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EXERCISE NO. PY2.11E
STUDY OF THE HEMOCYTOMETER
Hemocytometer:
It is a box, which contains the following three instruments:
a. An RBC pipette
b. A WBC pipette
c. An Improved Neubauer’s chamber
RBC pipette:
This consists of a capillary tube of glass, with a dilatation in the upper part.
A rubber tube is attached above the bulb of the pipette, which ends in a
mouthpiece.
The tube is called the stem – the dilatation, the bulb.
The bulb contains a glass bead, red in colour.
The stem is graduated into 10 divisions, the middle of which is marked 0.5.
The marking nearest to the bulb is marked 1.0.
The single mark above the bulb is inscribed as 101.
These markings indicate volume in respect of the total volume of the hollow
part. This does not follow any definite measurement system, so there is no
unit.
WBC pipette:
This is similar in structure to the RBC pipette, but with the following
differences –
The bulb is smaller
The glass bead is white in colour
The markings below the bulb are 0.5 & 1, but the marking above the
bulb is 10.
Improved Neubauer’s Chamber:
This is used to count cells, by laying them out in a very thin film.
It is a thick slide made of mineral glass.
In the middle is a central platform, divided into two parts by
a transverse gutter.
Each of the two platforms contain one laser-etched grid
markings, in which cells are counted.
On each side of the platform are grooves called moats,
whose outer sides are raised into platforms.
When using it, a cover slip is to be placed in such a way that it covers
the central platform and the gutters.
There remains a space 0.1 mm in depth between the cover
slip and the central platform.
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The counting area:
Each counting grid is 3 x 3 mm square.
Two vertical and two transverse lines divide it into 9 large squares.
So, each large square is 1mm x 1mm.
The 4 large squares at the four corners are meant for counting WBCs.
Each such square is further divided into 16 smaller squares measuring¼ mm x ¼ mm.
The central square is meant for counting RBCs.
The RBC counting area:
Its 1 mm x 1 mm area is divided by triple lines (0.01 mm wide) into 25 medium-sized squares.
So, each of these medium-sized squares have an area of 1/5 mm x 1/5 mm.
o Each of these squares is further divided into 16 smallest squares, each measuring1/20 mm x 1/20 mm.
o RBCs are counted in the central and the 4 smallest squares in the corners.
WBCs are counted in the areas marked W.
RBCs are counted in the areas marked 1, 2, 3, 4 & 5.
Under 10x magnification usually a large box is visible completely.
The central medium-sized box for RBC counting is visible under 100x magnification.
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Points to remember
Improvements in the Improved Neubauer Chamber:
Improved Naubauer Chamber Original Neubauer Chamber
The central large square (for RBC count) is divided into 5 The central large square is divided into 4 x 4 medium sized
x 5 medium sized squares squares
Makes calculations easier
Cleaning of the Naubauer Chamber:
It is first cleaned with soap water and then with alcohol.
Other uses of Hemocytometer:
The instrument is used for body fluids where the cell count is high.
Haematological use Non-haematological use
Absolute eosinophil count Sperm count
Platelet count Counting micro-organisms
Bone marrow cell count
WBC count in leukaemias
For counting items with a high count, the RBC counting area is used – RBC pipette is used for dilution.
Differences between RBC & WBC pipettes: discussed above.
RBC Diluting fluid (Hayem’s fluid):
Component Purpose
NaCl Maintains fluid isotonic to ICF (of RBCs)
Distilled water Maintains fluid isotonic to ICF (of RBCs)
Na2SO4 Chelates Ca2+ – prevents formation of clot & rouleaux
HgCl2 Prevents growth of bacteria & fungi.
Acts as an adhesive – fixes cells to the slide.
Alternative for Hayem’s fluid: solution of formalin in water.
WBC Diluting fluid (Turk’s fluid):
Component Purpose
Glacial acetic acid Fixative agent for WBC
Destroys RBC membrane (destroys RBCs)
Gentian violet Stains the WBC nuclei dark violet
Distilled water Makes the fluid isotonic
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Estimation of Haemoglobin by Acid Hematin Method
Instruments used:
Sahli’s hemoglobinometer Other things
Haemoglobinometer pipette N/10 HCl (in a dark colured bottle)
Haemoglobinometer tube A dropper
Glass rod (stirrer) Distilled water
Comparator box
Sahli’s hemoglobinometer is a box which contains the instruments mentioned above.
