Platelet count

TOTAL PLATELET COUNT USING HEMOCYTOMETER / NEUBAUER’s CHAMBER (MICRO DILUTION & MACRO DILUTION METHOD)

A BRIEF INTRODUCTION TO PLATELETS / THROMBOCYTES

The Platelets, also known as Thrombocytes, are the Oval, Round or rod-like cells present in the blood that helps mainly in the clotting of blood. On an average, the size of the Platelets is 2 – 4 µm (microns). The mature Thrombocytes are non-nucleated cells with a minute granules present in the cytoplasm and there is no pigment present in Platelets. The Average lifespan of Platelets is 3-10 days. Normally about 150,000 – 450,000 cells are present per cubic millimeter (mm3) of blood.

Platelet counts can be done manually or using automated cell counters. Since many laboratories use instruments that count platelets, red cells and leukocytes concurrently, a platelet count is a routinely reported result on many samples of dog and horse blood. However, platelet clumping will lower (and in some instances invalidate) the platelet count when determined by any method. This occurs especially in samples of cat and cow blood (see image below). In these instances, an accurate platelet count cannot be provided, but for most purposes, significant changes in platelet number can be detected during the microscopic examination of the stained blood smear (see section on platelet estimation). In such samples, close attention should be paid to the platelet smear estimate part of our hemogram. Platelet clumping is usually due to a sample collection problem and can be minimized by collecting blood from a large peripheral vein (cephalic or jugular), such that blood flows smoothly into the vacutainer or syringe, and using a 22 or 23 g needle (in a dog or cat). The blood should be mixed with the anticoagulant as soon as possible after collection, by gentle rotation or inversion. Platelet clumping increases with time, so platelet counts should be done as soon as possible after collection to maintain accuracy.

THE AIM / PURPOSE OF PERFORMING TOTAL PLATELETS COUNT

The purpose of performing Total Platelets count is to know whether or not you are suffering from Thrombocytosis a.k.a. Thrombocythemia (i.e. the increase in the no. of Platelets or Thrombocytes to more than 700,000/mm3) or Thrombocytopenia a.k.a. Thrombopenia (i.e. the Decrease in the no. of Platelets / Thrombocytes to less than 50,000/mm3).

Very large numbers of Platelets/ Thrombocytes are present in the Blood Specimen. Practically, counting this amount of Platelets directly under the microscope is highly impossible. So, the Platelets / Thrombocytes are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber.

Principle:

The blood specimen is diluted (usually in 1:20 ratio) with the help of Platelet diluting fluid (commonly the Rees – Ecker Fluid) which preserve and fix the Platelets / Thrombocytes and stains it. The Rees – Ecker fluid is isotonic to the Platelets / Thrombocytes and does not cause any damage to it whereas causes the lysis of red blood cells. After diluting the specimen in appropriate dilutions, the content is charged on Hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for Platelets count.

The composition of Platelet diluting Fluid (Rees – Ecker Fluid)

COMPONENTS	QUANTITY
Trisodium Citrate	3.8 gm
Brilliant Cresyl Blue	0.1 gm
40% Formalin	0.2 ml
Distilled Water	100 ml

The Final pH of the solution (at 25°C) varies from 6.8 – 7.0 which depends on the composition and companies who manufacture it.

Hemocytometer / Neubauer’s Chamber –

This is a special type of glass chamber that is used for the cell counting, especially for Blood cells. Nowadays, most commonly Improved Neubauer’s Chamber is used and in some laboratories, other types of chambers are also employed like Burkers chamber, Levy’s chamber and Fusch–Rosenthal chamber etc.

The Neubauer’s Chamber has ruled the area of total 9 square mm and the depth is 0.1 mm as when the coverslip is placed on the surface of the counting chamber, the space between the bottom of the cover glass and the base of grooved area measures 0.1 mm in depth.

The central 1 square is highly ruled which is divided into 25 squares. Each square of the Central Square is further subdivided into 16 small squares.

For Platelets Count, the cells are counted in the 5 squares of the Central square as 4 Corner squares of the Central square (divided into 25 squares) and 1 central square of the Larger Central Square (divided into 25 squares) which is same as for Red Blood Cells.

rulings in neubauer chamber for platelets - neubauer chamber - rulings in hemocytometer for platelets- hemocytometer — P – PLATELET COUNT ; W – WBC COUNT

Each square of the Central Square (divided into 25 squares) contains 16 small squares so the total no. of the area to be counted for Total Platelet Count –

16 × 5 = 80 small squares

Two Method has been developed for the Manual Estimation of Total Platelets / Thrombocytes Count Using Hemocytometer / Neubauer’s chamber –

Microdilution Method

Macrodilution Method

Here, I’ll explain both the methods but the Microdilution method is not preferred nowadays due to the use of Mouth pipettes. So let’s start with Microdilution method and then we’ll move to Macrodilution method….

