Are HTLA Antibodies Clinically Significant?

Transcription

1 CE Update Immunohematology II Are HTLA Antibodies Clinically Significant? Ruth Mougey, MT(ASCP)SBB T he term high titer, low avidity, or HTLA, is used to classify the serologic behavior of a group of antibodies that have been lumped together because of the unique difficulties encountered in working with them. Clear-cut identification of HTLA antibodies is beyond the resources of most laboratories but because these antibodies are not uncommon, all blood banks need to be able to recognize and cope with them. In the author's region, 4% of samples submitted to the reference laboratory have HTLA antibodies. The very term HTLA is ambiguous and misleading because not all socalled HTLA antibodies are high titered, especially if the patient has just been immunized. Although weak or low avidity reactions are typical of these antibodies, strong reactions do not preclude the specificity of an HTLA antibody. Since any blood group antibody that is just beginning to be produced can be weakly reactive, the recognition of low avidity and/or high From the Reference Laboratory, American Red Cross Blood Services, Madison, Wl titer is just a starting point in the process of resolving this type of problem. Some authors 12 have proposed the term "serologically difficult antibodies" or SDA as a better name than HTLA. While this is truly descriptive, it has not replaced the term HTLA. All the endless discussion on terminology merely points up the real problem with this group of antibodies: they are impossible for most laboratories to identify conclusively. Most other blood group antibodies exhibit clear-cut positive and negative test reactions, and the red cells of the antibody former can be typed to prove that they are antigen negative. This is not the case with HTLA antibodies, and obtaining reproducible results is difficult. For these reasons, there may not be agreement between reference laboratories about the specificity of a particular serum or a cell typing. Many reference laboratories have elected to focus their efforts on other aspects of the patient's problem rather than try to identify the exact specificity of HTLA antibodies. The purpose of this article is to discuss the clinical significance of these antibodies and how they may be resolved. Criteria for HTLA Antibody Workup 1. Weak or nebulous reaction with most panel cells. 2. Weak or nebulous reactions with most crossmatches. 3. Elimination of the presence of complement binding cold antibodies, anti-bg's, and rouleaux. 4. A history of previous pregnancy or transfusion. When an antibody problem fits these criteria, certain steps can be taken to confirm the presence of an HTLA antibody and to select the appropriate units of blood. Clinical Significance of HTLA Antibodies When discussing the clinical significance of HTLA antibodies, the whole dilemma of what "clinically significant or insignificant" means needs to be examined. How much red cell destruction is acceptable in a transfused patient? In some publications, it is apparent that any immune red cell destruction was considered LABORATORY MEDICINE VOL. 17, NO. 3, MARCH 1986 on 17 September 217

2 significant and, in many others, the cruder measure of no signs and symptoms of a transfusion reaction plus the expected rise in the hemoglobin value was used to document the benign nature of the patient's antibody. At the present time, the best way to critically study the destructive capability of an antibody is to perform a red cell survival, but long-term studies are difficult to do and many patients are unavailable for follow-up. There is some controversy in the interpretation of 51Cr studies in published case reports of patients with HTLA antibodies who were transfused. Mollison3 reviewed 51Cr survival studies of a number of cases and concluded that anti-cha, -Rg", -Xga, -Csa, -Yka, and -Kna were incapable of causing red cell destruction. These conclusions are in contrast to the review of Molthan" on patients' data from which it was concluded that the antibodies are biologically significant. Some of the differences in interpretation may be due to individual cases that were evaluated only at one hour and 24 hours as opposed to those cases in which the T5 of the transfused cells was determined. Overall, there seem to be data 3 indicating good short-term survival of transfused incompatible red cells and some data indicating long-term survival may be less than normal. 