The Difficult Crossmatch Part 1: Recognition and Resolution of Problems

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1 C/E Update: Blood Bank The Difficult Part 1: Recognition and Resolution of Problems by Thomas A. Noto, M.D. and F. Lynn Leverett, M.D. Category A1 continuing education credit is available to anyone who studies a C/E Update series and completes a written exam (prepared and distributed by the ASCP). For further information see page 52 in this issue. The problem crossmatch is a challenge to every one in the blood bank. Even the novice blood banker should regard this situation as a stimulating experience. For this reason, we shy away from such terminology as "troubleshooting the crossmatch". Rather, we stress "the approach;" a systematic review of data, observations and theoretical facts to provide a definitive resolution to the problem or difficult crossmatch. The objective of this presentation is twofold. Each step in a crossmatching procedure may provide clues that an incompatibility exists between recipient and donor. t is only appropriate, then, that the entire sequence of events leading to compatibility testing be reviewed first. This will include an outline of ABO blood grouping, Rh typing and a discussion of antibody detection and compatibility tests. Secondly, a series of flowcharts that define causes and solutions for the principal situations that result in incompatibility between recipient and donor will be presented. The entire flowchart scheme evolves around the results of the autocontrol and on observed temperaturedependent reactions. Based on the absence or presence of agglutination in the autocontrol, one is then capable of pursuing either cold or warm reactive alio or autoantibodies as the etiology of incompatibility (Table ). Because in most instances, the underlying cause of incompatibility is a single antibody or multiple Thomas A. Noto, M.D. is with The Department of Pathology at The University of Miami School of Medicineljackson Medical Center, Miami, Florida and F. Lynn Leverett, M.D., is with South Miami Hospital, Miami, Florida. antibodies in the recipient, Part 2 of the article discusses a simple approach to the use and interpretation of results of a red blood cell panel for antibody identification. Often, this simple task is confronted as an exercise in deciphering hieroglyphics. This should never be the case. Classification of Problems. DFFCULTES N ABO GROUPNG Correct ABO grouping is very important for safe transfusion because of the presence of antia and antib in the sera of most patients. Also, discrepancies in direct and reverse ABO grouping may be the first clue to, or forerunner of, an incompatible crossmatch. Resolution of these discrepancies may avert the problem crossmatch before it is encountered. A. Proper ABO Grouping n order to avoid problems in ABO grouping, it is imperative to utilize proper procedures for direct and reverse grouping. The technical details are given in the 1977 edition of the Technical Manual of the American Association of Blood Banks (AABB). 1 n general, the correct procedure for ABO grouping includes: 1. Strict adherence to the manufacturer's directions in the use of commercial products. (This statement applies to the use of all reagents in all phases of blood banking!) 2. Direct grouping of patient's cells with antia and antib sera. A full two minutes of observation is required with slide methods before a negative result is valid. 3. Reverse or confirmatory grouping with patient's serum and known group A, and B cells /78/0900/0008 $01.15 American Society of Clinical Pathologists Downloaded from

2 Table 1 Classification of coldreacting agglutinins and warmreacting agglutinins in respect to the autocontroi result. COLDREACTNG AGGLUTNNS WARMREACTNG AGGLUTNNS NEGATVE AUTOCONTROL POSTVE AUTOCONTROL NEGATVE AUTOCONTROL POSTVE AUTOCONTROL 1. Specific cold antibody 1. Anti 1. Single antibody to common antigen 1. Specific warm autoagglutinins 2. AntiH in A 1 or A^ recipient 2. Multiple antibodies 2. Rhhr warm autoagglutinins "nonspecific" 3. AntiA] in A2 or A2B recipient 3. Antibody to low incidence antigen 3. Pseudoagglulinins a. Rouleaux b. Albumin autoagglutininating phenomenon 4. Antibody to low incidence antigen 4. Antibody to high incidence antigen 5. Antibody to high incidence antigen 4. All individuals that appear as group O are tested with antia,b serum. B. Discrepancies Reactions of Red Cells With: AntiA AntiB AntiA.B AntiA, Reactions of Serum With: A, Cells B Cells 0 Cells Discrepancies between direct and reverse ABO grouping may result from any of the following: 1. Subgroups of A antigen 2. Unexpected specific antibodies 3. Cold autoagglutinins 4. Dysproteinemias 5. Bacterial contamination of typing cells a. Subgroups of A antigen. Group A 2 individuals comprise approximately 20% of the group A population. Group A 3 individuals comprise approximately 0.1% of the group A population. Approximately 