ACTIVITY NO.

Bone Marrow Film Examination for Platelet Maturation Series

The adult human produces 1011 platelets daily at steady state, a level of production that can
increase 20-fold or more in times of heightened demand. While our understanding of thrombopoiesis has
grown considerably since the advent of clonal assays of megakaryocytic progenitor cells in the 1970s and
the cloning and characterization of several hematopoietic growth factors that support the process in the
1980s and 1990s, as recently as 100 years ago platelets were referred to as the "dust of the blood" and we
had virtually no notion of their origin. Platelets were described by Addison in 1841 as "extremely
minute ... granules" in blood and were termed "platelets" (blutplättchen) by Bizzozero, who also observed
their adhesive qualities of "increased stickiness ... when a vascular wall is damaged".The same elements
were identified by microscopic examination of blood smears by Osler and by Hayem in the late
nineteenth century.Megakaryocytes have been recognized as rare marrow cells for nearly 2 centuries, but
it was the elegant camera lucida studies of Howell in 1890 and his coining of the term "megakaryocyte"
that led to their broader appreciation as distinct entities.In 1906, James Homer Wright suggested that
blood "plates" are derived from the cytoplasm of megakaryocytes, and the basic elements of
thrombopoiesis were established. Much has been learned since of the origins of thrombopoiesis.
The importance of megakaryopoiesis and thrombopoiesis for clinical medicine is immediately
apparent; morbidity and mortality from bleeding due to moderate to severe thrombocytopenia is a major
problem facing a wide range of patients. The origins of thrombocytopenia are beyond the scope of this
review, but include both iatrogenic and naturally occurring conditions that are frequently encountered in
clinical practice. The magnitude of the problem can be gauged by considering that in the United States
approximately 1.5 million platelet transfusions (representing the equivalent of the platelets from 9 million
units of blood) are administered yearly to patients to reduce their risk of severe bleeding. Unfortunately,
platelet transfusion therapy is less than ideal. At least 30% are associated with one or more complications,
usually by immune or cytokine-mediated febrile reactions, but occasionally by bacteremia, graft-versus-
host disease, or acute pulmonary injury. Moreover, an inadequate platelet response due to HLA
alloimmunization occurs in from 10% to 30% of individuals who require repeated platelet transfusions,
depending on the nature of their disease. And platelet transfusions are expensive; the approximate cost of
a standard platelet transfusion product (usually sufficient to raise the platelet count 30 to 50 x 109/L) is
$600, rising substantially if either a single donor apheresis product is desired or removal of leukocytes or
HLA matching is required. The distinguished physician and teacher William Osler stated, "We are still
without a trustworthy medicine which can always be relied upon to control purpura." In many ways,
Osler's dilemma is as true today as when first penned in 1892. Clearly, an understanding of
thrombopoiesis sufficient to allow therapeutic stimulation of marrow platelet production would prove
superior to the transfusion of allogeneic platelets.
Objectives:

 To examine the bone marrow film

 To describe the different cells of the platelet maturation series
 To distinguish megaryocytic series from other maturation series

Materials

 Bone marrow smear

 Peripheral blood smear
 Cedar wood oil
 Hematology Atlas
 Microscope

Procedure

1. Scan and examine the bone marrow and peripheral blood smears under the oil immersion film of
the microscope
2. Observe for the megakaryocytic and platelet maturation series
3. Describe and illustrate the results

Cells Descriptions
Megakaryoblast
Promegakaryocyte

Megakaryocyte

Platelets

Questions
1. Why are platelets small and irregular in shape?

2. The younger the cells the bigger its size. How come platelet maturation series is the
exemption to the rule?

3. Describe how will you name megakaryocyte according to the number of nucleus.

4. Draw platelets as seen on electron microscope.

ACTIVITY NO. 2
 Platelet Estimate

Platelet smear represents a subjective estimation of platelet numbers made during examination of
the stained blood film. As a rule, no attempt is made to provide an actual number, but rather a designation
into categories of "increased" (above reference intervals), "adequate" (within reference intervals), "low?"
(within low normal limits or mildly decreased), "low" (below reference intervals), and "very low" (<
30,000/µL) is made. For more precise enumeration, a platelet count should be requested.

Objectives:

 Perform platelet estimate with utmost accuracy and precision

 To correlate the platelet count with the clinical conditions of the patients
 To appreciate the importance of platelet count

Materials

 Blood smear
 Microscope
 Cedar wood oil
 Tally counter

Procedure

1. Scan the thin area, using the oil immersion lens.

2. Observe 10 fields, counting the platelets in each field, observing granulation and morphology.
3. Determine the average number of platelets observed per oil immersion field (OIF).
4. Report the platelet estimate and any abnormal morphology.
5. Interpret the platelet estimate as follows
a. Adequate: 8-20/OIF
b. Low Platelet: <7/OIF
c. High Platelet: >21/OIF

Results
Tally the results of the platelet count per field as follows:

Field Number of platelets

1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
TOTAL

Average
Interpretation

Illustrate the platelets as seen on oil immersion field

Questions:

1. Why is it necessary to perform platelet estimate?

2. What are the advantages and disadvantages of doing platelet estimate?

3. How would you rate the accuracy and precision of this method?
 ACTIVITY NO. 3

Direct Platelet Count (Rees and Ecker Method)

In an adult, a normal count is about 150,000 to 450,000 platelets per microliter (x 106/Liter) of
blood. If platelet levels fall below 20,000 per microliter, spontaneous bleeding may occur and is
considered a life-threatening risk. Patients who have a bone marrow disease, such as leukemia or another
cancer in the bone marrow, often experience excessive bleeding due to a significantly decreased number
of platelets (thrombocytopenia). As the number of cancer cells increases in the bone marrow, normal bone
marrow cells are crowded out, resulting in fewer platelet-producing cells.
Low number of platelets may be seen in some patients with long-term bleeding problems (e.g.,
chronic bleeding stomach ulcers), thus reducing the supply of platelets. Decreased platelet counts may
also be seen in patients with Gram-negative sepsis. Individuals with an autoimmune disorder (such as
lupus or idiopathic thrombocytopenia purpura (ITP), where the body’s immune system creates antibodies
that attack its own organs) can cause the destruction of platelets.
Certain drugs, such as acetaminophen, quinidine, sulfa drugs, digoxin, vancomycin, valium, and
nitroglycerine, are just a few that have been associated with drug-induced decreased platelet counts.
Patients undergoing chemotherapy or radiation therapy may also have a decreased platelet count. Up to
5% of pregnant women may experience thrombocytopenia at term.
Platelet consumption may be observed in renal diseases. Thrombocytopenic purpura (TTP) and hemolytic
uremic syndrome (HUS) are seen in renal failure and can result in fewer circulating platelets in the blood.
Similarly, a condition known as splenic sequestration, where platelets pool within the spleen, can also
cause a platelet decrease.
More commonly (up to 1% of the population), easy bruising or bleeding may be due to an
inherited disease called von Willebrand’s disease. While the platelets may be normal in number, their
ability to stick together is impaired due to a decrease in von Willebrand’s factor, a protein needed to
initiate the clotting process. Many cases may go undiagnosed due to the mild nature of the disease. Many
cases are discovered when a patient has to have surgery or a tooth extraction or when delivering a baby.
However, some cases are more severe and can be aggravated by use of certain drugs, resulting in a life-
threatening situation.
 Increased platelet counts (thrombocytosis) may be seen in individuals who show no significant
medical problems, while others may have a more significant blood problem called myeloproliferative
disorder. Some, although they have an increased number of platelets, may have a tendency to bleed due to
the lack of stickiness of the platelets; in others, the platelets retain their stickiness but, because they are
increased in number, tend to stick to each other, forming clumps that can block a blood vessel and cause
damage, including death (thromboembolism).