45 Again the question remains, how much red cell destruction is clinically insignificant? It is important to realize that when one gives blood that is incompatible, there may be some cell destruction. Cell survivals are most commonly used to show whether or not there will be any immediate, rapid destruction of incompatible red cells. There is some error in the test because very small amounts of blood are destroyed more rapidly than large amounts. Therefore, the actual destructive capability of an antibody may be overestimated. In many of the reports in the literature, the patients had a one-hour or one-hour and 24-hour study done. Because the results indicated no immediate severe reaction could be expected, the patients were then transfused with large numbers of incompatible units. Although no long-term survival studies could be done in these cases, it was noted that there were no signs and symptoms of a transfusion reaction, and it was typical that a positive di- Table: Serologic Characteristics of HTLA Antibodies Anti-Ch» Anti-Rg' Anti-Cs Anti-Yk Anti-JMH Anti-Kn«Anti-McC Antigen frequency (%)* 98(C) 97(C) 98(C) Reactivity with enzymes Inhibition by serum Reactivity with cord cells vs adult cellst W/N W/N 92 (C) 98 (B) Variable 98 (C) 99.7 (B) 99.8(C) 99(C) 98(B) SAW S/W S S/W S/W * C = Caucasian; B = black. fw = weaker; N = negative; S = same (cord cells weaker than, negative, or the same as adult cells). rect antiglobulin test (DAT) was seen over a long period of time.6 The interpretation could be made that although the donor red cells are sensitized by the HTLA antibody, they are not removed from the circulation as rapidly as would be expected by an antibody whose clinical significance is unchallenged. The decision to transfuse incompatible blood to patients with HTLA antibodies should be evaluated as to the purpose of the transfusions. If the patient needs transfusions for surgery or other situations of short-term loss, then the relatively good short-term survival of incompatible donor cells should be appropriate. On the other hand, if the patient will need repeated transfusions of blood, then the somewhat shortened survival of incompatible donor cells may be undesirable. There are other aspects to the selection of blood for patients with HTLA antibodies that are more important. Since it is difficult for most laboratories to conclusively identify HTLA antibodies, how can the laboratory be sure that that is what they are dealing with? As mentioned before, weak reactions do not always mean an HTLA antibody. Similarly, patients who are sensitized frequently make more than one antibody so that the presence of a second antibody that is clinically significant may go unsuspected. The decision to give incompatible blood to patients because they "only" have an HTLA antibody needs to be carefully considered in order to avoid these pitfalls. In the author's laboratory, when an HTLA antibody is suspected two things are done. First, the frequency of compatible blood is determined by doing 24 to 48 random crossmatches. Second, the patient's red cells are fully phenotyped to see what antibodies can be formed by the patient. Because it is separated from the clinical situa- tion, this reference laboratory prefers to offer compatible blood whenever possible to avoid the problems discussed previously. Since the strength of the HTLA antigens varies from individual to individual, it is usually fairly easy to find compatible blood if a 3-minute 37 C incubation is used. That is not to say that the same units would still be compatible if longer incubation and antibody enhancement reagents were used, because typically those units only have weaker than normal expression of the HTLA antigen. This is one way the variability of the HTLA antigens can be made to work for the blood bank. If many compatible units are found, then the HTLA antibody is probably too weak to identify. If all the donors are incompatible, then the antibody is strong enough to give consistent reactions with selected panel cells. Depending on the patient's transfusion needs, the least incompatible units may be set aside or more crossmatches may be done after the results of other tests are known. Once the antibody is confirmed as belonging to the HTLA class and no