Protocol for Invitrogen Countess

[David Kesner]

I posted earlier but don't see it anywhere so am trying again.

Does anyone have a protocol for whole bone marrow and leukemic blood WBC counts using the Invitrogen Countess? So far their technical department has not been any help. I know how to use the machine, but am looking for how labs prepare their samples for counting. Things like lysed or not, dilutions, etc.

David W. Kesner
ICDL
4355 Emerald Street
Suite 140
Boise, Idaho 83706
www.idahocytogenetics.com

[Alice]

Hi David,

This is Alice, a product manager for Life Technologies. I am sorry that technical support wasn't able to help you when you called. I have reached out to them again, and I have obtained some information that may be helpful to you:

Q Can the Countess™ instrument count white blood cells?

A The Countess™ instrument was able to count white blood cells from lysed whole blood and Ficoll

cell preparations.

 

Q Can the Countess™ instrument count whole blood cells containing non-lysed cells?

A Yes. You should dilute the blood sample by approximately 1:10,000 and count in “bead” mode. The

instrument cannot assess the viability of cells in a whole blood sample.

 

Directions for counting whole blood:

1) Try direction sample 10 uL with 10 uL with Trypan Blue, if getting an error message - over the 10e7

cells/mL maximum range.

2) Dilute blood in a physiological saline, PBS or other iso-osmotic buffer (with or without anticoagulant

as may be needed) suitable for whole blood.

Keep track of dilution and then mix 10 uL of this diluted whole blood with 10 uL of Trypan blue.

The Countess automatically multiplies the cell count by 2 to account for the 1:1 dilution with Trypan

blue. This would be the number of cells in the diluted sample and then, the end-user must also multiply by

the dilution factor used to dilute the sample to get total count of the undiluted whole blood.

 

Q Can the Countess™ instrument count PBMCs?

A Yes, the Countess™ instrument can count PBMCs. However, it CANNOT differentiate white blood

cell types.

 

Q Will the Countess™ instrument count RBCs (red blood cells)?

A Yes, the Countess instrument can count RBCs. Dilute the blood sample by approximately 1:10,000

and count in “bead” mode. The instrument cannot assess the viability of red blood cells.

Yes, the counter can count erythrocytes. We count a couple of different ways.

Quick check: load one side (also called “chamber”), read once

Precision check: load one side, read three times, average the three readings

Accuracy check/Hemocytometer comparison: load three to five sides, read each three times, average the side

readings, average the different chambers.

Please let me know if this is what you were looking for. Thanks!

Regards,

Alice