Manual Cell Count Flashcards
Why is manual cell counting necessary? (Principle)
May be necessary to use manual methods when counts exceed the linearity of an instrument, when an instrument is nonfunctional and there is no backup, in remote laboratories in Third World countries, or in a disaster situation when testing is done in the field
Manual cell counts are performed using?
Hemacytometer, or counting chamber, and manual dilutions made with calibrated, automated pipettes and diluents (commercially available or laboratory prepared).
The principle for the performance of cell counts is essentially the same for white blood cells (WBCs), red blood cells (RBCs), and platelet. However, they vary in?
dilution, diluting fluid, and area counted
What is the most common hemacytometer used?
Levy chamber with improved Neubauer ruling. Or the Improved Neubauer Counting Chamber/ Hemacytometer
The Improved Neubauer Hemacytometer is composed of two raised surfaces, each with?
a 3 mm x 3 mm square counting area or grid (total area 9 mm2)
The two chambers of the Improved Neubauer Hemacytometer is separated by?
an H-shaped moat
Each of the four corner
(WBC) squares is subdivided further into?
16 squares
The center square subdivided into ___ squares. And each of this square is further subdivided into ____
25 intermediary squares; 16 small squares
The distance between each counting surface and the coverslip is
0.1 mm (reciprocal is 10)
The total volume of one entire grid or counting area on one side of the hemacytometer is?
0.9 mm^3
Cite the general formula for manual cell counts.
Total count = cells counted x dilution factor /
area (mm^2 ) x depth (0.1)
or
Total count = cells counted x dilution factor x 10* / area (mm^2)
If one mm^3 is equivalent to one microliter (uL). How do you convert the count per uL to the count per liter (L)?
by multiplying uL by a factor of 10^6 or by 1,000,000,000
It refers to the number of WBCs in 1 liter (L) or 1 microliter (uL) of blood.
WBC or leukocyte count
Why is the diluting fluid of the WBC count hypotonic?
The diluting fluid lyses the nonnucleated red blood cells in the sample to prevent their interference in the count
The typical dilution of blood for the WBC
count is
1:20
Prior to cell counting, how do you clean the hemocytometer and coverslip that will be used?
You clean the hemacytometer and coverslip with alcohol and dry thoroughly with a lint-free tissue.
WBC counts should be performed within?
3 hours of dilution
When charging or filling both sides of the hemacytometer you hold the microhematocrit tube at a ____ angle and touch the tip to the coverslip edge where it meets the chamber floor
45-degree
What is the method used when counting cells under the microscope?
Battlement
How do you count all the cells (WBCs, RBCs, and Platelets) in the hemacytometer?
For WBC count- four corner squares.
For RBC count- five squares (four corner squares and the center square) in the center square of the grid.
For Platelet count- 25 small squares in the center square of the grid.
What is the preferred objective lens used for each cell count (WBCs, RBCs, Platelet).
For WBC count- LPF (10X objective lens, 100x total magnification).
For RBC count- HPF (40x objective lens, 400x total magnification)
For Platelet count- HPF (40x objective lens, 400x total magnification)
What type of microscope is used in Platelet cell count?
Phase-contrast microscope. A light microscope can also be used, but visualizing the platelets may be more difficult.
If the difference between the total cells counted on each side is more than 10% what does this indicates?
Indicates an uneven distribution, which requires that the procedure be repeated.
In WBC cell count, if 1:100 dilution was being utilized, how many squares/ area should you count?
9 large squares or 9 mm2 on both sides of the chamber
Characteristics of a platelet under phase-contrast microscope.
The platelets have a diameter of 2 to
4 um and appear round or oval, displaying a light purple sheen
What is often seen in the background during platelet cell count?
“Ghost” RBCs
How do you verify for the accuracy of manual cell count?
By performing a WBC/ RBC/platelet estimate on a Wright-stained peripheral blood film made from the same specimen used in manual counting
In platelet count, if fewer than 50 platelets are counted on each side, the procedure should be repeated by diluting the blood to ____. If more than 500 platelets are counted on each side, a ____ dilution should be made
1:20 and 1:200, respectively
Factors that may
cause the counts to be inaccurate.
Dirt in the pipette, hemacytometer, or diluting fluid
This phenomenon may occur when EDTA anticoagulant is used.
“platelet satellitosis”
This refers to the adherence of platelets around neutrophils, producing a ring or satellite effect
“platelet satellitosis”
How do you correct platelet satellitosis?
By using sodium citrate as
the anticoagulant.
Important fact to remember when using sodium citrate as anticoagulant to aid platelet satellitosis.
