Hemocytometer manual cell counting (1)

1. RAJESH MOHESS ,CLT.

3.  The hemocytometer is a specimen slide which is
used to determine the concentration of cells in a
liquid sample.
 It has a rectangular indentation that that creates a
chamber
 The device is carefully crafted so that the area
bounded by the lines is known, and the depth of
the chamber is also known.

4.  Given the known parameters it is possible to count
the number of cells or particles in a specific
volume of fluid, and thereby calculate the
concentration of cells in the fluid overall
 The hemocytometer is frequently used to
determine the concentration of blood cells (hence
the name “hemo-”)
 However, it can also be used for other samples,
such as sperm cells.

5.  The cover glass, which is placed on the sample, does
not simply float on the liquid, but is held in place at a
specified height (usually 0.1mm).
 Additionally, a grid is etched into the glass of the
hemocytometer.
 This grid, an arrangement of squares of different sizes,
allows for an easy counting of cells.
 This way it is possible to determine the number of cells
in a specified volume.

7.  The fluid containing the cells must be
appropriately prepared before applying it to the
hemocytometer.
Proper mixing:
The fluid should be a homogenous suspension.
Cells that stick together in clumps are difficult
to count and they are usually not evenly
distributed.

8. Appropriate concentration:
 The concentration of the cells should neither be too high or
too low.
 If the concentration is too high, then the cells overlap and
are difficult to count.
 A low concentration of only a few cells per square results in a
higher statistical error and it is then necessary to count more
squares (which takes time).
 Suspensions that have a too high concentration should be
diluted 1:10, 1:100 and 1:1000.
 A 1:10 dilution can be made by taking 1 part of the sample
and mixing it with 9 parts normal saline
 The dilution must later be considered when calculating the
final concentration.

9. Counting cells that are on a line:
 Cells that are on the line of a grid require
special attention.
 Cells that touch the top and right lines of a
square should not be counted
 Cells on the bottom and left side should be
counted.

11. Number of squares to count:
 The lower the concentration, the more squares
should be counted.
 Otherwise one introduces statistical errors.
 Cells should be counted on both sides of the
chamber.
 If the final result is very different, then this can
be an indication of sampling error.

13.  When a liquid sample containing immobilized cells is placed
on the chamber, it is covered with a cover glass, and capillary
action completely fills the chamber with the sample.
 Looking at the chamber through a microscope, the number
of cells in the chamber can be determined by counting.
 Different kinds of cells can be counted separately as long as
they are visually distinguishable.
 The concentration of the cells can be calculated from the
cells counted from the mixture using simple formulas

14.  Rulings cover 9 square millimeters.
 Boundary lines of the Neubauer ruling are the center lines of
the groups of three

15.  The central square millimeter is ruled into 25 groups of 16
small squares
 The ruled surface is 0.10mm below the cover glass
 One (1) Milliliter = 1000 cubic millimeters (cu mm)
 One (1) Microliter (ul) = One (1) cubic millimeter (cu mm)
 The number of cells per cubic millimeter =
Number of cells counted per square millimeter X
dilution (eg. 100 for WBC count) X
10 (depth factor)

16.  Depth: 0.1 mm

17.  Red square = 1 x 1 mm = 1 mm2 (AREA) = 0.1 cubic millimeter
(VOLUME - mm3)
 Green square = 0.25 x 0.25 mm = 0.0625 mm2 = 0.00625
mm3

18.  Yellow square = 0.2 x 0.2 mm = 0.04 mm2 = 0.004 mm3
 Blue square = 0.05 x 0.05 mm = 0.0025 mm2 = 0.00025 mm3

19.  There are different types of counting chambers
available, with different grid sizes.
 Know the grid size and height (read the instruction
manual) otherwise you’ll make calculation errors.
 The provided cover-glasses are thicker than the
standard 0.15mm cover glasses.
 They are therefore less flexible and the surface
tension of the fluid will not deform them.
 This way the height of the fluid is standardized.

20.  Moving cells (such as sperm cells) are difficult
to count.
 These cells must first be immobilized.
 The hemocytometer is much thicker than a
regular slide.
 Be careful that you do not crash the objective
into the hemocytometer when focusing

22.  The Unopette system is a system of prefilled
blood dilution vials containing solutions that
will preserve certain cell types while lysing
others.
 It utilizes a premeasured volume of diluent in a
chamber into which a specified amount of
blood is drawn

24.  The Unopette test system consists of a self-filling
capillary pipette
 It consists of a straight, thin-wall, uniform-bore
plastic capillary tube fitted into a plastic holder
 Also has a plastic reservoir containing a
premeasured volume of reagent for diluting

26.  The reservoir is punctured to open access to
the reagent
 The dilution is determined by the type of
capillary used since each type have different
volumes
 The diluted blood is added to a hemocytometer
chamber and cells are counted in a specified
area.

