Determination of the platelet count .pptx

How to determine platelet count

1. Determination of platelet count by DR RIFFAT SULTANA

2. Manual platelet counts Platelet counts can be done manually with a commercial diluting system, hemocytometer , and a microscope. These counts are less accurate than automated counts, because platelets can be difficult to distinguish from debris. Platelet clumping will also decrease the hemocytometer platelet count.

3. PLATELETS They are small and difficult to discern . Their adhesive character-attach readily to glassware, particles or debris in the diluting fluid . They clump easily . Not evenly distributed in the mixture of blood and diluting fluid . They readily disintegrate in the blood diluted with fluid making it difficult to distinguish them from debris . Therefore unless carefully done ,accurate counting of platelets becomes impossible

4. R equirement Microscope, hemocytometer , RBC pipette, cover slip, filter paper, disposable lancet alcohol, cotton wool,

5. Rees - Ecker  fluid - an aqueous  solution  of sodium citrate, sucrose, and brilliant cresyl blue used in platelet counts . Platelet fluid (Rees Ecker ) fluid comprising of 1.Nacl to make solution isotonic . 2. Saponin ( hemolysing agent)= 0.5 gm 3. Formaldehyde (antiseptic )= 1 ml 40% as preservative. 4. EDTA ( Anticoagulant)= 1.5 gm 5. Distilled water = 100 ml 3 . 6.Brilliant cresyl blue for staining. In stead of Rees Ecker ammonium oxalate solution can also be used

6. Composition of Rees Ecker solution 1.Nacl to make solution isotonic. 2.NaSO4 as an ant agglutinant. 3.Brilliant cresyl blue for staining. 4.Formaldehyde 40% as preservative. Distilled water. In stead of Rees Ecker ammonium oxalate solution can also be used.

7. Requirements:  RBC's pipette  Neubauer's Chamber  Cover slip  Petri dish  Filter paper  Pricking needle  Spirit swab  Microscope 

8. PROCEDURE Clean the tip of finger with alcohol and let it air dry. Perform a capillary puncture and wipe away the first drop of blood.Let another drop form. Take a clean and dry pipette place its tip in blood drop and draw the blood upto mark 0.5. Now place the tip in Rees Ecker Solution or Ammonium oxalate and draw the fluid upto mark 101.Shake the pipette well in order to mix the contents.Place the pipette safely on the table( for at least 10 min the pipette should be shaked ). Clean the hemocytometer with distilled water and dry it with soft piece of cloth.Place a clean cover slip over it.Focus it under low power objective.

9. Place the hemocytometer in a petri dish or box lined with moist filter paper to provide moist environment to platelets to prevent drying of platelets. This is done for about 20-30 minutes. Now focus the slide first under low power objective and then under high power objective count the number of platelets in 15 squares of 1/25mm2 that is 240 small squares. While counting apply the rule of L. Calculate the number of platelets /mm3 by using the formula

10. formula Out of the total 25 squares we have to look for 15 x16=240 16x25(total number of squares)=400 smallest squares. Total number of platelets in 240 squares=X. No of platelets in 1 small square= X/240. Volume of one small square=1/4000 mm3. So 1/4000mm3 will contain =X/240 cells. 1 mm3 will contain X/240 divided by 1/4000 which is equivalent to X/240 x4000. Dilution factor =2000(as the pipette is filled up to .5 mark) So the number of platelets /mm3 =X/240 x 4000 x200=X x3333.33

11. Platelets under high power objective lens

12. PRECAUTIONS Blood must be thoroughly mixed with the diluents by shaking the contents at least for 10 minutes. inadequate mixing results in clumping of platelets. The charged chamber should be kept for 15 minutes under petri dish. to prevent evaporation and for the cells to settle down. If other hematologic tests are to be done with platelet count ,and blood is used from the same puncture ,take blood for the platelet count first. The finger should not be squeezed excessively to collect blood .

13. Platelet count Platelet count is part of cellular part of blood. A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. Having more than 450,000 platelets is a condition called thrombocytosis ; having less than 150,000 is known as thrombocytopenia

