tentatively excluded. The pattern of reactivity for most examples of single alloantibodies should not overlap with any

3 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 351 other; eg, all of the K+ samples should not be the only ones that are also E+. To lessen the possibility that chance alone has caused an apparently definitive pattern, there must be sufficient number of red cell samples that lack, and sufficient red cell samples that carry, most of the antigens listed in Table Commercially prepared panels are generally issued every 2-4 weeks. Each panel contains different cells, with different antigen patterns, so it is essential to use the phenotype listing sheet that comes with the panel in use. Commercial cells usually come as a 2-5% suspension in a preservative medium; they can be used directly from the vial, although some workers prefer to wash the cells with saline and obtain a dry cell button to which the test serum is added. For solid phase methods, panels of dried red cell monolayers may be prepared and stored for long periods before use. 4 Enhancement Media Although the test system may consist solely of serum and cells, most workers use some kind of enhancement medium. Many different media are available, including low ionic strength saline (LISS), polyethylene glycol (PEG), and albumin. For the initial identification panels, most laboratories use the same enhancement method used in their routine antibody detection and crossmatch tests. Additional enhancement techniques may be employed for more complex identification. Enhancement techniques are discussed later in this chapter. Antiglobulin Reagents Most antibody identification tests include an antiglobulin phase. Either polyspecific or IgG-specific antiglobulin reagents may be used. Polyspecific reagents may detect, or detect more readily, antibodies that bind complement. While this may be advantageous in some instances, many workers prefer to use IgG-specific reagents to avoid unwanted reactivity due to in-vitro complement binding by cold-reactive antibodies. Table A Reagent Red Cell Panel for Alloantibody Identification Sample Rh Rhesus Kell Duffy Kidd P Lewis MNS # Phenotype C C w c D E e K Fy a Fy b Jk a Jk b P 1 Le a Le b MNS s 1 r r w R R R r r r r r r R Denotes presence of antigen; 0 denotes absence of antigen.

4 352 AABB Technical Manual Autologous Control It is important to know how a serum under investigation reacts with autologous red cells. This helps determine whether alloantibody, autoantibody, or both are present. Serum that reacts only with the reagent red cells usually contains only alloantibody, whereas reactivity with both reagent and autologous red cells suggests the presence of autoantibody, or autoantibody plus alloantibody. A patient with alloantibodies to antigens on recently transfused red cells may have circulating donor red cells coated with alloantibodies, that produce a positive autocontrol, usually in a mixed-field pattern. This may be misinterpreted as being due to autoantibody. A detailed history of recent transfusions should be obtained for all patients with a positive DAT. An autocontrol provides useful information in antibody identification studies, even if one has been previously performed, so that reactions with autologous and reagent red cells can be compared. When test methods used for antibody identification are different from or additional to the initial autocontrol method, an autologous control is essential. An autocontrol is not required in all circumstances, especially if the serum is not reactive by the method used. It may not be necessary to prepare enzyme-treated autologous cells when testing the serum against panel cells pretreated with enzyme. The autologous control, in which serum and autologous cells undergo the same manipulation as serum and reagent cells, is not the same as a DAT. If the autocontrol is positive in the antiglobulin phase, a DAT should be performed. If the DAT is positive, elution studies should be considered if the patient has been recently transfused, if there is evidence of immune hemolysis and/or if the results of serum studies prove inconclusive. A reactive DAT may also indicate the presence of autoantibody. If autoantibody is detected in the serum, adsorption studies may be necessary to establish that it is not masking coexisting alloantibodies. Basic Antibody Identification Techniques Traditional serologic methods based on agglutination and performed in test tubes or microplates remain the most commonly used and are the primary techniques discussed in this chapter. Other methods, which modify traditional test endpoints or are not dependent on agglutination, are also available. These include solid phase, flow cytometry, and gel or column techniques, as well as some automated systems. Such methods may be used in addition to or instead of the classical techniques discussed below. Interpretation of reaction patterns to identify antibody specificity are the same for all tests. Initial Observations Red cell panels are usually employed to investigate a serum already known to contain antibody, through reactivity detected in preliminary tests such as the antibody screening test or crossmatch. These initial tests provide important information about the test phases at which reactivity occurs, and the serum may sometimes be tested only by the technique with which the antibody was originally detected. For initial panels, however, it is common to use the same methods and range of phases used in the initial procedures; typically these include room temperature and 37 C testing, often with enhancement medium, and IAT. This ensures that the serum has been tested against cells of various phe-

5 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 353 notypes and antigen strengths, and guards against missing weaker reactions at some phases. Knowing the phase of initial reactivity may suggest possible specificities. Reactivity only at room temperature, for example, suggests the possibility of anti- M, -P 1, -I, -Le a,or-le b. The phenotype of the originally-reactive antibody detection cells may also provide clues as to specificity, or help exclude specificities. If multiple panels are available, this information can help in selecting the cells likely to be informative. If the patient has had antibodies previously identified, this may affect panel selection. For example, if the patient is known to have anti-e, it will not be helpful to test the serum against a panel of 10 cells, nine of which are e-positive. Testing a panel of selected e-negative cells will better reveal any newly formed antibodies. Sometimes the patient s phenotype influences the selection of reagent cells. If the patient is D-negative and the serum is reactive with D-positive cells in the screening test, an abbreviated panel of D-negative cells may be tested. This can both confirm the presence of anti-d and demonstrate the presence of additional antibodies. 5 Interpreting Results Interpretation of panel results can be a complex process combining technical knowledge and intuitive skills. Panel results will include a range of positive and negative results, each of which should be explained by the final conclusion. Positives and Negatives Both positive and negative reactions are important in antibody identification. Positive reactions indicate the phase and strength of reactivity, which can suggest specificities expected to react in that manner. Positive reactions also can be compared to the antigen patterns expressed by the panel cells to help assign specificity. Single alloantibodies usually yield definite positive and negative reactions that create a clear-cut antigen pattern with reagent red cell samples. For example, if a serum reacts only with cells 4 and 5 of the reagent red cell panel shown in Table 17-1, anti-e is very likely present. Both reactive samples express E and all nonreactive samples lack E. Negative reactions are also important in antibody identification, because they allow at least tentative exclusion of antibodies to antigens expressed on the nonreactive cells. Exclusion of antibodies is an important step in the interpretation process and must be done to ensure proper identification of all antibodies present. Exclusion or Crossing Out A widely used first approach to the interpretation of panel results is to exclude specificities based on nonreactivity with the serum tested. Such a system is sometimes referred to as a cross-out or rule-out method. Once results have been recorded on the worksheet, the antigen profile of the first nonreactive cell is examined. If an antigen is present on the cell and the serum did not react, the presence of the corresponding antibody may be at least tentatively excluded. Many workers will actually cross out that antigen from the listing on the panel sheet to facilitate the process. After all antigens present on that cell have been crossed off, interpretation proceeds with the other nonreactive cells and additional specificities are excluded. In most cases, this process will leave a group of antigens that still have not been excluded. Next, the cells reactive with the serum are evaluated. The pattern of reactivity for each nonexcluded specificity is

6 354 AABB Technical Manual compared to the pattern of reactivity obtained with the test serum. If there is a pattern that matches exactly, that is most likely the specificity of the antibody in the serum. However, if there are remaining specificities that have not been excluded, additional testing may be needed to eliminate remaining possibilities and to confirm the specificity identified. This requires testing the serum against additional cells. Once tentative identification has been established, testing the serum against cells selected for specific antigenic characteristics gives more information than using an additional, unmodified panel. For example, if the pattern of positive reactions exactly fits anti-jk a, anti-k and anti-s may still not be excluded. The serum should be tested against selected cells, ideally with the three phenotypes: Jk(a ), K, S+; Jk(a ), K+, S ; and Jk(a+), K, S. The reaction pattern with these cells should both confirm the presence of anti-jk a and include or exclude anti-k and anti-s. While the exclusion (cross-out) approach often identifies simple antibody specificities, it should be considered only a provisional step. As discussed below, some antibodies may be mistakenly excluded if, for some reason, no reactivity was obtained with a cell positive for the antigen. On occasion, similar patterns may be obtained for different specificities or when multiple antibodies are present. Computer programs are available that can interpret panel results with some success. Such programs can utilize information read by traditional manual methods and entered by the user, or can be coupled to automated test systems that directly transmit test results. Probability Conclusive antibody identification requiresserumtobetestedagainstsuffi- cient reagent red cell samples that lack, and that carry, the antigen that appears to correspond to the specificity of the antibody, to ensure that an observed pattern is not due to chance alone. Calculations The traditional way to calculate the probability that an antibody has been correctly identified is Fisher s exact method, in which the numbers of positive and negative results are compared with the numbers of cells that express or lack the corresponding antigen. 6 [See Table 17-2 (A).] For this method, a probability (p) value of 0.05 is generally accepted as a minimum value for considering an interpretation statistically valid. (See Table 17-3.) This means that chance alone would produce an identical set of results once in 20 similar studies. Most basic red cell panels have limited capacity for conclusive identification of some specificities, especially when multiple antibodies are present. To meet the p 0.05 statistical standard, tests with additional cells are often necessary. Fisher s exact method has been challenged as being too conservative and not addressing population frequencies for antigens for which the cells have not been typed. Harris and Hochman 7 have derived an alternative calculation to allow for such antigens. [See Table 17-2 (B).] Comparative p values by their method are also given in Table This calculation allows more liberal interpretation of results, and may require less actual testing to confirm specificity. Interpretation Most workers rarely use either statistical method in a conscious manner. A standard approach (based on Fisher s exact method) has been to require, for each specificity identified, three antigenpositive cells that do react and three an-

7 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 355 Table Calculation of Probability A. Fisher s Exact Method 6 The formula for calculating probability (p) is: (A+B)! (C+D)! (A+C)! (B+D)! N! A! B! C! D! B. Modification of Harris and Hochman 7 (A/N) A (B/N) B A = number of positive reactions observed with antigen-positive red cell samples B = number of positive reactions observed with antigen-negative red cell samples C = number of negative reactions observed with antigen-positive red cell samples D = number of negative reactions observed with antigen-negative red cell samples N = number of cells tested! = factorial, the product of all the whole numbers from 1 to the number involved. For example, 6! = =720 1! = 1 0! = 1 Consult references and suggested readings for further details on calculations. Table Probability Values No. Tested No. Positive No. Negative p (Fisher 6 ) p (Harris and Hochman 7 )

8 356 AABB Technical Manual tigen-negative cells that fail to react. This standard is not always possible, but works well in practice, especially if cells with strong antigen expression are available. A somewhat more liberal approach is derived from calculations by Harris and Hochman, whereby minimum requirements for a p value of 0.05 are met by having two positive and three negative cells, or one positive and seven negative cells (or the reciprocal of either combination). The possibility that the serum fails to react with antigen-positive cells (or of some false-positive results) must also be considered in determining specificity. Additional details on calculating probability may be found in the suggested readings by Race and Sanger and by Menitove. Phenotype of Autologous Red Cells Once an alloantibody has been identified in a serum, it is often helpful to demonstrate that the autologous red cells are negative for the corresponding antigen. For example, if serum from an untransfused individual appears to contain anti- Fy a but the autologous red cells have a negative DAT and type as Fy(a+), the data are clearly in conflict and further testing is indicated. Determination of the patient s phenotype can be difficult if the patient has been transfused recently, generally within 3 months. If a pretransfusion specimen is still available, these red cells should be used to determine the phenotype. Alternatively, the patient s own red cells can be separated from the transfused red cells and then typed. Procedures for this are given in Methods 2.15 and The use of potent blood grouping reagents, appropriate controls, and observation for mixed-field reactions often allows an unseparated specimen to be phenotyped. If there is little uncertainty about antibody identification, extensive efforts to separate and type the patient s own red cells are not necessary. Antigen-negative blood can be selected for transfusion; for patients with clinically significant antibodies, an antiglobulin phase crossmatch is required, 8 and a compatible crossmatch will provide additional confirmation of antibody specificity. Definitive testing can be done on the patient s red cells after a period without transfusion or, if this is not possible, after an interval during which only antigen-negative blood has been given. If the chronically transfused patient is not aplastic, any antigen-positive red cells detected after prolonged transfusion of antigen-negative blood would presumably be the patient s own. Complex Antibody Problems Not all antibody identifications are simple. The exclusion procedure does not always lead directly to an answer and additional approaches may be required. Figure 17-1 shows some approaches to identifying antibodies in a variety of situations when the autocontrol is negative. Additional approaches may be needed if the autocontrol is positive; these are discussed later in this chapter. Variations in Antigen Expression For a variety of reasons, antibodies do not always react with all cells positive for the corresponding antigen. Interpretation by exclusion may cause a given specificity to be crossed out if a cell is antigen-positive and the serum is nonreactive, despite the presence of the antibody. Sometimes, this prevents discernment of any pattern, but in some cases it may, by coincidence, yield a false pattern of specificity. Technical error or weak