other clinically significant antibodies are found to be present, the compatible or least incompatible units can be made available for transfusion. The blood bank physician and the patient's physician must decide on the appropriate course of action based on the test results and information obtained about the patient. There are some specific antibodies that may appear to be of the HTLA type, and it is important to exclude these when working with HTLA antibodies. Anti-Yt" and anti-lub are the most likely but anti-jr" and anti-lan have been reported7 to initially appear high titered and to have low avidity. Multiple antibodies directed against remnants of HLA antigens on red cells (Bg's) rarely mimic the weak LABORATORY MEDICINE VOL. 17, NO. 3, MARCH on 17 September 217

3 reactions of HTLA antibodies. The author's laboratory received an antido" that was thought to be an HTLA or Bg antibody by the referring laboratory because of its weak reactions. It was important to recognize the true specificity of anti-do" because those antibodies do cause transfusion reactions. Anti-Sda is an in vitro complement binding antibody that does not usually react with anti-igg antiglobulin reagent. This characteristic allows it to be readily distinguished from other antibodies of low avidity. The serology of HTLA antibodies is reviewed in the Table. It is difficult to put the antibodies into a table because there are so many exceptions that prove there are no rules. Generally speaking, the weaker the antibody reactions, the less likely the antibody is to react with cord cells and enzyme-treated cells. Anti-Cost-Stirling (Csa) and Anti-York (Yka) Anti-Cs* was first reported in 1965 by Giles and colleagues.8 The antibody is less common than other HTLA antibodies, and approximately 98% of Caucasians are Cs(a +). Anti-Yk* was described in and it was noted that although only 2% of random Caucasians are Cs(a-), 12.5% of Yk(a-) were Cs(a-). This phenotypic relationship is important to remember when using Y k ( a - ) and Cs(a - ) panel cells, since a result could easily be misinterpreted if the Cs* type of the Yk(a-) cell or the Yk" type of a Cs(a ) were unknown. The confusion would be compounded if a misidentified antibody former were negative for both Cs" and Yk*. This may be part of the reason why there is some disagreement between laboratories on the specificity of some HTLA antibodies. Anti-Yk" is the most common of the HTLA antibodies seen in Caucasians. Anti-Cs" is fairly rare. Anti-Chido (Cha) and Anti-Rodgers (Rga) In 1967, Harris and colleagues9 reported "A nebulous antibody responsible for crossmatching difficulties. Chido." The discovery by Middleton and Crookston1 that anti-ch" could be neutralized by plasma and was, therefore, also a plasma antigen was the first hint that this antibody was different from the other HTLA antibodies. Unfortunately, the subsequent discovery that the C h ( a - ) phenotype was associated with a certain HLA haplotype11 and that the antibody could be adsorbed with white cells led some workers to believe that the antigens were HLA associated and the term "red cell white cell antibody" came into vogue. 12 In fact, blood bankers tried to make all the HTLA antibodies they encountered fit this criteria, and even slight neutralization seen by titration was accepted as evidence of a red cell white cell antibody.1213 This state of confusion did not last long, and the discovery of antirodgers,14 a second plasma neutralizable antibody, led to the recognition of its association with certain HLA haplotypes; O'Neill and colleagues15 then correlated the Chido and Rodgers phenotypes with the electrophoretic polymorphism of the C4 component of complement and a person deficient in C4 was shown to be C h ( a - ), R g ( a - ). Tilley and colleagues16 confirmed these findings and showed that the Chido and Rodgers antigens were on the C4d fragment of C4. They also showed that the antiglobulin reactive anti-ch" and anti-rg" could be made to react as direct agglutinins if they were tested with cells heavily coated with C4d. In this case, it can be observed that it is not that the antibodies are of low avidity as much as that the antigens are of low density on the red cell, causing the serologic behavior characterized as HTLA. Since anti-ch* and anti-rg" can be neutralized, they are the easiest of all the HTLA antibodies to