Week 4 Lab: Hemocytometry and RBC Counting Flashcards
Hemo means?
blood
Cyto means?
cell
Meter means?
measurement/counter
It is an instrument used to count the blood cells.
Hemocytometer
Hemocytometer includes?
a) Neubauer’s slide
b) Cover slip
c) RBC pipette
d) WBC pipette
It is the name given to a thick glass slide used for hemocytometer.
Neubauer’s Slide
The Neubauer’s Slide has a/an _____ in the center.
H-shaped groove
The depth of the scales/depth between the Neubauer’s slide and cover slip is?
1/10mm or 0.1mm
True or false: The depth of the scales is always constant regardless of the manufacturer.
True
Neubauer’s slide with a cover slip over it is called a __________.
Neubauer’s chamber
True or false: You cannot use the Neubauer’s chamber to count other body fluids.
False. You can use it to count other body fluids, such as semen.
Each scale is _____ wide and _____ long.
3mm, 3mm
The whole scale is divided into __ big squares.
9
Each large square is ___ long and ___ wide.
1mm, 1mm
Area of 1 large square is?
1mm^2
The 4 corner large squares are for?
WBC count
The total area of the 4 large squares for WBC count is?
4mm^2
The 4 corner large squares for WBC count are further divided into how many smaller squares each?
16 smaller squares each
How many total smaller squares are used for WBC count?
64, because 1 large square is divided into 16 smaller squares, so 16x4 is 64.
Which large squares are used for WBC count?
4 corner large squares
Which large squares are used for RBC count?
4 central large squares
The central large square for RBC count is further divided into how many smaller squares?
25 smaller squares
The smaller squares for the RBC count are further divided into how many smallest squares?
16 smallest squares each
For RBC count, which squares should you include in the count?
total of 5 smaller squares: the 4 corner smaller squares and the central smaller square of the middle large square = 80 squares
For platelet count, which squares should you include in the count?
all smaller squares of the middle large square should be counted
What do you call the cells that are not counted because they are already dead?
Crenated RBC cells
RBC counting is done under which objective lens?
HPO/High Power Objective
True or False: High amount of light is needed when viewing the Neubauer’s slide.
False. Only low amount of light is needed.
WBC Square: Length of one smaller square?
1/4mm (0.25 mm)
WBC Square: Width of one smaller square?
1/4mm (0.25 mm)
WBC Square: Depth of one smaller square?
1/10mm (0.1 mm)
WBC Square: Volume of one smaller square?
1/160mm³ or 0.00625 mm³
RBC Square: Length of one smaller square?
1/5 mm (0.2 mm)
RBC Square: Width of one smaller square?
1/5 mm (0.2 mm)
RBC Square: Depth of one smaller square?
1/10mm (0.1 mm)
RBC Square: Volume of one smaller square?
1/250mm³ or 0.004 mm³
RBC Square: Length of one smallest square?
1/20mm (0.05 mm)
RBC Square: Width of one smallest square?
1/20mm (0.05 mm)
RBC Square: Depth of one smallest square?
1/10mm (0.1 mm)
RBC Square: Volume of one smallest square?
1/4000mm³ or 0.00025 mm³
Rules in Counting Blood Cells?
- Observe the boundary line of the square.
- Do not count cells touching bottom line and right line to avoid double counting.
- Count the cells touching halfway in and halfway out of the upper and left boundary lines.
- Never count cells touching halfway in and halfway out of the lower and right boundary lines of the square.
- Never count cells drying preparation.
- Never count cells in underfilled or overfilled chamber.
If double line, which line is the boundary of the square?
The outer line is the boundary of the square.
If triple line, which line is the boundary of the square?
The middle line is the boundary of the square.
Features of RBC pipette?
- It has a red bead
- It has graduations up to mark 101
- Size of bulb is larger
- Size of lumen is smaller
Features of WBC pipette?
- It has a white bead
- It has graduations up to mark 11
- Size of bulb is smaller
- Size of lumen is larger
In RBC counting, why do we need to dilute the blood?
because it will be more difficult to count if we don’t dilute the blood
How many units of fluid can an RBC pipette contain?
101 units of fluid
Function of the small red bead in an RBC pipette?
used to mix the blood and the diluting fluid
Diluting fluid used for RBC counting?
Hayem’s fluid
Blood is filled till mark ___ and Hayem’s fluid is then filled till mark ___.
0.5, 101
Importance of the discard step after mixing the blood and diluting fluid in the RBC pipette?
the 1st 3-4 drops contain only diluting fluid in the stem, therefore no RBCs are present
After the discard step, ___ portion out of 101 is discarded.
1
So, ___ part of blood is in ___ parts of fluid or, ___ part of blood is mixed in ___ parts of fluid. Thus, dilution factor for RBC counting is ___.
0.5, 100
1, 200
200
For WBC counting: ___ part of blood is mixed in ___ parts of fluid. ___ part of blood is in ___ parts of fluid. Thus, dilution factor for WBC counting is ___.
0.5, 10
1, 20
20
For Platelet counting: ___ part of blood is mixed in ___ parts of fluid, so dilution factor for platelet counting is ___.
1, 100
100
Magnification to see the general formation of slide.
4X (Scanner)
Magnification for WBC counting?
10X (LPO)
Magnification for RBC counting?
40X (HPO)
Unit of measurement for RBC counting?
cells per mm^3
SI unit for RBC counting?
x 10^12/L
The red cell count is the number of red cells in?
1cu.mm
The RBC count is one of the tests that are used for the diagnosis of?
anemia and polycythemia
Materials and Equipment for RBC Counting?
• Anticoagulated blood
• RBC Pipette
• Diluting Fluid
• Tally counter
• Counting Chamber
• Gauze pad
• Microscope
• Test tube
The hemacytometer should be in a _____ position.
horizontal
The total volume of each scale is?
9 cu mm
Formula for RBC count?
RBC count = # of RBC counted x area cf. x depth cf. x dil.cf
Formula for coversion of RBC count to SI unit?
RBC count- # of RBC count in millions/mm^3 x .000001 = _____ x 10^12/L
In RBC counting, we count cells in 5 smaller squares, and the area of one smaller square is 1/25mm^2, so what is the total area?
5 smaller squares x 1/25mm^2 = 1/5 mm^2
Variation in technique for polycythemia or erythremia?
blood is drawn to 0.3 mark of the RBC pipette and diluent up to 101, 1:333
Variation in technique for anemia?
blood up to 1 mark and the diluent to 101, the dilution is 1:100
Normal values of RBC for female?
3.6–5.6 x 10^12/L
Normal values of RBC for male?
4.2–6.0 x 10^12/L
Normal values of RBC at birth?
5.0–6.5 x 10^12/L
Physiologic Variation?
• Increased count in dehydration
• Increased count in exercise
• Newborn children have higher counts than adults
• Women have lower counts than male
• Individual living at higher altitudes have higher counts
Pathologic Variation?
• Increase erythrocyte count in polycythemia
• Increase in pulmonary tuberculosis and pulmonary fibrosis.
• Increase in acute poisoning
• Decrease in anemia and after hemorrhages
Characteristics and functions of Hayem’s fluid?
- isotonic solution for manual counting of RBC
- prevents agglutination
- maintains osmolarity
- prevents isotonicity, so that RBC maintains their shape & size