HEMA312LEC@ANT_BSMLS 3-B-2
MT Activity 1
1. Prepare a powerpoint presentation of the tests /assays for Primary hemostasis, Secondary hemostasis
and Fibrinolysis.
2. Content of each tests:
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
5. Reference value
6. Interpretation
Prolonged /Shortened result are seen in what conditions
3.Reference
HEMATOLOGY BOOKS BY WILLIAM, HOFF’S, TURGEON
Primary hemostasis
Platelet count - The platelet count is an important component of the complete blood count. In clinical
practice, abnormal platelet counts can occur unexpectedly. It is critical to understand the physiological
and technical factors that can influence platelet count, such as blood specimen collection method,
platelet sample preparation, instrument type, and calibration method. Failure to recognize these factors
has occasionally resulted in a misdiagnosis.
Specimen of Choice: The anticoagulant EDTA is used to collect venous blood. Blood from skin puncture
wounds varies, but it is acceptable if the blood is flowing freely and only the first few drops are used.
A. Direct Method - Platelets, like erythrocytes and leukocytes, are counted in a hematocytometer.
This will determine the amount of platelets in your blood. Thrombocytopenia is defined as a
platelet count that is lower than normal. This condition can cause you to bleed excessively after
HEMA312LEC@ANT_BSMLS 3-B-2
a cut or other type of bleeding injury. Thrombocytosis is defined as a platelet count that is
higher than normal.
This method employs a dilution of blood using an RBC pipet with the use of a hemacytometer.
1. Guy and Leake - Diluent contains sodium oxalate, 40% formalin (to preserve platelets), and
crystal violet (to stain).
Diluent Isotonic
Stain Crystal Violet
appearance of platelet Lilac refractile object
Diluting fluid:
a. Crystal Violet - 0.05 g (dye)
b. Sodium Citrate - 1.6 g (prevents clumping of platelets)
c. Distilled Water - 100 ml
d. 40% Formalin - 94.0 ml (preservative)
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
The determination is done by placing a small volume of isotonic diluent to the whole blood that was
treated with a red cell lysing reagent, such as sodium oxalate in Guy and Leake method, in a counting
chamber (hemocytometer) and counting platelets using phase-contrast light microscopy. The count is
then adjusted by the dilution factor.
4. Procedure
Procedure:
1. Platelets are counted in all the 25 intermediate squares of the central square. Platelets appear as
rounded, oval or coma-shaped highly refractile bodies measuring from 2-3u. The amount of light in the
microscope should be adjusted so that the platelets appear as highly refractile bodies.
HEMA312LEC@ANT_BSMLS 3-B-2
2. Computation:
5. Reference value
6. Interpretation
2. Rees and Ecker -
Isotonic
Reese Ecker Prevents platelet aggregation Sodium Oxalate
stain or dye BCB
appearance of platelet Bluish
Diluting fluid: Rees-Ecker Diluent
-does not lyse RBC and WBC
a. Brilliant Cresyl Blue - 0.15 g (dye)
b. Sodium Citrate - 0.4 g (prevents clumping of platelets)
c. Distilled Water - 100 ml
d. Formalin - 3 drops in 1:10 dilution (preservative)
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
1. Rinse RBC pipet first with RE diluting fluid by sucking in and out the diluting fluid (to prevent
disintegration of platelets).
HEMA312LEC@ANT_BSMLS 3-B-2
2. Count platelets in the 25 squares in the large central square on each side of the hemocytometer.
3. Computation:
(insert pic)
5. Reference value
Normal Value: 150,000-450,000/ mm3
6. Interpretation
3. Brecker Cronkite –
Hypotonic
1% Ammonium oxalate
Phase contrast microscopy
HEMA312LEC@ANT_BSMLS 3-B-2
1. Discussion
( to include also the importance of the test and when is the test being requested)
The reference method that uses phase-contrast microscopy with green (platelets appear dark) or gray
filter (platelets appear pink or purple). It also uses a flat-bottomed hemacytometer whose focus can be
easily adjusted and a thin coverslip (because thick coverslip retards refraction of light.).
