Standardization of the Flow Cytometric Determination of HLA Class I Antigens, 'Platelet-Specific' Glycoproteins and Activation Markers

Abstract

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10^7 and 10^8/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll- Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll- Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/1 resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the a-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the β(2)-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.

Demonstration by Flow Cytometry of the Numbers of Residual White Blood Cells and Platelets in Filtered Red Blood Cell Concentrates and Plasma Preparations

“…This receptor is highly upregulated in activated PLT. The demonstration of PLT in diluted whole blood using a rejec tion gate to screen out RBC and WBC is quick and accurate [13], A similar method using the MAB CD41 and CD42b was described by Dickerhoff and von Ruecker [14]. In dilu tion experiments, these authors found good linearity of FC measurements up to a DL of 50 xlO9 PLT/1.…”

“…Since impedance-based cy tometers for PLT counting fail at low PLT concentrations and counting with a microscopic chamber is unreliable be cause of the small size of PLT, the whole-blood method is the most reliable one available today. However, it can pro vide inaccurate measurements if the PLT are significantly activated in vitro or in vivo [13]. In this case, the activated PLT partially aggregate, are located in the area of the rejec tion gate set in the scatter dot plot and are excluded from the measurement.…”