27.  Unopette System discontinued
 The BMP LeukoChek is used to measure and dilute whole
blood for manual counting of leukocytes (WBC) and platelets
 It replaces the Unopette system
 Tested to CLIA guidelines
 Clinical Laboratory Improvement Amendments (CLIA) –
establish quality standards for all laboratory testing to
ensure the accuracy, reliability and timeliness of patient test
results regardless of where the test was performed

28.  For this procedure, whole blood is added to
ammonium oxalate (diluent), which lyses the
red cells while preserving platelets, leukocytes

29. PRINCIPLE
 Whole blood is added to the diluent, which lyses red cells but
preserves platelets, leukocytes
 When erythrocytes are completely lysed, the solution will be
clear red and counting can proceed.
 The diluted blood is placed in a hemocytometer according to
accepted technique.
 Cells are allowed to settle for 10-15 minutes before leukocytes
and platelets are counted.
 Under 100X magnification (x10 objective) using bright-light
microscopy, leukocytes appear refractile (can be seen as dark
dots)
 Under 400X magnification (x40 objective) using bright-light
microscopy, platelets appear oval or round and frequently have
one or more dendritic processes.

30.  BMP LeukoChek containing ammonium oxalate
Check expiration dates and do not use expired test kits.
Protect from sunlight.
 BMP LeukoChek capillary pipette, 20 μL.
 Hemocytometer : improved Neubauer ruling
 Hemocytometer coverslips
 Petri dish lined with filter paper that has been moistened and
two applicator sticks to hold the hemocytometer
 Microscope
 Hand counter
 EDTA whole blood

31. DILUTION RATIO
 Sample to total volume.......................1:100
 That is 1.98 ml of diluent to 20μl of sample

32.  1. Specimen should be well mixed and left on a
rocker for at least 5 minutes before using.
 2. Check BMP LeukoChek for clarity and
contents. If the BMP LeukoChek chambers
appear cloudy or the amount of reagent looks
questionable, do not use.

33.  3. With the reservoir on a flat surface, puncture the diaphragm of the
reservoir using the protective shield of the capillary pipette.
 A. Using a twist action, remove protective shield from the pipette
assembly.
 B. Holding the pipette and the tube of blood almost horizontally,
touch the tip of the pipette to the blood (fill with 20μl of blood).
 The pipette will fill by capillary action and will stop automatically
when the blood reaches the end of the capillary bore in the neck of
the pipette.

34.  C. Wipe the excess blood from the outside of the capillary pipette.
Be careful not to touch the tip of the capillary when wiping off excess
blood.
 D. Before entering the reservoir, it is necessary to force some air out
of the reservoir by squeezing it.
Do not expel any liquid and maintain pressure on reservoir.
 E. Place an index finger over opening of overflow chamber and
position pipette into reservoir neck.

 F. Release pressure on reservoir and then remove finger. The
negative pressure will draw blood into pipette.

35.  G. Rinse the capillary pipette with the diluents by squeezing the
reservoir gently two or three times.
This forces diluent up into, but not out of, the overflow chamber and
releases pressure each time to ensure the mixture returns to the
reservoir.
 H. Return protective shield over upper opening and gently invert
several times to mix blood adequately.

 I. Allow the BMP LeukoChek to stand for 10 minutes to allow RBCs to
hemolyze.
Leukocyte counts should be performed within 3 hours.

36.  4. Charge hemocytometer
 A. Mix the dilution by inversion and convert the BMP LeukoChek to
the dropper assembly.

 B. Gently squeeze BMP LeukoChek and discard first 3 or 4 drops.
This allows proper mixing, with no excess diluent in the tip of the
capillary.
 C. Carefully charge hemocytometer with the diluted blood, gently
squeezing the reservoir to release contents until chamber is properly
filled.
Be sure to charge both sides and not to overfill chambers.

37.  5. Place the hemocytometer in the pre-moistened Petri
dish and leave for 15 minutes.
This allows the sample to settle evenly.
 6. Cell count can now be performed

38.  A WBC count is performed with a Neubauer
hemocytometer.
 Using the X10 microscope magnification, count WBC
using the four outer large squares on the outer
sections of the counting chamber
 Count both sides of the chamber and average the
count.

