When an anti-coagulated blood sample is taken, how can we calculate the number of RBC, WBC, and platelets using the flow cytometry technique? Do we need to separate the components in the blood sample and then introduce the sample to the flow cytometer?
No need to separate the cells. You must dillute the sample with a diluent solution (we use Dice solution for fish blood). Just place a drop of the diluted blood on the chamber and follow the procedure below
https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx
Dear Kritika Ramchiary to you first question:
You can count the blood components by flowcytometry just by adding true count beads to the sample while acquiring.
Otherwise you will get the counts but not the concentration (numbers/uL). SO the true counts beads has a defined number in per microlitter volume. you add 10 microlitter beads to sample and take 900 beads/ ul is their defined value. so when your cytometer will count 900 beads in its column, it will consider it 1 ul volume and will give you value of L, M, N in per microlitter.
You can definitely count RBC as well by this method, but you know RBC are seen in Log scale while WBCs are seen on Linear scale in flowcytometer, so getting value of both the cell types need to be optimized. It might be done (does not seems impossible), if you want to do.
To your next question:
Yes RBC and WBCs can be differentiated by their scatter pattern as well as cell surface markers.
The so called hematology analyzers, commonly known as cell counters used in most clinical laboratories with 3 part and above versions are modified flow cytometers give very fast and precise counts of all blood cells.
Jitendra Kumar Shandilya, can you please eleaborate more or send me papers as to how to add true count beads to WBC, RBC, and platelets, and what will be the whole procedure to get the scattergram?
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