9 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 357 REAGENT RED CELL PANEL NEGATIVE AUTO CONTROL SOME CELLS POSITIVE (SAME STRENGTH AND PHASES) SOME CELLS NEGATIVE Suspect Single Antibody ALL CELLS POSITIVE (DIFFERENT STRENGTHS AND/OR PHASES) Suspect Multiple Antibodies SOME CELLS POSITIVE (DIFFERENT STRENGTHS AND/OR PHASES) AND SOME CELLS NEGATIVE Suspect Multiple Antibodies Test other selected cells to eliminate other specificities Test patient's cells to confirm they lack antigen Test selected cells to confirm and eliminate specificities Additional techniques may be useful (enzymes) Test patient's cells to confirm they lack antigens ALL CELLS POSITIVE (SAME STRENGTH AND SAME PHASE) Suspect Antibody to High-Incidence Antigen (See also Multiple Antibodies) WEAK REACTIVITY SEEN WITH SOME CELLS, ALL SPECIFICITIES ELIMINATED Suspect Weakly Reactive Antibody or Antibody Showing Dosage ONLY ONE CELL POSITIVE (DONOR UNIT OR PANEL CELL) Suspect Antibody to Low-Incidence Antigen or Antibody to HLA Antigen Test cells negative for high-incidence antigens May need help from Reference Laboratory for identification or confirmation Test patient's cells to confirm they lack antigen Enhancement techniques (enzyme panel, increase amount of serum used, increase incubation time) Test patient's cells to confirm they lack antigen Test cells positive for low-incidence antigens or known strongly positive for HLA antigens May need to refer to Reference Laboratory for identification or confirmation Figure Approaches for identifying antibodies (modified from Brendel 9 ).

10 358 AABB Technical Manual antibody reactivity are possible causes, and the strength of antigen expression on tested red cells should be kept in mind. Antibody specificities should, when possible, be excluded only on the basis of cells known to bear a strong expression of the antigen. Zygosity Reaction strength of some antibodies varies from one red cell sample to another. This may be due to the phenomenon known as dosage, in which antibodies react preferentially with red cells from persons homozygous for the gene that determines the antigen (ie, processing a double dose of the antigen). Red cells from individuals heterozygous for the gene may express less antigen and may react weakly or be nonreactive. Alloantibodies vary in their tendency to recognize dosage. Many antibodies in the Rh, Duffy, MN, and Kidd systems have this trait. Variation in Adults and Infants Some antigens (eg, I, P 1,Le a, and Sd a )are expressed to varying degrees on red cells from different adult donors. This expression is unrelated to zygosity; however, the antigenic differences can be demonstrated serologically. Certain antibodies, including those to I, Le a,le b,sd a,lu a, Lu b,vel,yt a,hy,mcc a,yk a,cs a,ch,and Rg antigens, react more weakly with cord red cells than they do with red cells from adults. Changes with Storage Blood group antibodies may react less well with stored than with fresh red cells. The M and P 1 antigens deteriorate during storage more rapidly than most others; the rate varies among red cells from different donors. Storage media can affect the rate of antigen deterioration. Fy a and Fy b antigens, for example, may be weakened when the cells are stored in a suspending medium of low ph and low ionic strength. The potential loss of antigen reactivity must be considered when using older cells for antibody identification. Red cells from donor units are often fresher than commercial reagent cells and have been stored in different media. Some antibodies give stronger reactions with suspensions of donor cells than with reagent cells; eg, antibodies in the Knops system and antibodies that react with HLA antigens on red cells often react best with fresh donor cells and may not react at all with stored reagent cells. After frozen storage, reagent cells may give weaker reactions with some antibodies. This can cause misleading patterns, especially when an antibody is interpreted as recognizing a high-incidence antigen, on the basis of nonreactivity with one or two thawed specimens. Certain antibodies react more strongly or weakly with cells from different commercial manufacturers, whose suspending media may differ in ph or other characteristics. Enhancement techniques often help resolve problems associated with variations in antigen expression (see Methods 3.2.3, 3.2.4, 3.2.5, and 3.5.5). The age and nature of the specimen must also be considered when typing red cells. Antigens on cells from clotted samples tend to lose activity faster than cells collected in citrate anticoagulants such as ACD or CPD. Red cells in donor units collected into these anticoagulants generally retain their antigens throughout the standard shelf life of the blood component. If red cells are used that are collected into EDTA they must be tested within 2 days of collection, although some studies have demonstrated significantly longer preservation of most antigens on EDTA-anticoagulated cells. 10