25% of the group A 2 B and 2% of group A 2 individuals have antia, in their serum (Fig. 1). As shown in Fig. 2, subgroups of A weaker than A 3 will not usually agglutinate with antia serum and will appear to be group O. Hence the need to confirm group O bloods with antia,b serum. The clinically significant subgroups of A will react with antia,b serum. Also note that the subgroups of A with antia, in the serum will reverse group as O. These procedures will confirm the subgroups of A: i. Use antia,b typing serum to detect the weak subgroups. ii. Use antia, typing serum (lectin or absorbed) to assure that cells are not group Aj. (AntiAi will Fig. 1. Confirmation of group A 2 patient with antia^ in serum. Group A, Group A 2 Group A 2 with antia, Group A 3 Group A 3 with antia, Group A4 or less Group A 4 or less with antia, Group A 2B Group A 2B with antia, Unknown Cells Reaction With: AntiA MF* MF* AntiB Unknown Serum Reaction With: A, Cells B Cells * MF = Mixed field agglutination. Small uniform clusters of red cells surrounded by free floating red cells. Fig. 2. Results of direct and reverse grouping in subgroups of A antigen. (continued on page 12) Downloaded from 9

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5 (continued from page 9) Reactions of Red Cells With: Reactions of Serum With: includes the patient's own red cells, the reagent reverse grouping cells and the antibody detection and identification cells. AntiA AntiB A, Cells B Cells _ Fig. 3. A discrepancy in a group AB type person due to an unexpected serum antibody. react only with A, cells. Results of antiaj serum with subgroups of A will be negative.) iii. Use the antibody screening test to exclude unexpected antibodies. b. Unexpected antibodies. t must be remembered that the Aj and B red cells used for reverse grouping have other blood group antigens on their surface. (These cells are rhrh(cde/cde), and most manufacturers try to provide Kellnegative cells as well.) Some of these other antigens will react with the appropriate antibody at room temperature. Notably the cold reacting antibodies, antip,, antim, and antile a antile b in the patient's serum, can cause a discrepancy between the direct and reverse grouping. Such a discrepancy in a group AB person might appear as presented in Fig. 3. A coldreacting antibody should be suspected with these results. f this is the case, then at least one of the antibody screening cells should agglutinate at room temperature. Remember that immune antibody responses begin with the synthesis of gm (coldreacting, 19S) antibodies and are followed by gc (warmreacting, 7S) antibodies. Therefore, a newly formed antikell or newly formed antihr' (antic) may react at room temperature. c. Cold agglutinins (antih and antil). AntiH is present in the serum of some individuals who are A! or A,B. This antibody does not pose the problems that antil does. AntiH reacts best with group O cells and less strong with A 2 and other A subgroups. The autocontrol is usually negative. High titers of antil can give discrepancies in ABO grouping. Since red cells of all adults contain the antigen, except genetic i adults, sera containing antil will agglutinate all adult cells. This Group Group 0 Group A Group B Red Cells Reaction With: AntiA AntiB Serum Reaction With: A, Cells B Cells Fig. 4. Examples of the ABO cell and serum groupings of persons with hypogammaglobulinemia. The ABO grouping in a patient with significant serum levels of antil will direct group as AB and reverse group as O. The presence of this strong cold agglutinin may interfere with Rh typing since the Rh control test is positive. n addition, the patient will have a positive direct antiglobulin (direct Coombs) test. These observations indicate the course of action to be taken. n this situation, correct ABO and Rh typing is obtained by washing the patient's red cells three to four times in warm saline. This procedure removes the antil from the surface of the patient's red cells and permits correct grouping and typing of the cells. (Reverse grouping is useless in the presence of antil). Even though one is able to appropriately type red cells, one should confirm the presence of antil. AntiH and antil can be identified with the "modified" panel for cold agglutinins described in Appendix A. d. Dysproteinemias. i. Rouleaux formation is most frequently caused by abnormally high concentrations of globulins in the serum. This is classically seen in plasma cell myeloma. Rouleaux may also be seen in patients who are receiving plasma expanders such as Dextran, etc. Rouleaux formation is not true agglutination and has a different microscopic appearance; i.e., the classical "stack of coins." Whole blood, with its high protein content, is used in direct grouping. Therefore, the patient's red cells will aggregate when placed on the glass slide at room temperature with the antia and antib grouping sera. Likewise, reverse grouping with the patient's serum will