Crossmatch Methods Research Articles

COMPARISON BETWEEN ENZYME-LINKED IMMUNOSORBENT ASSAY AND CYTOTOXIC CROSS-MATCH PROCEDURES FOR DETECTING IgG ANTI-DONOR ANTIBODIES1

Disadvantages inherent to complement-dependent cytotoxicity cross-match (CDC XM) methods are the requirements for complement and viable target cells, detection of antibodies (Abs) against non-HLA antigens, and subjective scoring. Cross-Stat (SangStat Medical Corp., Menlo Park, CA), a recently developed enzyme-linked immunosorbent assay XM procedure for the detection of IgG anti-donor HLA Abs, is theoretically devoid of these flaws. We compared results of Cross-Stat and our standard anti-human globulin (AHG)-enhanced CDC XM procedure on 524 sera from 230 transplant candidates, which were evaluated against 51 cadaveric donors. There was a significant correlation between AHG-CDC IgG XM and Cross-Stat results (P<0.001). For false negative sera, repeat AHG-CDC IgG XMs were still positive after platelet absorption, indicating that the Abs present were either non-HLA Abs or anti-HLA class II. Flow cytometry testing of false positive sera usually (42/62) substantiated Cross-Stat results, indicating that the discrepancy with AHG-CDC IgG XM is caused by greater sensitivity of Cross-Stat. Relative to the AHG-CDC XM, the sensitivity of Cross-Stat was 100%, the specificity was 93%, the positive predictive value was 73%, and the negative predictive value was 100%. A technical shortcoming of the Cross-Stat assay is that the frequency of indeterminate samples in the assays was 15%. Among 49 Cross-Stat negative vs. 13 Cross-Stat positive primary cadaveric renal allograft recipients (all AHG-CDC IgG-XM negative), there was no statistical difference in overall graft survival. Given the important theoretical advantages of enzyme-linked immunosorbent assay-based XM methods over the CDC XM, however, further testing of the clinical relevance of the Cross-Stat is warranted.

The clinical utility of flow cytometry in the histocompatibility laboratory

Flow cytometry crossmatching has become a routine part of many HLA laboratories, and applications for flow in HLA continue to increase. The flow cytometric crossmatch has established itself as an efficient and cost effective crossmatch method that has clear clinical implications for many patients. Using transplant outcome as our barometer, many laboratories have reported improved graft survival using flow cytometry. However, modifications to the methodology in the areas of quantification and specificity are needed before flow cytometry can completely replace cytotoxicity. The use of flow cytometry will continue to grow as more laboratories use it as their primary crossmatch methodology. Coincident with this will be growth in the area of antibody screening by flow cytometry. However, both of these two areas will require assistance from manufacturers in the form of standardized reagents and better automation and supportive software. Newer applications for flow cytometry make use of the ability to detect single HLA antigens either in disease states or for engraftment testing. These newer applications can also increase the utility of the flow cytometer in the clinical HLA laboratory. Last, the combination of molecular techniques (PCR or FISH) and flow cytometry may prove to be the most useful tool for documenting early bone marrow engraftment, rejection, disease relapse or documenting chimerism following transplant.

Proposed method to match physical test scores with the probable risk of a fatal fall

Since the elderly population employed in industries in Japan is gradually increasing year by year, it is estimated that the number of falls the elderly have, will also increase. To encourage elderly employees to accept new job assignments, especially those who do not wish to, evaluation methods are required that are reasonable and acceptable to them. On this point, exponential equations between age and fatality rates that were obtained in the author's previous study, can be used in understanding the effects of aging on fatal falls. To persuade elderly employees to be more aware of their increasing chances of a fatal fall, due to their decreasing ability and weakness of body to fall damage. The probable risk of a fatal fall can be assessed from individual ages. But most employees are reluctant to accept new job assignments from dangerous jobs to safer ones only for reasons of age. I suggest cross matching methods which highlight the relationship between these equations for fatality rates and physical scores. It should be noted that the single leg standing test introduced in this paper, of standing on a 90 mm wide flat beam with eyes closed is advantageous in that it can check ankle function, negate the effect of shoe heel height and shorten the measuring time. It can be concluded that the elderly who can stand for less than 2 seconds in a single leg standing test on a flat beam with eyes closed, for less than 8 seconds on a floor with eyes closed and jump less than 10 times in a jump step test must be very cautious when engaged in dangerous jobs where they are liable to fall.

A new method for determining anti-B cell antibodies and their specificity using flow cytometry

We describe a new flow cytometric B cell crossmatch method with improved sensitivity and specificity. It is based on the coating of B cell surface immunoglobulins with an unconjugated polyvalent anti-human immunoglobulin antibody. The method also provides a means for determining the specificity of anti-B cell antibodies and, potentially, for anti-HLA class II antibody specificity differentiation (DR, DQ or DP) in a binding inhibition test using mouse monoclonal antibodies directed against human HLA class II antigens.

