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Medical Laboratory Technology: Volume 1
Figure 14.5 Determination of coagulation time: (a) Mix appropriate reagents and plasma in small-size
test tubes (75 mm ×10 mm) placed in water bath; to this, calcium chloride is added, mixed
and instantly timed with a stopwatch, (b) Tilting the tube will identify the coagulation of
plasma, (c) Appearance of brin strands (jell formation) by moving a nichrome loop up
and down through the solution until the loop catches the brin strand; stop the stopwatch
as soon as the rst brin strand is visible. Note the time taken after the addition of
calcium.
1. Use a disposable capillary pipee with calibration of 10 µL and 50 µL. Calibration can
be done in the laboratory after buying long capillary tubes, which are cut into small
convenient pieces.
2. Draw the anticoagulant solution (citrate) into the capillary tube up to the 10 μL mark
before the skin puncture is done.
3. Puncture the ngertip (or the heel in babies) and aspirate the blood to the 50 µL mark.
4. Immediately empty the content of the pipee into a special test tube (9 mm × 100 mm),
by means of a rubber teat, for coagulation time determination.
5. Mix the blood sample and the anticoagulant thoroughly. Take triplicate samples in
separate pipees (50 µL each).
Note Separation of plasma is not done in the micromethod (capillary method). The
mixed blood is directly used for the coagulation study. Consider 50 µL of whole blood
as equivalent to 40 μL of plasma to compensate for the error. The volume of coagulation
reagent to be added varies accordingly in order to maintain the same proportion as the
macromethod (venepuncture).
Laboratory Investigation of Bleeding Disorders
359
Example
Coagulation reagent to be used is double the volume of plasma in either of
the method.
6. For macromethod: Mix 0.1 mL plasma with 0.2 mL of reagent.
7. For micromethod: Mix 50 μL of citrated blood (equivalent to 40 μL) with 80 μL of
reagent.
Equipment and supplies
• Arrangement for blood drawing: Obtain citrated plasma for all coagulation tests
• Water bath at 37°C: The water bath must be well lighted so that coagulation of the plasma
can be observed while the specimen is under the water surface. A shielded (protected
from glare) horizontal source of light with a uorescent lamp is ideal.
• Stopwatch or watch with second’s hand
• Capillary pipee or automated pipee to deliver 100 µL and 200 μL
• Graduated volumetric pipees: 5-mL with 0.1-mL graduation
• Test tubes (small size, 5-mL, 12 mm × 75 mm)
Reagents
• Calcium chloride solution: Use good quality calcium chloride reagent. Calcium chloride
solution supplies the missing factor IV while testing the anticoagulated specimen.
A. Stock solution of calcium chloride, 0.2 M
Calcium chloride (CaCl2) 22.2 g
Distilled water (q.s.) 1000 mL
Store the stock solution in a refrigerator at 4°C.
B. Working solution of calcium chloride, 0.025 M
Dilute the stock solution eight-fold before use: 1 mL of stock solution mixed with
7 mL of distilled water. Prepare as much as will be used for the day; discard after
the day’s work.
• Test reagents: These are commercially available. Purchase from a reliable supplier.
Many of these reagents are imported as they are not locally available.
Note Reagents should not be left at room temperature for prolonged period. It is
recommended that a small amount of reagent be taken from the stock bole at the
beginning of the day’s work and discarded after use.
• Control specimen: Every batch of reagent must be checked against the control plasma.
This is a part of the quality control programme in order to provide reliable results. Many
physicians prefer to have the answer as percent control plasma because the absolute
value varies considerably. Collect blood from a normal subject in the same way as done
with the patient. Plasma obtained from a normal subject is the control plasma. Control
plasma should be drawn every day unless dehydrated control plasma is purchased
from the manufacturer.
Additional information
Laboratories of most developing countries are still using the manual method for performing
the coagulation tests. As all coagulation tests must be performed at 37°C, a well-maintained
water bath or a heating block that keeps 37°C temperature is needed. Clot formation is
observed by tilting the tube in which pre-warmed plasma and reagents are mixed.
