ROUTINE LABORATORY EVALUATION OF

COAGULATION

William Henson - 1700s

- Blood ligated in the veins of a living animal clotted much more slowly than blood
that has been shed from the body, investigators have been avidly experimenting
with ways to measure the bloods capacity to clot.
Paul Morawitz - 1905
- Published a comprehensive explanation of the chemistry of coagulation.
- When plasma was placed in:
o Non-wettable surfaces coagulation did not occur
Paraffin-lined tubes
o Wettable surfaces coagulation did occur rapidly
Glass tubes
Doctors Roger Lee and Paul White - Lee and White whole blood clotting time (L-W)
Dr. Armand Quick - 1930
- Described a procedure called the prothrombin time (PT) test
- Extrinsic coagulation system
1950s - Studies of blood from haemophiliacs whose blood yielded normal
prothrombin times despite its inability to clot normally led to the development of the
partial thromboplastin time (PTT) test
- Intrinsic coagulation system

Tissue extract required for the extrinsic system to induce coagulation

o Factors I, II, V, VII and X
The Intrinsic system is stimulated by activating blood or plasma with negatively
charged surfaces to the presence of phospholipids supplied by platelets or
other reagents.
o Dominant
o All factors except VII and XIII

REFERENCE RANGES
Significantly affected by:
Patient Populations
Methodology
Reagent Systems
Instrument Systems
This will involve the performance of 20 to 40 determinations on plasma from healthy
volunteers in the same manner as patients samples are to be tested. Calculation of the
mean and 95% confidence limits produces a reference range
TESTS FOR THE INTRINSIC AND COMMON PATHWAYS
1. Lee and White Whole Blood Coagulation Time
1913

Based on the fact that when venous blood is put into a glass tube (foreign surface),
it will form a solid clot.
The time required for this response is a measure of the overall intrinsic
and common pathways of coagulation

2. Plasma Recalcification Time

Modification of the L-W

Uses citrated plasma (instead of whole blood), CaCl 2, glass or siliconized
tubes and either platelet-rich plasma (PRP), platelet-poor plasma (PPP)
or both.

This test is based on the fact that except for calcium, normal PRP contains all
the components of the coagulation mechanism necessary for generating
a fibrin clot.

Time required for blood to clot after Ca+2 is added - general measure of the
intrinsic and common pathways.

By using a parallel test on PPP, screening for a platelet function defect may also be
accomplished.

100 to 150 seconds - Reference ranges for PRP

130 to 240 seconds Reference range for PPP

o
Platelet-rich plasma should clot at least 20 seconds faster than platelet
plasma.

Disadvantages of the plasma Recalcification time

o Difficulty in standardizing the number of platelet
RPR and the length of time necessary to perform the test, which is
moreover is insensitive to moderate factor deficiencies
Errors in collection technique
It is important that the same size tube always be used for testing and
that the specimens be tilted uniformly
Because the procedure cannot be standardized, it is important to keep
as many variables constant as possible
The sensitivity of this test is somewhat improved by diluting the
plasma
This accomplishes three things:
o It adjusts the PRP closer to the actual in vivo platelet count
o It increases the sensitivity of the test system to factor
deficiencies
o It dilutes the natural inhibitors to coagulation that are
present. A normal control should be run with each test.

ACTIVATED CLOTTING TIME

1960
The lack of sensitivity and precision of the L-W and the plasma recalcification test
had led researchers to look for better methods of monitoring heparin therapy.
Although by 1964 the PTT test was recognized to be sensitive to heparin, the test
had not yet gained widespread acceptance as the heparin-monitoring procedure.
Dr. Paul Hattersley

Uses diatomaceous earth (diatomite) as an activator of the contact factors and

requires the blood to be kept warmed to a constant 37C by taking a special
incubator to the patient's bedside.

Principle
Whole blood contains all the components necessary to produce a clot when removed
from the veins and put into a glass tube. By adding an activator and keeping the blood at
a constant 37C, a more reliable and rapid screen of the intrinsic and common pathways
is achieved.
Reagents
Two evacuated tubes containing 12 mg of diatomite
Equipment
A portable heat block, thermometer and two stopwatches
Procedure
The two tubes containing diatomite are brought to 37C in a heat block at the patients
bedside. Using good venipuncture technique, at least 2 mL of blood is drawn into a tube
and discarded. The tourniquet is removed and the first tube with diatomite is attached to
the needle. When blood starts to flow into the tube, the first stopwatch is started. The
tube is filled, mixed and placed in the heat block. The procedure is repeated with the
second tube and the second stopwatch is started. After 60 seconds, the first tube is
observed by tilting it at 5-second intervals until a clot is formed, at which time the
second tube is observed using the same procedure. The appropriate stopwatch is
stopped at the first appearance of a clot in each tube. The duplicates should agree
within 10 seconds. The average time is reported.
Reference Range
75 to 120 seconds
140 to 185 seconds - Target range during heparin therapy
Interpretation
Prolongation of the ACT is indicative of one or more factor defects in the intrinsic or
common pathways or the presence of a circulating anticoagulant such as heparin.
PARTIAL THROMBOPLASTIN TIME

Hemophiliacs - have a prolonged clotting time, but when tissue

thromboplastin is added, as in the PT, the plasma clots just as normal plasma
does.
Expanded studies showed that thromboplastins, which are lipoproteins (protein +
phospholipid), may be classified as complete or partial (only phospholipid).
From these conclusions, investigators developed the non-activated PTT. This test is
more sensitive to abnormalities in the early stages than were previous tests of the
intrinsic system.
An important refinement of the PTT was the addition of negatively charged
activators to the system, resulting in significantly shorter clotting times.
Activated partial thromboplastin time (APTT)

 Reagents:
o Platelet substitute (phospholipid) prepared from brain or plant
phospholipids
o Activator kaolin, celite, micronized silica or ellagic acid
Principle
Measures all factors except VII and XIII.
Maximum activation of the contact factors is accomplished by addition of the
activator
Phospholipid is supplied to substitute for platelet factor 3 (PF3). From this point,
the APTT is essentially the same as a recalcification time of plasma
Specimen Requirements
Citrated platelet-poor plasma
Reagents
Phospholipid with activators (APTT reagent)
0.025 M CaCl2 (or as recommended by reagent manufacturer)
Controls
Commercial lyophilized controls are available in normal, midrange, and extended
ranges
It is recommended that a normal control and at least one abnormal control be
used. In-house preparations of pooled/frozen plasma may be used as controls.
Each laboratory must specify when controls are to be tested, what the satisfactory
control limits are, and, if duplicates are run, how closely the values should agree.
Equipment
12 x 75-min glass tubes, a heat block, and pipets - manual method
Instrument for electromechanical fibrin strand detection and appropriate
cups and pipets - fibrin strand method
For the photo-optical method, the specialized instrument and appropriate
accessories as listed by the manufacturer, are needed.
Procedure
Platelet-poor plasma (0.1 mL) is added to 0.1 mL of APTT reagent and incubated at 37C
for the period of time specified by the reagent manufacturer (approximately 3 to 5
minutes). After incubation, 0.1 mL of warmed CaCl 2 is added, and the time for clotting to
occur is recorded.
Reporting
Reported in seconds, to the nearest tenth
Reference Range
Differ according to the reagent, method, and instrument used. The reference
ranges may extend from a lower limit of 20 seconds to an upper limit of 45
seconds
Interpretation

