Title:	Example PBMC Hemacytometer Counting Standard Operating Procedure	
Origination Date:		Total Pages:		
Effective Date:		SOP Number		
Written By:		Supersedes SOP Dated:		

Approved by:	Name, Title	Signature	Date	

Revision History	Version/Date	Description of Change(s)	

			

Reviewed By:	Name, Title	Signature	Date	

	

			

Note: This is an example—not a required—SOP. This SOP must be tailored to laboratory-specific and/or study-specific requirements. This SOP cannot be used as it is. 

Table of Contents
1	Purpose
1	Scope
2	Background
3	Authority and Responsibility
4	Reagents and Materials
5	Instrumentation
6	Quality Control
7	Procedure
8	Cell Count Calculations
9	Maintenance and Cleaning
10	Definitions
11	References
12	Appendix

1	
Purpose
This standard operating procedure (SOP) gives instructions for determining cell counts using a hemacytometer, tally counter, and light microscope.
1	Scope
Using the cell counting method described in this standard operating procedure (SOP), the cell count and cell viability of a PBMC suspension can be determined.
2	Background
The improved Neubauer hemacytometer is a thick glass slide with a microscopic grid etched into a mirror-like polished surface.  The grid is composed of 9 large divisions, each having an area of 1mm2.  The large divisions are subdivided into .25mm2 and the center square is divided again into .0025mm2 squares.  The total area of the etched grid is 9mm2.  The raised edges of the counting chamber support a quartz cover slip that is exactly 0.1mm above the chamber.  The total volume of fluid that lies over the grid of the counting chamber is 9mm3.

To count peripheral blood mononuclear cells (PBMC) and determine viability, the cells are diluted in Trypan Blue which will pass through the cell membrane of non-viable cells giving them a blue appearance.  Healthy, viable cells will appear round and refractile under the light microscope.
3	Authority and Responsibility
3.1	[Complete with the title or titles of laboratory personnel with this authority] has the authority to establish, review and update this procedure.
3.2	[Complete with the title or titles of laboratory personnel with this responsibility] is responsible for the maintenance and control of SOP documentation.
3.3	[Complete with the title or titles of laboratory personnel with this responsibility] is responsible for the implementation of this SOP and for ensuring that all appropriate personnel are trained. 
3.4	All technicians are responsible for reading and understanding this SOP prior to performing the procedures described.
4	Reagents and Materials
Recommended vendors are listed. Unless otherwise specified, products of equal or better quality may be used when necessary.
4.1	Tally counter, 2 key
4.2	Quartz cover slips for hemacytometer
4.3	Lens Paper
4.4	95% ethanol (for 70% ethanol preparation if needed)
4.5	70% ethanol: 70 parts of 95% ethanol + 25 parts distilled water
4.6	Trypan Blue (0.4%) 
4.7	Glacial Acetic Acid (for red blood cell lyses during counting if needed)
2% acetic acid: 1 part glacial acetic acid + 49 parts of distilled water
4.8	Pipette Tips (sterile)
4.9	Small tubes for cell suspension dilutions such as Micro-centrifuge tubes, 1.5ml
5	Instrumentation
5.1	Light Microscope, 10 and 40x lens
5.2	Neubauer Hemacytometer with Coverslip (optional, plastic disposable hemacytometer.
5.3	Precision Pipette (for dilutions)
5.4	Biological safety cabinet, BSL 2.0 or higher
6	Specimens
The PBMC suspensions referred to in this SOP are those being counted prior to freezing as part of the PBMC processing procedure, or a PBMC suspension that has been thawed and counted as a post processing check of recovery and viability. 
7	Quality Control
7.1	Sample Quality Control (daily per specimen)
7.1.1	The PBMC cell suspension should be diluted and plated such that each 1mm2 counting square contains approximately 50-200 cells
•	Significant errors can occur if the cell count is too low or too high.  
•	Overlapping cells indicate the need for a greater dilution.  
•	Too few cells indicate the need for a smaller dilution.  
•	Re-dilute and recount as needed.
7.1.2	Cells should be evenly distributed, without overlapping or clumping. The cell counts between the four 1mm2 areas should agree within ± 15%.  If the cells are not evenly distributed or are clumped:
•	The chamber may have been loaded incorrectly.
•	The coverslip may have been bumped after loading. 
•	The hemacytomer/coverslip may have been dirty. 
7.1.3	Clean the chamber and coverslip with 70% ethanol and lens paper, reload and repeat count.  
7.2	Technologist Quality Control (monthly)
Perform comparison counts between technicians using the same sample. Technician results should agree within ± 20%.  The comparison counts should be documented including the interpretation (acceptable/not acceptable) and reviewed by the QA manager or supervisor.  Unacceptable comparison results should be resolved with the QA manager/supervisor immediately.
8	Procedure
Note:  Universal Blood Borne Pathogen precautions should be followed when handling blood and blood products.  Follow your laboratories safety SOP when performing this procedure.
8.1	Preparation of Cell Dilutions 
Cell dilutions should take place in a biological safety cabinet. 
8.1.1	Prepare a small aliquot of stained PBMC cell suspension for counting. 
For example, if preparing a 1:5 dilution: Using a sterile pipette tip remove 20μL of cell suspension and place in a microcentrifuge tube. Add 80μL 0.4% Trypan Blue and gently mix. 
Note: Adjust the amount of cell suspension and Trypan blue as needed for desired dilution. A dilution range of 1:2 to 1:20 will cover most PBMC cell suspensions. 
1:20- 20µL cell suspension + 380µL stain 
1:10- 20µL cell suspension + 180µL stain 
1:5- 20µL cell suspension +80µL stain 
1:2- 20µL cell suspension + 20µL stain 
8.1.2	Allow the cell suspension to sit in the Trypan Blue for at least one
8.2	Hemacytometer Plating
Refer to figure 1 to locate the hemacytometer sample introduction point and counting chambers.

