INTRODUCTION TO HEMOSTASIS, PRIMARY AND SECONDARY HEMOSTASIS, LABORATORY TESTS THAT ASSESS PRIMARY HEMOSTASIS

1. PREPARED BY:
PRINCESS ALEN I. AGUILAR,RMT
SOURCE: HO BY CUA
HEMATOLOGY BOOKS BY WILLIAM, HOFF’S, TURGEON

2. 1828The disorder was first described
in a thesis published by Hoff
1842 Platelets were described
1905 Theory of Blood Coagulation by
Paul Morawitz was accepted
1913  Lee and White WB CT was
performed
1930 PT Time was introduced by Quick
1940 Other tests for evaluating
hemostatic mechanisms, like platelet
count and bleeding time were introduced
1964 Cascade and Waterfall Theory of
coagulation was introduced
2ND Century A.D.  Hemophilia was first
recognized
12th Century A.D. Moises Maimonides
described 2 male siblings who died because
of excessive bleeding after circumcision
1803  Clinical description of families
with haemophilia was first published. The
disorder was given the name haemophilia
which means “love of haemorrhage” by
Schonlein
2ND Century A.D. 
Hemophilia was first
recognized

5. • Size: 20-50 µm in diameter.
• Cytoplasm: varying shades of blue
• Usually darker than the myeloblast.
• May have small, blunt pseudopods.
• Small to moderate amount. Usually a narrow
band around the nucleus. As the cell matures,
the amount of cytoplasm increases
• Usually nongranular.
• Nucleus:
• Round,oval or may be kidney shaped.
• Fine chromatin pattern
• Multiple nucleoli that generally stain blue
• N/C Ratio is about 10:1

6. • Size: 20-60 µm in diameter
• Cytoplasm: More abundant than
previous stage.
• Less basophilic than in the blast.
• Granules begin to form in the golgi region.
• Nucleus: Chromatin becomes more
coarse.
• Multiple nucleoli are visible.
• Irregular in shape; may even show slight
lobulation.
• N/C Ratio 4:1 to 7:1 depending on the
ploidy

7. • Size:30-90 µm in diameter
• Cytoplasm: abundant
• Pinkish blue in color
• Very fine and diffusely granular
• Usually has an irregular peripheral border
• Nucleus: small in comparison to cell size
• Multiple nuclei may be visible or the nucleus
may show multi-lobulation.
• Chromatin is coarser than in the previous
stage.
• No nuclei are visible.
• N/C ratio is 2:1 to 1:1

8. • Size: 40-120 µm in diameter
• Cytoplasm:
• contains coarse clumps of granules
aggregating into little bundles, which
bud off from the periphery to
become platelets
• Nucleus:
• multiple nuclei are present, or the
nucleus is multilobulated
• No nucleoli are visible.
• N/C Ratio is less than 1:1

9. • Size:1-4 µm in diameter
• Cytoplasm: light blue to purple.
• Very granular.
• Consists of two parts:
(1) the chromomere, which is
granular and located generally.
(2) the hyalomere, which surrounds
the chromomere and is non
granular and clear to light blue.

10. Stoppage of blood flow
Involves the interaction of blood vessels, platelets, the
coagulation mechanism, fibrinolysis and tissue repair
A complex process that:
Produces a clot to stop the bleeding
Keeps the clot confined
Dissolves the clot as the wound heals
CELLULAR ELEMENTS OF HEMOSTASIS:
Extravascular Tissue Factor  tissue surrounding the vessel
Vascular Intima  blood vessel through which the blood flows
Intravascular component  plasma proteins and platelets

14. A. PRIMARY HEMOSTASIS
Refers to the role of Blood
vessels (vascular system which
includes the arteries,veins,and
capillaries) and platelets in the
primary formation og platelet
plug in response to vascular
injury
TEST: BLEEDING TIME
1. PLATELET ADHESION  plt-
non plt substance;
Contact to plts with the
subendothelium and their
adhesion to it
Plts adheres to Collagen
Occurs in presence of vWF
vWF disease
Bernard-Soulier disease

15. A. PRIMARY HEMOSTASIS
3. PLATELET SECRETION
Release of granules
4. PLATELET AGGREGATION
Platelet to another platelet
Aggregate plt for oxygen
exchange
Requires fibrinogen (Bridge)
*Glanzmann’s thrombasthenia
2. PLATELET ACTIVATION
Morphologic and functional
changes in platelets (platelet
change shepe)
Form discoid to spherical with
pseudopod formation
Ca2+,actin and
myosin/thrombosthenin

20. B. SECONDARY HEMOSTASIS
Involves the enzymatic activation of series of
plasma proteins in the coagulation system
(coag factors) to form a fibrin meshwork (fibrin
clot)
TEST: CLOTTING TIME

