Serology of antibodies to second- and third-generation cephalosporins associated with immune hemolytic anemia and/or positive direct antiglobulin tests

BACKGROUND: First-generation cephalosporins rarely caused immune hemolytic anemia (IHA). Second- and third-generation cephalosporins, especially cefotetan and ceftriaxone, are increasingly associated with severe, sometimes fatal IHA. STUDY DESIGN AND METHODS: Samples from 53 patients with drug-induced IHA and/or positive direct antiglobulin test (DAT) were tested. Patients' sera were tested against drug-treated red cells (RBCs) and untreated or enzyme-treated RBCs, with and without the addition of drug solution. Eluates from patients' RBCs were tested against drug-treated and untreated RBCs. RESULTS: Forty-three patients had antibodies to cefotetan, 8 to ceftriaxone, 1 to cefoxitin, and 1 to cefotaxime. All patients had a positive DAT; only anticefoxitin and anti-cefotetan were demonstrable in RBC eluates. Sera containing anti-cefoxitin, anti-cefotaxime, and anti-cefotetan reacted with drug-treated RBCs (100%) and untreated or enzyme-treated RBCs in the presence of drug (98% or 100%, respectively). All of the ceftriaxone antibodies reacted with untreated or enzyme-treated RBCs in the presence of drug, but those tested did not react with ceftriaxone-treated RBCs. In addition to cefotetan-dependent antibodies, 19 (44%) and 14 (33%) of 43 sera contained drug-independent antibodies when tested with and without the presence of a polyethylene glycol potentiator, respectively. CONCLUSION: Cefotetan is by far the most common cause of drug-induced IHA. All cefotetan antibodies and the single examples of cefoxitin and cefotaxime antibodies reacted with drug-coated RBCs, and most, in contrast to the reactions of antibodies to first-generation cephalosporins (e.g., cephalothin), also reacted with RBCs (not treated with drug) in the presence of the drug. Ceftriaxone antibodies reacted only by the latter mechanism. Drug-independent antibodies (i.e., those reacting without any drug being present) were detected in 33 to 44 percent of patients' sera containing cefotetan antibodies, depending on the sensitivity of the method used.     

RBC antibody persistence

BACKGROUND: A person exposed to foreign blood group antigens may produce antibodies. The persistence of antibodies varies among people and among antibodies. A study was performed to investigate the persistence of clinically significant RBC alloantibodies over a period of 20 years. STUDY DESIGN AND METHODS: A retrospective examination was performed of all records of RBC antibodies in the transfusion laboratory computer database from 1978 through 1997. Records of patients who underwent at least one antibody investigation after an antibody had been detected were studied. The study included all antibodies against the Rh, K, Fy, Jk, and MNs blood group systems. An antibody was regarded as not persistent if, after previous detection, the screening or panel studies became negative for the antibody under study. Anti-D due to RhIg administration was excluded. RESULTS: An analysis was performed of 480 records consisting of 593 antibodies that fulfilled the criteria. Median antibody follow-up was 10 months (range, 1-240). In 137 patients, 153 (26%) antibodies became undetectable over the course of time. After initial negative screening investigations, 310 antibodies were formed. The antibodies that were still detectable had a median follow-up of 7 months (range, 1-193). A patient's age, sex, and antibody specificity were of no influence on the length of time that antibodies were detectable. Antibodies detected with a more sensitive screening technique were less persistent (p = 0.0002). For 28 patients, detection of antibodies was highly irregular. CONCLUSIONS: About 25 percent of all antibodies became undetectable over the course of time. The antibody screening technique used, rather than the antibody specificity, affected these results. To prevent delayed hemolytic transfusion reactions, precise antibody documentation is of great importance.     