Monitoring HIA Alloimmunization: Analysis of HLA Alloantibodies in the Serum of Prospective Transplant Recipients

The identification of serum HLA alloantibody in clinical transplantation can only be considered meaningful if the information derived from clinical tests can be used to predict crossmatch outcome. Drawing on the foundations and principles of blood transfusion medicine, this article presents a strategy for efficient, cost-effective patient HLA antibody monitoring that relies on a screening technique identical in sensitivity to the final donor crossmatch method. Application of this approach to the successful transplantation of highly immunized patients using HLA disparate cadaver renal allografts supports the contentions advanced within this article.

A cost‐effectiveness evaluation of platelet crossmatching and HLA matching in the management of alloimmunized thrombocytopenic patients

Effective platelet support for alloimmunized refractory thrombocytopenic patients may be provided by several potential strategies, the most common being HLA-matched single-donor platelets or crossmatch-compatible, pooled random- or single-donor platelets. This study used a detailed economic analysis to compare the cost-effectiveness of several techniques for platelet crossmatching and that of HLA-matched single-donor platelets. The crossmatch methods evaluated were a microlymphocytotoxicity test (LCT), an immunofluorescence technique (PSIFT), a radioactive antiglobulin test (PRAT), and an enzyme-linked immunosorbent assay (ELISA). The analysis was based on the need to support 100 refractory patients with acute leukemia with a presumed requirement of 500 transfusions. The relative costs for a successful crossmatch were: PRAT less than LCT less than LCT + PRAT less than PSIFT less than ELISA. In the comparison of the crossmatch methods, an increase in costs was generally associated with an increase in the number of successful transfusion episodes. However, decreasing marginal gains were seen. The HLA-matched single-donor platelets were relatively cost-inefficient in comparison to the crossmatch-compatible platelets. A theoretic sequence of tests for cost-effective provision of optimal platelet support in refractory patients was evaluated. Such considerations of cost are important in the selection of an optimal program for the management of alloimmunized refractory thrombocytopenic patients.

Radiolabeled red cell viability. II. 99mTc and 111In for measuring the viability of heterologous red cells in vivo.

The authors developed a double in vivo crossmatch method using 2 to 3 ml of potential donor blood labeled with either 400 microCi of 99mTc or 30 microCi of 111In-oxine. Data are presented for 19 crossmatches on nine patients, using one blood specimen labeled with 99mTc or two specimens, one labeled with 99mTc and the other with 111In. Normal values are given for standardization purposes. This method appears to have advantages over earlier in vivo crossmatch techniques using 51Cr-labeled RBCs. These advantages include the rapidity with which the in vivo crossmatch may be repeated, the ready availability of 99mTc and 111In-oxine, and the lower radiation absorbed doses with the shorter-lived radionuclides.

An Evaluation of Crossmatching, HLA, and ABO Matching for Platelet Transfusions to Refractory Patients

Refractoriness occurs in many patients receiving multiple platelet transfusions. We used a sensitive ELISA assay to assess the utility of crossmatching HLA-A,B matched single donor platelets in 51 consecutive, typical refractory patients. Of the 222 transfusions evaluated at 1 to 4 hours posttransfusion, only 17 of 54 (31%) with positive crossmatches had corrected platelet count increments of greater than or equal to 7,500/microL. In contrast, 95 of 168 (57%) of those with negative crossmatches had such increments (P less than .001). Regardless of the results of the crossmatch, HLA-A,B, and ABO matching had independent influences on transfusion outcome. The median corrected 1- to 4-hour increment for crossmatch negative transfusions was 13,300/microL for A/BU grade matches, 9,700 for BX, and 7,800 for C. Increments were 10,000/microL for ABO-identical transfusions and 5,900 for transfusions of platelets ABO incompatible with the recipient's plasma antibodies. When the donor platelets were ABO compatible, but the donor plasma contained ABO antibodies to the recipient's platelets, the increment was intermediate (8,200/microL). The most important factor in predicting platelet survival was the crossmatch, followed by HLA-A,B and ABO, each having independent predictive value. These data demonstrate that the predictive value of a negative crossmatch may be considerably less than that reported in previous studies with stable, less ill patients. In typical refractory patients, there appear to be mechanisms of platelet destruction that are related to HLA-A,B and ABO but are not detected with current crossmatch methods. We hypothesize that soluble plasma HLA-A,B and ABO antigens contribute to the destruction of donor and sometimes recipient platelets by an immune complex or other "innocent bystander" mechanism. With our crossmatching technique, HLA-A,B and ABO match grades remain relevant to platelet transfusion therapy in some refractory patients.