Diluting fluid: 1% Ammonium oxalate
-lyses RBC
2. Specimen
3. Principle
4. Procedure
Procedure:
HEMA312LEC@ANT_BSMLS 3-B-2
1. Well-mixed blood is diluted 1:100 in diluting fluid, and the vial containing the suspension is rotated on
a mechanical mixer for 10 to 15 minutes.
2. The hemacytometer is filled in the usual fashion, using a separate capillary tube for each side.
3. The chamber is covered with a Petri dish for 15 minutes to allow the platelets to settle in one optical
plane. A piece of wet cotton or filter paper is left beneath the dish to prevent evaporation.
4. Using this type of microscope platelets are seen very clearly against WBCs.
5. Platelets are seen as round to oval purple bodies sometimes with dendritic processes. Their internal
granular structure and a purple sheen allow the platelets to be distinguished from debris, which is often
refractile. Ghosts of the red cells that have been lysed by the ammonium oxalate are seen in the
background.
6. Allowable difference between each chamber is +/-10 platelets. If greater than 10, repeat mixing and
charging o countercheck the test.
7. Platelets are counted in 10 small squares, five on each side of the chamber. If the total number of
platelets counted is less than 100, more small squares are counted until at least 100 platelets have been
recorded-10 squares per side or all 25 squares in the large central square on each side of the
hemocytometer, if necessary. If the total number of platelets in all 50 of these small squares is less than
50, the count should be repeated with 1:20 or 1:10 dilutions of blood.
8. Computation:
5. Reference value
Normal Value: 150,000-450,000/ mm3
6. Interpretation
4. Unopette System -
Diluent 1% ammonium oxalate
Dilution 1:100
1. Discussion
HEMA312LEC@ANT_BSMLS 3-B-2
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
Principle: Blood is diluted in buffered ammonium oxalate which hemolyzes mature red cells and
preserves, platelets, leukocytes and reticulocytes. Platelets and Leukocytes may to be counted in a
standard hemacytometer
Diluting Fluid: 1% Buffered Ammonium Oxalate
Dilution: 1:100
4. Procedure
Procedure:
1. Add blood from capillary to diluent.
2. Rinse capillary.
3. Mix by inversion. Diluted sample is stable for 3 hours.
4. Let stand for 10 minutes to allow red cells to hemolyze.
5. Mix thoroughly by inversion.
6. Convert to dropper and charge hemacytometer. A hemacytometer with a Neubauer ruling is
recommended.
7. Cover hemacytometer with Petri dish to prevent dehydration while waiting 10 minutes for platelets to
settle.
8. Count platelets in all 25 small squares within a large center square within large center square of the
Neubauer ruling.
9. Multiply the total number of platelets counted by 1,000.
Note: If leukocyte is also to be counted, one must perform the leukocyte count before platelet
determination.
V. Tocantin’s
VI.Nygard’s
VII. Walker
HEMA312LEC@ANT_BSMLS 3-B-2
VIII. Van Allen’s Method – reported in percent
5. Reference value
6. Interpretation
B. Indirect Method
platelets are counted in relationship to rbc on a fixed stained smear
NOT RELIABLE because results depends on the distributions of the platelet in the rbc count
Platelets are counted simultaneously in a blood smear. Platelets are counted in relation to 1,000 RBCs in
the blood smear. Platelet Count (PC) /µL or PC/L is calculated based on RBC count obtained using
hemacytometer. This method is less reliable because the results are dependent on platelet distribution
and RBC count. The advantage is that it enables the study of platelet morphology.
1. Fonio’s Method (Dry Method) –
-14% magnesium sulfate
-Wright stain
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
Diluting fluid: 14% Magnesium Sulfate
-does not lyse RBCs
Procedure:
1. Disinfect the site of puncture.
2. Place a drop of diluting fluid on the disinfected area.
HEMA312LEC@ANT_BSMLS 3-B-2
3. Puncture through the drop of diluting fluid to a depth of 3 mm.
4. Transfer a drop of blood-MgSO4 mixture on a slide. (Ratio = 1:3)
5. Make a smear, dry and stain with Wright’s stain. (Meanwhile, do not forget to do the RBC count on
the patient.)