identify, and anti-ch* is probably the next most commonly encountered HTLA antibody after anti-yka. The use of C4 coated cells is also a rapid way of confirming the presence of one of these antibodies (see appendix 1). Since antich* and anti-rg* are reacting with the small amounts of C4 bound to the red cell, it is not too surprising that these antibodies have never been associated with red cell destruction, even with high-titered antibodies. A 1984 report by Lambin and colleagues17 described an unusual anaphylactic type of reaction in a patient receiving plasma. The patient was eventually shown to have an anti-rg* that could be demonstrated only by precipita- tion. The antibody was not a red cell agglutinin and resembled the type of antibody seen in IgA-deficient patients. This case is a good example of how a thorough investigation can determine an unusual cause of an allergic type of reaction. The Antibodies of the Knops-McCoy System In 197, Helgeson and colleagues18 reported a new high-frequency antigen, Kn*. The antibody of the patient Knops was reactive with 99.8% of the population and the red cells of Helgeson were also compatible. Helgeson's red cells were sent to many laboratories and soon other examples of antibodies that were only compatible with her cells were found. Many of these antibodies were later shown to be incompatible with the Knops red cells, however, and the term anti-kn"like was applied to these antibodies.19 Part of this puzzle was resolved by the article of Molthan and Moulds2 that described the McCoy antigen and its relationship to Kn*. Anti-McC* is nonreactive with the red cells of the Kn(a + ), McC(a-) phenotype and Kn(a-), McC(a-), or Helgeson type red cells. The patient Knops was Kn(a-), McC(a + ). Additional antibody specificities related to Knops-McCoy21 have been described that are also compatible with the Kn(a-), McC(a-) red cells. The relationship between the antigen Kn* and McC* is shown by the frequencies of people whose red cells are Kn(a-), McC(a ). If these antigens were unrelated, one would not expect that 69% of Kn(a - ) Caucasians would also be McC(a-). Similarly, 34% of McC(a-) blacks are Kn(a-). Some individuals who are Kn(a-) and McC(a-) have been shown to be negative for Yk* and Cs* as well. This overlapping of phenotypes has contributed to the confusion regarding the true specificities of some of these antibodies. One cannot identify HTLA antibodies on the basis of one or two antigen-negative cells and because the HTLA antigens tend to deteriorate during storage, negative reactions should be confirmed on fresh cells whenever possible. Anti-JMH Anti-JMH was first reported in LABORATORY MEDICINE VOL. 17, NO. 3, MARCH 1986 on 17 September 217

4 1977, although the antibodies had been known for a number of years. Sabo and colleagues22 were able to collaborate on a study that led to the naming of JMH after one of the first antibody formers. The antibody is usually found in elderly people, and the patients often have a positive DAT. The anti-jmh can occasionally be eluted from the cells and so this particular HTLA antibody behaves more like an autoantibody. There is some speculation that the antibody former's JMH antigen becomes suppressed in some way. The antigen is of high frequency and is destroyed by enzymes. To the author's knowledge, no transfusion reactions have been reported to be caused by anti-jmh, and it does not seem to cause any autohemolysis. Miscellaneous Anti-Gy" (Gregory) and anti-hy (Holley) are usually grouped with the HTLA antibodies, although there is good evidence that these antibodies are clinically significant.23 Like Kna and McC, there is a phenolytic association between Gy" and Hy. All Gy(a-) persons found to date have been Hy and have been Caucasian. All blacks that have been found to be Hy - have been Gy (a + w). Anti-Bg's are antibodies to the remnants of HLA antigens on red cells. The strength of the HLA antigen expressed on red cells varies considerably. For example, anti-bg8 reacts with cells from HLA-B7 positive persons, but not all HLA-B7 positive persons express the Bg" antigen on their red cells. In many instances, anti-yt" behaves like a benign HTLA antibody,23 although instances of hemolytic reactions have been reported. Its serologic characteristics seem to vary with the antibody former, and some caution should be observed when dealing