Hemolytic transfusion reaction due to anti-Tca

BACKGROUND: Anti-Tc(a) detects a high-incidence antigen in the Cromer blood group system. Cromer system antibodies have not usually been associated with hemolytic transfusion reactions or hemolytic disease of the newborn. CASE REPORT: Anti-Tc(a) (initially identified in the patient's serum in 1982) was not detected when she was admitted to the hospital with upper gastrointestinal. bleeding. Three units of red cells were administered. The patient was discharged, but was readmitted to the hospital after her hemoglobin fell to 7.1 g per dL. Antibody detection tests remained negative and three additional units were transfused. Over the next 7 days, her hemoglobin steadily fell to 5.5 g per dL. The level of lactate dehydrogenase rose to 1257, the plasma hemoglobin rose to >16 mg per dL, and the haptoglobin decreased to <6 mg per dL. Five days after transfusion, her direct antiglobulin test was weakly reactive with complement-specific antiglobulin reagents. Eluates were nonreactive. Anti-Tc(a) was detected in her serum; no other antibodies were detected. Differential typing failed to detect any circulating Tc(a+) red cells. The antibody was strongly reactive in a monocyte monolayer assay. CONCLUSION: Although Cromer system antibodies have generally not been proven to be clinically significant in transfusion therapy, the destruction of red cells from six units of transfused Tc(a+) red cells in this patient indicates that anti-Tc(a) may have destructive potential in some patients.     

Antibodies to private and public HLA class I epitopes in platelet recipients

BACKGROUND: Transfusions or pregnancies can cause immunization against private HLA determinants and public epitopes shared by more than one private HLA antigen. HLA antibodies are correlated with febrile transfusion reactions, lower platelet response following platelet transfusion, and an increased rate of renal transplant rejection. Until now, antibody specificities in alloantisera from platelet recipients have been poorly characterized. STUDY DESIGN AND METHODS: Consecutive serum screens from platelet recipients were analyzed for antibodies against private HLA class I antigens and public HLA epitopes using a serum analysis program based on the 2 x 2 table analysis of correlations. Serum screens of highly immunized patients and of patients with new alloimmunization events were reviewed separately. RESULTS: Of the serum screens from 566 platelet recipients, 1577 indicated alloimmunization (panel-reactive antibodies >5%). The program assigned a specificity in 1024 of these screens (64.9%) and at least once in 522 of 566 patients (92.2%). In 267 patients, antibodies detecting public epitopes in the combined A- or B-locus cross-reacting groups were found; other public markers were detected in 39 patients. Patterns of reactivity were remarkably less stable than in patient groups previously studied. In many patients, antibodies with apparent private epitope specificity preceded the identification of antibodies against a shared marker of the same cross-reactive group. However, the disappearance of antibodies (whether or not this was followed by a new antibody against a private or public marker belonging to another cross-reacting group) was also observed. CONCLUSION: The computerized analysis of microlymphocytotoxicity tests enhances the rate of antibody specification in sera from platelet recipients with lymphocytotoxic antibodies. The identified antibodies should be taken into account in the selection of platelet donors. The data confirm and extend previous observations on HLA class I antibodies and elucidate new alloimmunization events.     

K, Fy(a), and Jk(a) phenotyping of donor RBCs on microplates

BACKGROUND: In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time-consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fy(a), and Jk(a), as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population. STUDY DESIGN AND METHODS: One thousand thirty-one EDTA samples from blood donors were phenotyped for K, Fy(a), and Jk(a) by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed-ID, DiaMed). RESULTS: All typing results with the microplate method were correct. All reactions for K and Fy(a) typing could be interpreted by the computer. The results for Jk(a) were indeterminate in 1.4 percent (14/1031) of the samples. CONCLUSION: The PEG-IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.     