Haemoglobinometer pipette:
This has a single marking for 20µL of blood.
Haemoglobinometer tube:
It is circular or rectangular in profile.
It is is graduated in both percentage (10 to 140 %) and gram percent (2 to 22 gm %).
Comparator box:
It is a rectangular box containing a central tube for holding the Haemoglobinometer tube.
There are two coloured glasses on either sides, called the comparators.
The back of the box is covered by an opaque white sheet that diffuses light passing through
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Differential count of WBC
Leishman’s stain: composition.
Eosin: is an acidic dye – stains basic particles in a cell.
This stains RBCs & granules of eosinophils.
Methylene blue: is a basic dye – stains acidic particles.
This stains nuclei of leucocytes & granules of basophils.
Methyl alcohol (acetone free): fixes the stain to the slide (so, is a fixative).
It must be free of acetone, as acetone causes the cells to shrink and may even undergo lysis.
If Leishman’s stain is not available, the smear can be fixed with a few drops of methylated spirit.
Buffered water:
This is water with pH 6.8.
It causes ionisation of the dyes present in Leishman’s stain.
Measurement of ESR
Westergren’s pipette:
It is a 30 cm long glass pipette, open at both ends, with a central bore of 2.5 mm diameter.
It is graduated from 0 to 200 along the lower 2/3 of the length.
Blood (with anticoagulant) needs to be sucked into the tube up to the 0 mark.
o It is then attached to a Westergren’s stand – the 0 mark should point upwards.
Blood cells settle down with time – leaving plasma above.
o Reading is taken at the end of one hour – at the upper border of the cell column.
o The standard is to take the reading at 1 hour – this is enough time for all cells to settle.
Anticoagulant used with Westergren’s pipette is sodium citrate solution (3.8%).
Wintrobe’s tube:
It is a thick walled 11 cm long glass tube, circular in cross-section.
It has a central bore of 2.5 mm, closed at the bottom.
It is calibrated from 0-10 in both direction; above downwards and bottom upwards.
It is used for two purposes:
a. Measurement of ESR
b. Estimation of packed cell volume (PCV).
In the picture below: A is Westergren’s pipette B is Wintrobe’s tube.
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Estimation of ESR by Wintrobe’s tube:
Blood added with powder anticoagulant (double oxalate) is transferred into the tube with the help of a Pasteur
pipette. It is filled up to the 0 mark at the top.
o The Pasteur pipette is a capillary tube with a long nozzle.
The Wintrobe tube is kept in its stand.
Reading is taken at the end of 1 hour (from the 0 mark downwards).
ESR obtained with a Wintrobe’s tube is more accurate.
Normal value of ESR: readings are taken at the end of one hour.
Males Females
Westergren’s method 3-5 mm 4-7 mm
Wintrobe’s method 0-9 mm 0-20 mm
Estimation of PCV by Wintrobe’s tube:
Anticoagulated blood (as above) is taken up to the 10 mark.
It is then centrifuged for 30 minutes after closing the tube with a cork.
It forms three layers:
a. A layer of clear plasma on top.
b. A colourless layer of platelets & leucocytes in the middle (the buffy layer).
c. A large red layer below – formed by erythrocytes.
The hematocrit is the total volume of the lower and middle layers.
Other issues related to ESR:
ESR is as a maker for inflammation.
It indicates the degree of severity of an inflammatory condition; cannot diagnose the disease itself.
A single value of ESR is not of much significance. If the initial value is high, it must be followed up.
If the value keeps increasing, indicates increasing severity of disease.
As the value keeps decreasing; indicates decreasing severity of disease.
Three factors determine rise in ESR:
a. Increased fibrinogen in blood.
ESR increases in pregnancy as blood fibrinogen is higher.
b. Decreased viscosity of plasma.
ESR increases in fever, as blood viscosity decreases at higher temperature.
c. Decreased hematocrit (anemia).
ESR is higher in females (compared to males), as red cell mass is lower.
ESR is lower in neonates, as the red cell mass is higher.
Pathological conditions where ESR is raised: a few.
Infections like: tuberculosis and viral hepatitis.
After major trauma or surgery.
Acute rheumatic fever (inflammation affecting heart and joints).
Rheumatoid arthritis (inflammation affecting various organ systems including joints).
Certain cancers like lymphomas.