GUIDELINES ON MANUAL OPERATING PROCEDURE

FOR CLINICAL PATHOLOGY

BLOCK 14

TOPICS: HEMOSTASIS

SESSION 2
1. Bleeding Time (Ivy Method)
2. Platelet Count (Rees-Ecker)
3. PT (Prothrombin Time)
4. APTT ( Activated Partial Thromboplastin Time)
5. Blood Grouping
6. Cross Match

1. BLEEDING TIME (IVY METHOD)

Principle

A blood pressure cuff is placed on the patient’s arm above the elbow, inflated, and
maintained at a constant pressure throughout the procedure. One (or two) standardized
incisions are made on the volar surface of the forearm. The length of time required for
bleeding to stop is recorded as the bleeding time.

Reagent and Equipment

1. Blood pressure cuff

2. Bleeding time device
3. Stopwatch
4. Circular filter paper
5. Alcohol prep pads
6. Butterfly bandage

Procedure

1. Locate the area for the bleeding time. The patient’s arm should be extended with
the volar surface facing upward. Beginning at the middle finger move up the arm
in a straight line to 5 cm below the fold of the below. This area in the muscular
portion of the Volar surface of the forearm, 5 cm below the fold in the elbow, is the
standardized test site for the puncture. The area should be free of surface veins,
bruises, scars, and swelling. (shave the area if excessive hair is present)
2. Cleanse the site with an alcohol sponge and allow to dry
3. Place a blood pressure cuff on the patient’s arm above the elbow. Increase the
pressure to 40 mm Hg and hold this exact pressure for the entire procedure. The
incision must be made and the bleeding time started within 30 to 60 seconds after
the blood pressure cuff has been inflated.

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 4. Prepare the bleeding time device. Position it in the correct direction and
appropriate area on the arm, using only that amount of downward pressure so that
both ends are touching the skin and the device does not cause an indentation. (If
too much pressure is applied and the device depresses the skin, the incision will be
too deep)
5. Activate the trigger and start the stopwatch. Remove the device approximately 1
second after making the incision.
6. Blot the blood from the puncture site on a clean section of circular filter paper every
30 seconds. The filter paper must not touch the wound at any time.
7. When bleeding ceases, stop the watch and release the blood pressure cuff. Record
the results. Repeat the examination twice, report the average result.
8. Place a butterfly bandage over the puncture site, and advise the patient to keep the
bandage in place for 24 hours.

2. PLATELET COUNT (DIRECT METHOD; REES-ECKER)

Introduction

Platelet counts are important in helping to diagnose bleeding disorders. Platelets function
primarily in hemostasis (the stoppage of bleeding) and in maintaining capillary integrity.
Platelets are difficult to count. They are small, disintegrate easily, and are hard to
distinguish from dirt.

Principle of Method

Blood diluted with solution containing Brilliant Cresyl Blue so the platelet seem bright
blue. The platelets then are counted using hemocytometer. Results are doubled checked by
examination of the platelets on a Wright/Giemza stained smear.

Specimen
Whole blood (1 mL), using EDTA as the anticoagulant. Capillary blood may also be used.

Reagents and Equipment

1. Rees-ecker (to dilute Platelet):

Sodium citrate 3.8 g
Brilliant Cresyl Blue 0.1 g
Formaldehid 40% 0.2 ml
Distilled water 100 mL
Mix and Filter before use.
2. Erythrocyte pipe
3. Hemocytometer
4. Object glass, Wright/Giemsa stain
5. Microscope
6. Petri dish
7. Filter paper

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Procedure

1. Draw the blood up to exactly the 0.5 mark in the red count pipette and dilute to the
101 mark with red count diluting fluids, thus making 1:200 dilution of blood.
2. Mix the dilution for 3-5 minutes. Clean the counting chamber
3. Prepare the moist chamber as follows: obtain a petri dish and piece of filter paper
of approximately the same diameter as the petri dish. (Either the top or the bottom
petri dish may be used). Thoroughly moisten the filter paper and place in the top of
the petri dish so that it adheres to the dish.
4. When the diluted blood samples are adequately mixed, fill the one side of the
counting chamber with the dilution.
5. Place the moist chamber over the hemocytometer and allow the preparation to sit
for 15 to 20 minutes.
6. Count the platelets:
a. Carefully place the hemocytometer on the microscope stage.
b. Using low power (10 x objective) place the large center square in the middle
of the field of vision. Carefully change to the high dry objective (40x). The
platelets appear as small, round, oval, or elongate particles that are highly
retractile and stain a light bluish color.
c. Count the platelets in the 2 large squares in the corner square. The suggested
squares to use are those labeled with a W (Fig. 2)

Figure 2.

7. Calculate the number of platelets/L as shown below:

PLTs/L = # Cells counted x Correction for Dilution x 106

Correction for Volume

PLTs/L = # Cells counted x 200 x 106

2x1x1x0.1

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For example:
# Cells in two large squares = 400
Dilution = 1: 200
Volume counted = 2 large squares
Conversion to liter = x 106
PLTs/L = 400 x 1.0 x 200 x 106
0.2
= 4.0 x 1011

8. Scan a smear with the Wright or Giemza staining and estimate the platelet count to
cross check with the upper result.

Estimation plt count/mmc by indirect method (blood smear) = sum of plt on 20

immersion view x 1000

3. PT (PROTHROMBIN TIME)

Principle

The calcium in whole blood is bound by sodium citrate, thus preventing coagulation. Tissue
thromboplastin, to which calcium has been added, is mixed with the plasma, and the
clotting time is noted. (Factor VII reacts with the tissue factor [thromboplastin] in the
presence of calcium ions to activate factor X. The coagulation mechanism continues from
there.)
Reagent and Equipment

1. Water bath, 370C.

2. Thromboplastin-calcium chloride reagent.
3. Normal plasma control
4. Test tubes, 13 x 100 mm
5. Stopwatch

Specimen

Citrated plasma: 1 part 0.11 M sodium citrate to 9 parts whole blood (0.5 ml citrate + 4.5
ml whole blood). Testing should be performed within 2 hours after specimen is drawn
when the plasma is maintained at room temperature and within 2 to 5 days when the plasma
is stored at – 200C.

Oxalate Plasma: 1 part sodium oxalate and 9 parts whole blood.

Procedure

1. Centrifuge specimen as soon possible after blood collection to obtain platelet poor
plasma (10 minutes, 3000 rpm)

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 2. Testing should proceed immediately, or cap the centrifuged or plasma tube and
complete the procedure within 2 hours.
3. Pipette 0.2 mL of thromboplastin-calcium reagent into the appropriate number of
13 x 100 mm test tubes. Warm the test tubes in the water bath for at least 1 minute,
until they have reached 370C. The incubation period for this mixture is not critical
once it reaches 370C.
4. Incubate the plasma for 2 to 3 minutes, until it reaches 370C, plasma should be
incubated for no longer than 5 minutes after reaching 370C.
5. Forcibly pipette 0.1 mL of patient’s plasma into the test tube containing 0.2 mL of
thromboplastin-calcium mixture and simultaneously start the stopwatch.
6. Mix the contents of the tube, remove the tube from the water bath, and wipe dry.
Gently tilt the tube back and forth until a clot form, at which point the timing is
stopped. Record the coagulation time.
7. Each test and control plasma should be performed in duplicate when manual
methods are used.
8. Average the two clotting times and report the patient’s results along with the normal
range for the test as performed in your laboratory.

4. APTT (ACTIVATED PARTIAL THROMBOPLASTIN TIME)

The APTT is the most useful procedure for routine screening of coagulation disorders in
the intrinsic system, for detecting the presence of circulating anticoagulants (inhibitors),
and for monitoring heparin therapy. It measures those factors present in the intrinsic
coagulation mechanism except for platelets and factor XIII (factor VII is not measured
because it is in the extrinsic system).

Principle

The calcium in whole blood is bound by the sodium citrate anticoagulant to prevent
coagulation. The plasma, after centrifugation, contains all intrinsic factors except calcium
and platelets. Calcium, a phospholipids substitute for platelet (partial thromboplastin), and
an activator (to ensure maximal activation), are added to plasma. The time required for
plasma to clot is the activated partial thromboplastin time.

Reagents and Equipment

1. Water bath, 370 C.

2. Calcium chloride, 0,025 M:
Calcium-chloride anhydride 1.38 g
Aquades 500 ml
3. Partial thromboplastin containing an activator.
4. Normal control plasma
5. Test tubes, 13 x 100 mm.
6. Stopwatch.
7. Ice bath.

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 8. Micropipette 0,1 ml

Specimen

Citrated plasma: 1 part 0.11 M sodium citrate to 9 parts whole blood.

Procedure

1. Centrifuge specimen as soon as possible after collection to obtain platelet poor

plasma. Centrifuge at 3000 rpm in 10 minutes, then put the plasma in ice bath.
2. Incubate a sufficient amount of 0.025 M calcium chloride at 370 C.
3. Pipet 0,1 ml of control plasma (or patient’s plasma) into a 13x100 mm test tube,
put it in water bath for 1 minute.
4. Pipet 0,1 ml of partial thromboplastin (containing activator) into the test tube
containing the control (or patient’s) plasma.
5. Mix the contents of the tube quickly and place in a 370 C water bath for 2-3 minutes.
6. After exactly 3 minutes, forcibly pipette 0.1 ml of pre-warmed calcium chloride
into the tube, and simultaneously start stopwatch.
7. Mix the test tube immediately after adding calcium chloride. Allow the test tube to
remain the water bath while gently tilting the tube every 5 second. At the tube end
of 20 seconds, remove the test tube from the water bath. Quickly wipe off the
outside of test tube with clean gauze so that the content of the tube can be clearly
seen.
8. Gently tilt the test tube back and forth until a clot form, at which point the timing
is stopped.
9. When manual testing is performed, control and patient plasma specimens should
be run in duplicate, and the results averaged to obtain the final report.