 Fig. 1

8.2.1	Cell dilutions should take place in a biological safety cabinet.
8.2.2	Pre-clean the hemacytometer and the coverslip with 70% ethanol and lens paper.  Allow to dry.
8.2.3	Gently, but thoroughly mix the PBMC suspension.
8.2.4	Prepare a small aliquot of stained PBMC cell suspension for counting.
Example:  1:5 dilution
Using a sterile pipette tip remove 20µL of cell suspension and place in a microcentrifuge tube.  Add 80µL 0.4% Trypan Blue and gently mix.
Note:  Adjust amount of cell suspension and trypan blue as needed for desired dilution.  A dilution range of 1:2 to 1:10 will cover most PBMC cell suspensions
8.2.5	Allow the cell suspension to sit in the Trypan Blue for at least one minute before plating to allow for complete staining of the non-viable cells.
8.2.6	Do not count cells left standing in Trypan Blue for longer than 15 minutes.  After 15 minutes nonspecific staining of viable cells may occur.  Make a new cell-dye suspension before counting. 
8.2.7	If red cell contamination is present, a 1 to 1 mixture of Trypan Blue and 2% acetic acid can be used to dilute the PBMC cell suspension.  The acetic acid will lyse any red cells that may be present.
Example: 40µL Trypan Blue plus 40µL acetic acid is used to dilute 20µL of cell suspension for a final dilution 1:5.  Gently mix and allow to stand for 1 minute before plating.
8.2.8	Swirl, finger vortex, to thoroughly mix the now diluted specimen before plating.  A well-mixed specimen is critical for accurate counting.
8.2.9	With the coverslip in place, use a small pipettor, to place a drop (10µL) of cell suspension at the edge of the “V” shaped groove, sample introduction point, of the chamber (Fig.1).  
8.2.10	The chamber fills by capillary action and the cell suspension should flow smoothly into the chamber until it is completely filled.  Do not overfill.
8.2.11	If the chamber is accidentally overfilled or the coverslip is bumped, do not count.  Clear and clean the counting chamber and re-load.
8.2.12	Allow the sample to settle for 30 seconds, until the cells are all in the same plane when viewed through the microscope.
8.3	Counting PBMC
Figure 2 is a diagram of the grid on the hemacytometer counting chamber.
Refer to this figure as you read the section on counting PBMC.

               				 Fig. 2 

8.3.1	Place the hemacytometer on the microscope stage and focus on the cells and the grid using a low power lens, such as 10x.  
8.3.2	Scan the counting chamber noting cell distribution, unusual or excessive cellular debris or high numbers of red blood cells.  If the red blood cells are high enough to interfere with counting, make a new cell dilution using 2% Acetic acid/trypan blue diluent.	
8.3.3	Continue scanning.  Estimate the number of nonviable cells.  Are there greater than 5% nonviable cells?  Has the cell suspension been sitting longer than 15 minutes?  If so, make a new dilution.
8.3.4	When ready, move to a higher power lens for counting.  The correct power lens for counting PBMC will depend on the microscope being used and the experience level of the technologist.
8.3.5	Once the cells have settled, use a 2-key tally counter to count the cells in the four large corner squares (Fig. 2), separating the viable from the non-viable cells.  Each corner square is divided into a 4x4 grid and has an area of 1mm2.
Note:  Viable cells will appear as bright spheres surrounded with a halo.  Non-viable cells will be stained blue.
Note:  Red cells appear more spherical and slightly smaller than PBMC’s.  Small irregular shaped particles are platelets and debris.  Take care not to include red blood cells or platelets in the PBMC count.
To avoid over counting cells that lie on the outside grid lines of the large squares, a standard practice is to count cells that lie on the left and the top outer boarders of the large corner squares but not the cells similarly located on the right and the bottom.
9	Cell Count Calculations
The cell counts from the hemacytometer are used to calculate the number of cells/ml present and the per cent viable cells in the PBMC suspension.
9.1	To calculate the dilution factor:
DF = Volume PBMC + Volume Trypan Blue/Volume PBMC
Example:  (20L + 80L) / 20L = 5
9.2	To calculate cells/mL:
106 cells/mL = (CT/LSC) x DF x 104
CT = Total Cells Counted
LSC = Large Squares Counted
(each large square = 1mm2)
 DF = Dilution Factor
Example:  (80 cells/ 4mm3) x 5 x 104 = 1.0 x 106 cells/mL
9.3	To calculate per cent viable cells:
% Viable Cells = CV/(CV +Cnv) x 100
CV = Viable cell count
Cnv = Non-viable cell count
Example:  350/(350+50)x100 = 87.5%
10	Maintenance and Cleaning
10.1	After each use soak the hemacytometer and coverslip in 10% bleach for 30 minutes.  Rinse with tap water and then 70% ethanol.
10.2	Allow to air dry.  Lens paper may be used to wipe the surface of the counting chamber and coverslip.
Note:  Handle the hemacytometer and coverslip carefully to prevent scratches.  Scratches may visually interfere with counting and compromise the accuracy of the cell count.
10.3	Store the hemacytometer and coverslip in a covered container, such as a covered petri dish or box.
11	Definitions
Term	Definition	
PBMC	Peripheral Blood Mononuclear Cells	
SOP	Standard Operating Procedure	
12	References
12.1	Souza, Mde and Guarnacci, T, Determination of Cell Counts and Viability, SOP  TAVEG006, AFIMS Retovirology Laboratory, 315/6 Rajvithi Road, Bangkok 10400, Thailand.
12.2	Gaitan, Alicia and Montefiori, David C., CL Endpoint Assay, SOP HVTN00-E0016, Endpoint Neutralizing Antibody Assay Laboratory, Duke University.
12.3	Using a Hemacytometer, Cascade Biologics, Inc., Doc107.0.
12.4	Caprette, David R., Using a Counting Chamber, Rice University.
12.5	HIV Vaccine Trials Network (HVTN) Counting PBMC Suspensions Using a Hemacytometer, LP-HVTN-P0015-v1.0, 08 May 2009.
13	Appendix 
13.1	Hemacytometer QC Worksheet

Hemacytometer QC—Comparison Counts
Date		Sample ID		Sample Dilution		Reviewed By		
											
Initials	Tech#1:	Tech#2:	Tech#3:	Tech#4:	Group Results	
	Viable Cells	Non-Viable	Viable Cells	Non-Viable	Viable Cells	Non-Viable	Viable Cells	Non-Viable	Viable Cells	Non-Viable	
Sq#1
(cell/mm2)											
Sq#2
(cell/mm2)											
Sq#3
(cell/mm2)											
Sq#4
(cell/mm2)											
Avg Cell Count per Sq
(cell/mm2)											
	Lower limit of range (-20% mean) in cells/mm2			
	Upper limit of range (+20% mean) in cells/mm2			
Pass/Fail										
Comments/Corrective Action: