Bone Marrow Film Examination For Platelet Maturation Series: Activity No. 1
Bone Marrow Film Examination For Platelet Maturation Series: Activity No. 1
Bone Marrow Film Examination For Platelet Maturation Series: Activity No. 1
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:
Materials
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.
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:
Materials
Blood smear
Microscope
Cedar wood oil
Tally counter
Procedure
Results
Tally the results of the platelet count per field as follows:
Average
Interpretation
Questions:
3. How would you rate the accuracy and precision of this method?
ACTIVITY NO. 3
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:
Materials
Venipuncture set
EDTA tubes
RBC pipet
Counting chamber
Tally counter
Petri dish
Moistened filter paper
Microscope
Rees and Ecker fluid
Pipet shaker
Procedures
DCF = depth correction factor; DF = dilution factor; ACF = area correction factor
Results:
1. Tally the platelet counts in all five quadrants
Total
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:
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.
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
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
Total
2. Compute for platelet estimate. Show your calculation. Interpret the results.
Questions
2. What are the advantages of indirect method over the direct 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.
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 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
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
2. Illustrate the end point of the test showing the fibrin strands from the blood
Questions
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
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.
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?
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 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
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
Results
Questions
1. What is the clinical significance of increased and decreased clot retraction time?
b. Platelet count
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
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?
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).
1. In what conditions, aside from dengue will you see an increased capillary fragility?
3. Why would a decreased platelet count will inadvertently result into petechiae, ecchymoses and
purpura?
ACTIVITY NO. 11
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
Materials
Automatic Pipette
Test tube
Centrifuge tube
Water bath
Stop watch(timer)
Working reagent (Rabbit Brain Thromboplastin and Buffer with CaCl 2)
MANUAL PROCEDURE
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
MANUAL PROCEDURE
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.
Results:
NAME:
MANUAL SEMI-AUTOMATED
Prothrombin Time
PT % Activity
INR
Interpretation
Questions:
1. What are the coagulation factors affected by prolonged PT?
Objectives
Materials
Water bath
Stop watch(timer)
Working reagent (Ellagic Acid and Calcium chloride)
Test tube rack
Test tubes
Automatic pipette
Centrifuge
MANUAL PROCEDURE
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?
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
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
Results:
Questions: