Laboratory Evaluation of Platelets and Coagulation Pathways Capillary Fragility Testing

Laboratory Evaluation of Platelets o Rumple – Leede method is employed

 Quantitative (Platelet Count) o Measures the stability of the capillaries to increased pressure

◦ Platelet Estimates from Peripheral Blood films o Done through inflating the pressure cuff halfway between systolic
and diastolic and maintained for 5 minutes
◦ Manual Platelet Count
The petechaie is counted.
 Qualitative (Platelet Function)
 10=normal
◦ Bleeding Time Test  10-20= marginal
 >20= abnormal

Qualitative (Platelet Function) o Scale for reporting number of petechiae:

Bleeding Time Test  0 to 10 = 1+

 10 to 20 = 2+
o In this test, a superficial incision is made usually in the skin of 20 to 50 = 3+
the forearm 50 or more = 4+

o The time for the bleeding to stop in vivo measure the Clotting Time
interaction of platelets with the severed capillaries in the skin
o The time in which the liquid blood solidify after removal from the body
o Measure platelet adhesion and platelet aggregation
Methods:
o The test must be performed at room temperature
 Slide or Drop
o Make sure the patient’s platelet count is above the safe level  Lee and white method

o Not performed to patient that may have tendency to form

keloids

 Duke’s method- earlobe as puncture site (1-3 minutes)

 Ivy’s method- skin of the volar surface of the forearm is incised (1-7
minutes)
Quantitative (Platelet Count)  Indirect method

Platelet Estimates from Peripheral Blood Smear (PBS) 1. Dry/Fonio method

◦ Based on the knowledge that there is 1 platelet for every 10 - 40  PBS estimate ussing Miller disc
erythrocytes in a normal peripheral blood smear
 Diluent: 14% magnesium sulfate (do not lyse RBCs) with
◦ Thus an OIF having 100 erythrocytes have 3 – 8 platelets Wright or Giemsa stain

Manual Platelet Count 2. Wet/ Dameshek method

◦ Most commonly used dilution is 1:100 or 1:200 dilution loaded into a  Diluent: Rees Ecker reagents (sodium citrate+ sucrose+ BCB)
Neubauer hemocytometer chamber
Examine under OIO until 1000 RBCs counted

Requirements for the Manual Platelet Counting

Two Methods of Manual Platelet Count
 Specimen – if possible, must be an EDTA anticoagulated blood
 Direct method- platelets are counted in a counting chamber.
 Reagents and Equipments – diluents, Neubauer Counting Chamber,
1. Brecher- Cronkite method Thoma pipette either 1:20 or 1:200 dilution pipettes

 Induce RBC hemolysis and count with a phase-contrast  Procedure – must be read within all the 25 squares inside the central
microscope using a counting chamber large square

 Standard international method of visual counting Calculations :

 Diluent: 1% ammonium oxalic acid solution (lyse RBCs) (Cells Counted)(Dilution Factor)
=________________________________________
2. Rees-Ecker Method
(no. of sq.)(area of each sq.)(depth)
 Diluent: sodium citrate (prevents platelet clumping) with BCB

Reporting : µL or mm3 in Conventional Unit or

Liter in SI

Normal value: 150,000- 400,000/ cumm

150-400x109 /L
Laboratory Evaluation of Coagulation:
Partial Thromboplastin Time (PTT)
 Plasma Recalcification Time
o More sensitive to abnormalities in the earlier stages of coagulation than
 Activated Clotting Time
previous tests of the intrinsic pathway
 Partial Thromboplastin Time
o Used to screen for factor deficiencies of intrinsic and common pathways
 Prothrombin Time
o Used to monitor heparin therapy

Plasma Recalcification Time o Detects the presence of circulating anticoagulants (inhibitors)

o A modification of the whole blood clotting time using:
o Test for intrinsic pathway (12, 11, 9, 10, 8, 5, 2 and 1 also HMWK and
◦ citrated plasma PK)
◦ CaCl2
o Inhibitors (anti-thrombin, heparin)
◦ glass or siliconized tubes
***A prolonged PTT means that clotting is taking longer to occur than
◦ and either PRP,PPP or both
normal and may be due to a variety of causes. Often, this suggests that
o The test is based on the fact that except for calcium,
there may be a coagulation factor deficiency or a specific or nonspecific
normal PRP contains all the components of the coagulation
antibody (inhibitor) affecting the body's clotting ability. Coagulation factor
mechanism necessary for generating a fibrin clot
deficiencies may be acquired or inherited.

Activated Clotting Time

Prothrombin Time
o Used to monitor heparin therapy before PTT was discovered
o Based in principle that WB contains all the components necessary to o Based on experiments by the French physiologist de Bainville that
produce a clot when removed from the veins and put into a glass blood could be made to clot quickly by adding tissue factor
tube. By adding an activator and keeping the blood at a constant
o Screens for deficiencies of factors I, II, V, VII, and X
37˚C
o Used to monitor anticoagulant therapy with vitamin K antagonists

o Tests for extrinsic pathway deficiencies (7, 10, 5, 2, and 1)

Normal Values

PT, APTT, TT reaction