An evaluation of crossmatching, HLA, and ABO matching for platelet transfusions to refractory patients

Refractoriness occurs in many patients receiving multiple platelet transfusions. We used a sensitive ELISA assay to assess the utility of crossmatching HLA-A,B matched single donor platelets in 51 consecutive, typical refractory patients. Of the 222 transfusions evaluated at 1 to 4 hours posttransfusion, only 17 of 54 (31%) with positive crossmatches had corrected platelet count increments of greater than or equal to 7,500/microL. In contrast, 95 of 168 (57%) of those with negative crossmatches had such increments (P less than .001). Regardless of the results of the crossmatch, HLA-A,B, and ABO matching had independent influences on transfusion outcome. The median corrected 1- to 4-hour increment for crossmatch negative transfusions was 13,300/microL for A/BU grade matches, 9,700 for BX, and 7,800 for C. Increments were 10,000/microL for ABO-identical transfusions and 5,900 for transfusions of platelets ABO incompatible with the recipient's plasma antibodies. When the donor platelets were ABO compatible, but the donor plasma contained ABO antibodies to the recipient's platelets, the increment was intermediate (8,200/microL). The most important factor in predicting platelet survival was the crossmatch, followed by HLA-A,B and ABO, each having independent predictive value. These data demonstrate that the predictive value of a negative crossmatch may be considerably less than that reported in previous studies with stable, less ill patients. In typical refractory patients, there appear to be mechanisms of platelet destruction that are related to HLA-A,B and ABO but are not detected with current crossmatch methods. We hypothesize that soluble plasma HLA-A,B and ABO antigens contribute to the destruction of donor and sometimes recipient platelets by an immune complex or other “innocent bystander” mechanism. With our crossmatching technique, HLA-A,B and ABO match grades remain relevant to platelet transfusion therapy in some refractory patients.

Platelet Crossmatch Evaluation in Refractory Hematologic Patients

Although previous investigators have attempted to identify platelet crossmatching methods that could be used routinely for pretransfusion testing, such studies have excluded patients with underlying clinical conditions inherently associated with low corrected platelet increments. Because many refractory hematologic patients have such underlying conditions, we decided to assess the usefulness of platelet crossmatching (in addition to HLA matching) in determining the posttransfusion corrected platelet count increment in eight medically complicated patients who received a total of 35 single-donor platelet transfusions. In this limited study, the predictive value of a negative crossmatch was only 55%, whereas that of a positive crossmatch was 88%. The test used had a sensitivity of 65% and a specificity of 83%. The results of our study suggest that platelet crossmatching may be a useful additional study for predicting the outcome of transfusion--even in medically complex cases--if it can be done rapidly and relatively inexpensively.

Four crossmatch methods to select platelet donors

We employed four crossmatch techniques to select platelet donors for refractory patients. Forty-four donor-recipient pairs were studied in 32 patients. Analysis of effectiveness of platelet transfusions revealed that only 18 percent of transfusions gave a borderline response; the remainder were either effective or not effective at all. The corrected predictive values of three crossmatch tests were as follows: enzyme-linked immuno-specific assay, 81 percent; platelet immunofluorescence test, 73 percent; and lymphocytotoxicity, 70 percent (p greater than 0.05). The predictive value of these tests did not differ in HLA-matched versus unmatched platelet transfusions. Donor selection by lymphocytotoxicity compatibility did not appear to be useful if donors were selected by either of the other two methods. The fourth test, antiglobulin-modified lymphocytotoxicity, offered no advantage over lymphocytotoxicity. Our data suggest that platelet crossmatching assays are a useful adjunct to the selection process for the platelet donor in addition to ABO, Rh, and HLA matching.

Delayed hemolytic transfusion reactions. Evidence of the need for an improved pretransfusion compatibility test.

Delayed hemolytic transfusion reactions were diagnosed with a frequency of 1 per 4,000 units of whole blood or erythrocytes transfused, which represents an increased frequency of detection for those reported in other studies. The reasons for this increase, as well as the current detection of relatively milder reactions, appear to be related to the careful monitoring of the transfusion process, along with an increased clinical awareness of the problem and more sensitive laboratory detection methods. The increased frequency of detection emphasizes the need for more sensitive pretransfusion crossmatch methods to prevent those delayed hemolytic transfusion reactions that are the result of secondary immune responses.

The crossmatch profile and other extended crossmatch methods in clinical transplantation.

Preexisting humoral immunity to donor alloantigens was investigated in 67 potential renal transplant patients on chronic hemodialysis on a continuing basis over 28 months by 2,740 cytotoxicity tests. The cytotoxicity tests were enhanced by the use of six concentrations of recipient sera, and other techniques for better assessment of immunity. The six-well crossmatch concentration profile demonstrated 596 discordant results (21.8%) from those obtained by a single-well crossmatch. Among these were 196 positive profiles which were negative in the customary 1λ well. The profile method further demonstrated a low number of false positives (32) and resolved 100 of 116 doubtful reactions. Dilutions appeared more valuable than concentrations in determining the final crossmatch. No cases of hyperacute or accelerated rejection occurred in 20 cadaver and 9 related kidney transplants.