14. 6.  Coagulation Factors • Factor I : Fibrinogen • Factor II : Prothrombin • Factor III : Thromboplastin ( Tissue Factor ) • Factor IV : Calcium • Factor V : Labile factor or proaccelerin • Factor VII : Stable factor or proconvertin • Factor VIII : Antihemophilic globulin (AHA) • Factor IX : Christmas factor (AHB) • Factor X : Stuart- Prower factor • Factor XI : Plasma thromboplastin antecedent (AHC) • Factor XII : Hageman factor • Factor XIII : Fibrin-stabilizing factor 7.  Bleeding Disorders • Increased fragility of vessels – Aging – Medications (steroids) • Platelet defect (deficiency) – Low platelet count – Medication (aspirin, clopidogrel ) – Congenital platelet defect • Deranged clotting – Factor deficiency – Medication ( coumarin ) – Coagulation inhibitor 8.  Coagulation Factor Disorders • Acquired – Vitamin K deficiency – Parenchymal disease of liver • Hereditary deficiencies – Factor VIII : hemophilia A – Factor IX : hemophilia B – Factor XI : hemophilia C –Von Willebrand’s Disease 9.  Laboratory tests 1. Tests of the Vascular Platelet Phase of Hemostasis : • BBlleeeeddiinngg TTiimmee ((BBTT) 2. Tests of the Coagulation Cascade: • CClloottttiinngg TTiimmee (( CCTT) oorr CCooaagguullaattiioonn ttiimmee • AAccttiivvaatteedd PPaarrttiiaall TThhrroommbbooppllaassttiinn TTiimmee ((AAPPTTTT).. • PPrrootthhrroommbbiinn TTiimmee ((PPTT) • TThhrroommbbiinn TTiimmee ((TTTT) 10.  Laboratory tests 3. OTHERS: - Platelet Count - Capillary fragility test - Clot Solubility Test - Clot Retraction Test - Thromboplastin Generation Test - Factors, fibrinogen, anticoagulants assay - Platelet function tests (aggregation) - Tests for Liver function

15. 11.  1. Bleeding Time • It is the time taken from the puncture of the blood vessel to the stoppage of bleeding. • The bleeding time test is a useful tool to test for platelet plug formation and capillary integrity. • BT is more imp. than CT. • CT concerns the blood only i.e. how firm the the clot is formed, whereas BT involves the interaction of blood with the injured tissues. 12.  Duke Method • With the Duke method, the patient is pricked with a special needle or lancet, preferably on the earlobe or fingertip, after having been swabbed with alcohol. • The prick is about 3-4 mm deep. The patient then wipes the blood every 30 seconds with a filter paper. • The test ends when bleeding stops. The usual time is about 2-6 minutes. 13.  Ivy method ٠ Clean the anterior surface of the forearm with spirit. • The blood pressure cuff is placed on the upper arm and inflated to 40 mmHg. • A lancet or scalpel blade is used to make a shallow incision that is 1 millimeter deep on the anterior of the forearm. • The time from when the incision is made until all bleeding has stopped is measured and is called the bleeding time. Every 30 seconds, filter paper or a paper towel is used to draw off the blood. • Normal BT by this method is 3-6 minutes. 14.  Bleeding Time 15.  Bleeding Time • A prolonged bleeding time may be a result from decreased number of thrombocytes or impaired blood vessels. • Bleeding time is affected by platelet function, certain vascular disorders and von Willebrand Disease, not by other coagulation factors. • Diseases that cause prolonged bleeding time include thrombocytopenia, disseminated intravascular coagulation (DIC). • Aspirin and other cyclooxygenase inhibitors can prolong bleeding time significantly.

16. 16.  Bleeding Time • People with von Willebrand disease usually experience increased bleeding time. • Von Willebrand factor is a platelet agglutination protein, but this is not considered an effective diagnostic test for this condition. • It is also prolonged in hypofibrinogenemia . • Many experts regard the bleeding time as useless, in that it does not predict surgical bleeding. • Role in post Myocardial Infraction patients. 17.  2. Clotting Time • It is the time taken from the puncture of the blood vessel to the formation of a fibrin thread. • A. Capillary Glass Tube Method : Here the blood is collected in capillary tube & total time is noted to form FIBRIN THREADS on breaking tube every 30 seconds. N : 3-8 minutes • B. Lee & White method : Here venous blood is collected in 8 mm diameter glass tube, rocked in a water bath at 37°C & time is noted from the time of vene puncture till the blood stops flowing. N : 6-12 minutes. 18.  Clotting Time • Mechanism Involved is INTRINSIC Pathway. • CT depends on presence of all clotting factors. • It gets prolonged in : - 1. Deficiency of clotting factors – Hemophilia. - 2. Vitamin K Deficiency – Factor II, VII, IX & X. - 3. Anticoagulant overdose. • BT & CT is measured before surgery & liver or bone marrow biopsy. • PURPURA : BT increased, CT normal. •

17. A high Platelet Count ( thrombocytosis ) may indicate: Iron deficiency (anemia) Living conditions are too cold Liver disease or cirrhosis Leukemia Pathogenic infections, such as tuberculosis A low Platelet Count ( thrombopenia ) may indicate: Adverse antibody-antigen reactions, such as observed after a blood transfusion Lymphomas Disseminated intravascular coagulation (DIC) Bone marrow disease Hemolytic diseases, in the case of a newborn Burns, if present, are severe