11 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 359 No Discernible Specificity Factors other than variation in antigen expression may contribute to difficulty in interpreting results of antibody identification tests. If the reactivity obtained with the serum is very weak and/or if the cross-out process has excluded all likely specificities, alternative approaches to interpretation should be used. Antigens Present in Common Instead of excluding antigens on nonreactive cells, one can observe what antigens are common to the reactive cells. For example, if the cells reacting at room temperature are all P 1 -positive, yet not all the P 1 -positive cells react, the antibody could be an anti-p 1 that does not react with cells having a weaker expression of the antigen. (Such cells are sometimes marked on the panel sheet as +w.) With this in mind, one could use a method to enhance anti-p 1, such as testing at colder temperatures. If all the reactive cells are Jk(b+), but not all the Jk(b+) cells react, the reactive ones might all be Jk(a b+), with a double-dose expression of the antigen. Enhancement techniques, such as enzymes, LISS, or PEG, may then help demonstrate reactivity with all the remaining Jk(b+) cells. Typing the patient s cells to confirm they lack the corresponding antigen can also be very helpful. Inherent Variability Nebulous reaction patterns that do not appear to fit any particular specificity are characteristic of antibodies (such as anti-bg a ) that react with HLA antigens on red cells. These antigens vary markedly in expression on red cells from different individuals. Rarely, a pattern of clear-cut reactive and nonreactive tests that cannot be interpreted can result from the incorrect typing of reagent red cells. Unlisted Antigens Sometimes a serum reacts with an antigen not routinely listed on the antigen profile supplied by the reagent manufacturer; Yt b is one example. Even though serum studies yield clear-cut reactive and nonreactive tests, anti-yt b may not be suspected. In such circumstances it is useful to ask the manufacturer for additional phenotype information. If the appropriate blood grouping reagent is available, reactive and nonreactive red cell samples, as well as the autologous red cells, can be tested. However, these problems often have to be referred to an immunohematology reference laboratory. ABO Type of Red Cells Tested A serum may react with many or all of the group O reagent red cell samples, but not with red cells of the same ABO phenotype as the autologous red cells. This occurs most frequently with anti-h, -IH, or -Le bh. Group O and A 2 red cells have large amounts of H antigen; A 1 and A 1 B red cells express very little H (see Chapter 12). Sera containing anti-h or -IH react strongly with group O reagent red cell samples, but autologous A 1 or A 1 B red cells or donor cells used for crossmatching may be weakly reactive or nonreactive. Anti-Le bh reacts strongly with group O, Le(b+) red cells, but reacts weakly or not at all with Le(b+) red cells from A 1 or A 1 B individuals. Such antibodies should be suspected when the antibody serum, which uses group O red cells, is strongly reactive, but serologically compatible A 1 or A 1 B donor samples can be found without difficulty.

12 360 AABB Technical Manual Multiple Antibodies When a serum contains two or more alloantibodies, it may be difficult to interpret the results of testing performed on a single panel of reagent red cells. The presence of multiple antibodies may be suggested by a variety of test results. 1. The observed pattern of reactive and nonreactive tests does not fit that of a single antibody. When the exclusion approach fails to indicate a specific pattern, it is helpful to see if the pattern matches any two combined specificities. For example, if the reactive cells (see Table 17-1) are numbers 2, 4, 5, and 7, none of the specificities remaining after crossing-out exactly fits that pattern, but if both K and E are considered together, a pattern is discerned. Cells 2 and 7 react because of anti-k, cells 4 and 5, because of anti-e. If the typing patterns for no two specificities fit the reaction pattern, the possibility of more than two antibodies must be considered. The more antibodies a serum contains, the more complex the identification and exclusion of specificities will be, but the basic process remains the same. 2. Different red cell samples react at different test phases. When reactivity occurs at several phases, each phase should be evaluated separately. The pattern seen at room temperature may indicate a different specificity from the pattern of antiglobulin results. It is helpful to know which specificities are most likely to be seen at which phase (see Table 14-2). 3. Unexpected reactions are obtained when attempts are made to confirm the specificity of a suspected single antibody. If a serum suspected of containing anti-e reacts with additional samples that are e-negative, another antibody may be present or the suspected antibody may not really be anti-e. Testing a panel of selected e-negative cells may help indicate an additional specificity. 4. No discernable pattern emerges. Uniform or variable reaction strengths may be observed, and dosage or other variation in antigen strength does not provide an explanation. Additional approaches and methods of testing are usually indicated. Some helpful steps include: a. If strong positive results were obtained, use the exclusion method with nonreactive cells to eliminate some specificities from initial consideration. b. If weak or questionable positive results were obtained, test the serum against cells carrying a strong expression of antigens corresponding to any suspected specificities, and combine this with methods to enhance reactivity. c. Type the patient s red cells and eliminate from consideration specificities corresponding to antigens present on the autologous cells. d. Use methods to inactivate certain antigens on the red cells, eg, enzyme treatment to render cells negative for Fy a,fy b,s. e. Use adsorption/elution methods to separate antibodies. These and other methods that may be helpful are discussed below. Antibodies to High-Incidence Antigens If all reagent red cell samples are reactive, but the autocontrol is nonreactive, alloantibody to a high-incidence antigen should be considered, especially if the strength and test phase of reactions are

