Platelets Count

Sample for Platelets Count

1. This can be done on EDTA blood.
   1. This is stable for 5 hours at 23 °C and 24 hours at 4 °C.
2. Take capillary blood and dilute directly; this sample is stable for 3 hours.
3. Fetal blood is collected from the umbilical area percutaneous.
4. The platelets can be assessed on the DLC slide.

Indication for Platelets Count

1. To diagnose the cause of petechial hemorrhage in the skin.
2. To find the cause of spontaneous bleeding.
3. In women with heavy menses.
4. This is advised in a patient on chemotherapy.
5. This is advised in case of bone marrow failure.
6. Platelets count is of value in thrombocytopenia seen in:
   1. Uremia.
   2. Liver diseases.
   3. Malignancies.

Formation of Platelets 

1. Platelets are the smallest form of formed elements of blood.
2. Platelets are formed by the megakaryocytes in the bone marrow. Basically, these are the detached portion of the megakaryocytes.
3. Platelets are the smallest of the formed elements of blood.
   1. Platelets measure 3.0 x 0.5 µm in diameter.
4. These are non-nucleated, small, round, or oval, flattened disk-shaped structures.

Platelets function:

5. The glycoprotein of the platelet surface is important in the platelets reactions of:
   1. Adhesion.
   2. Aggregation.
   3. Leading to plug formation during hemostasis.
6. Glycoproteins are:
   1. Glycoprotein 1a.
   2. Glycoprotein 111a.
   3. Glycoprotein 1b.
   4. Glycoprotein 11b.
7. The binding site for 11b/111a is a receptor for the fibrinogen, which is important in platelet aggregation.
8. The membrane phospholipids known as factor 3 are important in converting coagulation factor X to Xa and prothrombin (factor 11) to thrombin (factor 11a).

Platelets distribution in the body:

1. The lifespan of platelets is roughly 7.5 days.
2. Of platelets, two-thirds are in the blood, and one-third are in the spleen.
3. Platelets activity needed for:
   1. Blood clotting.
   2. Vascular integrity.
   3. Vasoconstriction.
4. In hemostasis, the steps are:
   1. Platelets aggregation that occludes the breaks in the vessel wall.
   2. Initiates the clotting mechanism.

The platelet’s contents are:

1. The glycocalyx is the outer membrane.
2. α-Granules.
3. Lysosomes contain neutral proteases, bactericidal enzymes, and acid hydrolase.
4. Dense granules contain ADP, ATP, and Calcium.
5. β-thromboglobulin functions are:
   1. Inhibits heparin.
   2. It is chemotactic.
   3. Promotes smooth muscle growth for the repair of vessels.
6. Platelet factor 4, inhibits heparin.
7. Platelet-derived growth factor (PDGF)  functions are:
   1. It promotes smooth muscle growth.
   2. It takes part in atherosclerosis and lipid metabolism.
8. Thrombospondin functions are:
   1. It mediates cell-to-cell interaction.
   2. It promotes platelets-to-platelet interaction.
9. Fibrinogen leads to fibrin formation.
10. Von Willebrand factor (VWF) promotes platelet adhesion.
11. Factor V is a cofactor in fibrin formation.
12. Factor VIII is also a cofactor for fibrin formation.
13. Fibronectin  functions are:
    1. It is a cellular adhesion molecule.
    2. It promotes platelet spreading.
14. Plasminogen is the precursor to plasmin and has a function in fibrinolysis.
15. High molecular weight Kinogen has a role in activating the intrinsic pathway via contact.
16. α2-Antiplasmin inhibits plasmin.

Platelets activation:

1. This may be transient, reversible, or irreversible.
2. After activation, the changes are:
   1. Initially, there is a pseudopod formation.
   2. There is a contraction.
   3. Adhesion.
   4. Change in shape.
   5. There is a stickiness.
   6. There is aggregate formation.
   7. There is a platelet plug formation.
   8. There is the release of chemical secretions.
   9. The last step stabilizes the platelet plug by forming a fibrin mesh over the platelet aggregates.

NORMAL Platelets Count

Source 1 

• To convert into SI unit x, 10⁶ = x 10⁹/L

Source 2 

• Thrombocytopenia when The count is less than 100,000 /cmm
  Thrombocytosis when the count is more than 400,000 /cmm.
  Thrombocythemia when the count is above one million /cmm.