The automated coagulation analyzers range from relatively simple ones to more complex,
fully automated ones. The technology that has been used the longest is based on the detection
of a brin clot using a moving wire probe. Now, most instruments use electromechanical
methods or photo-optical density to detect clot formation. In the former, laser detects the clot
formation; in the laer, the change in transmied light is measured as the clot forms. Some
coagulation analyzers use only plasma for testing; others use whole blood.
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Medical Laboratory Technology: Volume 1
Determination of Prothrombin Time (Figure 14.6)
The haemostasis pathway is normally activated when damage occurs to blood vessel
endothelium or to body tissue. The extrinsic pathway converts Factor X, a proenzyme, to
enzyme Xa (activated) that in turn converts prothrombin to the enzyme thrombin (Figure
13.3). An enzyme, by denition, is a protein that is able to cause or accelerate changes in
other substances (called substrate) without being changed. Thrombin acts on brinogen and
triggers the formation of brin monomer that makes up the initial unstable clot. This in the
following phase is hardened by the formation of brin polymer.
Prothrombin (Factor II) is produced in the liver and is vitamin К dependent. A deciency of
vitamin К causes reduced amounts of the factor to be produced and can result in bleeding. The
prothrombin time is used as a coagulation-screening test to measure the extrinsic pathway.
Its major use is to monitor oral anticoagulant therapy since these anticoagulants decrease the
production of prothrombin and factors VII, IX and X in the liver.
Clinical significance
The prothrombin time (PT) or pro time in short, is one of the most frequently performed
coagulation tests. It evaluates the function of the extrinsic and common pathways of
haemostasis. It is used not only as a pre-surgery coagulation-screening test, but also to
monitor Coumadin (warfarin) anticoagulant therapy. Prothrombin results can be used to
guide the physician in regulating the patient’s anticoagulant dosage. The test was developed
by Dr. A. J. Quick, who named it prothrombin time because he thought it measured only
prothrombin. Even though it was later discovered that the test actually measures prothrombin
plus additional factors, it is still called the prothrombin time.
Prolonged PT: Deciencies of factors II, V, VII and/or X.
Other conditions: Coumadin therapy, heparin therapy, liver disease and Vitamin К
deciency.
Normal PT: Deciencies of factors VIII, XI or XII.
Normal value: 14 (±2) s
Reference values for prothrombin time
The accepted prothrombin time reference value is 10–13 s. However, the prothrombin time
(PT) is now reported as either the prothrombin ratio or the international normalized ratio
(INR). The prothrombin ratio compares the patient’s result with the mean of the normal
population of the facility. The INR is used to standardize prothrombin time reporting among
dierent laboratories. The manufacturer assigns an international sensitivity index (ISI) to each
reagent lot. This is because each lot of thromboplastin can have dierent sensitivity in the
prothrombin test. The ISI is used in the INR formula to compensate for varying sensitivities
of thromboplastin reagent. Before the INR was used, a patient could have a PT test performed
on the same day at dierent laboratories and have dierent results. The reference value for
the INR is 1.0–1.4.
An example of calculation for geing the INR is given here.
ISI (provided by the manufacturer) = 1.2
If the patient’s result is 24 s and the normal for the facility is 13 s, the INR will be
The reference value for INR is 1.0–1.4. The above result is positively ‘prolonged’.
Laboratory Investigation of Bleeding Disorders
361
Figure 14.6 Determination of prothrombin time: (a) Transfer the calcium chloride solution in
a test tube (75 mm × 10 mm) and place in water bath, (b) ln a separate test tube,
add requisite quantity of reagent and plasma and wait for temperature equilibrium,
(c) Transfer the requisite amount of calcium chloride solution to the plasma tube,
(d) Start the stopwatch instantly; mix the three solutions—reagent, plasma and calcium
chloride, and leave the tube in the water bath for 10 s, (e) Take out and start tilting
intermittently; stop the watch instantly when jelly-like brin strands appear before the clot
forms. Repeat the test, the nal results should be within ±2 s
Specimen
The specimen for the PT must be collected with minimal trauma to the vein and surrounding
tissue to prevent the release of tissue thromboplastin into the sample. The blood is drawn
into a tube that contains 3.2% solution of sodium citrate. The vacuum tube contains 0.5 mL
of the anticoagulant and is manufactured to draw 4.5 mL of blood. If the vacuum tube is not
available, it is essential that the proportion of anticoagulant to blood be one part anticoagulant
to nine parts blood (1:9) in order for the test results to be valid. The tube of blood must be
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Medical Laboratory Technology: Volume 1
centrifuged as soon as possible and the plasma to be transferred to a clean tube for use in the
assay. The plasma is usually assayed within 4 h of collection. Refrigerated blood can be used
up to 6 h. If a procedure calls for pre-warming the sample, it should not stand at 37°C for
more than 5 min, before being tested. The expiration date of collection tubes must be checked
and be valid because the anticoagulant can evaporate over time.