First two cases of immune hemolytic anemia associated with ceftizoxime

BACKGROUND: Second- and third-generation cephalosporins have been associated with immune-mediated hemolytic reactions. This report discusses two patients who developed clinically significant extravascular hemolysis while receiving the third-generation cephalosporin ceftizoxime (Ceftizox). This is believed to be the first time hemolysis has been described in patients receiving this drug. STUDY DESIGN AND METHODS: Immunologic workup of drug-dependent antibodies was performed on blood samples using drug-coated and immune complex methodologies. Antibody classes and titers were analyzed. RESULTS: Both the patients' sera contained anti-ceftizoxime that reacted with red cells only when ceftizoxime was added to the sera ("immune complex" method). The patients recovered without complications following discontinuation of the drug. Each patient had IgM and IgG drug-dependent antibodies.The drug-induced antibodies from each patient cross-reacted with cefotaxime, which is structurally similar to ceftizoxime, but cross-reacted either weakly or not at all with ceftriaxone, which has a more complex side chain. CONCLUSION: This report describes the first cases of immune hemolytic anemia associated with ceftizoxime. In drug-induced hemolytic reactions, prompt recognition and discontinuation of the drug may be important factors in reducing the chance of serious sequelae.     

Comparison of the performance of four microtube column agglutination systems in the detection of red cell alloantibodies

BACKGROUND: The purpose of this study was to compare the performance of four currently available microtube column agglutination systems in the detection of red cell alloantibodies to that of the standard tube low-ionic-strength solution (LISS) indirect antiglobulin test (IAT) (tube LISS-IAT). STUDY DESIGN AND METHODS: In a comparative study, 172 sera, previously demonstrated to contain red cell alloantibodies, were tested in parallel by the tube LISS-IAT and three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue, and Sanofi-Pasteur Scangel) and one affinity-adherence test system (Gamma ReACT). Tests were performed simultaneously by a single person on freshly thawed sera that had been frozen at -20 degrees C. RESULTS: The rate of detection of clinically significant alloantibodies (n = 154) in microtube column systems was very similar. One hundred forty-one sera (91.6%) reacted in the DiaMed-ID, 139 (90.3%) in the ReACT, 139 (90.3%) in the BioVue, and 142 (92.2%) in the Scangel. Only 117 (76.0%) of these sera reacted in the tube LISS-IAT. The detection rates for 18 antibodies of minor clinical significance (anti-M, -N, -P1, -Le(a), and -Le(b)) varied among the test systems: DiaMed-ID, 5 (28%); ReACT, 7 (39%); BioVue, 14 (78%); Scangel, 10 (56%); and tube LISS-IAT, 6 (33%). Antibody reactivity as determined by titer and score was very similar in all microtube column systems and higher in these systems than in the tube LISS-IAT. CONCLUSION: The sensitivity of all four microtube column systems in the detection of clinically significant red cell alloantibodies was similar and was markedly superior to that of the tube LISS-IAT. An individual cost-benefit analysis should be performed in every institution to decide whether a microtube column system should be applied. If so, the antibody screen in the microtube column agglutination system should ideally be performed in advance of the crossmatch to provide time to screen for compatible units.     

Immune hemolytic anemia induced by 6-mercaptopurine

BACKGROUND: Myelosuppression is the main hematotoxic effect of 6-mercaptopurine (6-MP), which is an antimetabolite chemotherapy drug. Immune hemolytic anemia associated with this drug has not been previously reported. CASE REPORT: A 67-year-old man with chronic myelomonocytic leukemia presented with anemia 2 weeks after 6-MP therapy had been initiated. Additional tests provided laboratory evidence of hemolysis. When treatment was stopped, the patient's condition and laboratory results showed a progressive improvement. RESULTS: The direct antiglobulin test was positive for IgG. The eluate and the serum were not reactive with panel red cells but reacted with 6-MP-treated red cells, while the normal serum pool was unreactive. The direct antiglobulin test was no longer positive by 20 days after the cessation of 6-MP therapy. CONCLUSION: This drug, 6-MP, should be added to the list of drugs that have been reported to cause immune hemolytic anemia by means of the so-called hapten mechanism.     

