Analysis of Urine and Body Fluids (Lab)

MODULE 7: Hemocytometer & Body Fluid Cell Count

HEMOCYTOMETER 4. While holding the pipette vertically, place the tip of

● Also known as a counting chamber, specialized the pipette in the tube containing 2% acetic acid
glass slide used in laboratory settings to count and and aspirate until the 11th mark
determine the concentration of cells in a liquid 5. Place the pipette horizontally and firmly hold the
sample opening at the tip of the pipette using your thumb.
● Primarily applied in hematology for blood cell Detach the sucking tube from the other end of the
counts, the hemocytometer may also be used in pipette and cover that opening with the index or
counting blood and other cellular components in middle finger
other bodily fluids 6. Vigorously mix the sample in the pipette by doing a
● Simple, cost-effective and standardized instrument figure of 8 motion with your hand for 45 seconds
in clinical diagnostics used to obtain accurate and
reproducible cell counts PREPARATION OF THE HEMOCYTOMETER
● The applications of the hemocytometer in the 1. Before loading your sample, make sure to wipe the
Clinical Microscopy laboratory include the hemocytometer using a clean gauze pad dipped in
determination of the number of sperm cells in a 70% isopropyl alcohol. Make sure that the
seminal fluid, and the determination of WBC count hemocytometer is dry before loading your sample.
in bodily fluids 2. Prepare a wet chamber by placing a damp gauze
pad (use water to dampen the pad) inside a petri
BODY FLUID CELL COUNT dish.
● Cell counts are integral to body fluid analysis. 3. After mixing the sample, hold the pipette vertically
● They provide information about the presence or with the index or middle finger covering the top of
absence of certain conditions, such as infections or the pipette. Discard the first four drops of the
even the viability of samples mixture onto a piece of gauze.
● The use of cell count is primarily applied in 4. Place a coverslip onto the counting chamber.
cerebrospinal fluid (CSF), seminal fluid analysis, 5. Gently release your index or middle finger from the
and synovial fluid testing pipette to allow a few drops of sample to flow.
● In CSF and synovial fluid testing, cerebrospinal 6. Place the tip of the pipette on the edge of the ruled
fluid is checked for the number of WBCs present to area of the counting chamber and gradually allow
rule out infections the mixture to seep under the cover glass. Fill this
● Moreover, for seminal fluid analysis, the number of area exactly. Repeat this step on the other side of
normal spermatozoa is counted to test for infertility the counting chamber.
● Cell counts are usually performed manually using 7. Place the charged hemocytometer inside the wet
the hemocytometer with the Improved Neubauer chamber and cover it. Incubate at room
counting chamber. temperature for 10 minutes.
● Depending on the cells being counted, only certain
squares are used. COUNTING THE CELLS AND COMPUTING THE
● Like any cell counting procedure, body fluid cell CELL COUNT
counts should be performed immediately after 1. After incubation, place the hemocytometer onto the
specimen collection because granulocytes and stage of the microscope. Keep the counting
even RBCs begin to lyse within one hour, and 40% chamber at a horizontal position at all times.
of WBCs disintegrate after two hours. 2. Adjust the microscope using the low- power
objective and with low light.
PROCEDURE 3. Identify the markings in the counting chamber.

DILUTION OF SAMPLE
1. Gently mix the test tube containing the sample by
inversion at least three times
2. Attach an aspirator to the sucking end of a Thome
WBC diluting pipette and then draw the sample up
to the 0.5th mark in the stem
3. For any excess samples in the pipette, gently
remove them by dispensing the contents in a clean
gauze

Irish Sabile 1
4. In proper focus, the white cells should look like - Normally, the number of RBCs and WBCs in body
small darks or black dots with a grainy or sandy fluids are low
texture. - Solution: Define lower limits + Protocol for manual
5. Scan the four large WBC squares for an even count with hemocytometer
distribution of the cells. If the distribution is uneven,
repeat the loading process. HEMOCYTOMETER PARTS AND DIMENSIONS
6. Beginning with the upper left large square, count all
white cells in the four large corner squares and add
the results to obtain the total number of cells
counted.
7. Count the cells on the opposite side of the counting
chamber and record the number of cells counted in
these four large squares. The total should be close
to the first count and should only have a 10%
difference.
8. Get the average of the count for the two chambers
and compute the cell count in cubic millimeters
using the formula below:

● 3x3mm counting grid subdivided into:

○ 9: 1x1mm2 squares
○ Each square is further divided into: 16, 100, or
HEMOCYTOMETER
400 smaller squares
- Manual or Automated
- Use: to perform manual cell counts on body fluids
- Cerebrospinal fluid, Synovial fluids, Pleural fluid,
Pericardial fluid, Peritoneal fluid, Peritoneal
dialysates, Bronchoalveolar lavages, Semen

SYNOVIAL FLUID AND SEMEN

- Highly viscous body fluids = Synovial fluid
- Fluids that fail to liquefy = Semen
- Pretreated before counting
- Clotted sample: INACCURATE cell counts
- Solution: “Specimen clotted (large clot); Results
may be inaccurate and must be interpreted with
caution”

3 Main types of Hemocytometer

● Fusch-Rosenthal
● Nageotte
● Neubauer (Improved)
● MAIN DIFFERENCE: Different design of calibrated
counting area

Disadvantages of Manual Cell Count

● Labor intensive
● Time consuming
● Needs advanced technical skills
● Poor precision
● Subject to error
● To solve this, Medtechs need to be technically ● 1 W square = 1 mm2 in area
proficient ○ The 4 “W” squares are best used for WBC
count
AUTOMATED HEMOCYTOMETER ● 1 R square = 0.04 mm2 in area
- Automated cell counting chambers: erroneous ○ The 5 “R” squares are best used for RBC count
results with low cell counts
AUBF Lab M7 2
 HEMOCYTOMETER PREPARATION
1. Mix specimen well (1-2
mins)
2. *Dilute specimen
(Optional)
3. Clean hemocytometer
with alcohol + wipe dry
4. Load/Charge
hemocytometer chamber
5. Incubate/leave chamber
undisturbed for cells to settle (10 mins)
6. Examine Microscopically
7. If even distribution, proceed with cell counting
8. If (+) overlapping/clumping
● Clean hemocytometer
● Mix specimen well
● Prepare new dilution
● Refill chambers

● Number of squares counted in each chamber

depends on the total number. of cells present. CELL COUNTS AND CALCULATIONS
● Accuracy in cell counts = directly related to cell
numbers. WBC COUNT/ TOTAL NUCLEATED CELL COUNT
○ Greater number of cells, Greater accuracy - Requested on almost all body fluids
● When not needed, high dilutions causes cellular - Determines the presence of infections
structures to disintegrate - 4 W Squares
● If extremely low cell counts: additional squares can
be counted RBC COUNT
● If High cell counts: fewer cells can be counted - Little clinical value
- 5 R Squares
How to Detect Errors in Preparation
● Dilutions: performed and analyzed in duplicate Cell
counts
● Compare each chamber
● Must agree less than or equal to 20%

Not True Replication

● Counting the same chamber twice
● Comparing chamber counts using the same diluted
sample

● Include cells touching the middle line of the top and

left sides of the square.
● Exclude cells touching the middle line on bottom
and right side of the square.

AUBF Lab M7 3
 CELL COUNTS AND CALCULATIONS

**From Cis and Trans ^_^

AUBF Lab M7 4