cause the reagent A! and B cells to aggregate, regardless of the isoagglutinins in the patient's serum. The patient will, therefore, direct group as AB and reverse as an O group. Here, a high index of suspicion, a look under the microscope and a bit of patient history can save a lot of time. Technical details for properly grouping and typing patients in the presence of rouleaux are described in Appendix B. /';'. Hypogammaglobulinemia. The expected isoagglutinins in this condition are very weakly reactive or are not present. Examples are given in Fig. 4. The direct group is that of an A, B or O, but the reverse grouping is that of an AB. Again, the clinical history is most helpful. "Physiologic" hypogammaglobulinemia is seen in newborns and the aged. Acquired hypogammaglobulinemia can be seen in the leukemias and lymphomas in any 12 LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER 1978 Downloaded from

6 age group. Congenital hypogammaglobulinemia occurs most frequently in children. n the senile and acquired hypogammaglobulinemias, the reactivity of the isoagglutinins may be enhanced by performing the reverse grouping at 4 C for six minutes. Proper interpretation requires the use of controls (patient's red cells with patient's serum and group O screening cells with patient's serum), because cold agglutinins could cause false positive results. n other types, serum protein electrophoresis or immunoelectrophoresis is required to establish the diagnosis. However, if these techniques are not available, the history suffices. Resolution of the problem is simply knowing why the isoagglutinins are not present. e. Bacterial Contamination of Typing Cells: T activation. All erythrocytes have a hidden surface antigen designated the T antigen. Bacteria and viruses can expose this antigen. Almost all normal adults have antit in their serum. f the A, or B reverse grouping cells are contaminated by bacteria, the T antigen is exposed. Therefore, the serum of all individuals will agglutinate the grouping cells, irrespective of whether antia or antib is present in their serum. The problem may be suspected when: 1. confirmatory cells are turbid or hemolyzed or, 2. discrepancies in direct and reverse grouping are observed in all patients of the same blood group. f bacterial contamination with resulting T activation is suspected, a fresh bottle of confirmatory cells should be used. f the discrepancy is resolved, no further action is necessary. f the discrepancy persists, reanalyze the situation, because the problem lies elsewhere. This problem is uncommon and can be completely eliminated by adherence to high standards in technique.. DFFCULTES N Rh TYPNG n addition to the information provided by ABO grouping, blood for the recipient is selected on the basis of Rh 0 (D) type. Serum or reverse typing here is not possible as in the ABO system because of the absence of "naturally occurring" isohemagglutinins to the Rh 0 (D) antigen. Unfortunately, incorrect Rh typing will not be detected by compatibility testing unless the recipient has been sensitized and has developed antirh (D) antibodies. n this section, only some of the problems encountered in Rh 0 (D) typing will be discussed. A. Significance of Rh Control Test Most erroneous results in Rh 0 (D) typing occurs as false positive results. Most of these may be detected Downloaded from with the Rh control (the patient's red CMS MSO with the Rh control reagent provided by the same manufacturer as the antirh 0 (D) being used). These false positive typings usually are caused by: 1. n vivo sensitization of patient's red cells by antibodies other than antirh 0 (D). 2. Serum protein abnormalities as seen in plasma cell myeloma. n these situations, the patient's cells will agglutinate spontaneously in high protein mediums in the absence of specific typing serum; hence, the reason for a positive Rh control test. Consequently, the results of Rh 0 (D) typing will be invalid when typing antiserum with a high protein medium is used. Correct typing requires a saline suspension of wellwashed red cells and the use of saline typing antiserum. However, even this technique does not always provide conclusive results. B. Problems Encountered 1. Patients With Direct Antiglobulin Test Rh Control A direct antiglobulin test should be performed whenever a positive Rh control test is observed. Most cases with a positive Rh control will have a positive direct antiglobulin test. This combination of test results which usually interferes with Rh 0 (D) typing occurs in: a. Autoimmune hemolytic anemia (autoagglutinins: warm or cold) b. Acquired hemolytic anemia due to drugs (antipenicillin antibodies; or red celllike autoantibodies induced by amethyldopa (Aldomet) 2 c. Hemolytic disease of the newborn (immune antibodies other than antirh 0 (D) in an Rhnegative infant). 1 n these conditions, false positive Rh 0 (D) typing is observed for reasons previously discussed. False negative Rh (D) typing may occur in hemolytic disease of the newborn when the infant is Rh 0 (D)positive. This type of reaction is caused by socalled "blocking" maternal antirh 0 (D) antibodiesthat saturate the Rh 0 (D) antigenic sites on the infant's red cells. This situation must be differentiated from false positive Rh 0 (D) typing that may occur in Rhnegative infants. 2. Patients With Negative Direct Antiglobulin Test Rh Control False positive Rh 0 (D) typing occurs in patients with elevated serum globulins that cause rouleaux formation of the red cells. This pseudoagglutina (continued on page 16) LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER

7 (continued from page 13) Table False positive results with Rh (D) typing. A. Rh Control Direct Antiglobulin Test 1. Autoagglutinins (Warm or Cold) 2. Drug induced problems Recommended Procedures a. Use saline typing reagents. b. With strong cold autoantibodies wash cells with warm (37C) saline prior to typing. a. Careful drug history from patient. b. Use saline typing reagents. c. Elution studies Eluate tested against ordinary screening cells without drug give negative results. Exception may occur with amethyldopa (Aldomet). important to suspect those patients who are truly Rh 0 (D)negative and may have experienced a large transplacental hemorrhage. These patients also will type weakly Rh 0 (D)positive because of the circulating Rh 0 (D)positive fetal cells. Microscopic observation of the agglutinated cells shows mixed field agglutination. The direct antiglobulin test may be positive if the patient has already been immunized. This history and laboratory records may be of additional assistance since the patient may have been typed previously as Rh negative. Likewise, the D u test will be in error. Table summarizes the situations resulting in false positive Rh 0 (D) typing and the procedures recommended in their investigation and solution. 3. Hemolytic disease of the newborn in Rh negative infants NOTE: This situation must be differentiated from false negative results due to "blocking antibodies" in RhD (D)positive infants. B. Rh Control Negative Direct Antiglobulin Test 1. Rouleaux s a. Elution studies preferred. Recommended procedures a. Wash cells thoroughly. b. Use saline typing reagents. tion becomes more evident when slide techniques performed at higherthanroom temperature are used. n this situation, the Rh control is positive, but the direct antiglobulin test is negative. Washing of the cells thoroughly with saline, and testing with saline typing antirh 0 (D) will usually determine the correct Rh type. 3. Weak Agglutination in a Truly RhNegative ndividual Weak agglutinations occur with antirh 0 (D) typing serum in patients who have received recent transfusions of blood of a different Rh type. These weak reactions represent "mixed field" agglutination and may be verified under the microscope. This is seen when the Rh 0 (D)negative individual is mistakenly or for some other reason transfused with Rh 0 (D) positive blood. The transfused Rh 0 (D)positive cells will react with antirh 0 (D) typing serum and form small agglutinates interspersed along the unagglutinated Rh 0 (D)negative cells. f sensitization has not occurred, both the Rh control and direct antiglobulin test will be negative. n this situation, true Rh (D) typing of the recipient is difficult to obtain in the presence of transfused donor blood. n any situation when the correct Rh 0 (D) type cannot be ascertained, it is best to select Rh 0 (D)negative donors for the recipient. Because the blood bank is sometimes called upon to provide blood for the postpartum woman, it is. PROBLEMS WTH ANTBODY DETECTON (SCREENNG) AND COMPATBLTY TESTS While the antibody detection test and the compatibility test are two distinct procedures, they share a common denominator. Both tests are designed to detect unexpected red cell antibodies in the serum of the recipient or of the donor. Actually, the antibody screening test complements the crossmatching procedure because, in some situations, it may detect antibodies missed in the compatibility test. Nevertheless, while it is not a substitute for a properly performed crossmatch, it is an important part of the compatibility test. This is emphasized in the current (1976 edition) Standards for Blood Banks and Transfusion Services, AABB, 3 which state: "Each blood sample submitted with a request for transfusion, shall be tested for unexpected antibodies within 48 hours prior to or concurrently with performance of the crossmatch." We recommend that both tests be performed simultaneously. This is possible since in most blood banks, the details of both procedures are similar. These procedures must provide in vitro conditions for the detection of antibodies with varying serologic features. Both tests must be designed so as to detect those antibodies that can cause decreased red cell survival in the recipient. The compatibility test involves the reaction of the recipient's serum with donor cells. The antibody detection procedure substitutes reagent red blood cells for donor's cells. The selected reagent cells must possess strongly reactive antigenic determinants so as to detect weak antibodies. n addition, the range of antigenic determinants on these cells must permit the detection of those antibodies that may decrease the survival of transfused donor cells. 16 LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER 1978 Downloaded from