13 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 361 uniform for all cells tested. Antibodies to high-incidence antigens can be identified by testing red cells of selected rare phenotypes, and by testing the patient s autologous red cells with sera known to contain antibodies to high-incidence antigens. Knowing the race or ethnic origin of the antibody producer can help in selecting additional tests to be performed. Cells that are null for all antigens in a system (eg, Rh null or K o ) or modified red cells (eg, dithiothreitoltreated cells) can help limit possible specificities to a particular blood group. If cells negative for particular highincidence antigens are not available, cells positive for lower-incidence alleles can sometimes be helpful. Weaker reactivity with Co(a+b+) cells than with common Co(a+b ) cells, for instance, might suggest anti-co a. Antibodies to high-incidence antigens may be accompanied by other antibodies to common antigens, which can make identification much more difficult. Serologic Clues Knowledge of the serologic characteristics of particular antibodies to highincidence antigens can help in identification. 1. Reactivity in tests at room temperature suggests anti-h, -I, -P 1,-P,-Tj a (-PP 1 P k ), some-lw, -Ge, -Sd a,or-vel. 2. Lysis of reagent red cells is characteristic of anti-vel, -P, -Tj a,and-jk3. It is also seen with some examples of anti-h and -I. 3. Reduced or absent reactivity in enzyme tests occurs with anti-ch, -Rg, -In b, or -JMH and is seen with some examples of anti-yt a and -Ge2 or -Ge3. 4. Weak, nebulous reactions in the antiglobulin phase are often associated with anti-kn a,-mcc a,-yk a,and-cs a. Complement-binding autoantibodies, such as anti-i or anti-ih, give similar results when polyspecific antiglobulin reagents are used. 5. Antibodies such as anti-u, -McC a, -Sl a, -Js b, -Hy, -Jo a, -Tc a, -Cr a, and -At a should be considered if the serum is from a Black individual because the antigen-negative phenotypes occur almost exclusively in Blacks. Makers of anti-kp b are almost always White. Anti-Di b is usually found among Asians, Hispanics, and Native Americans. Interpreting a Positive DAT When a patient produces antibody directed to a high-incidence antigen following transfusion, the posttransfusion redcellsmayhaveapositivedat,and both serum and eluate may react with all cells tested. This pattern of reactivity is identical to that produced by many warm reactive autoantibodies, which may also appear after transfusion; these two scenarios can be very difficult to differentiate. A posttransfusion alloantibody to a high-incidence antigen would be expected to produce a DAT of mixedfield appearance, because only the transfused red cells would be coated with antibody. In practice, however, weak sensitization and mixed-field sensitization can be difficult to differentiate. It may be helpful to use cell separation procedures to isolate autologous cells for testing. Chapter 14 discusses additional serologic characteristics of antibodies reacting with high-incidence red cell antigens. Problems with these antibodies often have to be referred to an immunohematology reference laboratory. Antibodies to Low-Incidence Antigens Reactions between a serum sample and a single donor or reagent red cell sample may be caused by an antibody to a lowincidence antigen, such as anti-wr a.if

14 362 AABB Technical Manual red cells known to carry low-incidence antigens are available, the serum can be tested against them, or the one reactive red cell sample can be tested with known examples of antibodies to low-incidence antigens. A single serum often contains multiple antibodies to low-incidence antigens, and the expertise and resources of an immunohematology reference laboratory will be required to confirm the suspected specificities. Serologic Strategies If antibody to a low-incidence antigen is suspected, transfusion should not be delayed while identification studies are undertaken. If antibody in the serum of a pregnant woman is thought to be directed against low-incidence antigen, testing the father s red cells can predict the possibility of incompatibility with the fetus, and identifying the antibody is unnecessary. If a newborn has a positive DAT, testing of the mother s serum or an eluate from the infant s cells against the father s red cells (assuming they are ABO-compatible) can implicate an antibody to a low-incidence antigen as the probable cause; identifying the antibody isusuallyoflittleimportance. Some reference laboratories do not attempt to identify antibodies to low-incidence antigens, since they are often only of academic interest and resources can better be devoted to problems of greater clinical importance. Identification may be made when time permits and suitable reagents are available. It is often practical to store individual specimens and then perform batch testing, to conserve rare frozen samples of cells or antibodies. Unexpected Positive Results When serum reacts with a panel cell designated as positive for a low-incidence antigen, further testing to exclude the antibody is usually unnecessary. For every antigen of low incidence represented on a panel there are many more that are not represented and that are also not excluded by routine testing. Reactivity against low-incidence antigens is not uncommon; although the antigens are rare, antibodies against some of the lowincidence antigens are much less rare. Presumably the testing is being performed because the serum contains some other antibody (-ies) and reactivity with the cell expressing the low-incidence antigen is a coincidental finding. This may complicate interpretation of the panel results, but rarely requires confirmation of antibody specificity or typing of donor blood to ensure the absence of the antigen. If typing is desired, a negative crossmatch with the patient s serum is sufficient demonstration that the antigen is absent. Many antibodies to low-incidence antigens are reactive only at temperatures below 37 C and are of doubtful clinical significance. When serum reacts only with red cells from a single donor unit or reagent cell, the other possibilities to consider are that the reactive donor red cells are ABO-incompatible, have a positive DAT, or are polyagglutinable. Antibodies to Reagent Components and Other Anomalous Serologic Reactions Antibodies to a variety of drugs and additives can cause positive results in antibody detection and identification tests. The mechanisms are probably similar to those discussed in Chapter 18. Most of these anomalous reactions are in-vitro phenomena and have no clinical significance in transfusion therapy other than causing laboratory problems that delay provision of a needed transfusion. Very rarely they may cause erroneous interpretations of ABO typing that could endanger the patient.