A prolonged APTT in the absence of heparin use indicates a factor deficiency, an

acquired circulating anticoagulant such as the lupus inhibitor or an antibody to
a specific factor such as factor VIII
Most commercial reagents will demonstrate a prolonged APTT if any factor measured by
the APTT is less than 40% to 50% of normal
Comment
Sources of error:
Sample collection and preparation
o The anticoagulant volume should be adjusted for individuals with
hematocrits greater than 0.55 L/L and less than 0.20 L/L
o Hemolysis may cause a falsely shortened APTT
o Platelets in the plasma sample may cause erratic results or falsely shorten
the APTT.
o Unexpected heparin contamination can spuriously lengthen the APTT.
Reagent preparation may be affected by improper storage, water impurities,
or proper dilution
Instrumentation
o Will cause error:
A failing light source
Fluctuations in temperature
Loss of calibration of tubing
Contamination
TESTS FOR THE EXTRINSIC AND CONMON PATHWAYS
PROTHROMBIN TIME
de Bainville - blood could be made to clot quickly by adding tissue factors.
The discovery that oxalate and citrate inhibit coagulation led to the assumption
that clotting was dependent on calcium.
One-stage PT - Dr. Armand Quick in 1935
- Indirect measure of prothrombin in plasma, dependent on the presence of
fibrinogen Subsequent discoveries of factors V, VII, and X showed that the PT is a
reflection of the activities of several factors. Thus, the PT used in today's
laboratory screens for deficiencies of factors I, II, V, VII, and X.
- Although the PT is no longer considered a measure of prothrombin itself. it is still
the
Test of choice for monitoring anticoagulant therapy by vitamin K antagonists
Three of the five factors measured by the PT (II, VII, X) are sensitive to and
depressed by these anticoagulants. Only factor IX, the other factor depressed by
vitamin K antagonists, is not detected by the PT.
Principle
When tissue extract or thromboplastin is added to PPP along with calcium, it reacts
with factor VII to convert factor X to X a. Factor Xa, along with factor Va,
phospholipid, and Ca+2 converts prothrombin to thrombin. Thrombin subsequently
converts fibrinogen to fibrin (see Fig. 52-1). The time from the addition of
thromboplastin/ CaCl2 to the formation of a clot is reported as the PT.

Specimen Requirement
Citrated platelet-poor plasma
Reagents and Equipment
Thromboplastin/CaCl2 (PT reagent)
Controls
o The equipment is the same as that used for the APTT.
Procedure
Aliquots of control and patient plasma are warmed according to the method being
used. The PT thromboplastin reagent is warmed by incubating it at 37C for 3 to 5
minutes, and 0.2 mL of PT reagent is added to 0.1 mL of plasma (patient or
control). The clotting time is recorded.
Reference Range
10 to 12 seconds - some photo-optical systems
12 to 14 seconds - manual methods.
Reporting
Several ways:
Patient time (in seconds) with the reference range
Patient time with the control time (in seconds)
Prothrombin ratio (the PT of the patient divided by the mean of the reference range
and multiplied by 100-rarely used in the United States)
Percent activity (outdated and not recommended)
The use of an international normalized ratio (lNR) has been proposed as the
standard method of reporting. It is popular in Europe but not widely used in the United
States
Interpretation
Prolongation of the PT indicates an abnormality of one or more common or
extrinsic coagulation factors
Using most commercial reagents, the PT is sensitive to factor deficiencies of less
than 40% to 50% of normal
Other Tests
1. Stypven time:
Utilizes the powerful coagulant properties of Russells viper venom (obtained
from the snake Vipera russelli)
This venom is capable of bypassing the action of factor VII and directly activating
factor X to Xa. When it is combined with dilute thromboplastin, a fibrin clot will
form through the reaction of factors Xa and Va, phospholipid, factor II and
fibrinogen.
Substitution caused problems in managing patients on anticoagulant therapy, as
the Stypven time produced shorter clotting times than did the PT and led to
serious overdosing, with resultant bleeding

The discovery of factors VII and X explained the discrepancy between the Stypven
time and the PT

2. Prothrombin-Proconvertin time.
Doctors Owren and Aas
Based on earlier observations that minor deficiencies can be more pronounced
when test plasma is diluted.
Plasma is tested at 1:10 dilution, and a reagent containing dilute thromboplastin
extract from bovine brain, CaCl2, and an excess of bovine factors V and l
(fibrinogen) is used.
The addition of labile factor V made the test more sensitive to those factors in
the extrinsic and common pathways that are affected by vitamin K antagonists