PEG adsorption of autoantibodies and detection of alloantibodies in warm autoimmune hemolytic anemia

BACKGROUND: The purpose of this study was to evaluate the effectiveness of PEG in the adsorption of autoantibodies and the detection of alloantibodies in patients with warm autoimmune hemolytic anemia (WAIHA). STUDY DESIGN AND METHODS: The study was divided into three parts. First, the effectiveness of antibody removal by a conventional adsorption procedure and that by the PEG adsorption method were compared by using commercial antisera adsorbed with antigen-positive RBCs. Second, patients' sera with known weak alloanti-E were used to test against screening cells after allogeneic adsorption to show nonspecific coating of antibodies and the dilution effect on the PEG adsorption procedure. Third, conventional and PEG one-cell sample allogeneic adsorptions of WAIHA patients' sera with and without alloantibodies were performed and compared. RESULTS: In the first part, seven of the eight polyclonal antisera gave a lower titer and score when tested against antigen-positive RBCs in the indirect antiglobulin test; this indicated that PEG is more efficient in removing antibodies. In the second part, similar reaction patterns were observed in antibody screening tests on unadsorbed sera and on sera after the first, second, and third adsorptions, which indicated that there was no alloantibody loss, nonspecific antibody coating, or dilution effect when PEG was added in the allogeneic adsorption procedures. In the third part, a 40-percent increase in efficiency in reducing the number of adsorptions and an 85-percent decrease in completion time of the adsorption procedures were obtained when PEG was used for adsorbing sera from 16 patients with WAIHA. Both conventional and PEG procedures were capable of removing autoantibodies to identify the specificities of alloantibodies. CONCLUSION: The PEG adsorption method is an effective, sensitive, and efficient method of enhancing auto- antibody adsorption and alloantibody detection. It can reduce the processing time and minimize the delay of urgent transfusions to patients with WAIHA and can indirectly enhance cost-effectiveness and decrease the labor-intensive testing required by these cases.     

Anti-G in a pregnant patient

BACKGROUND: Anti-G is a red cell (RBC) antibody of the Rh system. It has been described in pregnant women only in association with anti-D or anti-C; therefore, the ability of this antibody alone to cause hemolytic disease of the newborn is uncertain. One case in which this antibody caused no clinical sequelae is reported. CASE REPORT: The patient was a 35-year-old primigravida with type O, D-, C-, E-, c+ RBCs who was given 4 units of type O, D- allogeneic RBCs and 2 units of autologous RBCs 2 years antepartum. She was found to have anti-D and anti-C by an outside laboratory as part of a routine prenatal work-up. Further evaluation by our laboratory revealed the presence of anti-G and possible anti-C without anti-D. Titers at 22 weeks' gestation were 64 against r'r RBCs and 16 against R2R2 RBCs; these remained unchanged throughout the pregnancy. Amniocentesis performed at Weeks 28 and 32 showed no evidence of hemolytic disease of the newborn. A healthy 3.3-kg infant was delivered at 36 weeks' gestation. Prophylactic Rh immune globulin was administered antepartum and postpartum. The infant's RBCs were type O, D+, c+ C-, E-, and the direct antiglobulin test was positive. An acid eluate prepared from the baby's RBCs revealed anti-G. The total bilirubin was 5.5 mg per dL at birth, and the hematocrit was 66 percent. Total bilirubin peaked on Day 5 at 11.9 mg per dL, and no therapeutic intervention was required. CONCLUSIONS: Anti-G alone caused little if any fetal or neonatal hemolysis in this case. Although further study is needed, invasive fetal monitoring may be unnecessary if anti-G is the sole cause of fetomaternal RBC incompatibility.     

Donor-derived alloantibodies and passenger lymphocyte syndrome in two of four patients who received different organs from the same donor

