Hemocytometer Flashcards
Manual Cell Counting is used in the following circumstances:
Validity check of electronic methods for calibration purposes
Validity check of electronic counts in patients with profound leukopenia or thrombocytopenia
For blood specimens with platelet counting interferences
Back-up method
GENERAL PROCEDURES
- Specimen dilution
- Hemocytometer charging
- Microscopy (Viewing the ruled areas)
BLOOD DILUTION
Purpose
To avoid crowding of cells during manual counting
Name of pipette
Thoma pipette
Purpose of bead
For mixing
RBC Bulb units
100
WBC bulb unit
10
why the last calibration ends in 101 (for RBC) and 11 (for WBC)
One unit =_______
from the tip to the 1.0 mark
Size of the bulb
RBC - Larger
WBC -Smaller
Color of the bead
RBC - Red
WBC - White
Volume of the bulb
RBC - 100 units
WBC -10 units
Size of the bore
RBC - Smaller
WBC - Larger
Calibration
RBC - 0.5; 1; 101
WBC - 0.5; 1; 11
Dilution
RBC - 1:200
WBC - 1:20
Dilution factor
RBC - 200
WBC - 20
Diluting Fluids for WBC
2-3% Glacial Acetic Acid
1% Hydrochloric Acid
Turk’s Solution
The____ is the counting chamber used for manual counting of (3)
hemacytometer
white blood cells, red blood cells, and platelets.
The principle for counting all three cells are basically the same, only the (3) have differed.
dilution
diluting fluid
area for counting
Nowadays, the new improved_______ is the most commonly used hemacytometer for enumerating blood cells
Neubauer counting chamber
There are depressions, called “_____”,
“, that surround the platform.
A______ is positioned so that it covers both ruled areas of the hemocytometer.
This also allows the fluid in the chamber.
moats
coverglass
The two ruled areas of the hemocytometer have specific dimensions.
Each ruled area is consist of a large square (______). The total_____ area is divided into____ equal squares, each with____
3mm x 3 mm
9mm^2
nine
1mm^2
According to Rodak’s, all____ squares in the center are for platelet count).
25
-Depth of chamber =
0.1 mm
Before using the hemocytometer,____ of the blood sample are first made.
dilutions
T or F
Dilution for red cells, white cell, and platelet counts are done separately as each procedure uses different diluting fluids and equipment.
True
T or F
The diluting fluids and pipettes used for each type of cell have different characteristics that allow the counting of one cell type of interest only
True
RBC
Diluting Fluids
Hayem’s
Gower’s
Toisson’s
Bethel’s
Normal Saline Solution, 3.8%
Sodium Citrate
Formol Citrate
Characteristics of the Diluting Fluid
RBC - Isotonic solution
WBC - Will hemolyze red blood cells except the nucleated RBCs;
Hypotonic solution
Area counted
RBC - 0.2 mm2
WBC
(1:20) 4 mm2
(1:100) 9 mm2
Objective
RBC - 40x HPO
WBC - 10x LPO
Charging the hemocytometer
Clean the hemocytometer and coverslip with______.
Place the coverglass on the hemocytometer such that it covers______.
Charge the hemocytometer by touching the tip of the pipette to the point where the______ and ______ on one side meet.
Allow the fluid from the pipette to flow into one side of the hemocytometer by____ action.
The other side of the chamber is charged the same manner.
Before counting, the charged chamber should be allowed to stand for_____ in a moist chamber to allow settling of cells.
alcohol
both ruled areas
coverglass and the raised platform
capillary
10-15
minutes
Viewing the ruled areas
Mount the charged hemocytometer on the microscope with________ in place.
Bring the stage closer to the objective using course adjustment until the etched lines come into view.
Use the fine adjustment knob for sharp focus of the etched lines.
low power objective (10x)
The rule in counting
When counting, follow the _____pattern, where the______ and _____ are considered significant.
inverted L
upper and left boundaries
Rule in counting
• For three-line boundary…
do not count the cells that touch only the outermost boundary
Rule in counting
• If the cell touches both the mid and outermost boundary…
include it in the counting
Rule in counting
• For two-line boundary, count the cells that…
touch up to the second boundary line
Rule in counting
• For one-line boundary, count the cells that touch…
that single boundary line
Rule in counting
• Whether the box has three-, two, or one-boundary line, do not count any cells that…
touch the right and lower boundary lines
Rule in counting
• For the______ direction, start from the upper left corner box going to the right
serpentine
Why must RBC solution be ISOTONIC?
Prevent lysis of RBC
Why must WBC solution be ACIDIC or Hypotonic?
Will lyse ALL red blood cells except
nucleated RBCs
Enhances WBC nucleus
Dilution =
volume of solute
total volume of solution
RBC DILUTION
Computation for RBC Dilution
a. solute (pertains to the amount of blood sipped) -____ or _____
b. total volume of the solution (pertains to the volume capacity of the BULB) -_____
c. SOLVENT - just subtract_____ units from 100 units
0.5 units or 1.0 units
100 units
0.5
cells/uL =
number of cells counted x dilution factor
—————
area counted x depth * (.1mm)
Shortcut for solving
RBC = x1000
WBC = x50
DETERMINING THE ALLOWABLE VARIATION IN THE COUNTS
Whether RBC, WBC, or platelets, both sides of the counting chamber should be counted.
The counts of the two sides need NOT be exactly the same.
There is an allowable variation.
The counts of the two sides of the hemocytometer should agree by______
10% or less less.
To determine the allowable variation:
- Get the clifference of the two total counts
- Get the average of the two counts
- Divide the difference by the average and multiply by 100
ALLOWABLE VARIATION IN THE COUNTS
the example, the answer is 5.97%. Therefore, the variation between the total counts of the two sides of the counting chamber is______.
acceptable
When the outcomes in allowable variation is not acceptable,_________. In the case that the variation is still not acceptable after repeat charging, this warrants repeat______ or ______
repeat charging the hemocytometer
blood dilution or repeat blood collection.
NOTE: If the average count obtained is in decimal (e.g. 112.5), always…
round up to whole number
= 3.34 x 10^6uL
= 3.34 x 10^12/L
Conventional unit
S.I unit
Why discard first 3 drops?
Conc. cells
express with___ decimal points for RBC Count
express with___ decimal points for WBC Count
No decimal points for Platelet Count
2
1
For a Standard leukocyte count within the reference range, there should be NO MORE THAN____-CELL DIFFERENCE between the highest and lowest total number of cells found among the eight 1-mm-corner squares (2 chambers) counted.
15
For Standard erythrocyte count, there should be NO MORE THAN___-CELL DIFFERENCE between the highest and lowest total number of cells counted in the ten-0.04-mm^2 ( 2 chambers). Counts that do not meet these standards should not be reported. Rather, the procedure should be repeated.
20