with this antibody. zyme-treated panel cells and be able to perform serum neutralizations and antibody titrations. The blood bank also needs to have an adequate supply of donor blood to crossmatch, and the patient's red blood cells should be fully phenotyped, if possible. If these tests cannot be done, then the blood bank should refer the problem to another laboratory. The results of the enzyme panel must be evaluated. If the panel cells are all nonreactive, then the antibody may be anti-cha, -Yk, -JMH, or antirg". If the enzyme-treated panel cells are weaker and if some are negative, then the antibody is probably an antiyka. The results of the complete phenotyping must be analyzed to see which antibodies the patient can make. This information as well as crossing out on the nonreactive enzyme panel cells should be used to decide if any additional panel cells need to be tested. If HTLA antigen negative cells are available, the presence of additional antibodies may be excluded by negative reactions with those cells. AntiYt* and -Lub and other antibodies that may appear to be HTLA can be excluded in a similar fashion. If only the enzyme-treated cells are nonreactive, Lu(b-) and Yt(a-) cells should be tested. Resolving HTLA Antibody Problems If the results of the enzyme-treated panel are the same as the original panel, then an antibody in the KnopsMcCoy system, an anti-csa or, rarely, an anti-yka may be present. To exclude the presence of additional antibodies, it may be necessary to phenotype any compatible donor units and again to test any HTLA antigen negative panel cells available. The exclusion of other antibodies should be done on panel cells or donor cells from persons homozygous for the blood group gene whenever possible. If all the panel cells, crossmatches, and selected cells are incompatible, then the weak antibody is probably not an HTLA antibody but an antibody to an antigen of high frequency, in which case the problem may need to be referred to another laboratory. If the results of an initial investigation of an antibody problem indicate that it might be an HTLA antibody, then the blood bank needs certain resources to adequately resolve the problem. The blood bank should have or be able to prepare en- To further confirm the presence of an HTLA antibody, serum neutralizations and titers are useful (See Appendices 2 and 3 and Figs 1 and 2). Only anti-cha and -Rg" neutralize and newly formed antibodies will not be high titered. Mixtures of more than AHG AHG Neutralized Dilution Control Panel Panel Panel Cell* Fig 1. This panel shows the neutralization of an anti-ch* with serum, thus allowing the detection of an anti-e. Cells 1-1 are Ch*( + ); cells 4 and 5 are E +. one HTLA antibody are not uncommon, so the selection of the cells for the titers should be based on the phenotype of the patient's red cells and cells negative for one HTLA antigen that were incompatible with the patient's antibody (Fig 2). Summary As stated before, the important thing to be done when working with HTLA antibody problems is to find suitable blood for the patient and to exclude the presence of other antibodies. Establishing the identity of HTLA antibodies is unlikely without a large collection of rare cells, and the pitfalls are many. For the patient's sake, it is better to focus on the important issues than to apply a dubious identity and then try to make decisions based on that identity. AHGphase Neutralized Titer: AHGphase Control Titers Dilution #1 #2 #3 #1 #2 # ± ± + ± Cell #1 = Same Rh, MNSs Jk, Fy phenolypes as patient Cell #2 = Cs(a -) Yk(a-) Cell #3 = Kn(a -) McC(a -) Fig 2. These results show two antibodies are present. The only compatible cell is #2 after serum neutralization. The serum was later shown to have anti-yk11 and anti-ch*. LABORATORY MEDICINE VOL. 17, NO. 3, MARCH on 17 September 217

