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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
Laboratory Investigation of Bleeding Disorders
Chapter Outline
• Basic Screening Tests for Bleeding Disorders
▪ Bleeding time determination
▪ Determination of whole blood cloing time
▪ Clot retraction and lysis time
▪ Interpretation of clot morphology
• Coagulation Tests
▪ Determination of prothrombin time
▪ Determination of activated partial thromboplastin time
• Rapid Haemostatic Tests and Point-of-Care Instruments
• Tests for Fibrin Degradation Products (FDP) or D-dimer
• Protamine Sulphate Test
• Laboratory Diagnosis of Bleeding Disorders
▪ Substitution tests
• Therapy of Bleeding Disorders
• Review Questions
14
Ritwik Bhatia
Laboratory investigation of bleeding disorders is set up for patients who have a history
of spontaneous bleeding or excessive bleeding after trauma or surgery. The tests are also
undertaken for bleeding tendency in patients who have to undergo major surgery.
Acquired haemorrhagic disorders are more common than inherited disorders, and
platelet disorders are more common than the coagulation disorders. In patients with inherited
bleeding disorders, a family history must be investigated. Among the inherited coagulation
disorders, factors VIII and IX deciencies are the most common. They are transmied as
sex-linked disorders and aect males while females act as carriers of the recessive genes.
Other inherited disorders of coagulation such as factor XI deciency are transmied as
autosomal disorders and occur with equal frequency in males and females. The common
causes of acquired bleeding disorders are thrombocytopaenia (which can occur by a variety of
immune, non-immune, infectious and drag-induced, and malignant conditions), deciency of
vitamin К (due to hepatic or obstructive jaundice), therapy with the Coumadin group of drags
and others, disseminated intravascular coagulation (DIC), gammopathies and liver failure.
Various haemostasis tests are performed to identify bleeding disorders or to follow up
anticoagulant therapy. These tests are either done on the patient (like bleeding time) or the
patient’s plasma is taken for laboratory coagulation tests (like PT, APTT). In most cases,
citrated blood is used for geing the plasma. The principle behind these laboratory tests
Laboratory Investigation of Bleeding Disorders
calls for reagents, supplied by commercial companies that have ingredients at dierent
levels of coagulation pathways. Patient’s plasma supplies the missing factor(s) and the time
taken for the formation of clot determines the possible insuciency in the patient’s plasma
as compared to the normal plasma (normal time). The time taken for the formation of clot is
determined either manually or by an automated system.
Testing for the detection of brinogen degradation product (FDP) is called for when
there is high clinical suspicion for DIC or pulmonary embolism. In case of a positive nding,
conrmatory tests must be done to detect abnormalities in platelet function or deciency of
specic coagulation factors and defects in the brinolytic system. In any case of pancytopaenia
or severe thrombocytopaenia, where the diagnosis cannot be made with peripheral blood,
bone marrow aspirate must be obtained to exclude the possibility of acute leukaemias.
349
Basic screening TesTs for Bleeding disorders
Two basic screening tests—bleeding time and cloing time—are commonly used in the
coagulation laboratories of developing countries for the presumptive diagnosis of bleeding
disorders in a patient.
Bleeding Time Determination
Determination of bleeding time recognizes vascular defect and platelet disorder. The laer
is further conrmed through other tests described later. Prolonged bleeding time is generally
found with thrombocytopaenia (platelet count <50000 platelets/μL) and where there is a
platelet dysfunction. In case of von Willebrand’s disease, which is caused by a platelet defect
combined with factor VIII deciency, bleeding time is high with a normal platelet count.
Normal range
Duke method: 1–5 min
Ivy method: 5–11 min
Specimen
Performed on patient; no specimen necessary.
Principle
A standard incision is made in the skin of the patient and the length of time required for
bleeding to cease is recorded. There are two methods applied in the determination of
bleeding time—the Duke method and the Ivy method. The Duke method is now considered
to be obsolete because the results vary widely. Both the Duke method and Ivy method are
described in this book as many laboratories still follow the old Duke method. With Duke
method, the incision is made on the earlobe while in case of Ivy method, the incision is made
on the forearm under standardized conditions. The Ivy method is more reliable and should
be the method of choice.
Duke method
Duke method is easy to perform and requires minimal equipment or laboratory skill.
Supplies
• Alcohol sponge
• Sterile lancet
• Filter paper (circular) or bloing paper
• Stopwatch or a watch with seconds hand
Note Arrange all the necessary materials before starting the test. As the test is performed
on the patient, you must approach the patient with proper bedside manner and must be well
350
Medical Laboratory Technology: Volume 1
organized while performing the test. This improves public relations and builds condence in
the patient.
Procedure (Figure 14.1)
1. Gently clean the earlobe with an alcohol sponge. Do not rub. Allow it to dry completely.
2. Hold a glass slide behind the earlobe for support. Make a deep puncture with a sterile
lancet. Start the stopwatch and discard the glass slide. The blood should ow freely,
without squeezing the earlobe.
3. Using a circular lter paper, blot the drop of blood coming out of the incision every 30 s.
Do not allow the lter paper to touch the bleeding spot. Keep each subsequent drop a
lile further along the side of the lter paper; the drops become progressively smaller.
Figure 14.1 Determination of bleeding time by Duke Method. Ear lobe is the puncture site for this
method (a). Make a skin puncture on the ear lobe (b) and instantly start the stopwatch
(c). Collect the rst drop of blood on the corner of a blotting paper (d) and every 30 s
thereafter (e–g). When no more blood appears, stop the stopwatch and note the time on
the watch. Alternatively, count the number of drops collected and multiply it by 30. In the
illustration it is 7 × 30 = 210 s (i.e., 3 min and 30 s).
4. When bleeding ceases, stop the stopwatch.
5. Count the number of drops on the lter paper and multiply by 30 s.
6. Report to the closest minute.
Note If the bleeding continues for more than 10 min, discontinue the test and apply pressure
to the bleeding spot.
Ivy method
Ivy method is more reliable than the Duke method. It, however, requires some additional
equipment and skill to use sphygmomanometer.
Laboratory Investigation of Bleeding Disorders
Equipment and supplies
• Sphygmomanometer
• Sterile disposable lancet—capable of making an incision of 1 mm wide and 3 mm deep
• Filter paper (circular) or bloing paper (rectangular)
• Alcohol 70%
• Coon wool or surgical gauze
Procedure
1. Place the sphygmomanometer cu on the patient’s arm, above the elbow. Inate the
cu to 40 mm of mercury and hold this exact pressure for the entire period.
2. Choose an area approximately three ngers width below the bend in the elbow on the
volar surface. Clean the area with the alcohol sponge.
3. Hold the skin tightly by grasping the underside of the arm rmly and make two
separate punctures 5–10 cm apart, in quick succession, using a disposable lancet. Start
the stopwatch.
Note It is essential for you to be purposeful and precise as tentative jabs may not bleed
satisfactorily. Avoid any subcutaneous veins. The disposable lancet available for this
purpose can be inserted freely to its maximum depth without fear of penetrating too
deeply (B–D Microlance, Becton Dickinson Ltd., USA).
4. Blot the blood from each incision site on a separate piece of circular lter paper every
30 s. The lter paper should not touch the incision point at any time.
5. When bleeding ceases, stop the watch and release the blood pressure cu. Record
the bleeding time of the two punctures. The longer of the duplicate bleeding time is
probably a more accurate assessment of the result of the test than the average of the two.
6. Unless there is some technical reason to disregard the longer time (e.g., puncture of a
supercial vein), this should be reported as the result. The normal range for bleeding
time by Ivy’s method is up to 11 min. If the bleeding continues for more than 15 min,
apply pressure to the wound site, stop the bleeding and repeat the bleeding time on the
other arm. If the bleeding does not cease within 15 min, stop the bleeding by pressure
and report the result as greater than 15 min.
351
Determination of Whole Blood Clotting Time
When taken out of the body, normal blood clots within few minutes unless prevented by an
anticoagulant. The time taken for the whole blood to clot is its cloing time. Two methods are
applied for the determination of cloing time—Lee–White method and Capillary method.