Note
• The ratio of anticoagulant and blood is critical: one part anticoagulant (3.2% sodium
citrate) and nine parts blood.
• The lled tube should be immediately inverted to gently mix the blood and anticoagulant.
• While separating the plasma from the blood cells by centrifugation, the tube of blood
must be stoppered to prevent exposure to the air.
• All specimens, controls, and instruments must be at the proper temperature before the
test is performed.
Principle
The plasma and commercial reagents are warmed to 37°C in separate tubes. The plasma is
then forcefully added to the reagent while the tube is in the water bath (37°C). A timer is
started, and at the end of 10 s, the tube is picked up, held horizontally in good light and
gently tilted back and forth until a thickening appears (Figure 14.6). This is the brin clot and
the timer is stopped when it appears. The time for the clot to form is recorded in seconds. It
is recommended that the test be run in triplicate. Timing of the rst test will be approximate
and the remaining two should agree with each other. Abnormal and normal controls plasmas
are analysed with the patient samples.
Procedure (Figure 14.6)
Note If commercial preparations are used, follow the manufacturer’s instructions.
1. Check the temperature of the water bath (37°C) and place the test reagent to warm
up for 5 min. Do not take out large amounts of test reagent; keep the stock solution in
ice at the side of the water bath. Use of automatic pipee in dispensing reagents and
specimens is convenient (Figure 14.7). Alternatively, use capillary pipees.
2. Deliver 0.1 mL of citrated patient’s plasma into the
boom of a 5-mL test tube (75 mm × 10 mm) and
place it in the water bath at 37°C. The specimen
must not be le at 37°C for more than 1 min.
3. Add 0.2 mL of test reagent to the test tube
containing plasma by blowing from a pipee as
quickly as possible, with the tip of the pipee
just above the surface of the plasma and start a
stopwatch at the same instant.
4. Hold the tube with its lower end submerged in
water (37°C) and gently tilt the tube back and
forth until the rst strand of brin appears. Tilting
is done by gently inclining the tube from the
vertical to just short of the horizontal so that the
uid content (plasma) can be observed for the rst
signs of cloing.
Note If you are directly observing the clot, the
appearance of brin may well become evident by
sudden formation of a clot as the tube is tilted in
the water bath at the same time.
Figure 14.7 Automatic pipette
for dispensing small
quantities of reagent
and specimen
Laboratory Investigation of Bleeding Disorders
5. Repeat at least once for each specimen and record the mean time. The results should
agree with each other within ±1 s when the PT is less than 30 s.
6. Include the normal plasma (control) in each batch of tests. The result of the normal
plasma must be within the normal range (14 ± 2 s).
Reporting of results
Report the result in seconds and/or as percent of the normal.
363
deTerminaTion of acTivaTed ParTial ThromBoPlasTin Time
Clinical significance
The primary reason for requesting APTT test is to diagnose haemophilias that involve
deciencies of VIII, IX or XI. The test can also detect deciencies of factors V, X and XII;
however, it is relatively insensitive due to lack of factor VII. Activated partial thromboplastin
time (APTT) will also be prolonged in the presence of inhibitors of coagulation and in DIC
(disseminated intravascular coagulation). Hence, APTT is sensitive for various specic and
non-specic circulatory anticoagulants. It is widely advocated as the test of choice for the
control of heparin therapy.
Principle
Partial thromboplastin is the reagent used in performing the APTT. Partial thromboplastin,
the lipid portion of tissue thromboplastin, is manufactured from human or bovine brain
tissue or derived from soyabeans. Since partial thromboplastin performs the function of PF3
in the APTT test, platelet abnormalities will have no eect on the APTT.