15 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 363 Ingredients in the Preservative Solution Antibodies that react with an ingredient in the solution used to preserve reagent red cells (eg, chloramphenicol, neomycin, tetracycline, hydrocortisone, EDTA, or various sugars) may agglutinate cells suspended in that solution. Reactivity may occur with cells from several commercial sources or may be limited to cells from a single manufacturer. The autologous control is often nonreactive, unless the suspension of autologous red cells is prepared with the manufacturer s red cell diluent or a similar preservative. Such reactions can often be circumvented by washing the reagent cells with saline before testing. The role of the preservative can often be confirmed by adding the medium to the autologous control and converting a nonreactive test to a positive test. In some cases, however, washing the reagent cells does not circumvent reactivity and the resolution may be more complex. Ingredients in Enhancement Media Antibodies reactive with ingredients in other reagents, such as commercially prepared LISS additives or albumin, can cause agglutination in tests using reagent, donor, and/or autologous red cells. Ingredients that have been implicated include parabens (in some LISS additives), sodium caprylate (in some albumins), and thimerosal (in some saline preparations). LISS and other enhancement media may also dramatically increase the reactivity of some autoantibodies. Antibodies such as anti-i, -IH, or -Pr that ordinarily react only at colder temperatures may even react in the antiglobulin phase when these enhancement media are used. Omitting the enhancement medium, substituting plasma for serum, and/or using anti-igg rather than polyspecific antiglobulin serum will usually circumvent this reactivity. In some cases antibodies dependent upon reagent ingredients will also show blood group specificity, eg, paraben-dependent anti-jk a, caprylate-dependent anti-c. The autocontrol may be reactive if the patient s own red cells carry the antigen, but the DAT should be negative. Problems with Red Cells The age of the red cells can cause anomalous serologic reactions. Antibodies exist that react only with stored red cells, and these can cause agglutination of reagent red cells by all techniques, and enhanced reactivity in tests with enzyme-treated red cells. Such reactivity is not affected by washing the red cells, and the autocontrol is usually nonreactive. No reactivity will be seen in tests on freshly collected red cells, ie, from freshly drawn donor or autologous blood samples. Immunohematology Reference Laboratories When antibody problems cannot be resolved (or when specially typed blood is needed) immunohematology reference laboratories can provide consultation and assistance, through their access to rare donor files. (See Method 4.8.) The Patient with a Positive Autocontrol No Recent Transfusions Reactivity of serum with the patient s own cells may indicate the presence of autoantibody. (See Chapter 18.) If this reactivity occurs at room temperature or below, the cause is often anti-i or other cold autoagglutinin. Reactivity of the autocontrol in the antiglobulin phase usually signifies a reactive DAT and the possibility of autoantibody. If, in addition, the serum reacts with all cells