Because of the dilution in the citrate evacuated tubes, it is necessary to multiply the obtained platelet count by 1.1 for accuracy
These are rarely performed because of the inaccuracy of the count and questionable necessity
Manual RBC count
More accurate manual RBC procedures, Such as the _______ and _______, are desirable when automation is not available.
microhematocrit and hemoglobin concentration, respectively
Reported as percentage
Relative Count
Shows the real count/ number
Absolute Count
Dilution ratio for RBC
1:200
Dilution ratio for WBC
1:20
Dilution ratio for Platelet
1:100
The dilution factor is calculated by,
Dividing the the volume of the bulb units and blood units
Devices used for cell count
Thoma pipette (WBC and RBC), Improved Neubauer Hemacytometer, Compound microscope
Mark to which blood is drawn (RBC & WBC)
0.5 mark
Mark to which blood is drawn (Platelet)
1 mark
Mark to which diluting fluid is drawn in RBC Thoma pipette
101 mark
Area where both blood and diluting fluid combine
Bulb
It mixes the content; identifier of the type of thoma pipet
Bead
Mark to which diluting fluid is drawn in WBC Thoma pipette
11 mark
The presence of the bead within the bulb not only serves as an indicator for RBC and WBC thoma pipette but it also
Assists in the dilution action
When aspirating for blood, only aspirate in the _____ and wipe the excess blood ONLY within the _____ using tissue paper
0.5 mark and sides, respectively
Why do you need to refrain from wiping the tip of the pipette?
Because the tissue paper might sip the blood due to the capillary action.
For blood cell dilution, it requires specimens that are collected in
lavender top tube; EDTA
It represents the unit of the sample
Marks in the (long Calibrated stem) pipet. Ex 0.5 mark: 0.5 blood units
Refers to the units inside the bulb
Bulb units
The ratio of the blood units to the bulb unit
Dilution
Is done by dividing the Bulb units
by the blood units
Dilution factor
Using a hypertonic solution in RBC count results in
crenated cell
Using a hypotonic solution in RBC count results in
Cell lysis
Solutions or diluting fluids used for RBC count
Hayem’s
Gower’s
Dacie’s fluid
Solutions or diluting fluids used for WBC count
2% acetic Acid
1% Hydrochloric Acid
Turk’s Diluting fluid
Solutions or diluting fluids used for Platelet count
1% ammonium oxalate
Rees-Ecker fluid
Components of Hayem’s fluid are?
Sodium Chloride (Provides isonicity) 1g
Sodium Sulfate (Prevents Rouleaux) 5g
Mercuric chloride (antiseptic) 0.5 g
Distilled Water (Solvent) 200 ml
Components of Dacie’s fluid are?
Trisodium Citrate-3.13 g
formaldehyde (37%) 1 ml
distilled water 100 ml
Components of Gower’s fluid are?
Isotonic solution containing 12.5g of
sodium sulfate and 33.3 g of glacial
acetic acid in 200 ml distilled water
Components of Turk’s fluid are?
Acetic Acid- 3ml,
1ml of aqueous gentian violet,
and 100 ml distilled water
Components of Rees- Ecker fluid are?
3.8 grams of sodium citrate,
0.2 ml of 40% formaldehyde,
0.1 gram of brilliant cresyl blue in 100 ml aqueous solution
Diluting Fluid must be:
Cheap and Economical
Stable, Easy to Secure and Prepare
With Preservative Action
High Specific Gravity
With Buffer Action
Non-Allergenic / Non-corrosive
WBC Diluting Fluid must be hypotonic
RBC Diluting Fluid must be Isotonic
How do you mix the pipette?
Figure of 8 motion for 2-3 minutes
It is a tool used for manual cell counting. And was originally invented for quantifying blood cells.
Hemacytometer or Counting Chamber
In a normal adult, WBC ranges from about?
4,000-11,000/mm2
Its function is to provide immunity- the ability to resist infection
White Blood Cells
A WBC count above normal is called?
Leukocytosis
Physiologic causes of Leukocytosis
Exercise
Stress
Obstetric labor
Anesthesia
Pathologic causes of Leukocytosis
Infection
Hematologic Disorder (leukemia)
A WBC count below normal is called?
Leukopenia
Causes of Leukopenia include:
Viral Infection
Ionizing Radiation
Chemicals
Drugs
Hematologic problems (aplastic anemia)
When WBC is markedly elevated
(100-300x109/L) the dilution can be increased to
1:200
When WBC is also below 3 x10/L, the dilution can be reduced to
1:10
For patients with polycythemia, increase the dilution to ___. For anemia, reduce the dilution to ____.
1:300 and 1:100, respectively
