Fixation of platelet aggregate size distribution

Abstract

An experimental study has been carried out on the use of aldehydes for fixation of human platelet aggregate size distributions. The objective of the work was to develop a methodology of stopping aggregation and disaggregation processes for subsequent analysis. The results are intended to facilitate study of rates of aggregation and disaggregation as influenced by various stimuli. Platelet aggregation was induced in citrate-anticoagulated platelet-rich plasma (PRP) by addition of adenine dinucleotide (ADP) in final concentration ranging from .5 to 2 yM. The aggregated PRP specimens were diluted (158.5 to 1) in a counting medium (isoton) for size distribution analysis. An electronic particle counter was used to study the aggregate size distributions in the range 13-11 urn in equivalent spherical diameter. Parameters used to monitor the size distributions were cumulative volume and cumulative population of the aggregates, mean aggregate size, and volume available for aggregation from free (unaggregated) platelets. In preliminary studies evidence was obtained that glutaraldehyde was a more promising fixative than formaldehyde. Glutaraldehyde in appropriate concentrations caused no important problems in resuspension or in aggregate size change for times of fixation of several minutes. Dilution of aggregated PRP specimens in isoton for counting induced rapid disaggregation. However, it was found that this disaggregation could be avoided by use of glutaraldehyde in the isoton counting diluent. Glutaraldehyde addition to both the aggregated PRP specimen and to the isoton counting diluent to final concentration of .48 wt% was selected as the recommended procedure. Detailed studies were made of aggregate size distributions fixed at various times in the aggregation process. The results indicate that the fixative stops the reactions and stabilizes the distribution for times of 3 to 5 minutes. Thus, the procedure should be useful in studies on rates of platelet aggregation.