8 During storage, these reagent red blood cells may undergo a diminution of antigen strength. t is, therefore, recommended that these cells be tested daily, or at least prior to use, to assure adequate reactivity. The quality control testing of these cells should be done with a dilute antiserum. 4 For example, an antirh (D) serum, (commercial typing antiserum) diluted to give a 1 or 2 agglutination, may be used for this purpose. On occasion, one observes agglutination between the recipient's serum and reagent cells in the absence of a reaction with the donor's cells. Under such circumstances, an appreciation is acquired for the use of reagent cells for detecting antibodies. Aging of the donor's cells during storage weakens the antigenic determinants. This may account for the absence of agglutination described above. t is not advisable to ignore these observations, because such an antibody may decrease the survival of transfused donor's cells in the recipient. We believe that a control test consisting of the patient's serum with his own cells (the autocontrol) should be tested concurrently with the antibody detection test and the crossmatch. The result of the autocontrol test provides meaningful information when incompatibilities arise. n general, a positive autocontrol test usually signifies the presence of an autoagglutinin. A negative autocontrol test indicates that the patient is compatible with himself but that a specific antibody may be the cause of a positive antibody screening test or incompatible crossmatch. The problems detected with the antibody screening and compatibility tests are: A. Cold autoagglutinins B. Specific cold agglutinins C. Warm autoagglutinins D. Specific warm agglutinins E. Pseudoagglutinins Table Reactions of antibodies and serum factors in specific phases of the antibody detection and compatibility tests correlated with results of autocontrol test. Phase Room Temperature Saline ncubation Temperature 37C Albumin Anti Human Globulin (AHG) Antibody Detection and Compatibility Tests Autocontrol Negative Autocontrol Autocontrol Negative Autocontrol Autocontrol Negative Autocontrol nterpretation cold autoagglutinin specific cold agglutinin rouleaux albumin autoagglutinating phenomenon specific warm agglutinin warm autoagglutinin specific warm agglutinin Comments agglutination decreases or disappears with incubation (37C) autocontrol at 4C strongly positive agglutination decreases with incubation (37C) reactions accentuated in presence of albumin Other phases negative reactions may persist in antiglobulin phase agglutination with antiglobulin serum direct antiglobulin test positive may occur with certain drugs like Aldomet, etc. reactions due to antibodies detected only with indirect antiglobulin test other phases negative Rh 0 (D) and D u typing, screening the patient's serum with reagent red blood cells and crossmatching the donor cells with the patient's serum. Each action as programmed in these flowcharts (Figs. 59) is based on information obtained during pretransfusion testing. Table V nformation which can be derived from a comparison of the results of the compatibility testing and antibody detection tests. Table correlates the observations that may occur with these two important pretransfusion procedures in the above situations. Comparison of the results of compatibility testing with those of the antibody detection test performed on the recipient may provide additional information as outlined in Table V. Resolution of ncompatible es The flowcharts that follow represent the systematic course of action to be taken in solving the problem crossmatch. t is assumed that the reader has a thorough understanding and appreciation of the principles of pretransfusion tests presented in this article; including the ABO cell and serum grouping, Observation 1. All crossmatches and antibody de tection tests are positive. 2. Some or all crossmatches are incompatible; antibody detection test negative. 3. A single crossmatch is incompatible but others compatible; antibody detection test negative. 4. All or nearly all crossmatches compatible; antibody detection test positive. Possible nterpretation a. Antibody to a high incidence red cell antigen. b. Nonspecific agents, (ie, serum protein abnormalities) a. Antibody related to ABO system; antia, b. Deterioration of antigen on reagent blood cells. a. Antibody to a low incidence red cell antigen. a. Antibody to low incidence red cell antigen. n this situation, present in reagent cells; absent in donor's cells. b. Possibility of AntiH; reacts with group 0 reagent cells but not with Ai cells of donor. Downloaded from LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER

9 1. Specific cold antibody (antip,, M, Lea, [_eb, N) Discrepancy in ABO Possibly 75% of units incompatible Panel for identification Select donors negative for antigen after antibody identified AntiH in Ai or A B recipent Discrepancy in ABO No Compatible with Ai or A)B donors. ncompatible with A2 or 0 donors Modified panel for identification Donors must be Ai or A B 3. AntiA in A2 or A2B recipient Discrepancy in ABO Yes Negative ncompatible with all Ai or A B donors. Compatible with A2, A2B or 0 donors Prove patient a subgroup of A Donors must be subgroup A2, A2B, or group 0 4. Antibody to lowincidence antigen Discrepancy in ABO No Usually negative if incompatible, only one unit Panel f positive, usually only one cell Select compatible donor units Send serum and positive cells to consultant Antibody to highincidence antigen (Anti Sda, AntiEna, AntiVel) Discrepancy in ABO Yes! Panel All cells react Screen family (siblings first) for compatible blood Send patient's serum and cells to consultant Fig. 5. Coldreacting agglutinins with a negative autocontrol. All incompatible We wish to stress the important distinguishing features in the recognition and management of a recipient whose crossmatches are all incompatible.. ANTBODES TO HGH NCDENCE ANTGENS High incidence antigens are antigens present in over 98% of the population. They are present on Downloaded from by 18 guestlaboratory MEDCNE VOL 9, NO. 9, SEPTEMBER 1978 most of the reagent red blood cells used for antibody detection and identification. Antibodies to these antigens may be reactive in saline (i.e., antisd a, antien a, antivel) or reactive in the antiglobulin (AHG) phase of testing. All reagent and donor red blood cells agglutinate with the recipient's serum. The key to recognition is the negative autocontrol. The AHG reactive antibodies present (continued on page 20)

10 Anti Discrepancy in ABO Yes All incompatible at room temperature; Antihuman globulin phase may be incompatible "Modified" panel for identification Absorption of Anti \ Absorbed serum Antibody screen with absorbed serum with absorbed serum Fig. 6. Coldreacting agglutinins with a positive autocontrol. (continued from page 18) the greatest problems in transfusion management. n lifethreatening situations, it may be necessary to administer incompatible blood to these patients as a lifesaving measure. While most patients do not experience hemolytic reactions, others may have rapid in vivo erythrocyte destruction. 3. WARM AUTOAGGLUTNNS Compatibility testing and antibody identification may give the same results as that observed with antibodies to high incidence antigens. The key to recognition is the positive autocontrol. A. Specific Autoagglutinins Autoantihr"(e) may be present in autoimmune hemolytic anemia and in patients treated with methyldopa. There may be difficulty in Rh 0 (D) typing if the cells are heavily sensitized with autoantibody. The Rh (D) typing and the Rh control will be positive, as will tests for the other Rh factors. The patient has a positive direct antiglobulin test. The antibody detection test is positive and the panel identifies antihr"(e) in serum and eluate from the patient's cells. Salinereacting antiserums are used to Rh phenotype the patient's cells prior to any transfusion. A control of patient's cells is run simultaneously. f the control is positive, elution of the autoantibody from the patient's cells should be performed and phenotyping attempted on the eluted cells. The patient's serum will be "most compatible" with cells lacking the hr"(e) antigen. This can be demonstrated by titering the serum against selected Rh phenotypes, i.e., serum will be most compatible with R 2 R 2 (DcE/DcE) or r"r" (dce/dce) cells and least compatible with R,R, (DCe/DCe) or rr (dce/dce) cells. Most authorities in the field recommend that the donor blood for transfusion be selected on the basis of in vitro testing, utilizing titers where necessary and choosing the most compatible blood. (i ' 7,8 f the recipient lacks the rh"(e) antigen, the potential for allosensitization exists when R 2 (DcE) or r" (dce) cells are transfused. The detection of antirh"(e) and other alloantibodies in the patient with autoantihr"(e) is difficult. n a patient who has not recently had a transfusion, absorption of the autoantibody and testing of the absorbed serum against a panel may be helpful in demonstrating an alloantibody. Enzyme treatment of the patient's cells may enhance absorption of the autoantibody. Most of these patients respond well to steroid therapy and tolerate their anemia without requiring transfusion. Usually, only extenuating circumstances such as severe coronary ischemia, emergency surgery, or bleeding make transfusion mandatory as a temporizing measure. Since the in vivo survival of transfused hr"(e)positive cells is usually equivalent to that of the patient's own hr"(e) cells, one of us (F.L.L.) prefers to transfuse cells phenotypically matched for Rh antigens (and the Kell antigens, if possible). The phenotypically matched cells avoid the problems of allosensitization by these antigens and may avert problems if further transfusions are necessary. These transfusions are preceded by a carefully monitored in vivo crossmatch. B. "Nonspecific autoagglutinins These antibodies are Rhlike, i.e., react in albumin at 37 C, stronger in the antiglobulin phase and may or may not react with D and/or Rhnull cells. The antibodies seem to be directed toward an integral component of the Rh antigens. As with the autoantihr"(e), the recipient and donor are phenotypically matched where possible. The most compatible of these bloods is selected for transfusion. 20 LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER 1978 Downloaded from

11 1. Single antibody to common antigen Usually incompatible; depends on antigen frequency Panel Antibody to Rh antigens, Kell, Kidd, or Duffy identified Screen donors for antigen, if appropriate typing serum available Select donor units lacking corresponding antigen Multiple antibodies Most or all incompatible Panel Most or all cells react Check records for previous antibody studies "Sort out" antibodies by pattern and strength of reactivity with panel cells Phenotype recipient * Screen donor units to exclude those with specific antigens to antibodies identified with selected units Send serum to reference laboratory for definitive identification f compatible blood not available and patient actively bleeding and/or major surgery anticipated, consider transferring patient to large medical center. Send data and conclusions with the patient. Antibody to lowincidence antigen Usually negative if incompatible, only one unit Panel if positive, usually only one cell Select compatible donor units Send serum and positive cells to consultant Antibody to highincidence antigen All incompatible Panel All cells react Screen family (siblings first) for compatible blood Send patient's serum and cells to consultant Fig. 7. Warmreacting agglutinins with a negative autocontrol.. ALBUMN AUTOAGGLUTNATON PHENOMENON 9 All phases of antibody detection and compatibility testing requiring the addition of albumin show agglutination. This includes the autocontrol test when incubated with albumin. The direct antiglobulin test is negative. This antibody is not specific for a red cell antigen but is specific for the caprylatetreated, altered albumin. The albuminantibody complex is nonspecifically bound to the red cell membrane, and agglutination occurs. The agglutination of all red blood cells may at first be confused with an antibody to a high incidence antigen or to a warm autoagglutinin. Transfusion of blood in patients with this phenomenon is not a problem once the cause of the agglutination, principally in albumin testing, is recognized. Downloaded from LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER

12 1. Specific warm autoagglutinins Most or all incompatible Panel Antihr" (e) identified in serum and eluate from recipient's cells Phenotypically match donor units for crossmatch Select "most compatible" units of phenotypically matched donors Transfuse with caution 2. "Nonspecific" warm autoagglutinins All incompatible Panel Serum and eluate from recipient react in albumin and antihuman globulin with all cells Phenotypically match donor units for crossmatch Select most compatible units Transfuse with caution Fig. 8. Warmreacting agglutinins with a positive autocontrol. 1. Rouleaux ABORh 0 Typing Discrepancy in ABO Yes Rh 0 typing and Rh controlpositive Microscopic exam Wash cells for proper ABO and Rh 0 typing Antibody screen autocontrol crossmatch in all phases except antihuman globulin Avoid these results by recognizing problem in ABO and Rh typings Perform antibody screen, autocontrol, and crossmatch with saline replacement technique Direct antiglobulin Negative Albumin autoagglutinating phenomenon ABORh Typing Discrepancy in ABO No RH 0 typing and Rh controlnegative Antibody screen autocontrol crossmatch in albumin phase with possible carryover to antiglobulin phase Direct antiglobulin Negative Recognize Use saline techniques for antibody screen and crossmatch Use albumin without caprylate Fig. 9. Pseudoagglutination LABORATORY MEDCNE VOL. 9, NO. 9, SEPTEMBER 1978 Downloaded from

