Capillary fragility (resistance) test:

This test is used to determine the presence of vascular disorder, a circle 2.5
cm in diameter, the upper edge of which is 4 cm below the crease of the
elbow, is drawn on the inner aspect of the forearm, pressure midway between
the systolic and diastolic blood pressure is applied using sphygmomanometer
above the elbow for 15 minutes, and a count of petechiae within the circle is
made: 10, normal; 1020, marginal; more than 20, abnormal.

Assays for primary Haemostasis

Bleeding time
o Von Willebrand factor:
Antigen determination
Activity
oPlatelet count
oPlatelet aggregation
oActivation markers (b-TG, PF4, GP V)
oSpecialised tests for platelet function

Laboratory investigation of platelets and platelet function:

A number of simple tests are employed to assess the platelet dysfunction and
thrombocytopenia:
Blood count and blood film examination:
o Peripheral blood platelet count and skin bleeding time are the first-line
tests for platelet function.
o If the results of these two tests are within the normal limits, it is
unlikely that a clinically important platelet defect is responsible for the
bleeding tendency.
o Additional information may be obtained by inspecting a fresh blood
film which may show abnormalities of platelet size or morphology
which can be diagnostic importance.
o If the screening tests suggest a disorder of platelet function, further
investigations are organized.
o Drugs and certain foods may affect the platelet function tests (look for
the table below) and the patient must be asked to refrain from taking
such substances for at least seven days before the test.
o Platelet function tests can be divided into five main groups:
1. Adhesion tests
2. Aggregation tests
3. Assessment for the granular contents and the release reaction

1
 4. Investigation of the prostaglandin pathway
5. Test for platelet coagulant activity.

Blood count & film

Low platelet count Normal platelet count

1- Bone marrow examination 1- Bleeding time

2- Platelet antibodies 2- Platelet aggregation studies

3- Screening test for DIC
with ADP, adrenalin, collagen
and restocetin.
3- Other special platelet tests,
e.g. Adhesion studies, pool
measurement.
4- Factor VIII clotting assay.
vWF assay. vWF antibody
assay.

Screening tests:
As thrombocytopenia is a common cause of bleeding abnormality, patients
with suspected bleeding disorders should be initially have a blood count
including platelet count and blood film examination (in addition to
establishing the presence of thrombocytopenia, the cause may be obvious,
e.g. acute leukaemia).

Bleeding time test (BT):

When blood count, platelet count and blood film examination are normal the
bleeding time should be done to detect abnormal platelet function
(dysfunction).

Principle:
A standard incision is made on the volar surface of the forearm and the
time the incision bled is measured. Cessation of bleeding indicates the
formation of haemostatic plugs which are in turn dependent on the

2
 adequate number of platelet and the ability of the platelet to adhere to the
subendothelium and form aggregation.

Methods for BT:

a- STANDARDIZED TEMPLATE METHOD:
Materials:
1. Sphygmomanometer
2. Clearing swaps.
3. Template bleeding time device.
4. Filter paper.
5. Stop watch.

Methods:
o Place the Sphygmomanometer cuff around the patient's arm above the
elbow, initiate to 40 mm Hg and keep it at this pressure through the test.
o Clean the volar surface using the swap, chose an area with is devoid of
visible superficial veins.
o Press a sterile metal template with a linear slit 7-8 mm long firmly against
the skin and use a scalpel blade with a guard so arranged that the tip of the
blade protrudes 1 mm through the template slit.
o In this way make an incision 6mm long and 1mm deep. Start the stop
watch.
o Blot off gently but completely with filter paper at 15s intervals the blood
exuding from the cut.
o When bleeding has cease, stop the watch and carefully oppose the edges
of the incision and apply an adhesive strip to lessen the risk of keloid
formation.