The formation of a brin clot in the APTT can occur only if factors in the intrinsic pathway—
XII, XI, IX and VIII—and those in the common pathway—I, II, V and X—are present in
sucient amounts and are functional.
Normal range
35–40 s
It is best if each laboratory establishes its own normal range by periodically testing several
plasmas from normal patients. When the APTT is used to monitor heparin therapy, the usual
goal is to keep the patient’s APTT 1.5-2.0 times the APTT of the normal plasma control.
Specimen
Blood is collected using 3.2% sodium citrate anticoagulant. The proportions are one part
sodium citrate (0.5 mL) to nine parts (4.5 mL) blood, which is 1:9. Vacuum tubes are available
containing this volume and also in smaller sizes for paediatric use. After collection, the whole
blood sample is centrifuged and the plasma is removed and placed in another tube. The
plasma should be stored in a stoppered tube at 4°C until used for the test. The test should be
ran within 4 h of blood collection.
Reagents
Use commercial reagents and follow the procedure as instructed by the manufacturer. The
laboratory may have to supply calcium chloride solution (0.025 M). Citrated normal plasma
should be available for running the normal control.
• Calcium chloride solution: Use good quality calcium chloride reagent. Calcium chloride
solution supplies the missing factor IV while testing the anticoagulated specimen.
• Stock solution of calcium chloride, 0.2 M
Calcium chloride (CaCl2) 22.2 g
Distilled water (q.s.) 1000 mL
Store the stock solution in a refrigerator at 4°C.
• Working solution of calcium chloride, 0.025 M
364
Dilute the stock solution eight-folds before use: 1 mL of stock solution mixed with 7 mL of
distilled water. Prepare as much as will be used for the day and discard after the day’s work.
Procedure
The procedure given in the kit should be followed. The following is a general procedure
associated in most cases:
1. Pre-warm reagents at 37°C for at least 3 min but no more than 10 min. This includes
patient’s plasma, calcium chloride solution and the control plasma.
2. Prepare the patient’s samples by adding 0.1 mL of patient plasma and 0.1 mL of
thromboplastin reagent in a glass test tube (12 mm × 75 mm) that is held in a 37°C water
bath or on a heating block. These are allowed to warm and activate for 3 min.
3. To initiate the cloing reaction, blow in 0.1 ml of calcium chloride solution held in a
capillary pipee and start the stopwatch simultaneously.
4. Mix and leave undisturbed for 20 s.
5. Gently tilt the tube to observe the appearance of brin strands, the end point, which is
usually sharp.
Note The tube can be taken out of the water and tilted under the spotlight in order to
note the end point.
6. At the appearance of strands, stop the stopwatch and note the time.
7. Replicate all tests.
Medical Laboratory Technology: Volume 1
raPid haemosTaTic TesTs and PoinT-of-care insTrumenTs
Some medical situations require rapid haemostasis test results. These include circumstances
in which patients are receive heparin therapy or when conditions such as disseminated
intravascular coagulation (DIC), deep vein thrombosis (DVT) or pulmonary embolism are
suspected. Haemostatic tests used to help in diagnosis or in prescribing treatment for these
conditions include the activated cloing time (ACT) and APTT for monitoring heparin therapy.
Tests for brinogen/brin degradation products (FDP), such as the D-dimer test, are used
for suspected cases of DIC, DVT or pulmonary embolism. Several small portable coagulation
analyzers are available that can be used for ‘near-patient testing’ or at the point-of-care to
perform these tests injust a few minutes. These instruments can be useful in the rural seings of
developing countries. They are often baery operated and need only a small sample of blood.
Clinical significance
Heparin therapy: Heparin is an anticoagulant that inhibits the activated forms of factors
IX, Χ, XI and XII as well as platelet-release factor. It is prescribed to prevent thrombosis in
patient undergoing cardiac angioplasty, joint replacement and other procedures with risk of
clot formation. Heparin can also be prescribed in patient who has thrombosis or emboli. Since
patients vary in their response to heparin, and heparin from dierent sources has varying
activity, the blood levels of the anticoagulant must be closely monitored during therapy.