Objectives:

 To count platelets with utmost accuracy and precision

 To be able to correlate the results with the clinical conditions of the clients
 To calculate platelets from the patients correctly

Materials

 Venipuncture set
 EDTA tubes
 RBC pipet
 Counting chamber
 Tally counter
 Petri dish
 Moistened filter paper
 Microscope
 Rees and Ecker fluid
 Pipet shaker

Procedures

1. Extract blood from the patient and place it in EDTA tube.

2. Rinse diluting pipet with the diluting fluid to prevent platelets from adhering to the inside of
platelet
3. Draw blood up to mark “1” of the pipet and diluting fluid up to mark 101.
4. Mix in a pipet shaker for 1 minute.
5. Discard several drops before charging the counting chamber.
6. Allow the platelets to settle for 10 minutes by placing the counting chamber in a Petri dish lined
with moist filter paper to prevent evaporation.
 7. Count the platelets in the five quadrants of the RBC area.
8. Compute for platelet count using the formula:
Platelet Count =Cell counted × DCF × DF × ACF

DCF = depth correction factor; DF = dilution factor; ACF = area correction factor
Results:
1. Tally the platelet counts in all five quadrants

Quadrants Number of Platelets

Upper Left
Upper Right
Central
Lower Left
Lower Right

Total

2. Show your computation. Express the result in SI and interpret it.

3. Illustrate the appearance of platelets as seen in the counting chamber

Questions

1. Why are platelets difficult to count?

2. What are the purposes of the following?

a. Rinsing the pipet with the diluting fluid.

b. Placing the chamber in a petri dish with moistened filter paper

3. Differentiate the direct count over the indirect count

 ACTIVITY NO. 4

Indirect Platelet Count

Indirect platelet counts are based on the proportion of the number of red cells to the platelets. The
indirect platelet count is higher than the direct count because the red cells, which are used as a point of
reference in the indirect method, are not randomly distributed beneath the coverslip. The red cells are
concentrated at the edge of the coverslip so that the true ratio of red cells to platelets cannot be accurately
established. Indirect platelet counts based on the ratio in the central areas of the coverslip are too high.

Objectives:

 To perform indirect platelet count with utmost precision and accuracy

 To be able to calculate for the indirect platelet count correctly
 To appreciate the importance of doing the indirect platelet count

Materials

 Wright stain
 Tally counter
 14% Magnesium sulfate (MgSO4)
 Reese and Ecker
 Cedar wood oil
 Microscope

Procedure
Dameshek Method (Wet Method)

1. Perform a finger puncture. Wipe 1st drop of blood then, place a large drop of diluent over the
puncture site. (1:5 blood to diluent ratio)
2. Transfer a portion on a cover glass and invert on a slide.
 3. Allow to stand for 15 minutes.
4. Examine under OIO (diaphragm partly closed). Count platelets and RBCs until 1000 RBCs are
recorded. Platelets are lilac colored, tiny, glistening.

Fonio’s Method (Dry Method)

1. Perform a finger puncture. Wipe 1st drop of blood then, place a large drop of diluent over the
puncture site. (1:3 blood to diluent ratio)
2. Transfer mixture on 1 end of a clean slide.
3. Make a wedge smear with a spreader.
4. Air dry the smear.
5. Stain with Wright’s stain
6. Examine under OIO. Count platelets and RBCs until 1000 RBCs are recorded. (Do the counting
at 1/5 – 1/3 part from end of smear).

