Platelets – Part 2 – Platelets Count (Thrombocyte count)

Sample

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. Can take capillary blood and make a direct dilution, 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

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.

Pathophysiology

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 measures 3.0 x 0.5 µm in diamter.
4. These are non-nucleated, small, round or oval, flattened disk-shaped structures.

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

5. The lifespan of platelets is roughly 7.5 days.
6. Platelets two-third are in the blood and one-third in the spleen.
7. Platelets activity needed for:
   1. blood clotting.
   2. vascular integrity.
   3. vasoconstriction.
8. In hemostasis the steps are:
   1. Platelets aggregation that occlude the breaks in the vessel wall.
   2. Initiates the clotting mechanism.

9. The platelets 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 the heparin.
      2. It is chemotactic.
      3. Promotes smooth muscle growth for the repair of vessels.
   6. Platelet factor 4, inhibit the 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 platelets spreading.
   14. Plasminogen is the precursor to plasmin and has a function in the fibrinolysis.
   15. High molecular-weight Kinogen has a role in the activation of the intrinsic pathway via contact.
   16. α2-Antiplasmin inhibits plasmin.
10. 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 the shape.
       5. There is a stickiness.
       6. There is aggregates formation.
       7. There is a platelet plug formation.
       8. There is the release of chemical secretions.
       9. The last step is stabilizing the platelet plug by the formation of fibrin mesh over the platelet aggregates.

NORMAL

Source 1 

Age	x 103/µL
Fetal blood
18 to 20 weeks	242.1 ± 34.5
21 to 22 weeks	258.2 ± 53.6
23 to 25 weeks	259.4 ± 42.4
26 to 30 weeks	253.5 ± 36.6
Adult	150 to 400

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

Source 2 

Age	cmm (mm3)	x 109/L   (SI unit)
Adult/elderly	150,000 to 400,000	150 to 400
Premature infants	100,000 to 300,000	100 to 300
Newborn	150,000 to 300,000	150 to 300
Infants	200,000 to 475,000	200 to 475
Children	150,000 to 400,000	150 to 400

• 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 the size, shape, and clumping of platelets.
3. Direct count from the peripheral blood smears.
   1. Count platelets on oil objective in 10 fields and multiply with 2000, will give a rough idea about the count.
      1. Platelets in 10 field X 2000 = total platelets.
4. The manual method of platelet 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 and 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 central large square.

1. 7. The average number of platelets counted in 1 mm²  = X
   8. The dilution of the blood is 1:100.
   9. The depth of the chamber is 0.1 mm.

1. 10. Note: Can use the constant factor of 1000.
   11. To minimize the error to count platelets manually:
       1. Avoid the platelets clumping.
       2. Avoid the microclot formation.
       3. Run the test in duplicate and then get the average of the two results.
       4. If taking blood from the finger then don’t squeeze the finger.

What are the mechanism leading to thrombocytopenia:

1. This may be due to decreased production of the bone marrow. This may be due :
   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 destroying 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.
• Platelets counts > 50,000 /cmm usually show no bleeding.

 

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