Heterogeneity of antibody response to human platelet transfusion.

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.

New granulocyte antigens demonstrated by microgranulocytotoxicity assay.

Although granulocyte transfusions and bone marrow transplantation are becoming common clinical modalities, our knowledge of surface nonerythroid, nonlymphoid, non-HLA hematopoetic antigens remains very incomplete. Accordingly, we have systematically screened sera from recipients of multiple granulocyte and whole blood transfusions, and immunoneutropenic patients for antibodies directed primarily at granulocytes. The initial screens demonstrated that >50% of the sera from the above sources contained non-HLA cytotoxic and/or agglutinating antibodies. Preliminary clustering indicated seven possible new specificities detected by microgranulocytotoxicity. Calculations for Hardy-Weinberg goodness of fit based on a study of 98 unrelated donors plus informative families established that 5 of these were alleles of a single new locus termed Human Granulocyte Antigen (HGA)-3a, b, c, d, and e. Absorptions indicated that these antigens were present on mature granulocytes but absent from platelets, lymphocytes, monocytes, and myeloid precursors. A single antigen of another separate locus, HGA-1, was also identified. Absorptions revealed a quite different distribution for HGA-1 than HGA-3, this antigen being detected on monocytes and myeloblasts as well as on mature granulocytes. Independent segregation of the three loci from HLA, from the NA-NB and the 5a-5b antigens, and from themselves was confirmed in informative families.Finally, it seems likely that other antigens will be identified because several other sera that react with both monocytes and granulocytes have been detected.

Partial thromboplastin time test--proposed performance guidelines. ICSH Panel on the PTT.

The partial thromboplastin time (PTT) test is widely used as a screening test for the detection of hemophilia. It is also used to monitor patients on heparin anticoagulation. This proposal from a ICSH Panel proposes guidelines for the performance of this test, including comparable reference ranges, precision and sensitivity requirements.

Effects of cell membrane disruption on the relaxation rates of blood and clot with various methemoglobin concentrations.

The magnetic resonance imaging (MRI) characteristics of hemorrhage and clotted blood change with age. The effects of methemoglobin and cell membrane lysis, factors which in part may underlie this evolution of imaging characteristics, were studied using clotted and heparinized dog blood at various methemoglobin concentrations. Cell lysis did not alter the longitudinal relaxation rate (1/T1) in clotted or unclotted samples. Membrane lysis altered significantly the transverse relaxation rate (1/T2) in both clotted and unclotted samples. Lysed samples of oxygenated blood at 0% methemoglobin had significantly higher T2 values than intact samples. At 0% methemoglobin, clotted samples had slightly but significantly shorter relaxation times than unclotted samples. Within the samples studied, large changes in the state of oxygenation and methemoglobin content were observed in less than 24 h. Such changes necessitate frequent monitoring of these parameters if serial studies are to be done.

The calibration of automated instruments for accuracy in hemoglobinometry.

The surveys of the College of American Pathologists indicate a consistent bias between the manual "reference" method for hemoglobin determinations and the Coulter S measurements. The Coulter S hemoglobin values are invariably lower; the difference averages 0.3 g/dl. The commonly used calibration methods for the Coulter appear to be subobtimal. The hemoglobin calibration and quality assurance method proposed by Bull and colleagues is advocated as the presently most acceptable method. Commercial hemoglobin control materials should not be used for hemoglobin calibration.

An innovative method for the determination of normal values in hematology using peer group laboratories.

The author describes a proposed innovative method for the determination of normal values in hematology in which a large number of laboratories participating in the CAP proficiency testing program submit results of a previously performed, complete blood count. These data are compared with those derived from more formal studies of normal values. The advantages of this system, if validated, include its inexpensive simplicity and the possibility of determining normal values for relatively uncommon groups of patients such as neonates, infants, and small children.

The partial thromboplastin time in the CAP survey program.

Analysis of the data for the 1969 to 1973 CAP Surveys of proficiency in partial thromboplastin time (PTT) determination indicates more than desirable variability in this measurement. Non-activated procedures show greater interlaboratory variability than activated methods; therefore, they may be preferable for routine use. It is likely that many laboratories have not determined their own upper limit of normal for their PTT system and thus have received unacceptable evaluations in the Surveys. It also was determined that many laboratories do not closely follow the manufacturer's directions, especially in regard to incubation times and calcium concentration of the recalcification solution.

A delineation of performance criteria for the differentiation of leukocytes.

Performance criteria for manual differential leukocyte counting are tentatively set following analysis of the CAP interlaboratory survey programs in hematology. It is evident that intermediate levels of discrimination of five normal leukocyte types (neutrophils, eosinophils, basophils, lymphocytes, and monocytes) can be achieved by almost all laboratories. The most sophisticated level (differentiating band from segmented neutrophils and reactive from normal lymphocytes) is still somewhat controversial. Sampling and other variables when doing 100-cell differential counts at the intermediate level of discrimination are quite predictable, and CAP Survey results closely parallel the predicted results when uniform wedge of centrifuged smears stained in an acceptable manner are used. Automated differential counters should be expected to perform at a level of discrimination at least as good as that of human examiners.

A critical analysis of platelet counting methods.

A careful analysis of platelet counting methods, including an assessment of accuracy, was done using data from the College of American Pathologists Comprehensive Hematology Survey preserved platelet suspension specimen (H-32-1976). Comparisons between methods suggest systematic biases probably related to the calibration methods used for automated instruments. Falsely elevated counts occurred with manual methods and one light-dispersion system. Methods for calibration and continuing quality assurance of platelet counting are proposed.

The College of American Pathologists comprehensive blood bank survey program, 1973.

The 1973 Comprehensive Blood Bank Survey Program of the College of American Pathologists was administered to 2,200 laboratories and blood banks throughout the United States. The results showed a considerable increase in the accuracy of the testing and in the performance of reagents. Accuracy of ABO testing was 99.3%; Rh testing, 99%; crossmatching was 98.9% correct. Antibody detection was correct 96.3% of the time, with the exception of a cold-reacting anti-P1 which was found by only 62% of the participants. About the same number of laboratories also did not find the antibody during the crossmatch procedure. A sample from a donor with a positive antiglobulin test created much confusion and was misinterpreted in a number of ways. Reagents were found to be performing well with the exception of two, one [anti-hr'(C)] which produced a significant number of false positives, and another (anti-Kell) which produced numerous false negatives. The results seem to indicate that the anti-hr'(C) from several manufacturers is at fault for this error, while the anti-Kell errors showed no predilection for manufacturers and may represent erroneous testing by the laboratories or a failure to employ adequate quality control.

Quality assurance for multichannel hematology instruments. Four years' experience with patient mean erythrocyte indices.

Experience using the Bull patient moving average control system over a four-year period to control a Coulter multichannel hematology counting system is described. The system allowed the abandonment of commercial control materials at an annual saving of almost $3,000 without any deterioration in the quality of laboratory data generated. Guidelines or instrument trouble shooting are outlined. A catalog of all major control problem episodes that occurred during the four-year period is included.

The reticulocyte. An approach to definition.

Experiences of the CAP Survey Program in reticulocyte counts and morphologic identification of reticulocytes from 1971 to 1974 are reviewed. Problems of morphologic identification are reflected in an excessive variance of reticulocyte counts. Statistical sources of variations in counting are identified. The older, original descriptions and definitions of morphologic criteria are reviewed and discussed in relationship to Survey performance.

Unnecessary microscopy in routine urinalysis.

The consequence of omitting urine sediment microscopy in specimens with normal physicochemical testing was assessed in a retrospective review of laboratory and clinical data in 1,000 patients. The pathologic states of clinical significance most likely to be overlooked were Trichomonas infection and occasional cases of significant bacteriuria. However, the medical benefit of performing urine microscopy in these two groups of patients was not clear. The authors cautiously recommend reserving microscopy for urine specimens that show physicochemical abnormalities.

The comprehensive blood bank survey program of the College of American Pathologists, 1974.

Results of the 1974 College of American Pathologists Comprehensive Blood Bank Survey Program show that ABO and Rh typing are still holding their percentage of excellent (99.48%) accuracy. At least some of the errors still occurring are due to clerical mistakes in filling out the forms and not to technical inaccuracy. Antibody detection shows good accuracy but varies, depending on the complexity of the problems--the easier the problem the higher the rate of concurrence, while the more complex the problem the more variance in results. Crossmatching accuracy is 99% correct or better on any but the most complex problems. Some of the problems involving interpretation of autoimmune hemolytic anemia are discussed.

Factor VIII (antihemophilic factor) assay results in the 1976 College of American Pathologists Survey Program.

Data from the 1976 CAP Survey for the performance of Factor VII (antihemophilic factor) assays were analyzed. The results indicated an undesirable variation on both normal and abnormal specimens. Reagent-instrumentation systems and procedures were examined. The need for standardization of factor VIII assays is evident from the results of this study.

A method to examine the need for duplicate testing of common coagulation tests.

A statistical protocol for evaluating the need for duplicate coagulation testing was developed. It requires at least 32 sets of duplicate prothrombin times or partial thromboplastin times over the expected range of results. In addition to the statistical procedures, clinical evaluation of the duplicates is required.

The incorporation of red blood cell index mean data into quality control programs.

Patient red blood cell (RBC) index means, when used in quality control, form an independent standard that is as accurate and precise as preserved blood controls. If such patient data are routinely incorporated in intralaboratory and interlaboratory quality control programs, a substantial improvement in the present state-of-the-art is possible. Within the laboratory, each method serves to confirm the adequacy of the other. In interlaboratory control trials the combination makes it possible to specify the cause of most misanalyses. For similar reasons, the combination of both methods enables the manufacturer of quality control material to assess the adequacy of the manufacturing and value assignation process.

Pre-instrumental variables in coagulation testing.

A number of variables thought to affect measurement of prothrombin time (PT) and partial thromboplastin time (PTT) were examined in an effort to determine more precisely their effects on these measurements. On the basis of these studies, it is proposed that blood specimens be anticoagulated with one part 3.8% (w./v.) sodium citrate solution to 19 parts whole blood to avoid excessive anticoagulation of blood samples drawn from patients with polycythemia. Because of the smaller amount of plasma in such samples, relatively larger amounts of anticoagulant are used, and spuriously prolonged PT and PTT measurements commonly result. No deleterious effect on anemic specimens is evident when the smaller amount of citrate is used. Studies of the stability of these specimens indicate that unopened, vacuum-drawn specimens do not noticeably deteriorate for as long as 6 hours, even when kept at room temperature. Prothrombin time measurements remain constant for as long as 24 hours. However, a 10--15% lengthening of the partial thromboplastin time is evident after 24 hours of storage.

The effect of heparin on the activated partial thromboplastin time.

Data from the 1976 and 1977 CAP surveys were analyzed for response of the activated partial thromboplastin time (APTT) to heparin. Different sources and concentrations of heparin were used. The results indicate that the precision of the APTT is more dependent on instrumentation than on partial thromboplastin. This was true for all four of the heparinized specimens evaluated. A single exception was found with the "old" Dade reagent activated cephaloplastin. The mean difference in the activated partial thromboplastin times obtained with differing concentrations of heparin was entirely dependent on the partial thromboplastin reagent used. No significant difference in the results was found when equal concentrations of bovine lung and porcine intestinal mucosal heparin were compared.

Calibration methods for automated hematology instruments.

Recent CAP survey data have documented marked improvement in the reproducibility of hematologic tests. The favorable outcome is attributable largely to the widespread use of automated whole-blood analyzers. Calibration variability, however, detracts from this enhanced precision, and there are clear indications that improvement is necessary. Primary calibration methods for hemoglobin, hematocrit and cell counting procedures are presented. The methods of applying these primary calibration methods to automated whole-blood analyzers are delineated. The use of both preserved reference blood and statistical control technics for the identification of calibration loss is described.