21. Factor Trivial Name(s) Functions
Prekallikrein (PK) Fletcher factor
High molecular weight
kininogen (HMWK)
contact activation cofactor; Fitzgerald,
Flaujeac Williams factor
I Fibrinogen Polymerizes fibrin
II Prothrombin Serine proteases
III Tissue Factor Co-Factor
IV Calcium Mineral
V
Proaccelerin, labile factor, accelerator (Ac-)
globulin
Co-Factor
VI (same as Va) Accelerin
VII
Proconvertin, serum prothrombin conversion
accelerator (SPCA), cothromboplastin
Serine proteases
VIII
Antihemophiliac factor A, antihemophilic
globulin (AHG)
Co-Factor
IX
Christmas Factor, antihemophilic factor
B,plasma thromboplastin component (PTC)
Serine proteases
X Stuart-Prower Factor Serine proteases
XI Plasma thromboplastin antecedent (PTA) Serine proteases
XII Hageman Factor Serine proteases
XIII
Protransglutaminase, fibrin stabilizing factor
(FSF), fibrinoligase
Stabilizing Factor

22. Petechiae- purplish red pinpoint hemorrhagic spots in the skin caused by loss of capillary
ability to withstand normal blood pressure and trauma
Purpura – haemorrhage of blood into small areas of skin, mucous membranes, and other
tissues
Ecchymosis- form of purpura in which blood escapes into large areas of skin and mucous
membranes, but not into deep tissues
Epistaxis – nosebleed
Hemarthrosis – leakage of blood into joint activities
Hematemesis – vomiting of blood
Hemoptysis-Bloody sputum
Hematochezia- “red stool”; asstd with lower GIT bleeding
Hematoma –
Hemoglobinuria – hgb in urine
Hemorrhage- is a severe form of bleeding tha requires immediate intervention and transfusion
Melena- stool containing dark red or black blood; “ black tarry stool
Menorrhagia – excessive menstrual bleeding

27. INHIBITOR!

31. SPECIMEN COLLECTION: is a
prenalytic that may have serious
implications in laboratory testing.
According to studies done in recent
years, 32-75% of testing errors
occure during the pre-analytic
phase.
SPECIMEN : EDTA anticoagulated
WB- most commonly used
A. QUANTITATIVE PLATELET EVALUATION
PLATELET SATELLITOSIS
A form of pseudo-thrombocytopenia
Antibodies directed against GPIIb-IIIa react with
the leukocyte Fc gamma receptor III and attach
the platelet to neutrophils are the most
frequently involved; occasionally monocytes
Naturally occurring, ut exposure of antigen on
EDTA-treated platelets and leukocytes may
trigger the phenomenon.
Using sodium citrate as an anticoagulant should
correct this problem. Because of the dilution in
the citrate tubes, it is necessary to multiply the
obtained platelet count by 1.1

32. Platelets are counted in a
hemocytometer as in erythrocytes and
leukocytes
A. REESE-ECKER
DILUENT: isotonic
Sodium Citrate – prevent platelet aggregation
Formaldehyde- Preservative
BCB- Stain/dye
Microscopy: Light Microscopy
Appearance of platelets: BLUISH
B. BRECKER-CRONKITE- reference method
Diluent: Hypotonic= 1% ammonium oxalate
Microscopy: Phase-Contrast Microscopy (best
microscope for platelet morph evaluation)
I. DIRECT PLATELET COUNT
A. QUANTITATIVE PLATELET EVALUATION
C. GUY AND LEAKE
Diluent: Isotonic
 Sodium oxalate- prevent platelet
aggregation
Formaldehyde- preservative
CV- stain/dye
Mircoscopy: Light Microscopy
Appearance of platelets: LILAC
refractile object
D. UNOPETTE
Diluent: 1% ammonium oxalate
Dilution: 1:100

33. Platelets are counted in their relationship
to red cells on a fixed-stained smear. This
method is not reliable because the results
depend upon the distribution of platelets
and on the red cell count.
Stained by Giemsa (10 fields)
A. FONIO’S
14% Magnesium sulphate
Wright’s stain
B. DAMESHECK
BCB
Wright’s stain
C. OLEF’S
II. INDIRECT PLATELET COUNT
A. QUANTITATIVE PLATELET EVALUATION
Red cells must first be removed
from whole blood, either by
sedimentation or by controlled
centrifugation.
PRINCIPLE:
VOLTAGE PULSE COUNTING
ELECTRO-OPTICAL COUNTING
III. AUTOMATED PLATELET COUNT

34. Less than 100,000/uL  abnormally low
30-50,000/uL  bleeding possible with trauma
Less than 30,000/uL  spontaneous bleeding possible
Less than 5,000/uL  severe spontaneous bleeding
Note: normal platelet count + prolonged BT= QUALITATIVE PLATELET
ABNORMALITY, primary vascular abnormality and von Willebrand’s
syndrome
Low platelet count + normal BT: autoimmune thrombocytopenia
Low platelet count + very prolonged BT: Simultaneous quantitative and
qualitative platelet deficiency