Calculation
Platelet Count ( µL)=no . of platelets × RBC ( µL)/1000
Results

1. Tally the results of the platelet count per field as follows:

Field Number of platelets

DAMESHEK’s FONIO’s

1st
2nd
3rd
4th
5th
6th
7th
8th
9th
 10th
Total

2. Compute for platelet estimate. Show your calculation. Interpret the results.

Questions

1. Is the procedure accurate? Why?

2. What are the advantages of indirect method over the direct method?

3. Describe the procedure of Olef of indirect methods of platelet counts.

 ACTIVITY NO. 5

Bleeding Time – Duke’s Method

Bleeding time is a crude test of hemostasis (the arrest or stopping of bleeding). It indicates how
well platelets interact with blood vessel walls to form blood clots.
Bleeding time is used most often to detect qualitative defects of platelets, such as Von
Willebrand's disease. The test helps identify people who have defects in their platelet function. This is the
ability of blood to clot following a wound or trauma. Normally, platelets interact with the walls of blood
vessels to cause a blood clot. There are many factors in the clotting mechanism, and they are initiated by
platelets. The bleeding time test is usually used on patients who have a history of prolonged bleeding after
cuts, or who have a family history of bleeding disorders. Also, the bleeding time test is sometimes
performed as a preoperative test to determine a patient's likely bleeding response during and after surgery.
However, in patients with no history of bleeding problems, or who are not taking anti-inflammatory
drugs, the bleeding time test is not usually necessary.
For the Duke method, a nick is made in an ear lobe or a fingertip is pricked to cause bleeding. As
in the Ivy method, the test is timed from the start of bleeding until bleeding is completely stopped. The
disadvantage to the Duke method is that the pressure on the blood veins in the stab area is not constant
and the results achieved are less reliable. The advantage to the Duke method is that no scar remains after
the test. The other methods may result in a tiny, hairline scar where the wound was made. However, this
is largely a cosmetic concern.

Objectives:

 To perform the Duke’s method of bleeding time with utmost accuracy and precision
 To correlate the results of the bleeding time with the clinical conditions of the patients

Materials
  Blood lancet
 Filter paper
 70% alcohol
 Cotton balls
 Stopwatch

Procedure

1. Clean the site of puncture (finger or ear lobe) with cotton moistened with 70% alcohol. Allow to
dry.
2. Puncture the lower edge of the ear lobe to a depth of 3 mm. Wipe off the 1st drop with cotton
and start the stopwatch as soon as the blood appears.
3. At half-time intervals at the edge of small disc of filter paper is gently applied to the drop of
blood, care being taken not to touch the skin.
4. End point is reached when no more blood is absorbed by the filter paper.
5. Record the time.

Normal range: 1 to 3 minutes

Results
Tally the results:
Interpret:
Illustrate the filter paper that you used in the experiment
Questions
1. What is the clinical significance of prolonged or increased bleeding time?

2. Compare and contrast the Ivy’s method from the Duke’s method.

3. What are the advantages and the disadvantages of the Duke’s method?
 ACTIVITY NO. 6

Clotting Time – Slide Method

Clotting time is the time it takes for a blood sample to clot or coagulate in vitro, especially
through capillary tube method used to diagnose if clotting or coagulating disorders are present.

Objectives

 To perform the slide method with utmost accuracy and precision

 To correlate the results of clotting time with the clinical conditions of the patients
 To appreciate the importance of clotting time in the evaluation of hemostasis

Materials

 Blood lancet
 Slide
 Cotton
 70% alcohol

Procedure

1. Sterile the finger with cotton moistened with 70% alcohol. Allow to dry
2. Make a puncture. Wipe off the 1st drop of blood with a dry cotton and start the stopwatch as
soon as the blood appears.
3. Place a drop of blood on a clean dry glass.
4. After 2 minutes, draw a fine wire or pin through the drop of blood and gently lift the wire or pin.
5. Repeat at 30 seconds interval until tiny strands of fibrin clings to the wire or pin.
Normal range: 2-6 minutes

Results

1. Interpret the results:

2. Illustrate the end point of the test showing the fibrin strands from the blood
Questions

1. Give the clinical significance of prolonged clotting time.

2. Is the slide method accurate? Why?

3. What is being evaluated in the test for clotting time?

 ACTIVITY NO. 7

Clotting Time – Lee and White Method

Clotting time is defined as the time required for blood to form a clot, tested by collecting 4 mL of
blood in a glass tube and examining it for clot formation. The first appearance of a clot is noted and
timed. The normal coagulation time in glass tubes is 5 to 15 minutes. This simple test has been used to
diagnose hemophilia, but it does not detect mild coagulation disorders. Its chief application is in
monitoring anticoagulant therapy. It is rarely used in clinical practice. Also called coagulation time.
In the Lee-White method, blood in test tubes is maintained at a constant temperature and
examined regularly until clotting occurs; the test can be also be performed in capillary tubes. It is less
sensitive and now less often used than the activated coagulation time.

