CSF Hemocytometer procedure

Cerebral Spinal Fluid (Csf) Analysis: Specimen Processing And Cell Count Procedure Using The Cyto C-Chip Disposable Hemocytometer

Principle

A CSF is normally clear and colorless in appearance, but an elevated WBC count (>200uL) or RBC count (> 400uL), bacteria or increased protein levels will result in a turbid and/or colored appearance. CSF is collected in 3 to 4 sterile non-anticoagulated plastic tubes and sent to the lab for evaluation. Following visual inspection of the tubes they are distributed to the appropriate benches for testing. In Hematology, a hemocytometer is used to numerate the RBC’s and WBCs, and if required, cytospin slides are prepared via cyto-centrifugation and evaluated for cell type and morphology.

Materials

• Filtered 2% acetic acid with new methylene blue stain (Refer to appendix 4 for preparation)
• Disposable Neubauer Hemacytometer. (Refer to appendix #1 for use)
• Light microscope
• Petri plate with moist gauze
• Cell counters
• 12 x 75 glass tubes
• Gauze/Kim wipes
• Eppendorf pipettes
• Timers

Sample Type

• Cerebral spinal fluid collected in 3 to 4 sterile non-anticoagulated graduated plastic tubes.
• A min of 2 to 4 mL in each of the tubes collected.

Safety Considerations

• The 2% acetic acid is considered TOXIC AND CORROSIVE!
• Refer to MSD sheets for safety considerations, located in the reagent prep room.
• All specimens received in the lab must be regarded as potentially infectious so follow safe practices.
• Refer to regulations and safety precautions in the student lab manual located in each student lab.

RECEIVING CSF SPECIMENS IN THE LAB

1. Review requisition and confirm name, DOB and MRN match on all four tube labels
2. Macroscopically inspect all four tubes and document the volume, clarity, and color of the numbered specimens on the worksheet for reporting in LIS.
3. Distribute tubes to appropriate bench for testing:
   1. Chemistry: glucose, protein, LD, xanthochromia (may need to centrifuge to determine)
   2. Microbiology: Gram stain, C&S
   3. Hematology: WBC and RBC cell count, WBC differential
   4. Other testing: Cytology, molecular, flow cytometry, etc. If no fourth tube is received, you can use any other tube after testing is completed.

NOTE: If a small volume arrives, call the physician to prioritize testing.
Some labs may adjust the order of tubes for bench testing. Follow your SOP at your clinical site.

CELL COUNT PROCEDURE

1. Prepare the hemocytometer for testing (Refer to appendix 1).
2. Label the hemocytometer with patients’ information (Last name, first initial, and MRN #).
3. Mix the CSF using gentle inversion until thoroughly mixed. (about 8-10 times).
4. Pipette 10uL of “acid” treated CSF onto side A of the hemocytometer. To save time, this will be prepared in advance for the students. Note: RBCs are lysed leaving the WBC’s when treated with acid.
5. Pipette 10uL of “neat” CSF onto the side B of the hemocytometer. Note: Both WBCs and RBCs are counted on the nest side.
   1. Male note of which side is neat and acid.
6. Allow the hemocytometer to sit for 2 minutes before reading. This is important to allow the cells to settle out on the hemocytometer.
7. When ready to count, place the hemacytometer on the microscope stage. You may have to adjust the microscope lighting to see the cells on the grid clearly.
   1. Note: Some microscopes do not have an adjustable condenser lens.
8. Using a 10x objective, focus on the “Neat” side of the hemocytometer, and count all the cells in all nine squares. Document the results on the worksheet.
9. Move over to the “Acid” side and count the cells in all nine squares. Document the results on the worksheet.
10. Refer to Interpretation section below for calculating the WBC/RBC counts and document the results on the worksheet.
11. Document the results on the worksheet.

