A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 x 10(3)/microL (1-400 x 10(9)/L). Pooled analysis of the serial dilutions showed that RBC-platelet and RBC-RBC coincidence events became negligible at sufficiently high dilutions (i.e., > 1:1,000). All laboratories demonstrated excellent intra-assay and acceptable interlaboratory precision. Two antibodies (CD61 and CD41) were used for identifying platelets and individually gave acceptable results, but in a minority of samples, staining differences were observed. The optimum method thus uses a double-labeling procedure with a final dilution factor of 1:1,000. The study demonstrated that this method meets the criteria for a reference platelet count.

Six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol+). The other strains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol-)

Fibronectin (Fn) was found to bind to protein A-containing isolates of Staphylococcus aureus, but not to mutant strains devoid of this protein nor to clinical isolates of S. epidermidis. Fn was purified from human plasma by affinity chromatography on gelatin-Sepharose. After elution with 4 M urea, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified material detected no immunoglobulin contamination. This purified Fn was radiolabeled with 125I

The International Council for Standardization in Haematology (ICSH) and the International Society of Laboratory Hematology (ISLH) recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets. Fresh EDTA-anticoagulated venous blood specimens are measured within 4 hours of the draw. The specimen is prediluted (1:20) and the platelets labeled with two monoclonal antibodies specific to a cluster of differentiation common to all platelets. A final 1:1,000 dilution is made and at least 50,000 events with a minimum of 1,000 platelet events are counted with a flow cytometer to determine the RBC/platelet ratio. The platelet count is then calculated from this ratio and the RBC concentration of the original blood specimen.

No abstract