MICRODILUTION METHOD FOR THE ESTIMATION OF PLATELETS USING HEMOCYTOMETER

Materials Required for the Total Platelet Count by Microdilution Method

Blood sample (Capillary blood or EDTA anticoagulated specimen)
Platelet diluting fluid (preferably Rees – Ecker fluid)
Gauze piece or Cotton
WBC pipette
Hemocytometer a.k.a. Neubauer’s Chamber
Coverslip
Microscope

A Brief Introduction to WBC Pipette

WBC pipette is a graduated pipette that gives the dilution of 1:20. It has two markings at the bottom as 0.5 and 1 and the top of the pipette is marked 11. It has a round shape bulb which contains the White bead to mix the blood specimen and the diluting fluid. On the top, a rubber tube is attached to the pipette for sucking the blood specimen and diluting fluid.

When blood is sucked up to 0.5 mark and the diluting fluid up to 11 marks, gives the 1:20 dilution of Blood: Diluting fluid and When the Blood is sucked up to 1 mark and the diluting fluid up to 11, gives the 1:10 dilution of Blood: Diluting fluid which less commonly used. After sucking the Specimen & Diluting fluid, the content is gently mixed by rotating the pipette on its long axis to ensure thorough mixing of blood and diluting fluid.

Note: Nowadays Mouth pipetting is banned in most of the laboratories due to the high risk of getting infected with highly contaminated specimens of the patients. So instead of Microdilution method, the Macrodilution methods are employed in Laboratories…..

Specimen

Whole blood, anticoagulated with EDTA, or free-flowing capillary blood may be used.

Procedure of the Total Platelet Count by Microdilution Method

⇒ Fill the WBC pipette up to the 0.5 mark with the blood specimen and wipe out the pipette externally to avoid false high results.

⇒ Fill the same pipette with the Platelet diluting fluid (i.e. the Rees – Ecker Fluid) up to the mark 11.

⇒ Be cautious that there should be no air bubble in the pipette bulb.

⇒ Mix the Blood and Diluting fluid in the pipette by rotating the pipette (horizontally) between your palms.

⇒ Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer.

Note: Here a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm.

⇒ Now, put the WBC pipette, mix the solution present in it again and then discard 1-2 drops from the pipette before charging the chamber.

⇒ Gently press the rubber tube of the WBC pipette, so that the next drop of fluid is in hanging position.

⇒ Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

⇒ Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.

⇒ After charging, Put the charged chamber in a moist condition for 10-15 min so that the cells settle down in the chamber without getting dried & then focus the chamber under the microscope to calculate Platelets.

MACRODILUTION METHOD FOR THE ESTIMATION OF PLATELETS USING HEMOCYTOMETER

Materials Required for the Total Platelet Count by Macrodilution Method

Blood sample (Capillary blood or EDTA anticoagulated specimen)
Platelet diluting fluid (preferably Rees – Ecker fluid)
Hb pipette or Micropipette (0.02 ml or 20 µl)
Hemocytometer / Neubauer’s Chamber
Gauze piece or Cotton swab
Graduated Pipette (5 ml)
Test tubes
Cover Slip

Procedure of the Total Platelet Count by Macrodilution Method

⇒ Take 3.98 ml of Platelet diluting fluid i.e. Rees – Ecker Fluid in a Clean, Dry and Grease free Test tube.

Note: If you don’t have variable pipette in the lab which can measure 3.98ml or 3980 µl of Diluting fluid then take 4 ml of Diluting fluid with the help of 5ml Graduated pipette in the test tube and discard 20 µl of fluid using a micropipette or WBC pipette.

⇒ Now add 0.02 ml or 20 µl of Blood Specimen (1:200 Dilution) to the tube containing diluting fluid with the help of micropipette or WBC pipette.

⇒ Mix well for few minutes and ready your Hemocytometer / Neubauer’s Chamber.

⇒ Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer.

⇒ Now, take out the WBC pipette and fill it with the Diluted Specimen, mix the solution well and then discard 1-2 drops from the pipette before charging the chamber.

⇒ Gently press the rubber tube of the WBC pipette, so that the next drop of fluid is in hanging position.

⇒ Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately.

⇒ Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber.

Using Micropipette instead of WBC pipette for charging the Hemocytometer

⇒ You can also use a micropipette instead of WBC pipette for charging the Hemocytometer. So, with a micropipette, carefully draw up around 20 µl of the diluted specimen. Press the knob of the pipette to make a hanging drop at the tip of the micropipette.