35. Assess the ability of platelets to
aggregate after the addition of
aggregating agents
Sample: PRP (Platelet Rich Plasma)
Aggregating agents:
Collagen
ADP
Ristocetin
Epinephrine
Also: thrombin, arachidonic acid, serotonin
B. PLATELET AGGREGATION TEST
TEST CONSIDERATIONS:
No hemolysis
Fasting: 8 hours
pH: 6.5-8.5
Within 3 hours

36. The adhesiveness of platelets may be
measured in vitro by their ability to
adhere on glass surfaces with beads
Salzman method: test of the retention
of platelets within glass bead column
C. PLATELET RETENTION TEST (ADHESIVENESS)
Platelet adhesion may be tested in vitro by
their ability to adhere to glass surfaces. The
number of platelets in the blood, collected
through the glass bead collecting system, will
be lower than the number obtained by routine
venipuncture. (Brown)

37. C. PLATELET RETENTION TEST (ADHESIVENESS)
When blood is passed
through a glass bead
column, normal
platelets that have
access to normal vWF
will adhere and
aggregate to the beads
such that the effluent
from the column will
have a much lower
platelet count than the
starting sample
(Steininger).
DECREASED PLATELET
RETENTION
INCREASED PLATELET
RETENTION
Bernard Soulier Thrombotic disorder
Glanzmann Thrombasthenia Hyperlipidemia
vWD Carcinoma
Chediak-Higashi Oral Contraceptives
Myeloproliferative disorders Pregnancy
Uremia
Aspirin Administration
ABNORMAL PLATELET RETENTION TEST
REFERENCE INTERVALS:
• 26-60% (Brown)
• 70% or greater (Steininger)
• Each laboratory should establish its own reference range with the
equipment used.

38. Measures the entire platelet
function
actin/myosin/thrombosthenin
METHODS:
1. Qualitative Test
a. Hirshboek or Castor oil
method
NV: 15-45 mins
Formation of
dimpling/droplet like serum
on the surface of blood drop
b. Single Tube Method
D. CLOT RETRACTION TIME
2. Quantitative Test
a. Stefanni method- similar
with single tube method
Specimen: 3-5mL blood
Temp: 370C
Normal: begins within 1 hour,
complete within 18-24 hours
b. MacFarlane method
Specimen: 5mL blood
Temperature: 370C
Incubation time: 1 hour
NV: 44-67%

39. A. Tourniquet test (Positive pressure
test)
also called “Hess Test” or “Rumpel-Leede
test
a test used to measure Capillary fragility
most oftenly performed by the Physician
PRINCIPLE  The fragility of capillaries is
determined under increased pressure.
The pressure partially obstructs the
venous return from the arm and
increases intracapillary pressure. The
number of petechial hemmorhages
reflects the degree of capillary fragility
*Presence of numerous
Petechiae
• Increased in:
• Thrombocytopenia
• Decreased Fibrinogen
• Vascular purpura
• Vitamin K deficiency
• Von Willebrand’s disease
• Disseminated Intravascular
Coagulation (DIC)
E. CAPILLARY FRAGILITY TEST

40. • Equipments
Stethoscope
Sphygmomanometer
The test result may be graded roughly
as follows:
(0-10) 1+ = a Few petechiae on the
anterior part of the forearm
(10-20) 2+ = Many petechiae on the
anterior part of the forearm
(20-50) 3+ = Multiple petechiae over the
whole arm and back of the hand
(over 50) 4+ = Confluent petechiae on
the arm and back of the hand
B. Suction cup (Negative pressure
test) Second way to test Capillary
fragility
PROCEDURE:
1) Lubricate suction cup.
2) Apply suction cup to upper arm.
Keep negative pressure for 5 minute.
3) After 5 minutes, inspect skin under
the suction cup for Petechiae.
E. CAPILLARY FRAGILITY TEST

41. •Used to measure the
duration of bleeding after
measured skin incision.
•It depends on the elasticity
of blood vessel wall and on
the number and functional
capacity of platelets.
PRINCIPLE:
 It is performed by using a
sterile blood lancet to make a
measured skin puncture in the
earlobe or forearm. The time is
started immediately after the
puncture. Touching a piece of
filter paper to cut every 30
seconds until bleeding ceases
and record time and reported as
bleeding time.
F. BLEEDING TIME

42. The ff. are the drugs that
causes bleeding time prolonged:
Sulphonamides
Thiazide diuretics
Antineoplastic
anticoagulants,
Non-steroidal anti-inflammatory
drugs
Aspirin and aspirin compounds
Some non-narcotic analgesics
METHODS:
• Duke’s method (Template Bleeding
Time)
• Modified Ivy’s method best method to
assess platelet adhesiveness
• Coply Lalitch Method
• Adelson-Crosby Method
• MacFarlane’s Method  Same principle
with Adelson-Crosby Method but it only
uses earlobe as site of puncture
• Aspirin Tolerance test  Assesses the
effect of a standard dose of aspirin on the
duke’s bleeding
F. BLEEDING TIME