40.  When counting, the cells that touch the extreme lower and the
extreme left lines are included in the count.
Those on top and right are not included.
Count both sides of the chamber and average the numbers

41.  Use the following formulas to calculate the WBC.
 Cells/mm3 = Average No. of cells + 10% X depth
factor (10) X dilution factor (100) divided by the
Area (number of squares counted)
 Depth factor is multiplied by 10 to convert area to
volume in μl
 Area of each large square = 1mm, so for the 4
large squares = 4mm

42.  Example: If have 36 cells on one side and 44 cells
on the other side
Average = 80/2 = 40 cells + 10% (=4)
= 44 cells
 Cells/mm3 = (44 x 10 x 100)/ 4 (since 4 squares
counted)
 Cells/mm3 = 44,000/ 4 = 11,000
 Total WBC = 11,000 mm3

43. Normal Value:
 Adult: 4,000 – 10,000
 Newborn: 10,000 – 30,000

44.  Platelet counts are performed with a Neubauer
hemocytometer
 Counting is done using x40 dry phase contrast
objective.
Platelets will have a faint halo.
 The middle square of the hemocytometer chamber
is counted.
 It contains 25 small squares.

46.  Count the 25 squares in the middle of the counting chamber
No. of platelets/mm3 =
Multiply No. of platelets (+ 10%) X 1000
OR
 Count 5 of the 25 squares
 Take the average of both sides add 10%
 Multiply No. of platelets x 5000 = No. of platelets/mm3

47.  Example: If have 36 cells on one side in 5 squares and
44 cells on the other side in 5 squares
Average = 80/2 = 40 cells + 10% = 44
 Cells/mm3 = 44 x 5000 (since 5 squares counted)
 Cells/mm3 = 220,000
 Total Platelets = 220,000 mm3

48.  Normal Value:
Platelets: 150,000 to 400,000 mm3

49. LIMITATIONS
 1. Specimen should be properly mixed and have sufficient
volume of blood so there is no dilution of anticoagulant.
 2. The capillary tube must be filled completely and be free of
any air bubbles.
 3. After the hemocytometer is charged, it should be placed in
a pre-moistened Petri dish to prevent evaporation while the
cells are settling out.
 4. The light adjustment is critical. It is important for both
WBCs and especially platelets.
If the condenser is not in the correct position, it will fade out
platelets.
 5. Debris and bacteria can be mistaken for platelets.

50. LIMITATIONS
 6. Clumped platelets cannot be counted properly; the specimen must
be recollected.
The anticoagulant of choice is EDTA for preventing platelet clumping.
 7. Avoiding overloading of hemocytometer chamber.
 8. A highly elevated leukocyte or platelet count may make accurate
counting difficult.
In either instance, a secondary dilution should be made.
When calculating the total count, adjust the formulas to allow for
secondary dilution.
 9. All WBC and platelet counts are done in duplicate.
WBC counts should agree +/- 15%.
Platelet counts must agree +/- 25%.
If they do not agree, repeat counts

51.  Infections – most common is bacterial infections
It also occur in viral (lymphocytosis)
 Allergy and drug hypersensitivity
 Parasitic infections
 Inflammation: eg. Inflammatory bowel disease, RA, and
vasculitis
 Extremely low birth weight
 Malignancy and myeloproliferative disorders: eg. Leukemias,
lymphomas
 Increased release of WBC from bone marrow:- This occurs in
infection, stress, and hypoxia
it also occurs due to endotoxin stimulation and steroid
administration

52.  Viral infections – eg. HIV
 Medications – abx, diuretics
 Chemotherapy/Radiation therapy
 Hyperthyroidism
 Malignancy
 Leukemia
 Lupus
 Aplastic anemia

53.  Acute blood loss/Hemolytic Anemia
 Malignancy
 Splenomegaly
 Inflammatory conditions – RA, IBS, Celia
disease, Connective tissue disorder
 Pancreatitis
 Kidney disease

54.  Idiopathic thrombocytopenic purpura (ITP)
 Thrombotic thrombocytopenic purpura (TTP)
 Hemolytic uremic syndrome – heparin, sulfa drugs,
quinidine
 Bacteremia
 Autoimmune diseases
 Pregnancy
 Trapping of platelets in the spleen
 Reduced production of platelets
 Increased breakdown of platelets

55. THE END