Method to count platelets

1. By automated hematology analyzers.
2. Direct smear also gives information about platelets’ size, shape, and clumping.
3. Direct count from the peripheral blood smears.
   1. Count platelets on oil objective in 10 fields and multiply by 2000, giving a rough idea about the count.
      1. Platelets in 10 field X 2000 = Total platelets.

The manual method of platelets count:

1. Take 20 µL of blood.
2. Add 1.8 mL of 1% ammonium oxalate.
   1. Ammonium oxalate will lyse the RBCs and WBCs while Platelets will remain intact.
3. Leave for 15 minutes for complete lysis of RBCs.
4. Mount the Neubauer chamber.
5. Leave the chamber for 15 minutes in the high humidity.
6. Count the large central square labeled as P.

7. Calculations of the platelets:
   1. Area of 5 squares in the central area = 0.2 sq. mm
   2. The depth of the chamber is 0.1 mm.
   3. The volume of 5 squares in the central area = 0.2 x 0.1 µL   =  0.02 µL
   4. Dilution of the blood sample = 1:20
   5. If suppose P number of platelets is counted in 0.02 µL
   6. The number of platelets /µL = P x 20 x 1/0.2
   7.                                                     = P x 20 x 50
   8.                                                    = P x 1000
   9. Note: You can use the constant factor of 1000.

Another method of platelets counting:

1. Under 400 x magnification while adjusting the light.
2. Count all 25 squares in the large central area.
   1. Duplicate the counting procedure.

Platelets Count: Platelets count on the Neubauer chamber.

3. Multiply the counted platelets by 1000 = Total platelets count/cmm.
   1. Multiply this above number of platelets by 10⁶ to get the count in SI units.
4. Example:
   1. Suppose the platelet count in 25 squares (Central square) = 300 x 1000 = 300,000/cmm
   2. To convert  into SI units = 300,000/cmm x 10⁶
   3.                                             = 300 x 10⁹/L

Platelets counting formula

Source of errors in platelets counting:

1. To minimize the error of counting platelets manually:
   1. Mix thoroughly diluting fluid before filling the chamber.
   2. Avoid platelet clumping.
   3. Improper dilution.
   4. Not properly filling the chamber.
   5. Avoid microclot formation.
   6. Keep in mind the calculation errors.
   7. Contaminated counting fluid.
   8. In case of clumping of the platelets.
   9. Run the test in duplicate and then get the average of the two results.
   10. If taking blood from the finger, then don’t squeeze the finger.
   11. The count is lower on the skin puncture sample than on the venous blood sample.

Mechanisms of thrombocytopenia:

1. This may be due to decreased production of bone marrow. This may be due to the following:
   1. Bone marrow failure.
   2. Infiltration of the bone marrow by tumors or fibrosis.
2. Destruction or sequestration of the platelets by hypersplenism.
3. Antibodies destroy the platelets.
4. Destruction of the platelets by infection or drugs.
5. Increased utilization of disseminated intravascular coagulopathy.
6. In severe hemorrhage, which leads to loss of platelets.
7. A large blood transfusion leads to a dilutional effect.

Causes of thrombocytopenia or Decreased platelets:

1. Idiopathic thrombocytopenia ITP.
2. Hypersplenism.
3. Anemias like pernicious, aplastic, and hemolytic.
4. After a massive blood transfusion
5. An infection like viral and bacterial,
6. Chemotherapy treatment.
7. HIV infection.
8. Leukemias, carcinoma, and myelofibrosis. This is due to the infiltration of the bone marrow.
9. D I C.
10. Toxemia of pregnancy, eclampsia.
11. Antiplatelets antibody.
12. Renal failure.
13. Inherited diseases like Wiskott-Aldrich syndrome.
14. An autoimmune disease like systemic lupus erythematosus

Thrombocytosis or increased platelets:

1. Malignant tumors like leukemia and lymphoma,
2. Polycythemia vera.
3. Splenectomy.
4. Iron deficiency anemia.
5. Autoimmune diseases like Rheumatoid arthritis and SLE.
6. Hodgkin’s lymphoma
7. Chronic pancreatitis and inflammatory bowel disease.
8. Tuberculosis.

Critical value:

• The patient may develop spontaneous bleeding when the count is < 20,000 /cmm.
• Platelet counts > 50,000 /cmm usually show no bleeding.

Questions and answers: