Preanalytical and Analytical Issues in

Coagulation Testing
Objectives
1. Describe special precautions regarding specimen collection and
processing for coagulation testing and determine the
appropriateness of specimens.
2. State the type of anticoagulant tube used for coagulation testing.
3. Discuss the function of the sodium citrate anticoagulant in
coagulation testing.
4. Recognize the appropriate order of draw in terms of coagulation
testing.
5. site storage requirements for the prothrombin and activated
prothrombin time assays.
6. State the purpose of the blood to anticoagulant ratio in the sodium
citrate tube.
7. State the different analytical methods used for coagulation assays
tests.
Sample Collection
 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 90 % of calibrated volume
◦ Purpose of the anticoagulant is to bind or chelate
calcium to prevent clotting of specimen
Sample Collection
Sidenote: Samples with High hematocrits
◦ CLSI recommends adjusting anticoagulant
ratio for patients with hematocrits exceeding
55%
◦ High hematocrits may cause falsely prolonged
test results due to an over- anticoagulated
sample
Samples with High hematocrits
Site Selection
Untraumatic venipuncture is required
◦ Traumatic venipunctures release tissue factor
and initiate coagulation
Fingersticks/Heelsticksare not allowed
Indwelling IV line draws are discouraged
◦ Contain heparin
◦ Falsely increased results
Order of Draw
 The tube used for coagulation studies should be the first tube filled.
 The second one is SST.
 The third one is Heparin tube.
 The last one is EDTA Tube.
Storage Requirements
Prothrombin Time: PT
◦ Uncentrifuged or centrifuged with plasma
remaining on top of cells in unopened tube kept at
refrigerator or 18-24 oC 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
refrigerator or18-24 oC must be tested within 4
hours of collection
Storage Requirements
Other Assays
◦ Fibrinogen, Thrombin Time, Factor Assays
◦ Centrifuged with plasma remaining on top of
cells in unopened tube kept at 18-24 oC must
be tested within 4 hours of collection
Storage Requirements
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 oC for two weeks
 -70 oC for six months
Transportation of Specimen
Send specimen on ice OR deliver to lab
ASAP
Separate cells from plasma immediately
via centrifugation
 Any frozen plasmas must be transferred immediately to a 37°C
water bath, thawed for 4 to 5 min, and mixed gently. A slow thaw
at lower t should be avoided to prevent the formation of
cryoprecipitate, which reduces the FVIII:C, VWF, and fibrinogen
content of the supernatant plasma.
 The activity of coagulation factor V (labile factor) typically may be
10% to 20% lower in frozen-thawed plasma specimens than in fresh
specimens, and may lead to a falsely prolonged PT
Common Collection Problems
Error Consequence Comment
Short draw PT/PTT falsely prolonged Anticoagulant to blood ratio
<2.7 mL exceeds 1:9
Failure to mix specimen after PT/PTT falsely prolonged Blood clots form when
collection 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 PT/PTT falsely prolonged Must follow storage

temperature or held too long requirements

Chilling in refrigerator or PT falsely shortened Chilling to 4 oC activates factor

placing on ice VII.
Inadequate centrifugation PTT loses sensitivity for lupus Desire platelet poor plasma
anticoagulants and heparin.
Factor assays inaccurate
Prolonged tourniquet Falsely elevates vWF, factor Tourniquet causes venous
application VIII stasis,
Common Collection Problems (con’t)
Error Consequence Comment
Drawing coagulation tube PT/PTT falsely affected Contamination
PRIOR to other
anticoagulant tubes

Probing the vein PT/PTT falsely shortened Tissue thromboplastin is

released activating
coagulation

Heparin contamination from PTT falsely prolonged Heparin keeps the blood
line draw from clotting

Lipemia Test may not work Photo-optical methods

affected

Icterus Test may not work Photo-optical methods

affected
 Detection of clot formation in clot-
based assays
Electromechanical methods
 A steel ball rotates under the
influence of a magnet until the
formation of fibrin strands around
the ball stops it rotating. This is
detected by a sensor, and the
coagulation time is recorded.
Photo-Optical
 Scattered Light Detection for
Clotting Assays: The turbidity
during the formation of a fibrin clot
is measured as an increase in
scattered light intensity when
exposed to light at a specific
wavelength.
 Prothrombin Time/Protime (PT)
 Prothrombin time (PT) –
test contains
thromboplastin (TF) and
calcium chloride and
measures the extrinsic and
common pathways
(Normal=11-14 sec)
 It is prolonged in newborns
and then decreases
gradually till adult levels. It
is considered prolonged if
difference between patient &
control is > 2 sec.
A prolonged prothrombin time
Oral anticoagulants:oral anticoagulant therapy
with vitamin K antagonists such as Warfarin.
Deficiency of factor I, II, V, VII or X (Extrinsic
& common pathway factors), which may be
inherited or acquired, e.g. in liver disease or
DIC.
High levels of heparin.
Inhibitors to specific coagulation factors,
including the lupus inhibitor.
Vitamin K deficiency.
INR
The International Normalization Ratio
(INR) value is only used on patients who
are on oralanticoagulant therapy such as
Warfarin, Coumadin, Sintrom.
Because these patients must be
continuously monitored we should
standardize the reported PT results
Standardization ensures uniformity and
safety when a traveling patient is treated
in different geographical areas.
INR
INR=[PT (pt) /PT (Control)]
Normal (Adults): 0.8 – 1.3
PTT
Activated partial thromboplastin time
(APTT) -contact activators and a platlet
substitute and calcium chloride are added to
measure the intrinsic and common pathways
Normal usually 23-35 sec, may vary
depending upon analyzer used, reagents used,
and patient population.
An activator, such as kaolin, triggers the
pathway by activating the contact factors.
PTT
The APTT is prolonged with deficiencies
of the contact activation pathway (XII, XI,
IX, and VIII; also HMWK & PK) & the
common pathway (X, V, II, fibrinogen).
The PTT will be normal with pure factor
VII deficiency.
It is prolonged in liver disease, vitamin K
deficiency, therapeutic Warfarin &
heparin anticoagulation, DIC, Inhibitors
such as Lupus anticoagulants usually
PTT
A typical reference range for PTT is ~23
to 35 sec (< Control + 10 sec).
In adults it is prolonged if ratio
patient/control is > 1.2
In children it is prolonged if ratio > 1.3
In infants it is prolonged if >1.5 & in
Newborns if >1.6
PTT
The PTT is traditionally used to monitor
therapeutic anticoagulation with standard
(unfractionated) heparin. The usual
desired therapeutic range is prolongation
of the PTT to ~1.5 to 2.5 times the mean
normal result (ratio).
Important: check platelets count after 4
days of treatment.
Thrombin Time (TT)
 The thrombin time (TT) uses exogenous thrombin to
convert fibrinogen to fibrin. It is prolonged with
decreased fibrinogen (< 1g/L), elevated levels of FDPs,
and heparin (inhibitor of thrombin).
 It is not affected by deficiencies in the intrinsic or
extrinsic pathways.
 The TT can be used to monitor heparin therapy,
although the PTT is more often used.
 It may be used to monitor heparin therapy in patients
with lupus anticoagulant and a baseline prolonged PTT.
It is considered prolonged if > control + 5 sec.
Manual vs automated coagulation tests
For manual techniques, perform all tests
in duplicate. Duplicate clotting times
should not differ by more than 10%.
For automated tests with a between assay
coefficient of variation of less than 5%,
single replicates will normally be
acceptable, provided prolonged results
are checked
 Mixing Tests for further investigation of abnormal PT
& APTT
 Plasma samples found to have abnormal screening
tests (i.e. PT/APTT) may be further investigated to
define the abnormality by performing mixing tests.
 equal volume mixtures (termed 50:50) of patient and
control plasma.
 used to distinguish factor deficiencies from inhibitors
(non-specific lupus anticoagulants or specific factor
inhibitors such as antibodies directed against factor
VIII).
 factor levels of 50% of normal should give a normal
PT or PTT result.
 Mixing Study
◦ If the procedure corrects the prolonged PT or APTT,
the defect is in the procoagulant family.
 Mixing study corrects=Factor deficiency.
◦ If the procedure does not correct the PT or APTT, the
defect is due to an inhibitor
 Mixing study does NOT correct=Inhibitor

 Therefore, correction with mixing indicates factor

deficiency; failure to correct indicates an inhibitor.
Mixing Study
Mixing Tests for further investigation of
abnormal PT & APTT
If the PTT ratio remains above 1.2 for adults,
the incubated mixture “does not correct”. An
additional aPTT test should be done but after 1
to 2 hour incubation of the mixed plasma at
37C.
The reason behind this additional step is to
determine if there is a weak or time-dependent
& temperature-dependent circulating inhibitor.
Certain inhibitors such as factor VIII inhibitor
have a stronger inhibitory effect with prolonged
incubation.
 Factor Deficiency vs. Circulating
Inhibitor

Immediate PT or APTT Mixing study following

Deficiency or Inhibitor
after Mixing Study 2 hour incubation

Factor deficiency Correction Correction

Lupus-like anticoagulant No correction No correction

No correction
F-VIII inhibitor Correction
Mixing Tests for further investigation
of abnormal PT & APTT
Bleeding time
 The bleeding time (BT) is the best available screening
test of primary hemostasis.
 It is the time taken for a standardized skin cut of fixed
depth and length to stop bleeding.
 The Ivy method is the traditional method for carrying
out this test. In the Ivy method, a blood pressure cuff is
placed on the upper arm and inflated to 40 mmHg. A
disposable lancet is used to make two separate cuts into
the forearm usually 5-10 cm apart in quick succession.
 A stopwatch is started immediately and every 30
seconds filter paper is used to draw off the blood.
Bleeding time
 The time from when the incision is made until all
bleeding has stopped is called the bleeding time.
 The test is finished when bleeding has stopped
completely.
 An attempt to standardize the method [Template
Method] involves the use of an automatic blade which
makes a standard-sized incision [6mm in length x 1mm
in depth] on the volar aspect of the forearm.
 Historically measuring the Bleeding Time involved the
use of the ear lobe - the so-called Duke Method.
However, the ear lobe is highly vascular and is no
longer used.
Bleeding time
Affected by:
 Platelet count and function
 Function of vWF
 Vessel wall integrity

Normal range: Ivy’s incision method: up to 7 min. It is not necessary

to record endpoints if bleeding continues beyond 20 minutes.
Prolonged in:
 Thrombocytopenia (platelet count <100,000/μL)
 von Willebrand Disease
 Inherited or acquired disorders of platelet function (Bernard-Soulier
syndrome or Glanzmann’s disease; myelodysplasia)
 Antiplatelet agents (aspirin…),Uremia
 Collagen disorders (Ehlers Danlos syndrome)