BACKGROUND: Reported here is the occurrence of RBC alloimmunization in two of four patients who received different organs from an immunized donor. STUDY DESIGN AND METHODS: The donor, a 58-year-old woman, was group O D+, K-, and Fy(a-). Initially, her serum contained only a K antibody. After blood transfusion, a second antibody (anti-Fy(a)) could also be identified. The liver was given to a group O D+, K-, Fy(a+) patient; the pancreas and one kidney to a group O D+, K-, Fy(a+) patient; the heart to a group A D+, K-, Fy(a-) patient; and the other kidney to a group B D+, K-, Fy(a+) patient. RBC grouping and antibody screening were performed by standard techniques. Lymphoid microchimerism in the peripheral blood of the recipients was analyzed by flow cytometry and nested PCR. RESULTS: None of the recipients had irregular RBC alloantibodies at the time of transplantation. After the transplant, anti-K became detectable in the serum of the liver recipient, and anti-Fy(a) could be eluted from the RBCs of the liver recipient and the pancreas-kidney recipient. The latter patient also developed mild hemolysis, and his Hb dropped to 8 g per dL on posttransplant Day 9. Donor-derived lymphocytes were detectable by flow cytometry in the peripheral blood of the liver recipient and the pancreas-kidney recipient until Days 8 and 63, respectively, whereas no lymphoid chimerism could be demonstrated in the heart recipient. PCR chimerism analyses were positive in all three recipients over the whole observation period of 97 postoperative days. CONCLUSION: The amount of cotransplanted lymphoid tissue may correlate with the extent of peripheral lymphoid microchimerism and the antibody-formation capacity in solid organ transplantation.     

A MAR-like antibody in a DCWe/DCWe person

BACKGROUND: MAR (RH51), a high-incidence antigen in the Rh blood group system, is absent from RBCs with a double dose of CW or CX or a single dose each of CW and CX antigens, as well as from rare Rh phenotypes including D- - and Rh(null). The MAR antigen is associated with the presence of Ala36 and Gln41 on the RhCe protein. The original example of anti-MAR, described in 1994, was made by a DCWe/DCXe woman. It was possible that the antibody directed against a high-incidence antigen in the Rh system made by a DCXe/DCXe woman (CM) in 1983 was anti-MAR. CASE REPORT: A 52-year-old, multiply transfused, white woman (CJ) with pre-existing anti-c, -E, and -Jk(a) presented for preoperative work-up for her fifth open heart procedure. The strength of the reaction of her RBCs with anti-CW suggested a double dose of CW antigen. Her serum, which unexpectedly was strongly reactive with c-, E-, Jk(a-) RBCs by PEG indirect antiglobulin test, was incubated with E-c-, Jk(a-) RBCs, and an eluate was prepared. This eluate reacted 3+ with E-c-, Jk(a-) RBCs but did not react with Rh(null) (n = 5), - - (3), DCW- (2), Dc- (1), or DCWe/DCWe (1) RBCs. Two related DCXe/DCXe and two unrelated DCWe/DCXe RBC samples were weakly agglutinated. The patient's RBCs were negative with the original anti-MAR but reacted as strongly as the positive control RBCs with the antibody made by the DCXe/DCXe person. CONCLUSION: This is the first example of a MAR-like antibody made by a DCWe/DCWe woman. The specificity cannot be called anti-MAR, because some MAR-negative samples react, albeit weakly. The original anti-MAR, made by a DCWe/DCXe woman, did not react with DCWe/DCWe, DCWe/DCXe, or DCXe/DCXe RBCs. It is apparent that the specificity of "anti-MAR" differs slightly, depending on the CW/CX status of the antibody maker.     

Ultraviolet and visible light spectrophotometric approach to blood typing: objective analysis by agglutination index

BACKGROUND: A new blood typing technology based on ultraviolet (UV) and visible light spectroscopy (UV/visible spectroscopy) has been developed. Blood groups and types are determined by quantifying reproducible changes in the UV and visible light spectra of blood in the presence of agglutinating antibodies. STUDY DESIGN AND METHODS: Samples of red cells in the presence and absence of agglutinating antibodies were examined by UV/visible spectroscopy. Blood groups and types were determined by comparing the optical density spectra obtained between 665 and 1000 nm. These comparisons generate numbers (agglutination index) ranging from 0 to 100, with smaller numbers corresponding to lack of agglutination and larger numbers corresponding to agglutination. RESULTS: The optical density of agglutinated blood is dramatically different from that of unagglutinated blood. The agglutination index derived from the relative slopes of the spectra is an objective indicator of agglutination strength. An agglutination index greater than 17 consistently and accurately established blood group- and type-specific agglutination. CONCLUSION: The method accurately predicted A, B, and O blood groups, and D type in over 275 samples. Scattering theory-based calculations of relative volumes of red cells before and after agglutination show a direct correlation with the agglutination index and provide the theoretical basis of the analysis. This quantitative technique is reproducible and has the potential for automation.     

WBC subset analysis of WBC-reduced platelet components

BACKGROUND: WBC-reduced platelet components may be prepared by filtration or apheresis processing. Both methods have previously been shown to result in a residual total WBC content <5 x 10(6) per component. However, there may be differences in the efficacy of these techniques for removing certain WBC subsets. STUDY DESIGN AND METHODS: Two multiparameter flow cytometric assays were developed and validated to perform WBC analysis on WBC-reduced platelets collected with two apheresis instruments (Amicus and COBE Spectra) and on 6 units of filtered pooled random-donor platelet concentrates. RESULTS: All components contained <1 x 10(5) WBCs. The COBE Spectra and Amicus apheresis platelet components contained more WBCs than did filtered pooled platelets (p<0.05). Lymphocytes (T and B), monocytes, and granulocytes were identified in all components. Granulocyte content was lowest in the Amicus components and filtered pools. Monocytes were lowest in filtered pools. Amicus platelet components had fewer granulocytes and monocytes than the COBE Spectra platelets. Amicus and COBE Spectra components contained more lymphocytes than the filtered pools. CONCLUSION: Multiparameter flow cytometry can be used to quantify and characterize WBCs in WBC-reduced platelet components. WBC reduction by filtration or apheresis was highly effective. WBCs from each subset were identified in all components. Although filtered pools had the lowest numbers of WBCs, the very low numbers observed in all components suggests that the absolute quantitative differences in WBC subset content are of questionable clinical significance.     

Severe delayed hemolytic transfusion reaction secondary to anti-Ata

BACKGROUND: Anti-At(a) is a rare red cell (RBC) alloantibody found in the black population. It has been described as causing one case of mild hemolytic disease of the newborn, but its ability to cause hemolytic transfusion reactions is uncertain. CASE REPORT: The patient was a 60-year-old black female with a history of three uneventful pregnancies but no transfusions. On admission, her direct and indirect antiglobulin tests were negative, total bilirubin was 0.5 mg per dL, and lactate dehydrogenase was 224 IU per L. She received nine units of compatible RBCs in the perioperative period of a hemicolectomy. Her hemoglobin rose appropriately and stabilized at 12.6 g per dL by the 6th postoperative day. By Day 10 after surgery her hemoglobin had dropped to 6.8 g per dL, and her total bilirubin and lactate dehydrogenase had risen to 1.4 mg per dL and 783 IU per L, respectively. The direct and indirect antiglobulin tests were now newly positive with strengths of 3+. A warm hemolytic autoantibody was suspected. She was transfused two units of incompatible RBCs for a rapidly falling hemoglobin and symptomatic anemia. On Day 11, the total bilirubin rose to 3.5 mg per dL, and the lactate dehydrogenase was 1154 IU per L with a hemoglobin of 7.6 g per dL. Corticosteroids were begun. Studies of serum and an acid eluate revealed anti-At(a), but no other RBC antibodies. The patient stabilized, and further transfusion was avoided. CONCLUSION: Although anti-At(a) was previously described as being of uncertain clinical significance, this patient demonstrated the ability of the antibody to cause a severe delayed hemolytic transfusion reaction.     