6. Under OIO, count 250 RBCs per area in 4 areas to a total of 1,000
RBCs and count all the platelets seen.
7. Computation:
(insert pic)
5. Reference value
Normal Value: 250,000-500,000/ mm3
Advantage:
1. Easier to count RBCs and platelets
2. Size and shape of platelets can be observed
6. Interpretation
2. Dameshek Method (Wet Method) –
-BCB
-Wright stain
Microscopy Phase contrast microscopy
Diluting fluid: Rees-Ecker Diluent
-does not lyse RBC and WBC
a. Brilliant Cresyl Blue - 0.15 g (dye)
HEMA312LEC@ANT_BSMLS 3-B-2
b. Sodium Citrate - 0.4 g (prevents clumping of platelets)
c. Distilled Water - 100 ml
d. Formalin - 3 drops in 1:10 dilution (preservative)
e. Sucrose - 8.0 g
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
Procedure:
1. Disinfect the site of puncture.
2. Place a drop of diluting fluid on the disinfected area. This is done to avoid exposure to air and
disintegration of platelets.
3. Puncture through the drop of diluting fluid to a depth of 3 mm.
4. Transfer a drop of blood and diluting fluid mixture on the center of a coverslip. (Ratio = 1:5 – blood to
diluent) And invert over a glass slide and allow it to stand in a moist chamber for 15-45 minutes.
(Meanwhile, do not forget to do the RBC count on the patient.)
5. Examine the prepared smear under the oil immersion of a microscope.
6. Count 250 RBCs per area in 4 areas to a total of 1,000 RBCs and count all the platelets seen. Platelets
are lilac colored cells, tiny and glistening.
7. Computation:
(insert pic)
5. Reference value
Normal Value: 500,000-900,000/ mm3
HEMA312LEC@ANT_BSMLS 3-B-2
6. Interpretation
Automated Platelet Count types
-VOLTAGE PULSE COUNT
-ELECTRO-OPTICAL counting
C. Electronic Method -
1. Voltage-pulse counting -
2. Electro-optical Counting -
Normal Plt count prolonged Bt
-Qualitative Platelet defects
-Primary Vascular abnormality
-VWD
Low Plt Count Normal BT
-autoimmune thrombocytopenia due to anti platelet antibodies
Low plt count prolonged Bt
-simultaneous quantitative and qualitative platelet deficiency
III.Olef’s Method
The Olef’s method is the best method in the indirect procedures but
somewhat cumbersome.
Normal Value: 437,000-586,000/mm3
Platelet Adhesiveness
HEMA312LEC@ANT_BSMLS 3-B-2
A. Test for Adhesion of Platelets
1. In vivo method of Borchgrevink
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
5. Reference value
6. Interpretation
2. Salzman Method/Glass Bead Retention Dish
Test of the retention of the platelets with the glass bed column
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
5. Reference value
6. Interpretation
Decreased Plt retention
Bernard soulier
Glanzmann T
vWD
Chediak- H
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myeloproliferative disorders
uremia
aspirin
Increased plt retention
thrombocytic disorders
hyperlipidemia
carcinoma
oral contraceptive
pregnancy
3. Test for adhesion of platelets to collagen fibers
Sources of Error in Platelet Count
Determination of Clot Retraction
1. Discussion
( to include also the importance of the test and when is the test being requested)
2. Specimen
3. Principle
4. Procedure
5. Reference value
6. Interpretation
HEMA312LEC@ANT_BSMLS 3-B-2
Methods of Clot retraction time
Qualitative
-Hirsboek or castor oil method
-Single tube method
Quantitative
-Stefanini method
-Macfarlane method
A. Qualitative Test
1. Hirshboeck or Castor Oil Method (dimpling)
Qualitative method
NV: 15-45mins
pos- formation of dimpling or droplet like serum on the surface of the blood drops