5 C4d fragment of human C4. Nature anti-lan and -Jra to "HTLA" antibodies. 1978;276: Transfusion 1981;21: Giles CM, Huth MC, Wilson TE, et al: Three 17. Lambin P, Le Pennec PY, Hauptmann G, et al: Adverse transfusion reactions associated with a examples of a new antibody, anti-cs", which 1. Giles C: Serologically difficult red cell antibodprecipitating anti-c4 antibody of anti-rodgers reacts with 98% of red cell samples. Vox Sang ies with special reference to Chido and Rodgers specificity. Vox Sang 1984;47: ;1:45. blood groups, in Mohn JK, Plunkett RW, Cunningham RK, et al (eds): Human Blood Groups. 9. Harris JP, Tegoli J, Swanson J, et al: A nebu- 18. Helgeson M, aswanson J, Polesky HF: KnopsHelgeson, Kn, a high-frequency erythrocyte Buffalo, Karger, 1977, pp lous antibody responsible for crossmatching difantigen. Transfusion 197;1:137. ficulties (Chido). Vox Sang 1967;12: Rolih S: Definition and characterization of HTLA Middleton J, Crookston MC: Chido-substance in 19. Race RR, Sanger R: Blood Groups in Man, ed antibodies, in Recognition and Resolution of1. High Oxford, Blackwell Scientific, Titer, Low Avidity Antibodies. Washington, DC,plasma. Vox Sang 1972;23: Molthan L, Moulds J: A new antigen, McCa American Association of Blood Banks, 1979, pp 11 Middleton J, Crookston MC, Falk JA, et al: Linkage of Chido and HL-A. Tissue Antigens (McCoy), and its relationship to Kna (Knops) ;4: Transfusion 1978;18: Mollison PL: Determination of red cell survival using 51Cr, in Bell CA (ed): A Seminar on Im-12. Swanon J: Laboratory problems associated with 21. Molthan L: Expansion of the York, Cost, McCoy, group system: The new McCoy anleukocyte antibodies, in A Seminar on Recent Knops blood mune-mediated Cell Destruction. Washington, tigens McCc and McC". Med Lab Sci 1983;4:113Advances in Immunohematology. Washington, DC, American Association of Blood Banks, 1981, 121. DC, American Association of Blood Banks, 1973, pp pp Sabo B, Moulds JJ, McCreary J: Anti-JMH, an4. Molthan L: Biological significance of the York, 13. Issitt P, Issitt CH: Applied Blood Group Serol-other high-titer, low avidity antibody against a Cost, McCoy and Knops alloantibodies. Blood high frequency antigen, abstracted. Transfusion ogy, ed 2. Oxnard, CA, Spectra, 1975; Transfusion Immunohematology 1982;25: ;18:387. Longster G,a Giles CM: A new antibody specific ity, anti-rg, reacting with a red cell and serum 23. Vengelen-Tyler V: n-neutralizable HTLA an5. Gandhi JG, Moulds JJ, Szymanski 1: Shorttibodies and the antigens they define, in Recantigen. Vox Sang 1976;3: ened long-term survival of incompatible red cells ognition and Resolution of High-Titer, L Neill GJ, Yang SY, Tegoli J, et al: Chido and in a patient with anti-mccoy-like antibody. 15 O' Avidity Antibodies. Washington, DC, American Rodgers blood groups are distinct antigenic Transfusion 1984;24: Association of Blood Banks, 1979, pp Molthan L, Giles C: A new antigen, Yk» (York) components of human complement C4. Nature 1978;273: Judd WJ, Kraemer K, Moulds JJ: Rapid idenand its relationship to Csa (Cost). Vox Sang tification of Chido and Rodgers antibodies using 1975;29: Tilley CA, Romans DG, Crookston MC: LocaliC4d coated red cells. Transfusion 1981;21:189. zation of Chido and Rodgers determinants to the 7. Vengelen-Tyler V, Morel P: The relationship of References Appendix 1: Rapid Identification of Chido and Rodgers Antibodies Using C4d-Coated Red Cells24 red cells with trypsin. 4. Incubate at 37 C for 3 minutes. 5. Wash the red cells four times in isotonic saline, and dilute to a 5% concentration in Alsever's solution. These red cells will keep for several weeks in Alsever's solution at 4 C. 6. To one drop of C4d-coated (trypsin-treated-coated) red cells, 1. Dispense 1 ml of 1% sucrose into an evacuated tube conadd three drops of test serum. Similarly,testthe serum against taining 15 mg of K3 EDTA. the control (trypsin-treated non-c4-coated) red cells. 2. Add 1 ml of ACD/CPD/CPDA-1 whole blood, and mix well 7. Gently agitate the contents of each tube, and incubate at by inversion. room temperature for five minutes. 3. Incubate at 37 C for 15 minutes. 8. Centrifuge at 1, x g for 15 seconds. 4. Wash the C4-coated red cells three times in saline. 9. Examine the red cells macroscopically for agglutination. Grade and record the results. Materials for Rapid Identification of Ch and Rg Antibodies 1. Wash the red cells four times in isotonic saline, and com1. C4-coated red cells, as prepared in previously mentioned pletely decant the final supernatant fluid. technique, using blood from a known group O, Ch(a + )/Rg(a +) 11. To the dry red cell buttons thus obtained, add IgG according individual. to manufacturer's directions. 2. 1% trypsin. 12. Gently agitate the contents of each tube, and centrifuge at 3..1 M phosphate buffer at a ph of , x g for 15 seconds. 4. Serum to be studied. 13. Examine the red cells macroscopically for agglutination. Grade 5. n-c4-coated red cells (the same red cells used in. 1 and record the results. above, but not exposed to sucrose). 6. Anti-IgG. Materials for Preparation of C Id Coated Red Cells 1. CPDA-1 anticoagulated whole blood. 2. 1% (w/v) sucrose in distilled water. 3. Evacuated tube (16 x 1 mm, containing 15 mg of K3EDTA). 1. Wash the C4-coated red cells and non-c4-coated red cells three times in isotonic saline if suspended in a diluent other than saline. 2. Dilute one volume of the 1% trypsin solution with nine volumes of phosphate buffer, at a ph of Mix one volume of diluted trypsin with an equal volume of packed C4-coated red cells. Similarly treat the non-c4-coated tes: Agglutination of the C4d-coated red cells when the trypsin-treated control red cells are nonreactive is indicative of either anti-cha or anti-rg\ Agglutination of both the C4d-coated and the trypsin-treated control red cells is an invalid test result, due to either the presence of trypsin-dependent panagglutinins or an alloantibody with specificity unrelated to anti-ch" or anti-rg". There is no need to incubate the tests at 37 C prior to testing with anti-igg. 136 LABORATORY MEDICINE VOL. 17, NO. 3, MARCH 1986 on 17 September 217

6 Appendix 2: Titration of Serum Samples to Demonstrate the Presence of High-Titer, Low-Avidity (HTLA) Antibodies Materials 1. Selected red cell samples (see Fig 2). 2. Serum to be studied. 3. A polyspecific or anti-igg antiglobulin reagent. 4. 6% bovine serum albumin. 1. Prepare serai dilutions of test serum in 6% bovine serum albumin or pooled antibody free serum. Dilute from 1:2 to 1:2, Place three drops of each dilution into appropriately labeled 12 x 75-mm test tubes. 3. Add one drop of a 3% to 4% saline suspension of one of the selected red cell samples to one tube of each serum dilution (see Fig 1 for examples). 4. Gently agitate the contents of each tube and incubate at 37 C for one hour. 5. Wash the red cells four times in isotonic saline, and completely decant the final supernatant fluid. 6. To the dry red cell buttons thus obtained, add antiglobulin reagent according to manufacturer's directions. 7. Gently agitate the contents of each tube, and centrifuge at 1, x g for 15 seconds. 8. Examine the red cells for agglutination. Grade and record the results. Interpretation: As shown in Fig 2, HTLA antibodies will react far beyond the range at which most 2 + or weaker antibodies react. Appendix 3: Neutralized Panel for Detection of Additional Antibodies in Presence of Anti-Ch or Anti-Rg Materials 1. Patient serum. 2. Pooled antibody negative serum. 3. 6% bovine serum albumin. 1. Label two sets often tubes, 1 through 1. Label one set "test" and one set "control". 2. To the set labeled "test" add one drop patient serum and one drop pool serum to each tube. 3. To the set labeled "control" add one drop patient serum and one drop 6% bovine serum albumin to each tube. 4. Incubate at room temperature for 15 minutes. 5. Add one drop 4% panel cells to appropriate tubes. 6. Incubate at 37 C for ten minutes. 7. Wash panel cells four times and completely decant final wash. 8. Add polyspecific antiglobulin according to manufacturer's directions. 9. Mix and centrifuge at 1, x for 15 seconds. 1. Examine the panel red cells for agglutination. Macroscopically grade and record results. tes: If both the test and the control panel cells are positive, then the antibody is not anti-ch" or anti-rg", unless it is so high titered that a one to two dilution is too low a dilution. If so, a serum neutralization using serial dilutions as discussed in appendix 2 will show that the antibody can be neutralized. If both the test and the control panel cell are negative, then the antibody is too weak to be diluted. If a test panel cell is negative and a control panel cell is positive, then the antibody is neutralizable (anti-ch* or anti-rg"). If some of the panel cells are still reactive (see Fig 1), then a second antibody may be present. Category A-1 continuing education credit is available to anyone who studies a CIE Update series and completes a written exam. Exams will appear with the final article of each series in LABORATORY MEDICINE. After receipt of a completed answer sheet and exam fee at ASCP, a certificate of credit will be awarded to each participant. LABORATORY MEDICINE VOL. 17, NO. 3, MARCH on 17 September 217