Disseminated intravascular coagulation (DIC)
This is a life-threatening condition in which widespread thrombosis and secondary
haemorrhages occur due to a malfunction in mechanisms that maintain the balance between
cloing and dissolution of the clot (brinolysis). The result is pathological cloing and/or
excessive clot dissolution. Patients can develop DIC from injuries that cause widespread
damage to the vascular system, such as crush injuries sustained in construction work
or automobile accidents. Certain bacterial and viral infections can also cause DIC. The
haemorrhaging is caused by brinolysis combined with depletion of platelets and coagulation
factors.
Laboratory Investigation of Bleeding Disorders
365
Deep Vein Thrombosis (DVT) and pulmonary embolism
Formation of a thrombus or thrombi (plural), caused by slow blood ow or stasis in the large
veins, is the primary cause of DVT. This may happen in legs due to long periods of inactivity
of other parts of the body. Pulmonary embolism occurs when a dislodged clot is carried to
the lungs and blocks a pulmonary vessel. Thus, pulmonary embolism can be a complication
of DVT. Both DVT and pulmonary embolism are dicult to diagnose by clinical symptoms
alone. Pulmonary embolism is potentially a lethal condition and requires rapid diagnosis and
treatment for recovery.
Defective fibrinolysis
Under normal conditions, the brin clot formed from brinogen is converted from soluble
monomer to insoluble stable polymer. The laer plugs the hole, stops bleeding but the
insoluble brin clot is dissolved by plasmin (an enzyme) in order to keep the surface of the
blood vessel smooth without hindering blood ow. The process is known as brinolysis.
During this process various brin degradation products (FDP) are formed and are eventually
cleared from the body. Unfortunately, under certain clinical conditions, lysis of stable brin
clots also results in the formation of cross-linked FDPs called XDPs. The dérivâtes of XDPs
are proteins known as D-dimers. It is important to distinguish between FDPs and XDPs since
the presence of XDPs indicates a more serious clinical condition.
TesTs for fiBrin degradaTion ProducTs (fdP) or d-dimer
Fibrin degradation products (FDPs) and cross-linked DPs (XDPs) can be measured manually
by the immunologic method of latex agglutination. Alternatively various handheld analyzers
are available for near-patient testing. Both these tests can dierentiate between FDPs and
XDPs present in patient’s plasma. Tests for D-dimer are useful in diagnosing DIC, DVT and
pulmonary embolism.
In the latex agglutination tests, the latex beads are coated with a monoclonal antibody
specic for the D-dimer. When the patient sample is mixed with the test kit reagents on a special
slide, any D-dimer present is bound to the antibody on the beads and visible agglutination
occurs. Positive agglutination indicates the presence of D-dimer. Absence of agglutination
will be considered as normal where FDPs are present. The technique of latex agglutination
test is further described in the Immunology section. Many kits are available in the market and
results may be available within 3 min. In reporting the results, the degree of agglutination
may be mentioned in order to provide a semiquantitative measure of D-dimer in circulation.
The reference values for FDP are: any value <0.20 μg/mL is considered negative; any value
>0.20 μg/mL is considered as positive.
Specimens for the testing include citrated plasma, serum or urine, as instructed by various
manufacturers in their kits. Heparin or EDTA anticoagulants cannot be used. All reagents
must be brought to room temperature before tests are performed. The policies of the facility
and manufacturer’s package inserts must be followed concerning the number of controls to
be run. For the D-dimer latex agglutination tests, pipeing must be precise; the tests must be
read immediately at the specied time to avoid false-positive reactions caused by drying of
the latex.
Procedure
1. Following manufacturer’s instructions, mix the patient sample (serum, plasma, urine)
with test kit reagents on a special slide provided by the manufacturer with the test kit.
2. Mix the sample and reagent together in a circular gently motion. Simultaneously start a
timer for measuring the incubation period.
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Medical Laboratory Technology: Volume 1
3. Immediately at the end of the time period (recommended by the manufacturer), the
slide is inspected for agglutination.
4. The presence of agglutination is a positive result; absence of agglutination is a negative
result.
ProTamine sulPhaTe TesT
This is a simple test for the identication of the presence of FDR Formation of a clot occurs
in two steps. In the rst step, brin monomer is formed which is soluble and reacts with
protamine sulphate to produce a precipitate.