Passenger lymphocyte syndrome with severe hemolytic anemia due to an anti-Jk(a) after allogeneic PBPC transplantation

BACKGROUND: After allogeneic peripheral blood progenitor cell (PBPC) transplantation, a patient developed a severe hemolytic transfusion reaction due to passenger lymphocyte syndrome. CASE REPORT: A 50-year-old woman with secondary acute myeloid leukemia transforming from a myelodysplastic syndrome received an ABO-compatible PBPC graft from her HLA-identical sister. For prophylaxis of GVHD, the patient was treated with cyclosporine and methotrexate. Eighteen days after the transplant, the patient experienced a severe hemolytic transfusion reaction due to an alloantibody (anti-Jk(a)) produced by donor lymphocytes. RESULTS: The patient was typed as group A, Jk(a+) before transplantation; the donor was typed as group A, Jk(a-). On Day 18 after transplantation, the immunohematologic screening revealed a positive DAT (C3d 3+) and an alloanti-Jk(a). Hemolysis in the patient at that time was indicated by a drop in the Hb and an increase in the LDH level (maximum, 592 IU/L on Day 23). CONCLUSION: The course of hemolysis and the time of appearance of an alloantibody in this patient meet the criteria for passenger lymphocyte syndrome. In most cases, this syndrome is triggered by ABO system antibodies. This is the first reported case of passenger lymphocyte syndrome after PBPC transplantation that was due to an alloantibody that did not belong to the ABO system.     

Development of non‐ABO RBC alloantibodies in patients undergoing allogeneic HPC transplantation. Is ABO incompatibility a predisposing factor?

BACKGROUND: Data from the appearance of RBC antibodies other than ABO in patients undergoing HPC transplantation are limited. STUDY DESIGN AND METHODS: The incidence and specificity of non-ABO RBC alloantibodies are described in a series of 217 patients undergoing allogeneic HPC transplantation because of various hematologic malignancies. RESULTS: Eight patients (3.7%) developed 10 antibodies after transplant. None of these patients had previously been immunized. Seven patients had one RBC antibody and one patient had three RBC antibodies. Antibody specificity were anti-Jk(b) (2 patients), -Kell (2), -M (2), -Le(b) (1), and -D (1). Finally, two patients had a panagglutinin. The mean time between transplant and antibody detection was 23 days (range, 16-672). The source of the HPCs, the conditioning regimen administered, and the type of GVHD prophylaxis administered did not influence the rate of antibody formation. On multivariate analysis, ABO blood group incompatibility (p = 0.005) and patient's age (p = 0.02) were the only two variables significantly associated with the development of RBC alloantibodies. CONCLUSION: Patients undergoing allogeneic HPC transplantation are at risk of developing RBC-specific antibodies despite the immunosuppressive therapy administered. Antibody formation was more frequently observed in ABO-mismatched cases, which suggests a potential role of this incompatibility in facilitating antibody production.     

First results using automated epifluorescence microscopyto detect Escherichia coli and Staphylococcus epidermidisin WBC-reduced platelet concentrates

BACKGROUND: Many methods have been tested for the detection of bacterial contamination in platelets. However, only those using molecular biology or cell culturing consistently detect contamination at levels below 10(5) bacteria per mL. This report describes the initial investigation into an alternative method that offers the possibilities of high sensitivity and rapid response while using available laboratory equipment and supplies. This method relies on a fluorescent nucleic acid stain, which preferentially stains bacteria but not platelets, and automated epifluorescence microscopy for rapid analysis. Measurements in WBC-reduced platelet concentrates (PCs) contaminated with bacteria are reported at concentrations between 10(3) and 10(6) bacteria per mL. STUDY DESIGN AND METHODS: Staphylococcus epidermidis or Escherichia coli was inoculated into aliquots of WBC-reduced PCs on Days 2 through 5 of storage. Bacterially inoculated and control PCs were stained, platelets and residual WBCs were lysed, and 200 microL of sample was filtered onto black polycarbonate filters. All preparations were done in triplicate. An automated epifluorescence microscope examined approximately 2 percent of the area of each filter and used image analysis to select the fluorescent particles that should be counted as bacteria. RESULTS: Samples containing 3 to 5 x 10(3) bacteria per mL produced about three times as many fluorescent particles classified as bacteria as the controls. Lower concentrations of S. epidermidis were detected because of higher fluorescence intensity. Simultaneous preparation of six samples requires about 35 minutes. Analysis of each prepared sample takes 10 minutes, for a total preparation and analysis time of about 95 minutes for 6 samples. CONCLUSION: Low concentrations (<5 x 10(3) bacteria/mL) of deliberately inoculated S. epidermidis or E. coli can be measured quickly in WBC-depleted PCs by using a fluorescent nucleic acid stain, differential lysis, and automated microscopy. Continued refinement of the method, studies employing other bacterial strains, and further validations of assay performance are warranted.     