⇒ Now gently place the pipette tip against the edge of the cover glass and if required slowly expel the more liquid until the counting chamber is full. This process occurs by Capillary action, but care should be taken not to overfill the chamber. A volume of 10ml is sufficient to fill out the one counting chamber.

COUNTING THE PLATELETS / THROMBOCYTES UNDER THE MICROSCOPE

⇒ Focus the ruling using the 10x Objective lens and then Count the Platelets in 5 small squares of the central square as described above, using the 40x Objective lens.

⇒ Count the cells which are lying on the right and lower lines of the 5 small squares but not the opposite line. In case of marginal cells, count the cells on ‘L’ line that is either on Right and Lower lines or Left and Upper lines.

CALCULATIONS FOR THE TOTAL PLATELET COUNT USING HEMOCYTOMETER

⇒ After counting the cells under the microscope, we know the No. of Platelets in 5 squares of the central square. Let’s consider it as ‘N’ no. of cells.

⇒ Now, the volume of the fluid inside the chamber is the product of Area and depth of the Hemocytometer / Neubauer’s chamber.

⇒ The central area is the 1 sq. mm which is divided into 25 parts so the area is

25 squares = 1 sq. mm

⇒ Out of these 25 squares, the Platelets are counted in 5 squares. So the Area of 5 small squares is

5/25 i.e. 1/5

⇒ The depth of the Hemocytometer is 0.1 mm as described above in a short description of Hemocytometer.

Now Apply the Following formula to get the Total Platelets Count –

Total Platelets Count = N × Dilution / Area × Depth

N × 20 / (1/5 × 0.1)

Total Platelet count = N × 1000 / mm3

Using the Above formula we can calculate the Total No. of Platelets / Thrombocytes present in the Blood Specimen.

NORMAL VALUES OF PLATELETS / THROMBOCYTES

Normally about 150,000 to 450,000 platelets are present in Healthy individuals. These Values show variations in various physiological and Pathological Conditions.

ACTIVATED & INACTIVATED PLATELETS

PRECAUTIONS TO BE TAKEN WHILE PERFORMING TOTAL PLATELET COUNT BY HEMOCYTOMETER

⇒ Use of Mouth pipettes (WBC pipette) is banned in many countries. However, in case you have to use it, be cautious that you should not intake the diluting fluid or Specimen.

⇒ Accurately measure the amount of specimen and Diluting Fluid to avoid any error in the results.

⇒ In case you are performing this test by Microdilution method, mix the specimen and diluting fluid appropriately by gently rotating in between your palms.

⇒ Carefully charge the Hemocytometer or Neubauer’s chamber that it should not be overcharged and do not contain any air bubble in it.

Comments

Platelet counts should be performed within three hours after the dilution has been prepared.
The coefficient of variation (CV) for the 95% confidence limits is + 22%.
If blood is collected by a skin puncture, carefully remove the first drop of blood and collect free-flowing blood for the platelet count. This will minimize the occurrence of platelet clumping and adhesion of platelets to the puncture site.
If clumps of platelets are seen in the hemacytometer, the procedure should be repeated. Clumps may be due to inadequate mixing of blood or to poor technique in obtaining the blood specimen.
A Wright's-stained peripheral blood smear should be examined and the platelet estimate determined to confirm the hemacytometer platelet count. The platelet estimate should correlate with the platelet count + 25%. If a discrepancy exists, the platelet count and peripheral blood smear estimate should be repeated.
In acute leukemia, there is an increase of blast cells in the peripheral blood. Often fragments of cytoplasm about the size of platelets break off the blast cells. These cytoplasmic fragments are called hyaline bodies. They are the same size and density as platelets and may be counted as platelets by the hemacytometer or automated methods. It is very important that all platelet counts be confirmed by slide examination so that a false increase of platelets (due to the counting of fragments) is not reported.
Platelet satellitism will result in falsely decreased platelet counts. With platelet satellitism (Figure 8-1), platelets adhere to neutrophils when the blood sample is anticoagulated with EDTA and is not free to be counted. Platelet satellitism can be corrected by redrawing the blood sample using sodium citrate as the anticoagulant. The resulting platelet count must be multiplied by 1.1 to account for the dilutional effect of the citrate anticoagulant.
A second method for manual platelet counts is the Rees Ecker method. Using the erythrocyte diluting pipet, whole blood is diluted with a solution containing brilliant cresyl blue, which stains the platelets a light bluish color. The platelets are then counted using a standard hemacytometer and bright field microscopy.
Technical sources of error in hemacytometer cell counts are given in Web Table 7-4.