Clinical significance
A positive protamine sulphate test indicates a possible problem in brinolysis.
Reagents
• Protamine sulphate (Sigma Chemical Co., St. Louis, Mo., USA): 1% w/v solution in
imidazole buer. Store at 4°C. Make a fresh solution weekly. Some authors recommend
the use of barbitone buer, pH 7.3.
• Imidazole buer: In a 200-mL beaker place 0.68 g of imidazole, 1.17 g of NaCl and about
100 mL of distilled water. Dissolve ingredients and adjust the pH to 7.3 with 0.1 N HCl.
Note This requires approximately 37 mL of acid. After adjusting the pH, make the volume to
200 mL in a graduated cylinder or volumetric ask.
Procedure
1. Transfer 1 mL of plasma to the boom of a test tube (5-mL) and place in the water bath
(37°C).
2. Add 0.1 mL of 1% protamine sulphate to the plasma, mix and let the mixture incubate
for 3 min in the water bath.
3. Read against indirect lighting and a black background.
Grade the results as follows:
3+ Solid white clumps
2+ Fibrin strands as white material
1+ Coarse precipitate
± Fine precipitate
0 Clear solution
With a 2+ to 3+ reaction, the presence of brin monomer and early FDP is suspected.
laBoraTory diagnosis of Bleeding disorders
Most haemorrhagic disorders are inherited (e.g., haemophilia) and a few may be acquired
(e.g., liver disorder and prothrombin deciency). Classical haemophilia (haemophilia A,
caused by factor VIII deciency) is sex-linked and is found only in males, while females act
as carriers. This is also true for haemophilia B, caused by the deciency of factor IX. On the
other hand, haemophilia С (caused by factor XI deciency) is autosomal and is found in
both males and females. Medical history, family history and physical examination identify
Laboratory Investigation of Bleeding Disorders
367
bleeders; however, identication of the missing factor without the assistance of the laboratory
is not possible.
The following are some of the clinical conditions associated with bleeding disorders and
the corresponding laboratory tests, which may be able to identify the defect (Table 14.2):
1. Vascular defect: Bleeding time.
2. Coagulation factor deciency: Cloing time, PT and APTT. For specic factor deciency,
perform substitution tests.
3. Decreased platelet count or dysfunction of platelets: Platelet count, bleeding time, clot
retraction.
4. Excessive brinolysis: Related to the intake of certain drugs, diagnosed by clot lysis,
protamine sulphate test.
5. von Willebrand’s disease (vWD): It is a combination of disorders with vascular
defect, platelet defect and factor VIII deciency. It has an autosomal inheritance. This
is frequently diagnosed by abnormal bleeding time, normal platelet count, abnormal
cloing time and prolonged APTT.
6. Liver disorder and vitamin К deciency: The liver synthesizes most of the cloing
factors, and several of these are vitamin К dependent (II, VII, IX and X). Vitamin К being
fat soluble, its absorption into the body depends on the bile produced by the liver. Thus,
liver disorders and/or lack of vitamin К may lead to prolonged APTT and PT.
Substitution Tests
The crux of any coagulation deciency investigation is the proof that the patient’s plasma is
corrected by substitution of the missing factor. It is of particular importance for the dierential
diagnosis of the three major types of haemophilias (Table 14.1).
Table 14.1 Identication of common coagulation factor deciencies by substitution test
Decient
coagulation factor
II Pr Pr APTT not corrected by aged serum/
V Pr Pr PT corrected by adsorbed plasma
VII Pr Nr PT corrected by aged serum/plasma
VIII Nr Pr APTT corrected by adsorbed plasma
IX Nr Pr APTT corrected by aged serum/
X Pr Pr APTT corrected by aged serum/
XI Nr Pr (SI) APTT corrected by aged serum/
VWD Nr Pr (Md) APTT corrected by adsorbed plasma
Pr-Prolonged; Nr-Normal; Sl-Slight; Md-Mild
Results of basic coagulation tests Results of substitution tests
APTT PT
plasma or adsorbed plasma
plasma
plasma
plasma and adsorbed plasma
(variable)