Objectives

 To perform clotting time with utmost accuracy and precision

 To correlate the results of the clotting time with the clinical conditions of the patients
 To appreciate the importance of doing this test as an evaluation tool for hemostasis

Materials

 Syringe 5 cc
 3 test tubes (8 mm in diameter)
 Torniquet
 Cotton
 70% alcohol
 Test tube rack
 Normal saline solution (NSS)

Procedure
 1. Rinse the sterile syringe, needle and the three test tubes with NSS.
2. Label the test tubes – 1,2 and 3.
3. Withdraw 4 cc of blood, recording the time of the 1st appearance of blood in the syringe.
4. Remove the needle from the syringe and slowly place 1 ml of blood in each of the three test tubes
in order of their number.
5. After 3 minutes, slowly tilt the 1st tubes, at 30 seconds interval alternately tilt the 1st and the 2nd
tube until coagulation has taken place.
6. Time elapsed from the 1st appearance of blood in the syringe and clot formation in the 3rd tube is
the coagulation time.

Normal range: 7-15 minutes

Results

1. Interpret the result:

2. Illustrate and explain the procedure:

Questions

1. Compare and contrast the slide method from the Lee and White method.

2. What are the advantages and disadvantages of the tube method over the slide method?

3. Why is it that it is rarely being done in clinical settings?

4. Why do you think most clinicians would prefer to use the activated coagulation time instead of
the classical clotting time?
 ACTIVITY NO. 8

Clot Retraction Time – Mac Farlane Method (Tube Method)

Clot retraction time is directly proportional to the number of platelets and inversely
proportional and inversely proportional to the hematocrit and fibrinogen levels. When
fibrinolysis is active, the fibrin may be dissolved almost as it formed, and clot retraction will be
impaired.

Objectives

 To be able to perform tube method of clot retraction time with utmost accuracy and
precision
 To be able to correlate clinically the results of the test with the clinical condition of the
patient
 To be able to appreciate the importance of the clot retraction time to diagnose blood
disorders

Materials

 Centrifuge tube
 Cork stopper
 Coiled wire
 Test tube rack
 Syringe
 Incubator
Procedure

1. Place 5 ml of venous blood in a clean dry centrifuge tube.

2. Insert a coiled wire in the bottom of the tube (1 mm thick, 3 cm diameter coil).
3. Incubate at 37oC for one hour after clotting has occurred.
4. Gently lift the wire and allow the attached clot to drain for 2 minutes.
5. Read the volume of fluid remaining in the tube. Express this volume as a percentage of
the original volume of whole blood placed in the tube.

Normal Value: 48-64% (average of 55%)

Degree of Retractibility

1. Normally the coagulum commences to retract within the 1 st one hour and form clot with
complete retraction in 24 hours.
2. The normal clot is firm, difficult to break up with blunt instruments, and difficult to
flatten out. Defective clots are soft, friable and easily crushed.
3. In thrombocytopenia purpura, a coagulum is formed in the normal time, but it does not
retract.
4. In hemophilia, the coagulum forms very slowly, but the clot when formed retracts
normally.

Digestion of clots

1. Normally there should be no digestion of clots.

2. Digestion of clot, giving it a worm-eaten appearance, is seen in cirrhosis of the liver.

Results

1. Interpret your results:

2. Illustrate your results:

 3. Show your computation:

Questions

1. What is the clinical significance of increased and decreased clot retraction time?

2. Explain the relationship of clot retraction to:

a. Fibrinolysis

b. Platelet count

c. Packed cell volume

 ACTIVITY NO. 9

Hirschboeck Method (Castor Oil Method)

Materials
 Castor oil
 Slides
 WBC Pipet

Procedure
1. Puncture sterilized finger, wiped off the 1st drop, fill pipet up to mark 1.
2. Slowly, allow the blood to flow down the pipet until a big drop accumulate at the tip.
3. Slowly lower the pipet until the drop of blood touches the castor oil and allow the blood
to be suspended just below the meniscus.
4. Start the time and observe for a sign of visible dimpling after 10 minutes until a definite
serum is visibly seen around the suspended blood. Record the time

Normal Range: 15-45 minutes

Questions

1. What is the clinical significance if the clot retraction time is below 15 minutes and above
45 minutes?

2. How will the platelet count and hematocrit affect the clot retraction time?

3. Compare and contrast theMacFarlane method from the Hirschboeck method.

 ACTIVITY NO. 10

Capillary Fragility Test-Rumpel and Leede Method

The Rumpel and Leede test is also known as the tourniquet test and it determines the fragility of
the capillary. The use of the term-tourniquet test, however, is no longer being advocated because the
procedure does not necessarily use tourniquet in inducing capillary resistance.

The test is based on the principle that thrombocytopenic patients would have capillary resistance,
hence, producing petechiae in the skin. Thus, it could be used as a screening procedure in diseases where
platelets are decreased such as dengue.

Objectives:

 To perform the procedure of the test with utmost accuracy and precision.
 To correlate the results of the test with the clinical condition of the patient.
 To identify markers of capillary resistance such as petechiae, ecchymoses and purpura.

Materials:

 Sphygmomanometer
 Ruler
 Timer

Procedure

1. The Rumpel Leede test is to be examined a medical investigation around the stability of the
capillaries as well as the efficiency of the platelets.
2. For the execution of the test a blood pressure seal is put around an upper arm and inflated so far
that the pressure lies between the systolic and diastolic blood pressure.
 3. Over the exact value disagreement prevails in the literature. Thus e.g. recommended the pressure
to 10 mmHg above the diastolic blood pressure to adjust, in other place a pressure is indicated by
20 mmHg below the systolic pressure.
4. The seal becomes, gives it also here different statements, after 5 - 10 minutes again removes.
Show up in the arm below the congestion petechiae like that are positive the test.

Positive Results

The test is positive if there are more than 20 petechiae per square inch (a petechiae is a small red
or purple spot on the body, caused by a minor hemorrhage).

Illustrate a positive result

Questions

1. In what conditions, aside from dengue will you see an increased capillary fragility?

2. Differentiate petechiae, ecchymoses and purpura.

3. Why would a decreased platelet count will inadvertently result into petechiae, ecchymoses and
purpura?
 ACTIVITY NO. 11

Prothrombin Time (PT)

Principle of the method:

When calcium thromboplastin is added to citrated plasma, the factors of extrinsic coagulation
system are activated; the time to formation of a fibrin clot is then measured.

Clinical Significance
Since its original description by Quick in 1935, the Prothrombin time (PT) or Quick test has
remained an important test for detect disorders of blood coagulation, it is the common coagulation
procedure performed in routine laboratories, apart from the APTT.
The PT is particularly sensitive to detect of the extrinsic coagulation pathway (Factors II, VII, X
and Fibrinogen) as well as its inhibitors. It is an indicator of hepatic disease. It is also the most
commonly used test for monitoring oral anticoagulant therapy. PT is commonly used for monitoring
heparin anticoagulant therapy. Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.

Objectives

 To be able to perform Prothrombin time with utmost accuracy and precision.

 To be able to correlate clinically the results of the test with the clinical condition of the patient
 To be able to appreciate the importance of the Prothombin Time to diagnose blood disorders.

Materials

 Automatic Pipette
 Test tube
 Centrifuge tube
  Water bath
 Stop watch(timer)
 Working reagent (Rabbit Brain Thromboplastin and Buffer with CaCl 2)

MANUAL PROCEDURE

1. Reconstitute the Thromboplastin reagent according to the manufacturer’s insert

2. Pre-warm the reagent at 37˚C and the sample.
3. Add 200 ul of PT-Working Reagent and warm it in the water-bath for 5 mins. at 37˚C.
4. Add 100 ul of citrated plasma and mix.
5. Start the timer
6. Stop the timer if GEL FORMATIN is present.

Reference Value:
PT (seconds): 11-14 sec.
INR Ratio: 0.8-1.2
Calculations:
PT of the patient ∈sec
INR RATIO=
PT of normal plasma ( pool% )∈ sec

PT of normal plasma (pool %)∈sec

PT % Activity= ×100 %
PT of the patient ∈sec

MANUAL PROCEDURE

1. Reconstitute the Thromboplastin reagent according to the manufacturer’s insert

2. Pre-warm the reagent at 37˚C and the sample.
3. Set the thrombotme
4. Add 200 ul of PT-Working Reagent and warm it in the water-bath for 5 mins. at 37˚C.
5. Add 100 ul of citrated plasma and mix.
6. Start the timer
7. Stop the timer if GEL FORMATIN is present.

SEMI-AUTOMATED PROCEDURE
 1. Use the <TEST> button to select, for example, the “PT Test double”.
2. Break the cuvette racks at the point of fracture and place the double cuvettes in the two
incubation rows (4,5).
3. Add one ball to each cuvette using the ball dispenser.
Note: In this regard, the ball dispenser should be placed on the cuvette in such a manner that no
balls can bounce back.
4. Pipette 100μl of patient plasma into the double cuvette.
5. By exerting brief pressure on the right-hand cuvette, the incubation timer is started. At the
beginning of the incubation time a beep sounds and the right-hand cuvette of the corresponding
incubation row is illuminated in red. 10 seconds before the end of the incubation time the cuvette
starts flashing, a beep indicates that the incubation time has lapsed and the illumination is turned
off.
6. Once the incubation time has ended, place the cuvette in the measuring channel and press the
<RESET> button. The message “Adjust” will flash in the display. This means that the measuring
system is adjusting itself to the sample turbidity, and cannot start. Once the adjustment has taken
place, the display will show “0.0”. The measurement can now start.

Note: Using gentle pressure, insert the cuvettes into the measuring channel until they engage.

7. Draw up the PT reagent (200μl) and pipette it into the cuvette.

8. The measuring time will be stopped once coagulation occurs. Once both measuring times have
been determined, the second values for the individual measuring channels will be displayed
briefly, and the display will then automatically show the calculation value - e.g. % activity or INR
- required by the parameters. The second value and sample number can be accessed again by
pressing the <TEST> button.
9. Remove the cuvettes from the measuring channel and start the next measurements

Results:

NAME:

MANUAL SEMI-AUTOMATED

Prothrombin Time
PT % Activity
INR
 Interpretation

Questions:
1. What are the coagulation factors affected by prolonged PT?

2. Give the clinical significance of a prolonged PT?

3. What ids the INR? Gives its importance.

 ACTIVITY NO. 12

Activated Partial Thromboplastin Test

Principle of the method

When phospholipids complex and calcium chloride (CaCl2) are added to citrated plasma, the
factors of intrinsic coagulation system are activated; the time to formation of a fibrin clot is then
measured.
Clinical Significance
The time measurement of APTT is the most common coagulation procedure performed in routine
laboratories, apart from the PT. The APTT is particularly sensitive to defects of the
intrinsic coagulation pathway (Factors VIII, IX, X, XII). It is commonly used for monitoring
heparin anticoagulant therapy. Clinical diagnosis is should not be made on a single test result; it should
integrate clinical and other laboratory data.

Objectives

 To be able to perform the method of Activated Partial Thromboplastin Time

 To be able to correlate clinically the results of the test with the clinical condition of the patient
 To be able to appreciate the importance of the activated partial thromboplastin time to diagnose
blood disorders.

Materials

 Water bath
 Stop watch(timer)
 Working reagent (Ellagic Acid and Calcium chloride)
 Test tube rack
  Test tubes
 Automatic pipette
 Centrifuge

MANUAL PROCEDURE

1. Pre-warm all the reagents and the sample at 37˚C.

2. Add 100 ul of plasma in the tube +100 ul of R1 (ellagic acid) warm at 37˚C in 5 mins.
3. After performing the step 2, add R2 CaCl2) 1oo ul then mix.
4. Start the timer
5. Stop the timer if GEL FORMATION is present.

SEMI-AUTOMATED PROCEDURE

1. Use the <TEST> button to select, for example, the “PTT Test double”.
2. Break the cuvette racks at the point of fracture and place the double cuvettes in the two
incubation rows (4,5).
3. Add one ball to each cuvette using the ball dispenser.
Note: In this regard, the ball dispenser should be placed on the cuvette in such a manner that no
balls can bounce back.
4. Pipette 100μl of patient plasma and 100μl of PTT reagent (Ellagic acid) into the double cuvette.
5. By exerting brief pressure on the right-hand cuvette, the incubation timer is started. At the
beginning of the incubation time a beep sounds and the right-hand cuvette of the corresponding
incubation row is illuminated in red. 10 seconds before the end of the incubation time the cuvette
starts flashing, a beep indicates that the incubation time has lapsed and the illumination is turned
off.
6. Once the incubation time has ended, place the cuvette in the measuring channel and press the
<RESET> button. The message “Adjust” will flash in the display. This means that the measuring
system is adjusting itself to the sample turbidity, and cannot start. Once the adjustment has taken
place, the display will show “0.0”. The measurement can now start.

Note: Using gentle pressure, insert the cuvettes into the measuring channel until they engage.

7. Draw up the Calcium chloride reagent (100μl) and pipette it into the cuvette.
8. The measuring time will be stopped once coagulation occurs. The second value and sample
number can be accessed again by pressing the <TEST> button.
9. Remove the cuvettes from the measuring channel and start the next measurements
Reference Values:
APTT (in seconds) 25 - 38 secs.

Results

NAME:

MANUAL SEMI-AUTOMATED

Prothrombin Time
Interpretation

Questions

1. What are the coagulation factors affected by prolonged Activated Partial Thromboplastin Time?

2. Give the clinical significance of a prolonged Activated Partial Thromboplastin Time.

3. What is the importance of the activity factors in aPTT? Enumerate the different activity factors.

4. Differentiate PTT from aPTT.

 ACTIVITY NO. 13

Mixing Study

A common coagulation test used to distinguish between a coagulation factor deficiency, a factor
inhibitor and lupus anticoagulant. The test is performed when a patient has an explained prolongation of a
coagulation screening assay, such as the APTT. The mixing study is usually done by mixing equal
volumes of a patient plasma and pooled normal plasma and then repeating the aPTT on the mixture.
Principle
The basic principle is that the normal plasma contributes a sufficient concentration of clotting
factors to “correct” for a factor deficiency. A mixing study that corrects the aPTT is characteristic of
factor deficiency, whereas as mixing study that does not correct the aPTT indicates a factor inhibitor.

Objectives

 To be able to perform the method of 7 points Mixing studies

 To be able to correlate clinically the results of the test with the clinical condition of the patient
 To be able to appreciate the importance of the mixing studies to evaluate if there is a factor
deficiency or other coagulation disorder.

Materials

 Water bath
 Stop watch(timer)
 PT reagents
 aPTT reagents
  Test tube rack
 Test tubes
 Automatic pipette
 Centrifuge
 Thrombotimer

Procedure

1. Dilute citrated plasma with normal plasma 7 times using the dilution method below.

Patient
100 90 80 50 20 10 0
plasma
Normal
0 10 20 50 80 90 100
plasma

NOTE: Use normal human plasma from pre-determined samples and not normal control
2. Perform aPTT and PT using the diluted sample (Immediate mix result)
3. Incubate the diluted samples for 2 hours in 37C
4. Perform again the aPTT and PT using the incubated diluted sample (Incubated mix result)
5. Graph the results and observe the pattern.

Graph results

Boat-shape graph – signify a change in concentration

 If a change in shape is not observed, there is factor deficiency

 If there is a change in shape, this indicates the presence of inhibitors
 Inverted boat signifies the presence of lupus anticoagulant.

Results:
Questions:

1. What is the importance of Mixing Studies?

2. Give the advantages and disadvantages of 7-points mixing study.

3. Differentiate the two types of Mixing studies.