Background: Platelet receptor glycoprotein expression is assessed in our laboratory by flow cytometry. The statistical components collected for analysis are percentage of gated cells and mean fluorescent intensity (MFI).

An arbitrary cut off of >80% is considered to demonstrate full expression but is limited by lack of standardisation and the subjective nature of interpretation. The Cytoflex™ flow cytometer (Beckman coulter) has been recently validated. Among other features, the Cytoflex™ offers the capability to detect 13 colours and exhibits major sensitivity for small particles. Use of DURAClone ™ technology has improved the turn around time, offered antibody standardisation from batch to batch and allowed for a range of glycoproteins antibodies to be expressed in a single tube.

Aims: Our study aims to assess if MFI data obtained during platelet glycoprotein analysis could be used to support diagnosis of platelet receptor dysfunction.

Methods: EDTA whole blood from patients with suspected glycoprotein deficiency is collected. Following incubation, 20,000 platelet events are counted on the Cytoflex™. The gate strategy is applied and the platelet population subsequently interrogated for each different antibody on the panel. The results are collected by quantifying the % of positive antibody expression and the MFI.

Results: We have collated MFI values from 20 control values to assess if a reference cut-off may be viable. Results from genetically confirmed patients with Glanzmann’s Thrombasthenia (GT) and Bernard Soulier syndrome (BSS) were compared (Table 1).

The MFI data obtained from control samples (with exception of the platelet activation marker CD62p) were shown to display normal distribution by Shapiro-Wilk analysis. A potential threshold was set at Median – 2xS.D.

Conclusion(s): The presented data suggests that MFI may be considered as an additional reporting parameter in the diagnosis of altered platelet glycoprotein expression. A combination of MFI and % positive antibody expression may prove a more robust diagnostic tool.

Table

Table 1: Results from patients with Glanzmanns’ Thrombasthenia -GT- and Bernard Soulier Syndrome -BSS- are compared to reference values obtained from 20 donors. The % expression of glycoprotein CD61 and CD42a are shown to be decreased in the GT and BSS patients respectively. Interestingly although the GT patient showed full expression of the CD41 glycoprotein, the MFI suggested a reduced density of glycoprotein per platelet. Similarly in the BSS patient the CD42b MFI density is borderline. CD42a showed reduced receptor expression across the gated platelets with borderline MFI.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/platelet-glycoprotein-analysis-by-flow-cytometry-could-further-diagnostic-information-be-gained-from-the-mean-fluorescence-intensity-values/