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1. General Approach in
Investigation of Haemostasis
Lecture 1: Introduction

2. Preanalytical Variables including
1. Sample Collection.
2. Site Selection.
3. Storage Requirements.
4. Transportation of Specimen.

3. Haemostasis
 Hemostasis is a complex interaction
between vessels, platelets and
coagulation proteins that, when working
properly, stops bleeding while
maintaining blood flow in the vessel.
 Specific tests are available to evaluate
platelet function, coagulation proteins,
natural occurring inhibitors and
fibrinolysis.

4.  Proper sample collection is of utmost importance for reliable
test results to evaluate the bleeding patient, thrombosis or
fibrinolysis (preanalytical phase)
 All these tests are influenced by sample collection, sample
processing and sample storage.
 The laboratory will not evaluate samples that are hemolyzed,
clotted, contain fibrin strands or improperly stored.
 Reference Laboratory Services will immediately notify the client
of any problems with the sample.
 When blood is withdrawn from a vessel, changes begin to take
place in the components of blood coagulation. Some occur
almost immediately, such as platelet activation and the initiation
of the clotting mechanism dependent on surface contact.
Sample Collection

5.  Anticoagulant of choice
 3.8% or 3.2% Sodium Citrate
 3.2 % Preferred as the standard measure due to stability
and closeness to the plasma osmolality
 Anticoagulant/blood ratio is critical (1:9)
 Exact amount of blood must be drawn. No short draws
are acceptable, this will falsely increase results due to
presence of too much anticoagulant
 CLSI guideline is +/- 10 % of fill line
 Purpose of the anticoagulant is to bind or chelate calcium to
prevent clotting of specimen
* CLSI : Clinical and Laboratory Standards Institute
Sample Collection

6.  Other anticoagulants, including oxalate, heparin, and EDTA, are
unacceptable.
 The labile factors (factors V and VIII) are unstable in oxalate,
whereas heparin and EDTA directly inhibit the coagulation
process and interfere with end-point determinations.
 Additional benefits of trisodium citrate are that the calcium ion is
neutralized more rapidly in citrate, and APTT tests are more
sensitive to the presence of heparin.
Sample Collection

7.  According to the latest CLSI (formerly NCCLS) guideline on
coagulation testing, it is important to adjust the sodium
citrate volume when a patient’s hematocrit is greater than
55%.
 Examples of patients who may have elevated hematocrit
values are newborns or people with polycythemia vera.
 NCCLS* recommends adjusting anticoagulant ratio for
patients with hematocrits exceeding 55%
 High hematocrits may cause falsely prolonged test results
due to an over- anticoagulated sample
 Formula correction achieves a 40% hematocrit
* National Committee for Clinical Laboratory Standards
Sample Collection : Samples with High
hematocrits

8. X = (100–PCV)*vol./(595–PCV)
Where:
X= volume of sodium citrate
Vol =volume of whole blood
drawn
PCV= patient’s hematocrit
Examples:
Patients Hct= 60%, V= 5 mL
X=(100-60)*5 / (595-60)
= 40*5 / 535 = 0.34 ml
Patient Hct = 25%, V=5 ml
X=(100-25)*5 / (595-25)
= 75*5 / 570 = 0.65 ml
HCT
Citrate
(ml)
0.20 0.70
0.25 0.65
0.30 0.61
0.55 0.39
0.60 0.36
0.65 0.31
0.70 0.27

9.  Untraumatic venipuncture is required
 Traumatic venipunctures release tissue factor and initiate
coagulation
 Fingersticks/Heelsticks are not allowed
 Indwelling IV line draws are discouraged
 Contain heparin & diluted blood
 Falsely increased results
 Order of Draw
 Evacuated tube system
 Blue top is 2nd
 If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red
top)
Site Selection

10.  Prothrombin Time: PT
 Uncentrifuged or centrifuged with plasma remaining on top of
cells in unopened tube kept at 2-4 o
C or 18-24 o
C must be
tested within 24 hours of collection
 Activated Partial Thrombin Time: APTT
◦ Uncentrifuged or centrifuged with plasma remaining on top of
cells in unopened tube kept at 2-4 o
C or 18-24 o
C must be
tested within 4 hours of collection
 Other Assays
 Fibrinogen, Thrombin Time, Factor Assays
 Centrifuged with plasma remaining on top of cells in
unopened tube kept at 2-4 o
C or 18-24 o
C must be tested
within 4 hours of collection
Storage Requirements

11. TEST
PLASMA
STABILITY AT
RT
CENTRIFUGE TO
PREPARE
PLATELET-FREE
PLASMA
REFRIGERATION
(Or transport on ice)
FREEZE
PLATELET-
FREE
PLASMA
PT 24 hours Do not refrigerate
If >24 hour delay
in testing
PTRX 24 hours Do not refrigerate
If >24 hour delay
in testing
PTT 4 hours
If >4 hour delay in
testing
PTTRX 2 hours
Within one hour of
collection
If >2 hour delay in
testing
TT 4 hours
If >4 hour delay in
testing
OTHER
ASSAYS
4 hours
Within one hour of
collection
Within one hour
of collection
Storage Requirements

12.  Other general notes
 Perform coagulation tests ASAP
 Specimen may deteriorate rapidly (especially
factors V and VIII)
 If the testing is not completed within specified times,
plasma should be removed from the cells and placed
in a frost free freezer
 - 20 o
C for two weeks
 -70 o
C for six months
Storage Requirements

13.  Send specimen on ice OR deliver to lab ASAP
 Separate cells from plasma immediately via
centrifugation
Transportation of Specimen

14.  Platelet –Poor plasma (PPP)
 Platelet-Poor plasma is necessary for coagulation testing to
prevent activation of platelets and release of PF4, a
heparin inhibitor.
 The plasma platelet count must be < 10,000 /mm3.
 Specimen has been centrifuged for 15 minutes @ 2500 x g
 Why is PPP essential?
1. Contains platelet factor 4 (heparin neutralizer)
2. Contains phospholipids (affects lupus anticoagulant and
factor assay testing)
3. Contains proteases (affect testing for vWF)
Platelet Poor Plasma

15. Platelets Poor Plasma preparation:
To prepare platelet-Poor plasma
 Centrifuge the blue top evacuated tubes (CLSI, formerly
NCCLS recommendation is 1500 rpm for 15 minutes).
 Using a plastic pipette, immediately remove the top 2/3
of the plasma to a plastic aliquot tube.
 Centrifuge this plasma sample and remove the top ¾ of
the plasma to a second plastic aliquot tube with a fresh
plastic pipette.
 Freeze the specimen within one hour of collection.

16.  Platelet-Rich plasma (PRP)
 Used in platelet function studies
 200-300 x 10 9 /L
 Specimen must be centrifuged for 10 minutes @ 200 x
g
Platelets Rich Plasma (PRP)

17. Common Collection Problems
Error Consequence Comment
Short draw
<2.7 mL
PT/PTT falsely
prolonged
Anticoagulant to blood ratio exceeds
1:9
Failure to mix
specimen after
collection
PT/PTT falsely
prolonged
Blood clots form when anticoagulant
& blood do not mix
Excess vigorous
mixing
PT/PTT falsely
shortened
Hemolysis and platelet activation
cause start of cascade
Hemolysis PT/PTT falsely
shortened
Reject specimen
Improper
storage: wrong
temperature or
held too long
PT/PTT falsely
prolonged
Must follow storage requirements
Chilling in
refrigerator or
placing on ice
PT falsely shortened Chilling to 4 o
C activates factor VII.

18. Error Consequence Comment
Inadequate
centrifugation
PTT loses sensitivity for
lupus anticoagulants and
heparin.
Factor assays
inaccurate
Prolonged tourniquet
application
Falsely elevates vWF,
factor VIII
Tourniquet causes
venous stasis,
Drawing coagulation
tube after to other
anticoagulant tubes
PT/PTT falsely affected Contamination
Probing the vein PT/PTT falsely shortened Tissue thromboplastin is
released activating
coagulation
Heparin contamination
from line draw
PTT falsely prolonged Heparin keeps the blood
from clotting
Lipemia Test may not work Photo-optical methods
affected
Common Collection Problems

19. Principles of Laboratory Analysis
 The more detailed investigations of coagulation proteins
also require caution in their interpretation depending on
the type of assay performed. These can be divided into
three principal categories, as described in the following
sections.
1. Immunological
2. Assays Using Chromogenic Peptide Substrates
(Amidolytic Assays)
3. Coagulation Assays
4. Other Assays

20. Immunological
 Include immuno-diffusion, immuno-electrophoresis,
radioimmunometric assays, latex agglutination tests, and tests
using enzyme-linked immunosorbent assays (ELISA).
 Fundamentally, all these tests rely on the recognition of the
protein in question by polyclonal or monoclonal antibodies.
Polyclonal antibodies lack specificity but provide relatively high
sensitivity, whereas monoclonal antibodies are highly specific
but produce relatively low levels of antigen binding.

22.  latex agglutination kit: Latex microparticles are coated with
antibodies specific for the antigen to be determined. When the latex
suspension is mixed with plasma an antigen–antibody reaction takes
place, leading to the agglutination of the latex microparticles.
 Agglutination leads to an increase in turbidity of the reaction
medium, and this increase in turbidity is measured photometrically
as an increase in absorbance.
 Usually the wavelength used for latex assays is 405 nm, although for
some assays a wavelength of 540 or 800 nm is used. This type of
assay is referred to as immuno- turbidimetric.

23. Notes:
 Do not freeze latex particles because this will lead to irreversible
clumping.
 An occasional problem with latex agglutination assays is
interference from rheumatoid factor or paraproteins. These may
cause agglutination and overestimation of the protein under assay.

24. Chromogenic Assay
 Chromogenic, or amidolytic, methodology is based on the use of a
specific color-producing substance known as a chromophore.
 the chromophore normally used in the coagulation laboratory is
para-nitroaniline (pNA), which has an optical absorbance peak at
405 nm on a spectrophotometer.

25. Coagulation Assays
 Coagulation assays are functional bioassays and rely on
comparison with a control or standard preparation with a known
level of activity.
 In the one-stage system optimal amounts of all the clotting factors
are present except the one to be determined, which should be as
near to nil as possible.
 The best one-stage system is provided by a substrate plasma
obtained either from a patient with severe congenital deficiency or
artificially depleted by immuno-adsorption.

26. Coagulation Assays
 Coagulation techniques are also used in mixing tests to identify a
missing factor in an emergency or to identify and estimate
quantitatively an inhibitor or anticoagulant.
 The advantage of this type of assay is that it most closely
approximates the activity in vivo of the factor in question.
However, they can be technically more difficult to perform than
the other types described earlier.

27. Other Assays
 Using snake venoms (The Taipan venom time employs a reagent
isolated from the venom of the Taipan snake (Oxyuranus
scutellatus) that directly activates prothrombin in the presence of
phospholipid and calcium.)
 Aassay of ristocetin cofactor (used to diagnose von Willebrand
disease )
 The clot solubility test for factor XIII.
 DNA analysis is becoming more useful and more prevalent in
coagulation. However, this requires entirely different equipment and
techniques