Normal range:
2.5-9.5 mins

B. IVY'S METHOD:
o The test is similar to the template method, but instead of a standardized
incision two separate punctures, 5-10 cm apart are made in quick
succession using a disposable lancet ( ay lancet cutting depth of 2.5 mm
and width of just over 1 mm is suitable.
o A source of inaccuracy of ivy's method is depth is not be standardized.

Normal range:
2-7 min.

C. DUKE METHOD:

3
Old method, based on madding a small cut using a lancet in the earlobe.
Blood flows from the puncture; the time it takes for the bleeding to stop is
measured.

Materials:
o Sterile blood lancet.
o Ether
o 1 slide
o Filter paper.
o Stopwatch

Method:
1. Gently clean the lobe of the ear with cotton dipped in ether. Do not rub.
2. Allow to dry
3. Puncture the earlobe deeply.
4. Start the stop watch.
5. The blood should flow freely, with out need to squeeze the earlobe.
6. After 30 seconds, collect the first drop of the blood on the corner of the
filter paper.
7. Do not touch the skin with the paper.
8. Wait 30 seconds, collect the second drop of the blood in the same way.
9. Continue to collect blood by the same way.
10.When no more blood appears, top the watch.

Normal range:
1-5 minutes.

PLATELTE COUNTS USING BLOOD FILM:

BLOOD FILM SPREADIGN AND STAINING.

Materials:
- Dust free clean glass microscope slide.
- Spreader which should be a microscope slide and one corner removed
at each end.

Method:
1- Place a small drop of well mixed blood on the slide about 1-2 Cm from
one end.

4
 2- Posses the spreader at an angle of 45o about one cm in front of the drop
of the blood.
3- Move the spreader back to make contact with the drop of blood, and
then move it quickly and smoothly along the line of contact of the
spreader with the slide.
4- Allow film to dry completely in the air.

Note:
The edge of the spreader should be very smooth, and narrow than that of the
slide. The spreader must be cleaned and dried if it had been used for
spreading more than five films.

Staining procedures:
1- Place dried film on the staining rack with film facing upwards.
2- Dip the well dried film in reagent one for 5 seconds.
3- Dip the film in reagent two for 5 seconds
4- Dip the film in reagent three for 5 seconds
5- Clean under side of the slide and leave to dry upright.
6- Use the 40X lens to choose a proper area.
7- Use the oil immersion lens to investigate the morphology.
8- Count the mean number of platelets in 10 fields at least, multiply by
20,000
9- The result is expressed in No./cmm

Diagnosis of platelet defects:

Laboratory tests might include:

1. Full blood count.

2. Liver enzymes.

5
 3. Renal function, vitamin B12 levels, folic acid levels.

4. Erythrocyte sedimentation rate.

5. And peripheral blood smear.

6. If the cause for the low platelet count remains unclear, bone marrow
biopsy is often undertaken, to differentiate whether the low platelet
count is due to decreased production or peripheral destruction.

Platelets dysfunction (thrombocytopathy):

These are characterized by:
1. A prolonged bleeding time
2. Normal platelet count
3. And disordered platelet aggregation.

Heamophilia A:

Laboratory features:
Prolonged activated partial thromboplastin lime (APTT).
Normal prothrombin time (PT).
Normal bleeding time.
Plasma factor VIII reduced (<1% of normal in severe cases, and up to
10% in mild cases).
Carriers have factor VIII approximately 50% of normal. DNA analysis is
helpful in carrier detection and counselling.
Von Willebrand factor level is normal.
A family history is frequently present, although not essential.
Recently, genetic testing has been made available to determine an
individual's risk of attaining or passing on Haemophilia.
A very small minority of patients have antibodies against factor VIII
that impair its functioning. Management of these patients is more
complicated.

Heamophilia B:
o Diagnosis and treatment are similar to haemophilia A. except that
factor IX concentrate is used for treatment.

6
VWD:

Diagnosis:
APTT is prolonged. PT normal.
Factor VIII and vWF levels arc reduced (Factor VIII levels are also
performed as factor VIII is bound to vWF which protects the factor
VIII from rapid breakdown within the blood. Deficiency of vWF can
therefore lead to a reduction in factor VIII levels).
Bleeding time is prolonged.
Defective platelet function, reduced aggregation with ristocetin,
Mild thrombocytopenin may occur
When suspected, blood plasma of a patient needs to be investigated for
quantitative and qualitative deficiencies of vWF.

Normal levels do not exclude all forms of vWD: particularly type 2.

A platelet aggregation assay will show an abnormal response.

Other tests performed in any patient with bleeding problems are a full
blood count (especially platelet counts), APTT (activated partial
thromboplastin time), prothrombin time, thrombin time and fibrinogen
level.

Diagnosis of Haemophilia A, B & Von-Willebrand's Disease

Haemophilia Haemophilia VW
A B Disease
Inheritance Sex-linked Sex-linked Dominant
Main sites of Muscles, joints, Muscles, joints, Mucous
post trauma or post trauma or membranes,
haemorrhage operative. operative. skin cuts, post
trauma or
operative
Platelet count Normal Normal Normal
Bleeding Time Normal Normal Prolonged
PT Normal Normal Normal
PTT Prolonged Prolonged Prolonged
or normal
Factor VIII Low Normal Low
Factor IX Normal Abnormal Normal
Restoctin induced Normal Normal Impaired
platelet aggregation
Platelet aggregation Normal Normal Abnormal

7
 vWF Normal Normal Low

Diagnosis of DIC:
Although numerous blood tests are often performed on patients prone to DIC,
the important measures are: full blood count (especially the platelet count),
fibrin degradation products or D-dimer tests (markers of fibrinolysis),
bleeding time and fibrinogen levels.

1. Decreased platelets
2. Elevated FDPs or D-dimers (which are produced when fibrin
undergoes degradation when blood clots are dissolved by fibrinolysis).
3. + 4 Prolonged bleeding time and decreased fibrinogen are markers of
DIC.

LABORATORY ASPECTS OF HAEMATOLOGICAL

COAGULATION.

A. screening tests Normal

1. Prothrombin Time (PT) 10-14 sec
2. Activated Prothrombin Time Test (APTT&PTTK) 30-40 sec
3. Thrombin Time test 10-12 sec.

B. Special factors Assays

C. Tests for Fibrinolysis:
1. Euglobulin Clotlysis time
2. Fibrin degredation products(FDPs).

Methods used are:

1. Automatic
2. Manual.

SAMPLING OF BLOOD:
1. Plastic syringe.
2. Needle of an adequate gauge.
3. Proper vein.
4. Clean venipuncture.
5. Suction equal to filling rate.

8
 6. Avoid unduly and prolonged tourniquet.
7. Rapid transfer to sample tube of an exact amount of blood.
8. Blood should not be squirted into the tube.
9. Adequate mixing of blood.
10. Proper storage and quick transport to laboratory.

SAMPLE TUBE SHOULD BE:

1. Plastic or siliconized glass.
2. Should be storage in a refrigerator.
3. Should be in date.
4. Anticoagulant should be seen to be present and clear in
appearance.

Processing of Sample in the Laboratory.

1. After clerical samples are finally checked for the presence of

clots
2. Sample is then spun at 1500g in a centrifuge and plasma
separated into clean sterile plastic stoppard tube.
3. Separated plasma should be kept in melting ice (0o C) until
tested (ideally within 4 hours of obtaining sample).

Interpretation of Results:

XII VII

XI

IX

VIII

9
 II

FIBRINOGEN

1. PT ( Normal), APTT (Prolonged)

2. PT ( Prolonged), APTT (Prolonged)
3. PT (Prolonged), APTT (Normal)
4. PT ( Normal), APTT (Normal)

Collection and Handling of blood in coagulation lab.

Precautions:
Gloves
Avoid injury.
Sharp object disposal
Samples to be sent in closed plastic bags.

Samples:
Sodium citrate tubes used primarily for coagulation testing must be filled to
the required volume of the tube in order to maintain the appropriate
concentration of citrate anticoagulant in the plasma to be tested.
o Coagulation testing is highly sensitive to a numberless of specimen
collection and handling variables.
o Because important diagnostic and therapeutic decisions are based on
the results of coagulation tests, a procedural guideline is required to
fully address these variables.
o The following information applies to all Routine Coagulation tests (i.e.,
Prothrombin Time, APTT, Fibrinogen, etc.).

COLLECTION TUBES
1. It is highly recommended that blood specimens for coagulation testing
be collected by venipuncture using a vacuum collection device that
collects the specimen directly into an evacuated.

10
2. 3.2% tri-sodium citrate (light blue-top) is the proper anticoagulant. This
laboratory requires the use of 3.2% tri-sodium citrate for all coagulation
testing. If any 3.8% citrate tubes are received, the test is cancelled and
the physician is notified. No other anticoagulants are acceptable for
coagulation testing.
3. Light blue-top tubes (citrate) are available in a 4.5ml full draw tube or a
2.7ml and 1.8 ml draw to accommodate pediatric testing volumes.
4. These tubes are pre-calibrated to draw the specified amount of blood,
resulting in the proper 9:1 ratio of blood to anticoagulant.

NEEDLE SIZE
o 20-21 gauge needles are recommended to avoid clotting or hemolysis.
o For the pediatric patient, a 21- to 23-gauge needle may be used.
o A winged blood collection set of the same gauge or measure can also
be used.
o Small Syringe sizes are discouraged because of the increased risk of
hemolysis. Additionally, with larger syringes, there is an increased
chance that clotting may occur.
o If a syringe is used, a small volume syringe is recommended.

INDWELLING CATHETER COLLECTION

o Under certain circumstances, blood specimens for coagulation testing
may be drawn from an indwelling catheter. In this case, the line should
be flushed with 5 mL of saline and the first 5 mL of blood or six dead
space volumes of the catheter discarded.
o Collection of blood through lines that have been previously flushed
with heparin should be avoided, whenever possible. If the blood must
be drawn through an indwelling catheter, possible heparin
contamination and specimen dilution should be considered.
o If heparin contamination is suspected, the laboratory should be
notified.

TECHNIQUE
o Specimens should be obtained from a single venipuncture with
minimal tissue trauma. The blood should flow freely into the container.
o Traumatic venipuncture and/or slow-flowing draws should be avoided.
Either may result in an activated or clotted sample.
o Prolonged venostasis may raise the levels of factors VIII:C and IX, or
it may activate the fibrinolytic system.

11
 o Regardless of the device used for specimen collection, all tubes should
be gently inverted IMMEDIATELY at least five times to mix. DO NOT
shake or mix vigorously.
o If only a coagulation specimen is drawn, a pilot tube should be
collected and discarded. If multiple specimens are collected, the
coagulation specimen should be collected into the second or third tube.

COLLECTION PROBLEMS
UNDERFILLING: Inadequate filling of the collection tube will decrease
the required blood:anticoagulant ratio (9:1), and may lead to falsely
prolonged results.

OVERFILLING: Overfilling will cause an incorrect blood to anticoagulant

ratio which may result in slightly decreased results or the specimen may clot
or contain fibrin due to inadequate anticoagulant for the blood volume drawn
which will result in the specimen being rejected.

Diagnosis of Factor V Leiden mutation

o Suspicion of factor V Leiden being the cause for any thrombotic event
should be considered in any white patient below the age of 45, or in
any person with a family history of venous thrombosis.
o This disease can be diagnosed by watching the aPTT as activated
protein C is added. With a normal patient, adding APC increases the
aPTT. In patients with factor V Leiden, adding APC to plasma of
Factor V leiden will fail to prolong APTT.

o There is also a simple genetic test that can be done for this disorder,
and will give a quick diagnosis.

First line tests used to investigate acute haemostatic failure.

PT APTT TT Platelet Condition.

count.
1. N N N N Disorder of Platelet function, Factor XIII

12
 deficiency, Disorder of vascular
haemostasis, Normal haemostasis.
2. Long N N N Factor VII deficiency, Early oral
anticoagulant.
3. N Long N N Factor VIII:C, IX, XI, XII, PK, HMWK
deficiency, vWD, circulating
anticoagulant.
4. Long Long N N Vit.K deficiency, Oral anticoagulants,
factor V, VII, X and II deficiency.
5. Long Long Long N Heparin, Liver disease, Fibrinogen
deficiency, Hyperfibrinolysis
6. N N N Low Thrombocytopenie.
7. Long Long N Low Massive transfusion, Liver disease
8. Long Long Long Low DIC, Acute liver disease.

Screening Tests for Haemostasis

Screening tests. Defect.

B.T. Prolonged. Platelet ( or dysfunction)+ VWD
APTT prolonged. Factors defect (XII,XI,VIII,IX,X,V,II I)
PT prolonged. Factors defect (VII,X,V,II,I)
TT prolonged Fibrinogen defect (factor I), high FDPs.
Reptilase Time Fibrinogen defect (factor I), high FDPs
Prolonged Not affected by heparin therapy
FDPs High o DIC
o Snake bite.
o Thrombolytic therapy.
o Dysfibrinogenaemia.
Platelet count Low Thrombocytopenia.
Platelet count Normal Platelet dysfunction.
Preparation of the sample.

1- Preparation of Platelet Poor Plasma (PPP).

o PPP is prepared by centrifugation at 2000g for 15 min.
o It should be kept at room temperature if it is to be used for Prothrombin
Time test, factor VII assays or platelet function test, and at 4 OC for other
assays.
o The test should preferably be completed within 2 h of collection.

13
 o The sample may be frozen at -40 OC for several weeks without a
significant loss of activity for most of the haemostatic components to be
assayed.

2- Control blood samples:

o Control blood samples, obtained from a healthy subject, are treated as
described above.
3- Aged plasma:
o Citrated Platelet Poor Plasma from healthy person and incubated at 37
O
C for 2-3 days.
o The Prothrombin Time at the end of this time should not exceed 90 s.
o This plasma is deficient for factor V and VIII:c.
o It can be transferred into small container and stored at -40 OC. it will be
stable for several weeks.
4- Adsorbed plasma:
o Prepared using special reagents, this type of plasma is deficient of
factor II, VII, IX and X.
o It can be transferred into small container and stored at -40 OC. it will be
stable for several weeks.

One stage Prothrombin Time (PT):

Principle:
The test measures the clotting time of plasma in the presence of an
optimal concentration of tissue extract (thromboplastin) and
indicates the overall efficiency of of the extrinsic clotting system.
Although originally thought to measure the Prothrombin, the test is
now known to depend also on reactions with factors V, VII and X,
and on the fibrinogen concentration of the plasma.

Reagents:
Patient and control citrated plasma (PPP). Note that the plasma stored at 4OC
may have a shortened Prothrombin time as a result of factor VII activation in
cold.
PT reagent: Thromboplastin + CaCl2 0.025 mol/l.

Method:
1. keep thromboplastin at 37 OC for 10 min

14
 2. Place 0.1 ml of plasma of into a glass tube placed in a water-bath and
add 0.1 ml of the thromboplastin.
3. Wait for 3 min to allow the mixture to worm.
4. Add 0.1 ml of warmed CaCl2 and mix the contents of the tube.
5. Start the stop-watch and record the end point.
6. Carry out the test in duplicate on the patient's and control plasma.

Normal range:
11-16 seconds.

Interpretation:
The common causes of prolonged PT when investigating acute
haemostatic failure are:
1. The administration of oral anticoagulant drugs.
2. Liver diseases, particular obstructive.
3. Vitamin K deficiency.
4. Disseminated Intravascular Coagulation.
5. Coagulation factor deficiency VII, X, V , Prothrombin or
fibrinogen deficiency.

ACTIVATED PARTIAL THROMBOPLASTIN TIME

This test is also known as partial thromboplastin time with Kaolin (PTTK),
and the Kaolin Cephalin Clotting Time (KCCT).

Principle:
The test measures the clotting time of plasma after the activation of
contact factors but without added in tissue extract (thromboplastin)
and so indicates the overall efficiency of of the intrinsic clotting
system.
To standardize the activation of the contact factors, the plasma is
first pre-incubated with Kaolin. A standardized pholspholipid is
provided to allow the test to be performed in platelet poor plasma.
The test not only depend on the contact factors and VIII and IX, but
also on the reactions with factors X, V, Prothrombin and Fibrinogen.
It is also sensitive to the presence of circulating anticoagulants
(inhibitors) and heparin.

15
Reagents:
Patient and control citrated plasma (PPP).
APTT reagent, (phospholipid + Kaolin)
CaCl2 0.025 mol/l.

Method:
Keep the reagent in 37 OC for 10 min
Add 0.1 ml of PTT reagent.
Add 0.1 ml of plasma and incubate the mixture for 3 min at 37 OC
Add 0.1 ml of pre-warmed CaCl2 and mix the contents of the tube.
Start the stop-watch and record the end point.
Carry out the test in duplicate on the patient's and control plasma.

Normal range:
30-40 seconds.

Note :
The actual times depend on the reagents used

Interpretation:
The common causes of prolonged PTTK in acute haemostatic
failure are:
1. The administration of heparin or contaminated with heparin.
2. Liver diseases.
3. Disseminated Intravascular Coagulation.
4. Coagulation factor deficiency or deficiency, haemophilia A,
haemophilia B, and factor IX and factor XII, PK, HMWK, V ,
Prothrombin or fibrinogen deficiency.
5. Massive transfusion with stored blood.
6. A circulating anticoagulant.

Thrombin Time

PRINCIPLE:
Thrombin is added to the plasma and the clotting time is measured. The TT is
affected by the concentration and reaction of the fibrinogen, and by the
presence of inhibitory substances, including fibrinogen/fibrin degradation
products (FDP) and heparin. The clotting time and the clot appearance are
equally informative.

16
Reagents:
o Patient and control citrated plasma (PPP).
o Thrombin Solution reagent: bovine thrombin

Method:
Keep the reagent in 37 OC for 10 min
Put 0.1 ml of plasma and incubate the mixture for 3 min at 37 OC
Add 0.1 ml of TT reagent and mix.
Start the stop-watch and record the end point.
Carry out the test in duplicate on the patient's and control plasma.

Normal range:
14 20 seconds.

Interpretation:
1. The common causes of the prolonged Thrombin Time are:
2. Hypofibrinogenaemia, as found in DIC and more rarely in congenital
defect or deficiency of this factor.
3. Raised concentration of FDPs as encountered in DIC or liver disease.
4. Presence of heparin which interferes with the thrombin-fibrinogen
reaction.
o Transparent bulky clot is found if fibrin polymerization is abnormal,
as in the case in liver disease and some congenital dysfibrinogenaemia.
o A gross elevation of the plasma fibrinogen concentration may also
prolong the thrombin time.

Mixing Study.

In the cases of prolonged PT and APTT, a 50:50 mixture of normal and test
plasma should be tested. If the result of the prolonged test using the mixture
was reduced by more than 50% of the deference between the two individual
clotting times, that means that the normal plasma corrects the prolonged time,
the patient may has a deficiency of one or more clotting factors. If, the normal
plasma fails to correct the prolonged test, an inhibitor or anticoagulant may
be present.

17
18