13 Adult Cells Group Cord Cells Group Patient Cells (Control) the uncommon genetic i cell when included on a commercial panel. Anti AntiH AntiH A, A ± 0 * Rouleaux Appendix B 1. ABO Grouping and Rh Typing in the Presence of Rouleaux Antii NOTE: Strength of reactivity shown here is relative and not absolute. Fig. 10. Modified Appendix A Modified Panel for Cold Agglutinins panel This panel is used to identify the cold agglutinins antil, antih and antii. These agglutinins have a common property of reacting strongest at 4 C with decreasing reactivity at increasing temperatures. However, they react differently with red cells of different antigenic composition. A few basic facts about each antibody are necessary in order to understand the rationale for this panel. 1. Anti reacts with all adult red cells except genetic i. Adult cells contain varying amounts of antigen, so the strength of reactivity may vary with individual red cells. t does not react or reacts very weakly with cord red cells. 2. AntiH reacts strongest with group O, adult or cord red cells. t reacts at intermediate strength with group A, cells and little, if at all, with group A, cells. The reactivity is directly proportional to the amount of H substance on the red cell. 3. AntiH reacts best with cells that have both and H antigens. 4. Antii reacts only with red cells having the i antigen, ie, cord cells, and genetic i cells. This panel consists of reagent group A, and group A 2 red cells, the antibody screening cells as the adult group O cells and group O cord cells that are received from the delivery room. This panel of cells will give a pattern of reactivity characteristic for each antibody Fig. 10. A. Procedure 1. Make a 25% suspension of each of the red cells to be tested. The cord blood cells should be saline washed four times prior to making this suspension. 2. Add one drop of each of the 25% cell suspension to separate, appropriately labeled tubes. 3. Add two drops of patient's serum to each tube. Gently mix. 4. ncubate at room temperature (RT) for 15 to 30 minutes. 5. Centrifuge and observe for agglutination. 6. Transfer tubes in a rack or serofuge head to a refrigerator at 4 C for six minutes. Remove rapidly. 7. Centrifuge and observe for agglutination. 8. Grade strength of agglutination after gently swirling the cell button off the bottom of the tube. 9. Compare results with those shown in the table to identify the cold agglutinin. Note: Anti, antih and antih react with all reagent commercial screening and panel cells at 4 C. An exception occurs with a. Direct ABO grouping. Wash patient's red cells three to four times with saline. Do the direct grouping by test tube method. f agglutinates appear macroscopically, observe under the microscope. f agglutinates have the stacked coin appearance, add two drops of saline and gently mix. Rouleaux will disperse; true agglutination will not. b. Reverse ABO grouping. Do the reverse grouping by the test tube method. Centrifuge the tubes, aspirate serum and replace with two drops of saline. Mix cellsaline mixture and respin. Gently resuspend the cells in the saline. True agglutinates will remain as compact, irregular clumps. c. Rh typing. Wash red cells with saline three to four times. Perform Rh typing with saline antirh (D) typing serum following the manufacturer's directions. 2. Saline Replacement Technique for Antibody Detection, dentification and ing Tests n these tests, the patient's serum with the rouleaux forming properties is mixed with red cells from other sources. Therefore, difficulty may be experienced in these procedures when the cells are not washed free of the patient's serum. Specific antibodies may be present in the patient's serum in addition to rouleaux. n spite of rouleaux, these antibodies will react with their corresponding antigens, at the appropriate temperature, in the appropriate medium. This reaction is specific with antibody binding to antigen in a union that will not be disturbed by the addition of saline. The saline will, however, disperse the rouleaux, leaving true agglutinates intact. These principles can be utilized in the saline replacement technique in all phases of antibody and compatibility testing. A. Procedure The antibody detection, identification and crossmatching tests are performed as usual with these additional steps when rouleaux is encountered. 1. Respin the serum and cell mixture. 2. Aspirate and discard the supernatant serum. 3. Add two drops of saline to the cells and gently mix. 4. Spin the salinecell mixture. 5. Observe for agglutination by gently swirling the cell button off the bottom of the tube. References 1. Technical Manual of the American Association of Blood Banks, 1977, 7th edition. Washington. D.C. 2. Garratty G. and LD. Petz, 1974, The nvestigation of Drugnduced Problems in the Blood Bank. A Technical Workshop presented by the Committee on Workshops of the American Association of Blood Banks. Washington, DC. 3. Standards for Blood Banks and Transfusion Services, 1976, American Association of Blood Banks, 8th edition. Washington, D.C. 4. Myhre, B.A., Quality Control in the Blood Bank. American Society of Clinical Pathologists. Commission on Continuing Education, Chicago. (Catalog # ). 5. Moulds, J.J., Multiple and High ncidence Antibodies. Part A. Detection and dentification. Transfusion with "lncom Downloaded from LABORATORY MEDCNE VOL 9, NO. 9, SEPTEMBER