16 364 AABB Technical Manual tested, autoadsorption or other special procedures may be necessary to determine whether autoantibody in the serum is masking any significant alloantibodies. If the serum is not reactive or shows only weak reactivity, an eluate may demonstrate more potent autoantibody. If the DAT is negative, but the autocontrol is positive by IAT, an alternative explanation is required. Such results are unusual and may indicate antibody to a reagent constituent causing in-vitro reactivity with all cells, including the patient s own. It may also indicate that the wrong cells were added to the test! Cold Autoantibodies. Potent cold autoagglutinins that react with all cells, including the patient s own, can create special problems, especially when reactivity persists at temperatures above room temperature. Cold autoagglutinins may be benign or pathologic. (See Chapter 18 for a more detailed discussion.) There are two divergent objectives in testing a serum with a potent cold agglutinin. One is to determine if the thermal amplitude is high enough (usually 30 C or above) that the antibody has clinical significance; to do this, in-vitro autoadsorption of the serum must be avoided. Keeping the freshly collected blood warm (37 C) until the serum is separated usually provides an appropriately informative specimen. The second objective, the common one outside of cold agglutinin syndrome, is to circumvent the coldreactive antibody and allow detection of more important antibodies. Procedures for the detection of alloantibodies in the presence of cold-reactive autoantibodies are discussed in Chapter 18 and include: 1. Prewarmed techniques, in which red cells and the serum to be tested are incubated at C before they are mixed (see Method 3.3). 2. The use of anti-igg rather than polyspecific antiglobulin serum. 3. Cold autoadsorption, to remove autoantibodies but not alloantibodies. 4. Heterologous adsorption with rabbit red cells. Dealing with Warm Autoantibodies. Patients with warm-reactive autoantibody present in their serum create a special problem, because the antibody reacts with virtually all cells tested. If such patients are to be transfused, it is important to detect any clinically significant alloantibodies that the autoantibody may mask. Techniques are discussed in Chapter 18 and Methods 6.4, 6.5 and 6.6. Reactivity of most warm reactive autoantibodies is greatly enhanced by such methods as PEG and enzymes, and to lesser extent by LISS and albumin. It may be advantageous to perform antibody detection tests without the enhancement media usually employed. If tests are nonreactive, the same procedure can be used for crossmatching, without the need for adsorptions. Recent Transfusions If the autocontrol is positive in the antiglobulin phase, there may be antibodycoated cells in the patient s circulation, causing a positive DAT, often of mixedfield reactivity. Elution may be helpful, especially when tests on serum are inconclusive. For example, a recently transfused patient may have a positive autocontrol and serum that reacts weakly with most but not all Fy(a+) red cells. It may be possible to confirm anti- Fy a specificity by elution, which concentrates into a small fluid, volume the immunoglobulin molecules present in small numbers on each of the red cells in the starting preparation. It is rare for transfused cells to make the autocontrol positive at other test phases, but it can occur, especially with a newly developing or cold-reactive alloantibody. If the positive DAT does not have a mixed-field appearance and, especially, if

17 Chapter 17: Identification of Alloantibodies to Red Cell Antigens 365 the serum is reactive with all cells tested, the possibility of autoantibody should again be considered. Detection of masked alloantibodies may require allogeneic adsorptions. Accurate phenotyping of red cells may be difficult if the DAT is reactive in any patient, whether or not there has been recent transfusion. A positive DAT will cause the cells to be reactive in any test requiring addition of antiglobulin serum and with some reagent antibodies (notably those in the Rh system) that include an enhancement medium. Many monoclonal reagents can give valid phenotyping results despite a positive DAT. Selecting Blood for Transfusion Once an antibody has been identified, it is also important to decide its clinical significance. Antibodies reactive at 37 C and/or by IAT are generally considered clinically significant and those reactive at room temperature and below are not; however, there are many exceptions. For example, anti-ch, anti-rg, and many of the Knops and Cost antibodies have little or no clinical effect, despite reactivity by IAT. Anti-Vel, -P, and -Tj a (-P+P 1 +P k )may react only at cold temperatures yet may cause significant cell destruction in vivo. Comparison with documented cases in the literature and consultation with immunohematology reference laboratories should provide guidance about previous examples of similar specificities. Phenotyping Donor Units Red-cell-containing components selected for transfusion to a patient with an antibody should, whenever possible, be tested and found to be negative for the appropriate antigen, unless the antibody is known to lack clinical significance. Even if the antibody should be no longer detectable, the red cells of all subsequent transfusions to that patient should lack the antigen, to prevent a secondary immune response. The transfusion service must maintain records of all patients in whom significant antibodies have been previously identified. 8 An antiglobulin crossmatch procedure is required if the serum contains, or has previously contained, a significant antibody. A potent example of the antibody should be used to identify antigen-negative blood. Often, this is a commercial antiserum, but to save expensive or rare reagents, units can first be tested with the patient s serum. The absence of antigen, in nonreactive units, can then be confirmed with the commercial reagent. Sufficiently potent antibodies in patients specimens, that have reactivity as good as, or better than, commercial antibodies, can be stored frozen for future use. If the antibody is of unusual specificity or one for which commercial reagents are not available, a stored sample can be used to select units for transfusion at a later time, especially if the patient s later specimens lose reactivity. If a patient s serum is to serve as a typing reagent, it should be well characterized and retain its reactivity after storage, and appropriate negative and weakly positive controls should be used at the time of testing. The FDA has established the following criteria for licensing some reagents, 11 and these criteria may be useful guidelines for evaluating inhouse antisera. 1. Anti-K, anti-k, anti-jk a, anti-fy a, and anti-c w : dilution of 1:8 to give at least 1+ reaction. 2. Anti-S, anti-s, anti-p 1,anti-M,anti- I, anti-c (saline), anti-e (saline), and anti-a 1 : dilution of 1:4 to give at least 1+ reaction. 3. Most other specificities: undiluted, must give at least a 2+ reaction.