Cell counting

Human and murine platelets from whole blood can be immunophenotyped by the labelling of GpIb and GpIIb/IIIa to determine the presence of the respective clinical syndromes Glanzmann's Thrombasthenia and Bernard-Soulier syndrome and also the counting of platelet numbers.

 

The homozygote double recessive and heterozygote conditions of Glanzmann's Thrombasthenia has a >90% and approximately 50% reduced expression of GpIIb/IIIa respectively. Bernard-Soulier's syndrome showing a marked reduction in GpIb expression that can be readily shown by flow cytometric analysis after immunophenotyping of GpIb.

Protocols :

Platelet labelling Protocol

1. Dilute citrated whole blood 1:10 in PBS.
2. Diluted whole blood (50 ml) was incubated with 5 ml of FITC conjugated monoclonal antibody (CD42a, or CD41 or CD61) and 5 ml PE conjugated Glycophorin-A at RT for 15 mins. This enables separation of platelets from noise and red blood cells.
3. This preparation was then diluted with PBS (300 ml) and analysed by flow cytometry.
4. Instrument settings were FSC and SSC parameters adjusted to LOG settings to enable detection and separation of platelets from electronic noise. An event stop gate was placed around the platelet cloud and a minimum of 10,000 events collected.

Cell counting protocol

1. Make a dilution of latex beads in PBS.
2. Count beads on a Haemocytometer.
3. Add a known number of beads (20,000) accurately to a known volume of sample.
4. Draw an event stop gate on bead population to stop at 2,000 events.
5. Other gated events are a proportion of the bead count.

Example of cell counting