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COAGULATION
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
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.
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
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
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