Lee–White method
Lee–White cloing time is a screening test for determining the coagulation defect. The
method is not very sensitive but because of the ease in performing the test, it is used along
with a baery of other screening tests. Only severe cloing factor deciency is recognized
by this method. In case of prolonged cloing time (more than 10 min), the patient should be
subjected to more detailed tests for the identication of missing coagulation factors. Presence
of a circulatory anticoagulant, like heparin (used as a therapeutic drug), will also cause
abnormally long cloing time. This procedure is therefore used to monitor heparin therapy.
However, APTT and PT are beer tests for this purpose.
Two methods will be described here, venepuncture method and capillary method. The
laer is not considered to be as sensitive but it is easy to perform.
Normal range
4–9 min
352
Specimen
The test is done at the side of the patient with freshly drawn blood without any anticoagulant.
Principle
Venous blood is collected in a clean glass tube (acid-washed) without any anticoagulant. The
length of time that it takes for the blood to clot or coagulate at 37°C is the cloing time or
coagulation time. In case of alternative capillary method, following skin puncture, blood is
taken into a plain capillary tube and the stopwatch is started. The formation of brin string is
noted by breaking the capillary tube at regular interval. The time taken for the rst appearance
of the brin string is noted as the coagulation time.
Medical Laboratory Technology: Volume 1
Capillary method
This method is insensitive and unreliable, and should be used only if venous blood cannot
be obtained. It is essential to obtain a rapid, free ow of blood, requiring no squeezing or
manipulation of the puncture wound. If manipulation is required, the test must be abandoned.
Equipment
1. Disposable lancet
2. Capillary tubing (10 to 15 cm length and 1.5 mm diameter) without anticoagulant
Procedure (Figure 14.2)
1. Warm up the nger for skin puncture.
2. Make an incision with a sterile disposable lancet to a depth of 3 mm. As soon as the
blood is visible, start the stopwatch.
3. Wipe o the rst drop of blood and allow the ensuing drop of blood to ow into two
capillary tubes held side-by-side.
4. After 2 min, break o the capillary tube, 1 to 2 cm from the end, with the help of a glass
le.
5. When a thin string of brin can be seen between the broken ends of the capillary tubes,
stop the watch and note the time.
6. When reporting the time, mention that the capillary tube method was used.
Venepuncture method
This is more reliable and sensitive than the capillary method. It, however, requires special
arrangements for venepuncture and a temperature-controlled water bath.
Equipment
• Arrangement for blood drawn by venepuncture—coon wool, surgical gauze soaked in
70% alcohol (or other disinfectant), plastic syringe (10-mL) and 20 gauge needle
• Test tubes: acid-washed (10-mL), marked to hold 1 mL uid
• Water bath at 37°C or wide-mouthed thermos containing water at 37°C. The laer is
taken to the bedside of the patient
Laboratory Investigation of Bleeding Disorders
353
Figure 14.2 Capillary method of determining clotting time. (a) Perform skin puncture, draw blood
in a capillary and start the stopwatch. (b) Break the capillary in portions and (c, d) stop
the watch when ne strands of brin are seen between the broken ends. (e) Stop the
stopwatch instantly and note the clotting time.
• Stopwatch
• Coon wool
Procedure (Figure 14.3)
1. Collect over 2 mL of venous blood by routine venepuncture. Use a plastic syringe and
20 gauge needles. Venepuncture must be carried out quickly and without any
interruption. Start the stopwatch as soon as the blood enters the syringe.
2. Remove the needle from the syringe and ll each of the two tubes to the 1-mL mark.
3. Plug the tubes with non-absorbent coon wool and place them in the water bath at
37°C. Some laboratories perform the test at room temperature but the results are not
consistent. It is prolonged in cold temperature and hastened in hot weather.
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Figure 14.3 Determination of clotting time and clot retraction time by Lee-White method (see text for
details). A. Clotting time (α–f): Perform venepuncture and start the stopwatch as soon as
blood appears in the syringe (a). Take o the needle and transfer the blood specimen (b) to two
clean small-size test tubes. Note the whole blood clotting time sequentially in both tubes (c–f).
B. Clot retraction time (g–i): Transfer the clotted blood to a water bath or leave at
room temperature (g, h); examine the clot formation after the specied time. Note the
morphology of the clot formed—normal (]), lack of retraction or partial retraction indicates
abnormalities. Platelet deciency (k-1, 3), brinogen deciency (k-2), abnormal plasma
protein (k-4), and haemophilic condition (k-5). When lysis occurs (k-6), red cells sink to
the bottom.
4. After 3 min, remove the rst tube from the water bath and tilt the tube gently to an angle
of 45° to see whether the blood has cloed. If the blood has not cloed, return the tube
to the water bath and examine it at 30 s intervals to see if it has cloed.
5. When the blood is cloed, the tube can be tilted through an angle of 90° without spilling
the contents. As soon as the blood is cloed, immediately examine the second tube. The
blood in the second tube usually clots very soon after the blood in the rst tube.
Laboratory Investigation of Bleeding Disorders
6. Stop the stopwatch and note the time.
7. The coagulation time is the cloing time of the second tube.
8. Bring the cloed blood to the laboratory and continue the test for the determination of
clot retraction and lysis time.
Common sources of error
Cloing time can vary widely if the technique is faulty. One of the most common errors is
taking an inappropriate volume of blood for cloing time determination. Volumes less than
1 mL of blood will give a shorter time. Other technical errors include faulty venepuncture,
inaccurate incubation temperature, air bubble in the syringe, incorrect timing and unnecessary
agitation of blood specimen. Always use clean glassware and a plastic syringe. The laer
eliminates the use of siliconized glassware.
355
Clot Retraction and Lysis Time
A clot forms as the end product of blood coagulation. The clot under normal conditions
undergoes contraction, where serum is expressed from the clot and the clot becomes denser.
A substance released by the platelet, called thrombosthenin, is responsible for clot retraction.
Normal clot retraction starts within 30 s after the blood has cloed, and is about 50% at the
end of 1 h. Clot retraction is requested in case of suspected haemorrhagic disorders related to
platelets. In case of thrombocytopaenia, poor clot retraction is noted, which is also found in
the case of poor platelet function with normal platelet count (thrombasthenia).
In order to observe brinolysis, incubation of the clot is continued. Lysis is enhanced in case
of brinolytic diseases. The test is rather insensitive but requires no special eort to perform it
in addition to cloing time determination. Abnormal cases must be further pursued in special
laboratories to detect brin degradation products (FDPs).
Normal value
Clot retraction: When measured after 1 h of blood collection (blood kept at 37°C). Abnormal
clot retraction is reported when less than 50% retraction occurs at 1 h.
Lysis time: The lysis time for a normal clot is about 72 h. Fibrinolysis is considered abnormal
when lysis is seen within 24 h.
Principle
Retraction of the clot is recognized by the fact that the clot leaves the sidewall of the test tube
(Figure 14.3). Although clot retraction starts within 30 s after cloing, it is clearly seen after
1 h. The brin clot dissolves due to brinolysis which is a natural process. With brinolysis,
the clot, initially formed as a result of coagulation, becomes uid (dissolves) and the red cells
sink to the boom of the test tube.
Specimen
This test is in continuation with the cloing time determination.
Equipment
As with cloing time determination, continue the process of incubation in the water bath
(37°C).
Procedure (Figure 14.3)
1. Continue to incubate the tubes in the water bath after determining the cloing time
(Lee–White method).
2. Examine the clot after 1 h and 24 h.
3. Report the clot morphology (size and consistency) and nature of retraction (normal,
doubtful or poor). If the clot has partly left the side wall of the tube, and is about 50%
retracted, the retraction is considered to be normal.
356
4. After reporting the clot morphology and retraction, examine if there is any lysis. Lysis
is recognized by the dissolution of clot; the red cells sink to the boom of the tube.
Observation for lysis continues until 72 h (normal). Report the lysis time. Shortened clot
lysis time (1 to 48 h) indicates increased brinolytic activity.
Medical Laboratory Technology: Volume 1
Interpretation of Clot Morphology
In case of normal retraction, the red clot is well separated and it is aached to the sides of
the tube. There may be a small deposit of red cells at the boom of the tube. Lack of a rm
clot or formation of a small clot at the boom of the tube with a thin strand of brinogen
indicates possible brinogen deciency. Special biochemical methods will be able to assess
the brinogen content.
In thrombocytopaenia and defects of platelet function, the clot does not retract completely
from the side and hardly any serum is exuded. Thrombocytopaenia is detected by the platelet
count. In case of haemophilia, there may be no clot at all, or a yellow clot at the surface which
forms very slowly over the red cell deposit at the boom. Abnormal plasma protein, formed
during lymphoproliferative disorders, may also aect the morphology of the clot. There may
be yellow-coloured cloed plasma at the surface, beneath which is the poorly retracted red
clot.
coagulaTion TesTs
Coagulation studies require special laboratory preparation and care in specimen (blood)
collection. If the specimen is not collected or stored properly, the results will not be reliable.
It is important to remember that some of the coagulation factors commonly sought (e.g.,
factor VIII) are labile and will disappear from a normal specimen if it is left unaended at
room temperature for some time. This leads to erroneous results. In addition, the amount of
anticoagulant used or the container used for collecting specimen will also aect the results of
coagulation studies.
Specimen collection for coagulation studies
Citrated plasma is the principal specimen used in coagulation studies (Figure 14.4). Blood is
collected by venepuncture, and during the collection of blood, avoid undue venous stasis and
frothing (formation of air bubbles) in the plastic syringe. Immediately after the collection of
blood, it should be placed in ice for transportation. A portable wide-mouthed thermos can be
used for cooling the specimen. EDTA-anticoagulated blood is used only for the platelet count
to the boom.
Supplies (Containers)
Thoroughly cleaned penicillin boles of 10-mL capacity with clean stoppers can be used.
To prepare containers for blood collection, rst make a marking on the bole at the 5 mL
level. Then transfer 0.5 mL of the sterilized citrate solution into the container. Following
venepuncture, when the whole blood is brought to the mark of 5 mL, the dilution will be 1:10.
Reagent
Anticoagulant solution (trisodium citrate, 0.106 M):
Trisodium citrate (Na3C6H5O7 2H2O) 31.3 g
Distilled water (q.s.) 1000 mL
Laboratory Investigation of Bleeding Disorders
Figure 14.4 Preparation of plasma specimen for coagulation tests: (a) Blood specimen, collected by
venepuncture, is anticoagulated with citrate, (b–d) Plasma is separated for coagulation
tests, (c) Use of Pasteur pipette with teats is recommended instead of mouth pipetting
357
Dissolve the citrate in about 800 mL of water placed in a 1-L volumetric ask and then
dilute the solution to the mark. Divide the solution in convenient volumes (e.g., 10 mL) into
small boles ( 15-mL capacity) with stopper and sterilize boles by autoclaving. The sterilized
boles can be kept at room temperature.
Procedure
Venepuncture procedure (Macromethod)
Venous blood collected by venepuncture should be used whenever possible. It yields more
blood for multiple tests.
1. Keep the blood collection boles containing 0.5 mL of citrate solution on the table, lid
open and ready for use.
2. Draw blood by the usual venepuncture technique without undue stasis, in a 5 mL plastic
syringe ed with a short 19 to 20-gauge needle.
3. Remove the needle and transfer the collected blood specimen into the blood collection
bole up to the 5-mL mark.
Alternatively Take 0.5 mL of citrate solution into a 5-mL plastic syringe prior to
collection of 4.5 mL of venous blood. Pull the plunger further back and then gently mix
the blood with the citrate. The empty space created by the withdrawal of the plunger
allows the blood to mix.
4. Pour the anticoagulated blood into a test tube and centrifuge for 10 min at about 2500
rpm.
5. Promptly separate the plasma, which is used in the coagulation tests. If the plasma
cannot be used immediately, it should be refrigerated. Plasma left at room temperature
loses the labile factors; this may lead to erroneous results (Figure 14.5).
Skin puncture procedure (Micromethod)
This method is applied for infants and small children and for patients where venepuncture
is dicult. Plasma is not separated from the specimen collected by skin puncture. Instead,
whole blood is used after mixing the anticoagulant (citrate) with the blood specimen.