2. Single Tube Method
B. Quantitative test
1. Stefanini Method
Quantitative
-similar to single tube meyjod 3-5ml of blood 37c
2. Macfarlane Method
5ml of blood
Incubation time 1hr
Nv 44-67%
Capillary Fragility/Resistance Test
1. Tourniquet or Rumpel-Leede or Hess Test positive pressure technique.
Capillary fragility test (two types)
HEMA312LEC@ANT_BSMLS 3-B-2
-Tourniquet test (rumpel leede test)
-Petechiometer method or Negative pressure technique
Rumpel leede test
aka: Tourniquet test, hess teSt, Positive pressure test measure the capillaries to resist pressure
corrrelates with the degree of thrombocytopenia
Principle of Tourniquet test
obstruction of venous blood will increase capillaries pressure then will
results in intravasation of blood which are manifested by petechiae
Bleeding Time
1. Duke’s Method
aka template bleeding time uses feather lancet and filter paper
2. Modified Ivy’s Method
best method to assess platelets adhesiveness
3. Coply Lalitch Method and Adelson-Crosby Method
4. Macfarlane’s Method
same principle as adelson crosby method but it only use EAR LOBE as the puncture siye
5. Aspirin Tolerance Test
assesses the effects of a standard dose of aspirin on the duke's bleeding time
Bleeding time is prolonged in the following conditions:
• When the blood platelets are greatly reduced.
o Thrombocytopenic purpura - dec. In platelets
HEMA312LEC@ANT_BSMLS 3-B-2
o Acute Leukemia
o Aplastic anemia
• Injury of capillary wall
o Scurvy
o Toxins (infection, chemical, snake venom)
• PT deficiency
o Destructive disease of the liver
o HDN
• Slightly prolonged severe anemia
Factors affecting Bleeding time
-elasticity of the tissue
-ability of the blood vessels to constrict and retract
-mechanical and chemical action of platelet in the formation of hemostatic plugs
Abnormal Bleeding time usually occurs in?
Thrombocytopenia
Hypofibrinogenemia
vWD disorders
Connective tissue disorders
Secondary hemostasis
HEMA312LEC@ANT_BSMLS 3-B-2
Test of Phase II Coagulation
A. The Coagulation Test
B. Partial Thromboplastin Time
C. Activated Partial Thromboplastin time (APTT)
D. Differential Tests of Activated Partial Thromboplastin Time (DAPTT)
E. Differential Partial Thromboplastin Time (DPTT)
F. Plasma Prothrombin Time (PROTIME)
G. Serum Prothrombin Time or Prothrombin Consumption Test (PCT)
H. Thromboplastin Generation Test
Test of Phase III Coagulation
A. The Coagulation Test
1. Micro Methods
a. Slide or Drop Method
b. Capillary or Dale and Laidlaw’s Method
2. Macro Methods
a. Lee-White Method or Whole Blood Clotting Time
b.
3. Activated Coagulation Time of Whole Blood
4. Plasma Recalcification Time
5. Activated Recalcification Time
B. Thrombin Time
C. Fibrindex Test
D. Fit-test (Immunological Test)
E. Fibrinogen Titer Method
F. Assay of Plasma Fibrinogen
Fibrinolysis
1. Whole Blood Clot Lysis Time
2. Euglobulin Clot Lysis Time
3. Protamine Sulfate Turbidity Test
4. Latex D-dimer Assay
5. Prothrombin Fragment 1.2 Test (F-1.2)
HEMA312LEC@ANT_BSMLS 3-B-2
6. Tanned Red Cell Hemagglutination Inhibition Immunoassay
Lab evaluation: test for inhibitors of coagulation
1. Plasma Antithrombin Test
2. Plasma Thrombin Time
3. Assay for Lupus Anticoagulant (Tissue Inhibition Test)