Trends in the incidence of delayed hemolytic and delayed serologic transfusion reactions

BACKGROUND: An increasing incidence of delayed hemolytic and delayed serologic transfusion reactions (DHTRs/DSTRs) has been seen at the Mayo Clinic since 1978. Recently, the average length of stay (LOS) for inpatients and the average number of red cell transfusions per inpatient (TPI) decreased, and the albumin and papain technique for RBC antibody detection was replaced by a polyethylene glycol technique. These changes may have affected the incidence of DHTRs/DSTRs. STUDY DESIGN AND METHODS: The diagnoses of DHTR and DSTR made at the Mayo Clinic from 1993 through 1998 were reviewed. These data were compared with previously published Mayo Clinic data from 1980 through 1992. The LOS for inpatients and the average TPI were also obtained from hospital data. RESULTS: The incidence of DHTR/DSTR increased from 1 in 1899 in 1980 through 1992 to 1 in 1300 in the 1993 through 1998 period (p < 0.05). Similarly, DSTR increased from 1 in 2990 in 1980 through 1992 to 1 in 1612 in the 1993 through 1998 period (p < 0.05). The incidence of DHTR showed a trend toward decrease, from 1 in 5405 in 1980 through 1992 to 1 in 6715 in 1993 through 1998. No alloantibody specificities were statistically associated with DHTRs in 1993 through 1998, unlike in the 1980 through 1992 period. Moreover, the incidence of Jka antibodies increased in 1993 through 1998, while the incidence of other alloantibodies remained stable. Average LOS and TPI declined by 24.5 percent and 8.8 percent, respectively, between the two periods. CONCLUSION: Recently, a trend toward a decrease in the incidence of DHTR and a significant increase in DSTRs has occurred at the Mayo Clinic. These changes are most likely due to a combination of factors, including a decrease in average LOS and the adoption of the polyethylene glycol antibody detection system.     

DNA from blood samples can be used to genotype patients who have recently received a transfusion

BACKGROUND: The use of hemagglutination to phenotype red cells from recently transfused patients or of red cells that are coated with IgG can be time-consuming and difficult to interpret. Because the molecular bases of many blood group antigens are known, it was investigated whether polymerase chain reaction (PCR) analysis of DNA, from white cells in blood from transfused patients, could be used to predict the blood group antigen profile of a patient. STUDY DESIGN AND METHODS: To prevent problems arising from potentially poor-quality DNA in clinical samples, primers that flanked the polymorphism of interest and that replicated a relatively short PCR amplicon were used. The PCR products, with or without digestion with the appropriate restriction enzyme, were analyzed on gels. Samples were collected from 60 patients who had received from 2 to 50 units of RBCs in the 7 days before sample collection. RBCs from some of these patients were coated with IgG. Analyses for RHD/non-D, RHE/RHe, KEL1/KEL2, FYA/FYB, FY-GATA, JKA/JKB, and GYPA M/N were performed by using assays that had been validated with DNA prepared from untransfused volunteers of known phenotype. The genotyping assays were performed without knowledge of the expected result. RESULTS: The predicted genotype after analysis of the 60 patient samples was that expected from the results of phenotyping. In all cases, the molecular analysis gave a single result; no evidence of chimerism was obtained. CONCLUSION: In each case, the molecular genotype results were in agreement with the blood group antigen as determined by historical phenotyping, phenotyping after hypotonic washing, detection of alloantibodies in the patient's serum, or elution of alloantibody(ies). Under the conditions of these assays, reliable determination of a blood group allele can be made by PCR-restriction fragment length polymorphism testing.