Procedural Note

1. Although the acetic acid is in a dilute form, avoid breathing in any fumes.
2. Always keep the acetic acid covered when not using it.
3. Ensure that the hemacytometer is free of dust and dirt before using.
4. If there is going to be a delay in counting you need to keep the chamber in a moist environment to avoid drying out. You can moisten gauze in a petri dish and keep the hemacytometer there until you are ready to count the cells.
5. Count the entire surface (all nine large squares) following the counting protocol indicated in the quality section.
6. Perform the procedure in a biological safety cabinet.
   1. Note: We are using non biological hazardous material to prep the CSF specimen, so no need to set up in a biological safety cabinet.

QUALITY CONTROL

1. Slides for evaluation of cellular morphology must be made ASAP to avoid cellular degradation and maintain cellular integrity.
2. Ensure all samples are properly labelled and match accurately with the requisition.
3. Tubes should contain about 2-4 mL of CSF and will be labelled in the numerical order in which they are collected. Note: volume will vary.
4. Perform the hematology examination within 1 hour of collection, as per CLSI guidelines.
5. Results for both WBCs and RBCs are reported in x 106/L.
6. To get the total number of RBCs counted, subtract the acid side from the neat side.
7. The total number of WBCs counted is the total of all cells counted on the acid side.
8. Only count the cells that touch the top and left side of the squares to maintain consistency in your count. (Refer to appendix 3).

EXPECTED VALUES

INTERPRETATION

Formula

• Number of cells x dilution divided by the volume. With CSF, no dilutions are done unless indicated.

Example

• A total of 16 cells counted on the acid side and a total of 25 cells counted on the neat side.
• Calculate the WBC and RBC counts.
• 25-16 = 9 – therefore there are 16 WBC and 9 RBC
• Note: 9 Large Squares counted (entire surface) = volume is 0.9
• 16 (WBC) /0.9(volume) 9(RBC)/0.9(volume)
• WBC =17.7 x 106/L RBC = 10 x 106/L

Note: both these values are high!

• Refer to Hemocytometer measurements

SOURCES OF ERROR

1. Dust or dirt in the counting chamber can result in erroneous results.
2. Contaminated diluting fluid may cause erroneous results.
3. A high WBC count may make it difficult to obtain a reasonable count, so a secondary dilution may be required. Note: use the lowest possible dilution.
4. You can run the CSF on the analyzer to obtain a RBC count, if present in excessive amounts on the hemocytometer. Follow lab protocol.
5. Not maintaining a consistent counting routine may lead to erroneous results.
   • See Solid Circles are Counted.
6. Not allowing the chamber to settle before counting will result in erroneous results.
7. Using the wrong tube for testing may lead to erroneous results. This may apply more to microbiology.

How to Use a Hemacytometer

Preparing the Hemocytometer.

1. Remove the disposable hemocytometer from the package.
2. Inspect it to make sure there are no cracks or dirt, etc.
3. Label the Hemocytometer with patient name (last name, first initial) and the MRN number.
4. Fill both sides of the Hemocytometer with the specimen/dilution.
5. The entire surface of the chamber is counted for CSF specimens (all nine squares).
6. Once the count is completed, discard the hemocytometer in the biohazard waste container.

Hemocytometer Measurements

• With CSF all nine squares are counted.
• For other fluids and EDTA specimens: White blood cells are counted in the four corner squares marked with a “W,” using a 10x low power objective.
• Platelets, not shown here, are counted in the entire center square (all twenty-five small squares), using a 40x dry objective. Not practice anymore.
• RBC counts are no longer performed manually.

Solid Circles are Counted, and the Open Circles are Not Counted

• This is a common counting strategy used in many labs to ensure accuracy in counting WBCs Count cells touching the top and left, but not the ones touching the right or bottom.
• Note: This only applies to the outside borders of the nine squares, not all squares.

Procedure for Preparation of 2% Acetic Acid with Methylene Blue

1. Pour 490 mL of the Distilled Water into a 500mL cylinder.
2. Carefully add 10mL of glacial acetic acid to the distilled water, bringin the total volume to 500mL.
3. Cover with the parafilm and mix thoroughly.
4. Add a very small amount of New Methylene blue powder to color the solution a very pale blue. The tip of a wooden applicator stick is suffient.
5. Label a glass flask/bottle with appropriate safety information
6. Filter the solution into a labelled glass container.
7. Refrigerate the reagent until required.