MLS 12Aa: Hematology 2 (Laboratory)

MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS RECAP

9 are small, anuceate cytoplasmic fragments of megakaryocytes
Concept Map § anucleate means absence of nucleus
9 produced in the bone marror
9 1 mature megakaryocyte = 2,000 to 4,000 platelets
9 maturation period of 5 dyas
9 has a life span of 8-11 days or 9-12 days
9 gray-blue with purple granules in Wright’s stain
§ nucleus is not stained because it has no nucleus
9 2-3 um in diameter
9 1/3 is found in the spleen, 2/3 found in the blood

î ROLE OF PLATELETS IN THE BODY

o true blood cells
9 maintain blood vessel integrity by initiating vessel wall
repairs

o together with the blood vessel, play an essential role on the

primary hemostasis in the formation of the primary platelet plug
9 rapidly adhere to the surfaces of damaged blood vessels
î Platelets 9 form aggregates with neighboring platelets to plug the
o otherwise known as "thrombocytes" vessels
o plays an essential role in the body particularly in the: 9 secrete protesins and small molecules that
9 primary hemostasis for the formation of primary platelet trigger thrombosis, or clot formation
plug
9 thus monitoring its number and function is of great o major cells that control hemostasis
importance 9 a series of cellular and plasma-based mechanisms that:
î In the advent of automation in Hematology, it is still knowledgeable for the
• seal wounds
students to learn about the manual technique.
• repair vessel walls
î As future Medical Technologists, you are expected to be bound with the basics
• maintain vascular patency (unimpeded blood flow)
of platelet morphology and appearance as well as quantifying them in
numbers.
î CHARACTERISTICS
o 2 to 4 um in diameter
PLATELETS AND PLATELET COUNT
o round or oval
o anucleate
Platelets (Thrombocytes)
9 for this reason some hematologists prefer to call platelets
“cell fragments”
o slightly granular

Peripheral blood smear containing platelets (H) î their small size makes them appear insignificant
o but they are essential to life
and normal blood cells.
o are extensively studied for their complex physiology

î uncontrolled platelet and hemostatic activation are responsible for:

o deep vein thrombosis
o pulmonary emboli
o acute myocardial infarctions (heart attacks)
o cerebrovascular accidents (strokes)

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 MLS 12Aa: Hematology 2 (Laboratory)
o peripheral artery disease DIRECT METHODS IN PLATELET COUNT
o repeated spontaneous abortions (miscarriages) î most accurate way of platelet count (because it quantifies platelets directly)
î quantifies platelets directly
î THROMBOCYTOSIS î uses a counting chamber (or hemocytometer)
o elevated platelet counts
9 signal inflammation or trauma Direct Methods
9 but convey modest intrinsic significance î Guy and Leake’s method
î Brecher-Conkrite method
î THROMBOCYTOPENIA î Walker & Sweeney’s method
o low platelet count î Unopette method
9 a common consequence of drug treatment and may be life- î Van Allen’s method
threatening î Nygard’s method
9 usually accompanied by easy bruising and uncontrolled î Tocantin’s method
hemorrhage î Kristenson’s method as Modified by Leinpert
• because the platelet is responsible for normal
blood vessel maintenance and repair Brecher-Conkrite Method
9 accounts for many hemorrhage-related emergency î recommended method for manual platelet count
department visits
î principle:
î accurate platelet counting contributes to patient safety o diluent contains 1% ammonium oxalate which completely lyses RBC
o because it provides for the diagnosis of thrombocytopenia in many
disorders or therapeutic regimens î phase contrast microscope
o enhance the refractiveness of the platelets
Platelet Count o makes the identification easier
î aka thrombocyte count o recommended microscope for manual platelet count
î the number of platelets in 1 L or 1 mL of whole blood
Reese-Ecker Method
î this test is used to: î most commonly used in direct platelet count
o diagnose bleeding disorders
o monitor conditions affecting the bone marrow î uses Reese-Ecker diluting fluid
o assess quantitative platelet disorders o contains:
9 brilliant crescyl blue --- stains
î can be performed using direct and indirect methods platelets as light blue
9 citrate
î manual platelet count 9 formaldehyde
o performed to assess the number of circulating platelets in the blood
î ordinary light microscope can be used
Materials: î HPO is used
î EDTA-anticoagulated blood o the 25 squares of the large central square is counted
î Reese-Ecker diluting fluid
î Pipette shaker 1 Rinse the RBC pipette with the Reese-Ecker diluting fluid.
î Neubauer counting chamber 2 Centrifuge and filter the diluent.
î Glass slides 3 Draw blood to the 0.5 or 1 mark of the RBC pipette.
î Wright’s stain 4 Draw diluent (centrifuged/ filter) to the 101 mark of the RBC pipette.
î Microscope 5 Place on the pipette shaker for 5-10 minutes.
6 Expel the first 5 drops and load them on both sides of the Neubauer
counting chamber.

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 MLS 12Aa: Hematology 2 (Laboratory)
7 Allow to stand for 15 minutes in a covered Petri dish with moist filter î counting area for direct platelet count
paper to allow platelets to settle.
8 Platelets are counted using the 40X objective lens (400X total 9 the whole of the large central square (is
magnification). outline in blue) is the platelet counting area
9 Count the number of platelets in the 25 small squares in the
2
center square of the grid. 9 it comprises 1 mm as its area
9 This area of the square is 1 mm2.
9 Platelets should be counted on each side of the 9 the large central square is divided into 25
hemocytometer smaller squares and each square is further
9 The difference between the totals should be less than 10%. divided into 16 squares
9 Average and compute for the Platelet Count.
9 this means that you have to count the
î In manual identification of platelets, they appear as: platelets in the entire 25 squares of the large
o highly refractile central square
o round bodies
o approximately 1/10 the size of the surrounding cells 9 upon obtaining the number of platelets in both the upper and lower chambers of
o diameter of 2 to 4 um the hemocytometer, you can now proceed into solving for the platelet count
o appear round or oval
o displaying a light purple sheen when phase-contrast microscopy is Computation Formula:
used

î shape and color

o help distinguish the platelets from highly refractile dirt and debris
Normal Range: 150 – 450 x 109 platelets/uL
î “ghost” RBCs often are seen in the background
EXAMPLES
î Given:
o Platelets in upper chamber = 214
o Platelets in lower chamber = 220
o Dilution Factor = 1:100
o Number of squares counted = 1

note:
9 assuming that you obtained these values, check first if the platelet
counted in both the upper and lower chambers must agree within
10%
9 also, take note of the dilution used
9 the number of squares counted is uniform for platelet count since the
note: entire large central square is counted
2
9 the appearance of platelets in two of the 25 small squares in the center o it constitutes to 1 mm as its area
square of a hemocytometer when performing direct platelet count

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 MLS 12Aa: Hematology 2 (Laboratory)
o ANSWER:
9 120,000/ul
9 platelet count is below the normal range

î Given:
o Platelets in upper chamber= 485
o Platelets in lower chamber= 479
o Dilution Factor= 100

o ANSWER:
9 217,000 platelets/ul
9 platelet count is within the normal range

note:
9 this is how to compute for platelet count
9 substitute the obtained values in the general formula
9 first, average the number of cells counted by adding them both and
dividing by 2
o multiply the result to the dilution factor o ANSWER:
9 dilution factor can be obtained by getting the reciprocal of the dilution 9 482,000 platelets/uL
used 9 platelet count is above the normal range
o in this case its 100 because the dilution used is 1:100
9 upon reaching the denominator of the formula, we have fixed values
o number of squares counted which is 1
2
o area of one square which is 1mm NOTES TO REMEMBER WHEN PERFORMING DIRECT
o depth of the chamber which is 0.1 mm METHOD OF PLATELET COUNT:
9 take note of the values and units used
9 in calculating, simplify the values in both the numerator and diluting fluid 1% Ammonium Oxalate
denominator of the formula most common dilution 1:100
fewer than 50 platelets are adjust the dilution to 1:20
î Given: counted on each side
o Platelets in upper chamber= 111 more than 500 platelets are adjust the dilution to 1:200
o Platelets in lower chamber= 115 counted on each side
o Dilution Factor= 100 objective 40x phase contrast microscopy
2
area counted 1mm

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 MLS 12Aa: Hematology 2 (Laboratory)
Other Direct Methods for Platelet Count: 1 Disinfect the puncture site.
2 Diluting fluid is placed over the puncture site.
Reagents: 3 Puncture through the drop of diluting fluid.
9 40% HCHO 4 Transfer the mixture of blood and diluting fluid on the center of the
9 Sodium citrate coverslip and invert over a glass slide.
9 Crystal Violet 5 Allow it to stand in a moist chamber for 15-45 minutes.
6 Examine the prepared smear under OIO.
Procedure: 7 Count 250 RBCs per area in 4 areas to a total of 1,000 RBCs and
9 Rise RBC pipette with Reese- Ecker fluid. count all the platelets seen.
9 Draw blood to 0.5 mark and diluting fluid
Guy and Leake’s
1 to 101 mark.
Method Modified Fonio's Method
9 Mix well by shaking.
î similar to Damechek but mixture is smeared on a slide, dried and stained
9 Charge and allow to settle for 5 to 15
minutes in a moist chamber.
1 Stain a perfect smear using Wright’s stain.
Computation: 2 Under OIO, count platelets in 10 consecutive fields
9 Assuming that each field will have 3-10 platelets per 100
RBCs.
3 Multiply the number of platelets counted to 2,000 to obtain the platelet
count.
Phase Microscopy the recommended method for manual platelet
2 Method/ Brecher- count uses 1% ammonium oxalate & utilizes the
Cronkite same procedure with Reese- Ecker Normal Value: 150, 000 - 350, 000 platelets/mm3
3 Walker & Sweeney’s Method
Dameshek and Fonio’s Method Computation
4 Unopette Method (1:100 dilution factor)
5 Van Allen’s Method (reported in percent)
6 Nygard’s Method
7 Tocantin’s Method
8 Kristenson’s Method as Modified by Leinpert Modified Indirect Platelet Count
î mostly used by laboratories
INDIRECT METHODS IN PLATELET COUNT o to counter check the results of platelet count as released by
î Platelets are counted in relation to 1000 RBCs in the peripheral blood smear automated analyzers
o we don’t need to use a counting chamber anymore
o instead, platelets are counted by evaluating or studying blood î a medical technologist should prepare a good quality smear
smears î the smear is stained using Wright’s stain
î using the battlement method of counting, count platelets in 10 consecutive
Indirect Methods OIF
î Dameshek method o assuming that each field contains 100 RBCs
î Fonio’s method
î Olef’s method Computation Formula:
î Cramer and Bannerman method
î modified indirect platelet count

Dameshek Method
î capillary puncture is done
î diluting fluid is placed over the puncture site
î mixture (blood and diluting fluid) is placed on a cover slip and inverted on a
slide
î platelets are counted in 1000 RBCs

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 MLS 12Aa: Hematology 2 (Laboratory)
Examples
î Suppose you counted a total of 115 platelets on 10 consecutive oil immersion
fields.
o by using the 2,000 factor, the platelet count would be
230,000/mm3 (Number of platelets x 2,000)
o this value will be reported as normal
9 since the platelet count falls on the 150, 000 - 350, 000
platelets/mm3 range

î A medical technologist counted a total of 60 platelets on 10 consecutive oil

immersion fields. Calculate the platelet count and interpret.

Reporting of Platelet Estimate

*normal can also be called adequate

Platelets in a blood smear

î have an average diameter of 2 to 4 um
ANSWER: î with younger platelets being larger than older ones
9 132, 000 platelets/mm3 î in contrast to megakaryocytes, platelets have no nucleus
9 platelet count is below the normal range î cytoplasm is light blue with evenly dispersed, fine red-purple granules

î A platelet estimate was performed on a peripheral blood smear. A total of 177

platelets was obtained. How would you report for the platelet count?

177 x 2, 000

ANSWER:
9 354, 000 platelets/mm3
9 platelet count is above the normal range

Platelet Estimate note:

î The average number of platelet in 10 consecutive OIO fields when multiplied 9 appearance of platelets in peripheral blood smear when performing
to 20,000 estimates the platelet count per uL or mm3. indirect platelet count

note: Other Indirect Methods for platelet count:

9 in some laboratories and hospitals, platelet count is not reported in
numbers, sometimes results are released descriptively according to Reagents:
laboratory protocol and platelet estimate is one method used 9 Brilliant Crescyl Blue
9 here, the average number of platelets counted on 10 OIF are multiplied 9 Sucrose
by 20,000 9 0.4 g Sodium Citrate
9 each platelet estimate falls on a certain range and has a corresponding 9 40% HCHO
reporting 1 Dameshek Method
Procedure:
9 Place a gtt of diluting fluid over the
puncture site.
9 Puncture through the drop to prevent the
disintegration of platelets.

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 MLS 12Aa: Hematology 2 (Laboratory)
9 Transfer a drop of mixture on the center of CONDITIONS THAT AFFECT PLATELET COUNT
the coverslip.
9 Invert over a glass slide. THROMBOCYTOPENIA
9 Allow to stand on a moist chamber for 15 î Thrombocytopenia
minutes. o the decrease in number of circulating platelets
9 Examine the smear under the o platelet value is lower than 100,000/ uL
microscope.
9 Count 1000 RBCs and all the platelets î this can be seen in cases of:
seen within the count. o thrombocytopenia purpura
o aplastic anemia
Computation: o acute leukemia
o pernicious anemia
o Gaucher’s disease
1, 000 o sometimes following chemotherapy and radiation

Normal Value: Megakaryocyte

9 250, 000- 500, 000 platelets/mm3 hypoplasia
Decreased platelet

THROMBOCYTOPENIA
Reagent: production
9 14% MgSO4 Ineffective
thrombocytopoiesis
Increased platelet
Procedure:
destruction
9 Place a gtt of diluting fluid over the
puncture site.
9 Puncture through the drop to prevent the Abnormalities in
disintegration of platelets. distribution or dilution
2 Fonio’s Method 9 Transfer a drop of mixture on a glass
slide.
9 Make a smear and dry.
note:
9 Stain with Wright’s stain.
9 most of the diseases on the first classification results to chromosomal
9 Examine the smear under the
abnormalities or genetic defects that leads to megakaryocytic hypoplasia
microscope.
in the bone marrow
9 Count 1000 RBCs and all the platelets
o this may be caused by congenital, neonatal or acquired condition
seen within the count.
9 another mechanism which is ineffective thrombocytopoiesis is manifested
3 Olef’s Method by megaloblastic anemia which is a disorder in the production of red blood
Crammer & Bannerman cells
4
Method o in here, platelets are also adequately produced
Procedure: o in megaloblastic anemia, megakaryocytes even if normal in amount
9 Prepare a good quality smear. in the bone marrow, there would be still a decreased platelet count
9 Stain with Wright’s or Giemsa. due to impaired DNA synthesis
Modified Indirect 9 Using the crenellation method in counting, o an impaired DNA synthesis results to a grossly abnormal formation
5
Platelet Count count the platelets in 10 consecutive OIO of the megakaryocytes, thus resulting in impaired platelet or
fields using 10x eyepiece and multiply by decreased platelet production
2000 to obtain the platelet count.

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 MLS 12Aa: Hematology 2 (Laboratory)
More specific diseases under the megakaryocytic hypoplasia in the bone Increased or accelerated platelet destruction are caused by the following
marrow: diseases:

Decreased platelet
THROMBOCYTOPENIA

production
Immune
Increased platelet
destruction
Non-immune
Abnormalities in
distribution or dilution

note:
9 in here, there is an increased or normal platelet production thus platelet
count is normal
9 where does thrombocytopenia occur?
o it occurs when the production capacity is no longer adequate to
compensate for the increased rate of destruction
o this increased platelet destruction can be caused by immune
responses in the body, mechanical damage or consumption as seen
on non-immunologic process
note:
9 in hypersplenism, there is increased platelet sequestration and as a
consequence, there will be a decreased number of platelets in the venous
blood or in the circulation
9 another example if found in cases of massive blood transfusions where
platelets viability is impaired due to storage or temperature conditions which
causes temporary thrombocytopenia

THROMBOCYTOSIS
î Thrombocytosis
o an abnormally high platelet count, typically 450,000/ uL
o increased in circulating platelets

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 MLS 12Aa: Hematology 2 (Laboratory)
o this can be seen on cases of:
9 polycythemia vera WHY ARE PLATELETS DIFFICULT TO COUNT?
9 idiopathic thrombocytopenia (drawbacks)
9 CML î they easily disintegrate
9 splenectomy î small, colorless, refractile bodies
î difficult to distinguish from debris
î unevenly distributed in the blood
THROMBOCYTOSIS î have a tendency to attach to foreign surface
Reactive î have a tendency to clump with each other
thrombocytosis
these conditions may lead to a variety of sources of error

Myeloproliferative
disorders
SOURCES OF ERROR

9 can cause the platelets to clump on the

note:
Inadequate mixing and
9 reactive thrombocytosis can result to elevated platelet count due to
poor collection of the
inflammation, trauma or underlying related conditions
specimen
o the term reactive also means secondary
9 in myeloproliferative disorder, there is an excessive production of mature
erythrocytes, granulocytes and platelets with thrombocytosis as its hallmark Dirt in the pipette,
o platelet production remains responsive to thrombopoietin (TPO) and hemacytometer, or
there is an increased production rate diluting fluid
The phenomenon of
“platelet satellitism” may
occur when an EDTA
anticoagulant is used
hemacytometer
9 if the problem persists after the predilution, a new
specimen is needed
9 a skin puncture specimen is less desirable
because of the tendency of the platelets to
aggregate or form clumps
9 may cause the counts to be inaccurate
9 this refers to the adherence of platelets around
neutrophils, producing a ring or satellite effect
9 using sodium citrate as an anticoagulant should
correct this problem
9 because of the dilution in the citrate evacuated
tubes, it is necessary to multiply the obtained
platelet count by 1.1 for accuracy

9 they easily disintegrate

Aside from the above 9 they are small, colorless, refractile bodies
mentioned sources of 9 difficult to distinguish from debris
errors, platelets are 9 unevenly distributed in the blood
difficult to count because: 9 have a tendency to attach to foreign surfaces

î glassware must be scrupulously cleaned

o because debris and dusts are easily mistaken for platelets

î diluting fluid must be filtered just before use

o in order to remove particles

î if venous blood is used, platelets must be counted within 3 hours

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 MLS 12Aa: Hematology 2 (Laboratory)
o because whenever there is a delay, it will cause platelet DIRECT METHOD
disintegration and platelet clumping Brecher-Cronkite Reese-Ecker
Diluent 1:1 or 1% ammonium Reese-Ecker diluting fluid
î blood should be rapidly diluted oxalate - brilliant crescyl blue
o in order to prevent platelet clumping - formaldehyde
- citrate
î blood should be thoroughly mixed with the diluent by shaking Most common dilution 1:100 1:100
o because inadequate mixing may result in platelet clumping Pipette used RBC pipette RBC pipette
Procedure EDTA anticoagulated blood is used
î charged hemocytometer must be kept in a moist chamber
o to prevent evaporation and for cells to setlle down

î platelet satellitism
o occurs when EDTA anticoagulant is used
o refers to the occurrence of platelets around neutrophils producing a
ring or satellite
o use sodium citrate as an anticoagulant and after obtaining the
platelet count, multiply the value by 1.1 for accuracy

Recommended phase-contrast compound

microscopy microscope,HPO (40x)
Formula

SUMMARY (SYNCHRONOUS)
RBC pipette VS WBC pipette
PLATELETS

Other name Thrombocytes

Distribution in the body 1/3 in spleen
2/3 blood (circulation)
Maturation period 5 days
Life span 8-11 days or 9-12 days
Function essential for primary hemostasis in the
formation of primary platelet plug Procedure:
Size 2-4 um in diameter 1. rinse the RBC pipette with the Reese-Ecker diluting fluid
9
Normal range 150 – 450 x 10 platelets/uL • to avoid adherence of platelets to the RBC pipette
• this is because platelets tend to adhere on a glass
METHODS OF PLATELET COUNT
î 2 methods 2. centrifuge and filter the diluent
o direct method • to remove the debris in the diluting fluid
o indirect method

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 MLS 12Aa: Hematology 2 (Laboratory)
• remove the diluting debris because debris are sometimes mistaken
as platelets

3. draw blood to the 0.5 or 1 mark of the RBC pipette

4. draw the diluent (centrifuged/filter) to the 101 mark of the RBC pipette

5. place on the pipette shaker for 5-10 minutes

• to mix the diluent and the blood
• in the absence of pipette shaker, stop the opening of the RBC pipette
and shake it with your hand

6. expel the first 5 drops and load them on both sides of the Neubauer counting Formula:
chamber
• to make sure that the mixture in the bulb is sampled to the
hemocytometer and not the initial sample from the calibrated stem
because the solution in the calibrated stem is unmixed and relatively
cell-free 

constants:
• to avoid falsely decreased platelet count 2
î area of 1 square = 1mm
î depth =0.1mm
7. allow to stand for 15 minutes in a covered Petri dish with moist filter paper to
î no. of squares counted = 1
allow platelets to settle
• prevent evaporation of sample while waiting for the cells to settle
normal range:
• moist filter paper is used to avoid evaporation and drying of the
î 150 – 450 x 109 platelets/uL
counting chamber
if dilution factor is not given:
8. Platelets are counted using the 40X objective lens (400X total magnification).
Count the number of platelets in the 25 small squares in the center square of
the grid. This area of the square is 1 mm2. Platelets should be counted on
each side of the hemocytometer and the difference between the totals should
be less than 10%. î total volume of sln in the bulb = 100 uL
9. Average and compute for the platelet count
INDIRECT METHOD
note: Procedure
9 platelets are colored blue because the Reese-Ecker diluting fluid has brilliant Dameshek uses coverslip (wet preparation)
crescyl blue which stains platelets as light blue
9 platelets are highly refractile and round bodies
9 platelets display a light purple sheen

Rules in counting platelets: (same with RBC and WBC count)

î count all cells seen inside central large square
î count all cells touching the top and left margin line of the counting area 

î in cases that there are two margin lines, the reference line is the inner line Fonio’s uses glass slide
î in cases that there are three margin lines, the reference line is the middle line
î follow the crenillation technique or the battlement method in counting to
prevent recounting the cells.

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 MLS 12A: Hematology 2 (Lecture)

MLS 12Aa: Hematology 2 (Laboratory) Causes of Relative / Reactive / Secondary Thrombocytosis
î hemorrhage or surgery
o low platelets after 2-6 days note:
§ platelets 2 m
9 hig

o return to pre-hemorrhagic levels 10-16 days after blood loss 9 in chronic inflamm
increases
note: 9 there is no specific ex
Formula 9 primarily during surgery, it is expected to have low platelet count due 9 platelets are also indi
to hemorrhage 9 aside from inflammat
(Dameshek 9 during surgery there is blood loss and bleeding platelets also increas
and Fonio’s) 9
9
after 10-16 days gulpi mataas ang platelet number
eventually, it will return to its normal number î exercise-induced (stress)
o release of platelets fro
î postsplenectomy o returns to its pre-exer
Formula o spleen sequesters 1/3 of platelets of exercise
o increase by 30% to 50%
(modified § reach or exceed 1 million/uL note:
o reaches a maximum 1-3 weeks and remain elevated for 1-3 months 9 during exercise, phys
Fonio’s) o rebalancing of the circulating platelet pool to incorporate the splenic back to the circulation
platelet pool count
9 platelets move from s
Indirect method note:
9 if spleen is removed, platelets will stay in the circulation resulting to
9 platelet count will be

î Platelets are counted in relation to 1000 RBCs in the peripheral blood smear high platelet number î rebound
o reaches a peak 10 to
o in viewing the smear, estimate if the RBC count in the field is 1000 î iron deficiency anemia § alcohol or m
o 50% of mild IDA secondary to chronic blood loss § therapy for t
o platelet count may be 2 million/uL
î in Dameshek method, it is allowed to stand for 15 minutes because it is a wet o role of IRON in regulating thrombopoiesis
§ TPO
note:
9 results when you a
preparation note: decreases platelet nu
9 iron can normally regulate TPO 9 avoiding the drug or c
9 if you are iron deficient, there is no enough iron to control or regulate 9 example: alcohol inta
TPO which will eventually result to elevated platelet number from the bone marrow
Modified Fonio’s method 9 iron and TPO is inversely proportional to each other prompt increase in p
î used in the laboratory commonly in hospitals » if there is less iron, there is continuous function of TPO (high
platelet number) 9
number will be back t
example: methotrexa
î a medical technologist should prepare a good quality smear » presence of normal iron, TPO will be regulated (normal also inhibits the relea
treatment is over, pla
platelet number)
o uses whole blood EDTA eventually go back to
î inflammation and disease
î the smear is stained using Wright’s stain o thrombocytosis as indication of inflammation Thrombocytosis Related with M
î using the battlement method of counting, count platelets in 10 consecutive o activation of the inflammatory process
o early evidence of a tumor and various carcinomas
î
î
not in response to TPO
platelets are usually >1000 x 1
OIF o thrombocytosis in the absence of active bleeding
note:
o example: Kawasaki disease
§ inflammation of the walls of small and medium-sized 9 this disease has an u

MLS 12A: Hematology 2 (Lecture) §

arteries throughout the body
coronary artery thrombosis and aneurysms
because it does not r

PADAYON, RMT!!!
PLATELET ESTIMATE Causes of Relative / Reactive / Secondary Thrombocytosis § platelets 2 million/uL
î hemorrhage or surgery 9 higher risk of cardiovascular complications
î important because it tells us what is the platelet levelo of lowthe patient
platelets after 2-6 days note:
î results are qualitative o return to pre-hemorrhagic levels 10-16 days after blood loss 9 in chronic inflammation and disease, platelet number/count
increases
note: 9 there is no specific explanation why this happens
9
primarily during surgery, it is expected to have low platelet count due 9 platelets are also indicators of inflammation (the same with ESR)
Review module 3 in Hematology 2 lecture for thrombocytosis and thrombocytopenia
to hemorrhage 9 aside from inflammatory process, if there is tumor and carcinomas,
THROMBOCYTOSIS 9 during surgery there is blood loss and bleeding platelets also increases
9 after 10-16 days gulpi mataas ang platelet number
î CAUSES: 9 eventually, it will return to its normal number î exercise-induced (stress)
o release of platelets from the splenic pool or hemoconcentration
o myeloproliferative neoplasms (MPNs)î / essential
postsplenectomy
thrombocythemia o returns to its pre-exercise baseline level 30 minutes after completion
o relative/secondary thrombocytosis o spleen sequesters 1/3 of platelets of exercise
o increase by 30% to 50%
§ platelets increase for compensation§ for reach the following
or exceed 1 million/uL note:
reasons o reaches a maximum 1-3 weeks and remain elevated for 1-3 months
o rebalancing of the circulating platelet pool to incorporate the splenic
9 during exercise, physical activity or stress, platelets in the spleen go
back to the circulation which results to increased platelet number or
9 hemorrhage or surgery platelet pool count
9 platelets move from splenic pool to circulation pool
9 postsplenectomy note: 9 platelet count will be back to normal *kung nagpahuway ka*
9 iron deficiency anemia 9 ifhigh spleen is removed, platelets will stay in the circulation resulting to
platelet number î rebound
9 inflammation and disease o reaches a peak 10 to 17 days after withdrawal of the following
î iron deficiency anemia § alcohol or methotrexate
9 exercise induced stresso 50% of mild IDA secondary to chronic blood loss § therapy for the underlying condition
9 rebound o platelet count may be 2 million/uL
o role of IRON in regulating thrombopoiesis note:
§ TPO 9 results when you are taking a certain chemical or drug which
note: decreases platelet number
9 iron can normally regulate TPO 9 avoiding the drug or chemical will rebound the platelet number
9 if you are iron deficient, there is no enough iron to control or regulate 9 example: alcohol intake --- alcohol will inhibit the release of platelets
TPO which will eventually result to elevated platelet number from the bone marrow--- when you withdraw alcohol, there will be a
9 iron and TPO is inversely proportional to each other prompt increase in platelet number (but after a few weeks, platelet
» if there is less iron, there is continuous function of TPO (high number will be back to normal)
platelet number) 9 example: methotrexate --- this is a treatment for cancer patients --- it
» presence of normal iron, TPO will be regulated (normal also inhibits the release of platelets from the bone marrow --- if the
platelet number) treatment is over, platelet number will promptly increase (and will
eventually go back to normal after a few weeks)
î inflammation and disease
PADAYON, RMT !!! o thrombocytosis as indication of inflammation
o activation of the inflammatory process î not in response to TPO QUITAG,BULQUERIN, DEALA
Thrombocytosis Related with MPNs (Myeloproliferative Neoplasms)
12
9
o early evidence of a tumor and various carcinomas î platelets are usually >1000 x 10 L (1 million per microliter)
o thrombocytosis in the absence of active bleeding
o example: Kawasaki disease note:
 MLS 12A: Hematology 2 (Lecture)
MLS 12Aa:

Hematology 2 (Laboratory)
Myeloproliferative Disorders § could be antibody mediated or not related to antibody
î essential or primary thrombocythemia § non-immune if no involvement of antibody
THROMBOCYTOPENIA
MLS 12A: Hematology 2 (Lecture)
î chronic myelogenous leukemia
î REASONS: î polycythemia vera
î chronic idiopathicproduction
myelofibrosisof platelets
§

§
immune if involvement of antibody
o increased sequestration
the problem could be the spleen

o impaired or decreased
§ chemotherapy, drug, alcohol abuse § 1/3 of the platelet population is on the spleen
Essential or Primary Thrombocythemia
Myeloproliferative Disorders § could be antibody mediated § increased destruction
or not related of platelet = increased sequestration
to antibody
î essential or primary aplastic
§î thrombocythemia
characterizedanemia

by peripheral blood platelet counts >1million/uL § non-immune o if no involvementofofdistribution
abnormalities antibody or dilution
î chronic myelogenous TAR

§î leukemia
uncontrolled proliferation of marrow megakaryocytes § immune if involvement § ofdilution
antibody – there could be a false decrease in the number of
î polycythemia vera § replacement
o because ofofnormal
a mutated marrow
gene o increased sequestration platelets because of increased plasma volume
î chronic idiopathic myelofibrosiso one quarter have a mutation in the Janus-associated kinases (JAK1 § the problem could be§ theplasma spleen level might be elevated (hemodilution)
§ megaloblastic anemia

and JAK2) § 1/3 of the platelet population
o increased is on the spleen
consumption
§ Bernard
Essential or Primary Thrombocythemia Soulier

î acquired and prevent in middle-aged and older patients § increased destruction § of ifplatelet
there= increased
is increased sequestration
activation of platelets thereby
î May-Hegglin
î characterized by§peripheral blood platelet
hemorrhage, Anomaly

plateletcounts >1million/uL
dysfunction and thrombosis o abnormalities of distribution or dilution consuming the platelets it will result to decreased platelet
î uncontrolled proliferation
§ ofomarrow
Wiskott-Aldrich megakaryocytes
hemorrhage syndrome
is present (many platelets but dysfunctional) § dilution – there could be number a false decrease in the number of
in the circulation
o because of a mutated o geneactivation of platelets resulting to thrombosis platelets because of§increased platelets plasma volume
that are utilized in the formation of clot cannot be
o increased platelet destruction
o one quarter have a mutation in the Janus-associated kinases (JAK1 § plasma level might be elevated (hemodilution)
recycled and eventually, this will be disintegrated
§
and JAK2) Immune causes:involvement of antibodies o increased consumption
î Blood Picture: § if there will be more formation of clots, because platelets are
o9 platelets
î acquired and prevent in middle-aged immuneandmay thrombocytopenic
older patients
be: purpura (ITP)
 § if there is increased continuously activation ofactivated,plateletsthisthereby
could result to its consumption
î hemorrhage, platelet dysfunction 9 HDN and thrombosis
§ / heterogenous
HTR
 size (vary in consuming the platelets
§ it will result to(gakaubos)
consumption decreased platelet
--- result to decreased platelet
o hemorrhage is present (many platelets but dysfunctional) number in the circulation
9 neonatal
o activation of platelets resulting
sizes) alloimmune / autoimmune number
§ to thrombosis
bizarrely
thrombocytopenia shaped § platelets that are utilized§ in the formation
platelets of clot cannot
if continuously be they are being utilized
activated,
§ may be clumped on blood recycled and eventually, this will
and be disintegrated
consumed (thrombocytopenia)
î Blood Picture: 9 drug-induced films thrombocytopenia
 § if there will be more formation of clots, because platelets are
o platelets may be: 9 post§ transfusion
may be agranular purpura or continuously activated, this could result to its consumption
§
§ heterogenous
Non-immune
sizes)
size (vary in
cause: no
hypogranular involvement
(wala unod) of antibodies
§
WHY consumption (gakaubos) --- result to decreased platelet
ARE PLATELETS DIFFICULT TO COUNT?
number
9 §
pregnancy have and a clear, light blue
preclampsia î platelets have a activated,
tendencythey to are
clump with each other
§ bizarrely shaped § platelets if continuously being utilized
appearance (dapat purple)
o increased
§ may be sequestration
clumped on blood and consumed whole blood is the optimum in platelet count through venipuncture
o (thrombocytopenia)
o presence of megakaryocyte fragments or nuclei
§ filmshypersplenism o we cannot rely so much in capillary puncture because there are some
§ may be agranular or
§î hypothermia
Bone Marrow: small vessel bleeding patients
in the who areattributed
skin not bleedersto thrombocytopenia manifests as
hypogranular (wala unod)
o abnormalities
§
o of megakaryocytes
have a clear, distribution
light blue or dilution
hemorrhages of different sizes
o increased consumption
appearance large (normal size is 30-50 um)
(dapat§ purple) SOURCES OF 9 ERROR
A – petechiae
AND PRECAUTIONS
o presence of megakaryocyte cellular or
§ fragments atypia
nuclei 9 B – purpura
§ TTP (thrombotic § thrombocytopenic purpura)
tend to form clusters (clump within the bone marrow) î glassware must
9 C – ecchymosis be scrupulously cleaned
î Bone Marrow:
§ HUS (hemolytic uremic syndrome)
 o becausethrombocytopenia
small vessel bleeding in the skin attributedo to simple
debris and dusts
bruising are easily mistaken for platelets
manifests as
o megakaryocytes9 O157:H7 E.coli hemorrhages of different sizes o plateletso whenever tend to adhere
there in glassware
will be involvement that
of clot is the
within whytissue,
the RBC pipette
§ § largeDIC(normal size is 30-50THROMBOCYTOPENIA
(disseminated intravascular coagulation)
um) 9 A – petechiae must be it will be called
rinsed firsthematoma
with the diluting fluid
§ î general mechanisms 9 B – purpura
cellular9atypia prolonged
impaired(clump
APTT and
or decreased
PT
§ o clusters
tend to form within theproduction of platelets
bone marrow) Impaired and Decreased Production
9 C – ecchymosis
9 increased D-dimer
primarily, the levels
problem is in the bone marrow (origin) î diluting fluid must be filtered just before use
§ o Conditions related:
simple bruising
9 decreased § fibrinogen
problem in the levels
production or proliferation of the o will
î chemotherapy,
o whenever there in order
bedrug, toalcohol
involvement remove abuse
of particles
clot within the tissue,
THROMBOCYTOPENIA megakaryocytes thus, lesser platelets are formed or it î aplastic
will be called to remove debris and dirt
o anemia
hematoma
î general mechanisms produced î TAR
§
o impaired or decreased production decreased production of megakaryocytes which
of platelets will affect
Impaired î replacement of normal marrow
and Decreased Production
§ primarily, the problem isthe
in number
the boneofmarrow
platelets
(origin) Conditions related:
îî ifmegaloblastic
venous blood is used, platelets must be counted within 3 hours
anemia
§ problem o in increased platelet destruction
the production or proliferation of the Bernard
î chemotherapy, drug,îalcohol because whenever there is a delay, it will cause
oabuse
Soulier platelet
megakaryocytes §thus,there could
lesser be autoantibodies
platelets are formed andor alloantibodies
î that is anemia
aplastic î May-Hegglin disintegration
Anomaly and platelet clumping
produced directed to the individual platelets î TAR î Wiskott-Aldrich syndrome
§ decreased production of megakaryocytes which will affect î replacement of normal marrow
PADAYON, RMT!!!of platelets
the number
î blood should be rapidly dilutedBULQUERIN, BAES, DEALA, QUITAG
î megaloblastic anemia 3
o increased platelet destruction î Bernard Soulier o in order to prevent platelet clumping
§ there could be autoantibodies and alloantibodies that is î May-Hegglin Anomaly
directed to the individual platelets î Wiskott-Aldrich syndrome
î blood should be thoroughly mixed with the diluent by shaking
PADAYON, RMT!!! o because
BULQUERIN,inadequate mixing may
BAES, DEALA, QUITAG 3 result in platelet clumping
o that is why shaking of the blood and diluent mixture 5-10 minutes

î charged hemocytometer must be kept in a moist chamber

o to prevent evaporation and for cells to settle down
o achieved by placing a moist filter paper in a petri dish

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 13

 MLS 12Aa: Hematology 2 (Laboratory)
î platelet satellitism 4. Explain the effect of platelet satellitism and clumping on complete blood
o occurs when EDTA anticoagulant is used count results.
o refers to the occurrence of platelets around neutrophils producing a 9 Platelet satellitism and platelet clumping can cause pseudo-
ring or satellite thrombocytopenia (falsely decreased)
o remedy: use 3.2% sodium citrate as an anticoagulant and after
obtaining the platelet count, multiply the value by 1.1 for accuracy 5. Identify conditions that may influence the increase and decrease in
platelet counts.
FOLLOW UP QUESTIONS 9 increase in platelet count (thrombocytosis)
1. State the importance of platelet count. o myeloproliferative neoplasms (MPNs) / essential
9 used to screen for or diagnose various diseases and conditions that thrombocythemia
can cause problems with blood clot formation o hemorrhage or surgery
9 may be used as part of the workup of a bleeding disorder, bone o postsplenectomy
marrow disease, or excessive clotting disorder o iron deficiency anemia
9 used to assist in the diagnosis of bleeding disorders and to monitor o inflammation and disease
patients who are being treated for any disease involving bone o exercise induced stress
marrow failure o rebound
9 decrease in platelet counts (thrombocytopenia)
9 this test is used to: o chemotherapy, drug, alcohol abuse
o diagnose bleeding disorders o aplastic anemia

o monitor conditions affecting the bone marrow o TAR

o assess quantitative platelet disorders o replacement of normal marrow
o megaloblastic anemia

2. Identify the different methods in performing manual platelet count.
o Bernard Soulier

Students need to identify platelets in both direct and indirect methods.
o May-Hegglin Anomaly

9 direct methods
o Guy and Leake’s method o Wiskott-Aldrich syndrome
o Brecher-Conkrite method – recommended o immune thrombocytopenic purpura (ITP)

o Reese Ecker methods – most common o HDN / HTR

o Walker & Sweeney’s method o neonatal alloimmune / autoimmune thrombocytopenia
o Unopette method o drug-induced thrombocytopenia

o Van Allen’s method o post transfusion purpura
o Nygard’s method o pregnancy and preclampsia
o Tocantin’s method o hypersplenism
o Kristenson’s method as Modified by Leinpert o hypothermia
9 indirect methods o TTP (thrombotic thrombocytopenic purpura)
o Dameshek method o HUS (hemolytic uremic syndrome)

o Fonio’s method o DIC (disseminated intravascular coagulation)
o Olef’s method
o Cramer and Bannerman method 6. Identify the standard reference values for platelet count.
o modified indirect platelet count – mostly used 9 Direct method: 150 – 450 x 109 platelets/uL
9 Indirect method:
3. Identify the potential sources of error when performing manual platelet o Fonio’s: 150, 000 - 350, 000 platelets/mm3
count. o Dameshek: 250, 000- 500, 000 platelets/mm3
9 Inadequate mixing and poor collection of the specimen
9 Dirt in the pipette, hemacytometer, or diluting fluid
9 The phenomenon of “platelet satellitism” may occur when an EDTA
anticoagulant is used

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 14

 MLS 12Aa: Hematology 2 (Laboratory)
PROBLEM SOLVING 3. Compute for the platelet count.
1. A 1:100 dilution of blood is made with 1% ammonium oxalate as the diluent. Number of platelets in the upper chamber = 477
The large central square on both sides of the hemocytometer is counted, for Number of platelets in the lower chamber = 481
a total of 260 cells. What is the total platelet count? Blood is drawn up to 1 mark.

given: given:
number of cells counted: 260 number of cells counted: 477 + 481 = 958
dilution factor: 1:100 number of squares counted: 1
number of squares counted: 1
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑖𝑛 𝑡ℎ𝑒 𝑏𝑢𝑙𝑏
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 =
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑 𝑎𝑠𝑝𝑖𝑟𝑎𝑡𝑒𝑑
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑜𝑛𝑒 𝑠𝑞𝑢𝑎𝑟𝑒 𝑥 𝑑𝑒𝑝𝑡ℎ
100 𝑢𝐿
260 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 =
𝑥 100 1
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 = 2
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 1: 100
130 𝑥 100
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 =
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚 # 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 =
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑜𝑛𝑒 𝑠𝑞𝑢𝑎𝑟𝑒 𝑥 𝑑𝑒𝑝𝑡ℎ
answer: 13,000 platelets/uL (decreased)
958
𝑥 100
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 = 2
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚
2. Blood is diluted 1:200, and a platelet count is performed, 180 platelets were
counted in the large central square on one side of the hemocytometer and 186 479 𝑥 100
on the other side. What is the total platelet count? 𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 =
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚
given: answer: 479,000 platelets/uL (increased)
number of cells counted: 180 + 186 = 366
dilution factor: 1:200
number of squares counted: 1

# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 =
# 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑜𝑛𝑒 𝑠𝑞𝑢𝑎𝑟𝑒 𝑥 𝑑𝑒𝑝𝑡ℎ

366
𝑥 200
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 = 2
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚

183 𝑥 200
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠/𝑢𝐿 =
1 𝑥 1𝑚𝑚< 𝑥 0.1𝑚𝑚

answer: 366,000 platelets/uL (normal)

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 15

 MLS 12Aa: Hematology 2 (Laboratory)
PLATELET FUNCTION TESTS î Functional platelets:
î Platelet function tests o adhere to subendothelial collagen
o designed to detect qualitative (functional) platelet abnormalities in 9 normal adhesion requires intact platelet membranes and
patients who are: functional plasma VWF
9 experiencing the symptoms of mucocutaneous bleeding 9 VWF is important in the adhesion of platelets on the
damaged collagen
o a platelet count is performed, and the blood film is reviewed before
platelet function tests are begun o aggregate with one another
9 because thrombocytopenia is a common cause of 9 normal aggregation requires that platelet membranes and
hemorrhage platelet activation pathways are intact
9 the plasma fibrinogen concentration is normal
î Qualitative platelet abnormalities
o are suspected only when bleeding symptoms are present and the o secrete the contents of their agranules and dense granules
platelet count exceeds 50,000/uL 9 secretions are released normally from platelet granules

o although hereditary platelet function disorders are rare, acquired

defects are common Platelet Aggregometry & Lumiaggregometry
î Acquired platelet defects are associated with: î platelet adhesion, aggregation, and secretion are assessed using in vitro
o liver disease platelet aggregometry
o renal disease o in vitro means outside
o myeloproliferative neoplasms o testing the quality of platelets outside the body
o myelodysplastic syndromes
o myeloma î Aggregometer
o uremia o an instrument designed to measure platelet aggregation in a
o autoimmune disorders suspension of citrated whole blood or PRP (platelet rich plasma
o anemias
o drug therapy

î Platelet morphology is often a clue

o Bernard-Soulier syndrome
9 the blood film reveals mild thrombocytopenia and large gray
platelets

o immune thrombocytopenic purpura or thrombotic thrombocytopenic

purpura
9 presence of large platelets on the blood film associated with
elevated mean platelet volume
9 often indicates rapid platelet turnover
note:
o giant or dysplastic platelets are seen in: 9 platelet aggregometer has:
o myeloproliferative neoplasms o incubation
o acute leukemia o pipette
o myelodysplastic syndromes o computer
o barcode
Platelet Aggregometry and Lumiaggregometry o reading
î Principle:
o functional platelets adhere to SEC (squamous epithelial cells),
aggregate with one another, and secrete the contents of their
α-granules and dense granules

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 16

 MLS 12Aa: Hematology 2 (Laboratory)
Specimen for Pletelet Aggregometry and Lumiaggregometry Start the stirring device and the recording computer. The stirring device turns
the stir bar at 800 to 1200 rpm, a gentle speed that keeps the platelets in
6
Whole Blood Platelet-rich Plasma suspension. The instrument directs focused light through the sample cuvette
9 3.2 % sodium citrate 9 3.2 % sodium citrate to a photodetector.
9 18°C to 24°C until testing 9 18°C to 24°C until testing As the PRP is stirred, the recorder tracing first stabilizes to generate a
7
9 within 4 hours of specimen 9 centrifuged for 30 mins with baseline, near 0% light transmittance.
collection the stopper in place After a few seconds, the operator pipettes an agonist (aggregating agent:
9 mixed 1:1 with normal saline 8 induces the aggregation of platelets) directly into the sample to trigger
aggregation.
PLATELET AGGREGOMETRY
î in a normal specimen, after the agonist is added
Platelet Aggregometry Using Platelet-Rich Plasma o the shape of the suspended platelets
9 changes from discoid (inactivated) to spherical (activated)

o the intensity of light transmittance

9 initially (and briefly) decreases
9 then increases in proportion to the degree of shape change

o percent light transmittance

9 is monitored continuously and recorded
9 as platelet aggregates form
• more light passes through the PRP
• the tracing begins to move toward 100% light
transmittance

î platelet function deficiencies are reflected in diminished or absent aggregation

o many laboratory directors choose 40% aggregation as the lower limit
of normal
9 40% is normal
î PRP aggregometry is performed using a specialized photometer 9 below 40% is abnormal
o called a light-transmittance aggregometer
9 (PAP-8E Platelet Aggregation Profiler; Bio/Data Corp.,
Horsham, PA)
9 we use this because we measure the percent light
transmittance

î when using a PRP specimen, centrifuge for 30 mins with the stopper in place
before testing

After calibrating the instrument in accordance with manufacturer instructions:

The operator pipettes the PRP to instrument compatible cuvettes, usually 500
1
uL
2 Drop in one clean plasticized stir bar per sample
3 Place the cuvettes in incubation wells
4 Allows the samples to warm to 37°C (normal body temp.) for 5 minutes
Transfer the first cuvette, containing specimen and stir bar, to the instrument’s
5
reaction well

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 17

 MLS 12Aa: Hematology 2 (Laboratory)
note:
9 5 phases of aggregation
1. baseline at 0% aggregation
§ there is the resting platelet
§ platelet is in discoid shape
2. shape change after the addition of the agonist
§ platelet shape changes from discoid to spherical
3. primary aggregation
§ light transmittance eventually increases
§ then secretion of alpha and dense granules
4. release of adenosine diphosphate and adenosine
triphosphate
5. second-wave aggregation that forms large clumps
§ secondary aggregation
9 % aggregation of measured by amount of light transmittance
through the test sample
9 PRINCIPLE:
o % aggregation in the y-axis
o time in minutes in the x-axis (usually 5 mins)
o % aggregation is directly proportional to the % light
transmittance
o the higher the % aggregation, the higher the % light
transmittance (normal platelet quality)
o lower than 40% light transmittance means that there is
something wrong with the platelet (it cannot aggregate well)
Whole-Blood Platelet Aggregometry
î platelet aggregation is measured by electrical impedance
o using a 1:1 saline–whole blood suspension
9 (Model 700 Whole Blood/Optical Lumi-Aggregometer;
Chrono-log Corp., Havertown, PA)
PADAYON, RMT !!!
The operator pipettes aliquots of properly mixed whole blood to cuvettes and
1 adds equal volumes of physiologic saline. Suspension volume may be 300 to
500 uL.
2 Drop in one stir bar per cuvette.
3 Place the cuvettes in 37°C incubation wells for 5 minutes.
4 Transfer the first cuvette to a reaction well.
5 Pipet an agonist directly into the specimen.
Suspend a pair of low-voltage cartridge-mounted disposable direct
6
current (DC) electrodes in the mixture.
note:
9 steps 1-5 is the same with platelet rich plasma aggregometry
9 nag dugang lang si step 6
î as aggregation occurs,
o platelets adhere to the electrodes and one another, impeding the
direct current (DC)
î rise in impedance is directly proportional to platelet aggregation
o amplified and recorded by instrument circuitry
î whole-blood aggregometry tracing
o closely resembles a PRP-based light-transmittance aggregometry
tracing
o the difference is that whole-blood aggregometry tracing uses
electrical empidance while in PRP aggregometry uses light
transmittance
PLATELET LUMIAGGREGOMETRY
î Chrono-log Whole Blood/Optical Lumi-Aggregometer
o may also be used for simultaneous measurement of
9 platelet aggregation
9 secretion of adenosine triphosphate (ATP)
• from activated platelet dense granules
î the procedure for lumiaggregometry
o differs little from that for conventional aggregometry
o simplifies the diagnosis of platelet dysfunction
î as ATP is released,
o it oxidizes a firefly-derived luciferin-luciferase reagent (Chrono-
lume; Chrono-log Corp.) to generate cold chemiluminescence
proportional to the ATP concentration
o a photodetector amplifies the luminescence, which is recorded as a
second tracing on the aggregation report
o lumiaggregometry may be performed using whole blood or PRP
QUITAG,BULQUERIN, DEALA 18
 MLS 12Aa: Hematology 2 (Laboratory)
1 the operator adds an ATP standard to the first sample Agonist Typical Final Platelet Membrane
2 then adds luciferin-luciferase and tests for full luminescence Concentration Receptors
the operator then adds luciferin-luciferase and an agonist to the second PAR1 and PAR4
3 thrombin 1 unit/mL
sample GP Iba and GP V
4 the instrument monitors for aggregation and secretion simultaneously ADP 1-20 µM P2Y1 and P2Y12
thrombin is typically the first agonist used because thrombin induces full epinephrine 2-10 µg/mL a2-adrenergic receptor
5
secretion collagen 5 µg/mL GP Ia/lla and GP VI
the luminescence induced by thrombin is measured, recorded, and used for arachidonic acid 500 µM TPa and TPb
6
comparison with the luminescence produced by the additional agonists GP lb/IX/V in association
normal secretion induced by agonists other than thrombin produces ristocetin 1 mg/mL
7 with VWF
luminescence at a level of about 50% of that resulting from thrombin note:
9 GP – glycoprotein
9 PAR – protease-activatable receptor
9 P2Y – platelet membrane ADP-receptor
9 TP – thromboxane receptor

Thrombin (or TRAP)

note:
9 scanning electron micrograph of resting and activated platelets
o resting or inactivated platelets are discoid just like RBCs
o activated platelets are spherical and has pseudopods
PLATELET AGONISTS
Platelet Agonists (Activating Agents) Used in Aggregometry
î optical PRP-based aggregation method
o employed most frequently in clinical practice
ADP
î the agonists used are:
1. thrombin or synthetic thrombin receptor-activating peptide (TRAP)
2. adenosine diphosphate (ADP)
3. epinephrine
4. collagen
5. arachidonic acid
6. ristocetin
î Small volumes (2 to 5 mL) of concentrated agonist are used
o so that they have little dilutional effect in the reaction system
î platelet membrane receptors
o receptors found in platelets where agonists bind
9 cleaves two platelet membrane protease-activatable receptors (PARs), PAR-
1 and PAR-2, both members of the seven-transmembrane repeat receptor
family
9 cleaves glycoprotein (GP) 1ba and GP V
9 internal platelet activation is affected by membrane-associated G proteins and
both the eicosanoid and the diacylglycerol
9 thrombin-induced activation results in full secretion and aggregation
9 reagent thrombin is stored dry at –20°C and is reconstituted with physiologic
saline immediately before use.
9 leftover reconstituted thrombin may be divided into aliquots, frozen, and
thawed for later use
9 thrombin has the disadvantage that it often triggers coagulation (fibrin
formation) simultaneously with aggregation
9 the use of TRAP avoids this pitfall
9 binds platelet membrane receptors P2Y1 and P2Y12, also members of the
seven-transmembrane repeat receptor family
9 ADP-induced platelet activation relies on the physiologic response of
membrane-associated G protein and the eicosanoid synthesis pathway
9 the end product of eicosanoid synthesis, thromboxane A2, raises cytosolic
free calcium, which mediates platelet activation and induces secretion of ADP
stored in dense granules
9 the secreted ADP activates neighboring platelets
9 the most commonly used agonist, particularly in aggregometry systems that
measure only aggregation and not luminescence
9 at ADP concentrations near 1um, platelets achieve
only primary aggregation, followed by disaggregation
9 the graph line deflects from the baseline for 1 to 2 minutes and then returns to
baseline

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 MLS 12Aa: Hematology 2 (Laboratory)
9 primary aggregation involves shape change with formation of 9 the operator dilutes arachidonic acid with a solution of bovine albumin for
microaggregates, both reversible immediate use
9 secondary aggregation is the formation of full platelet aggregates after release 9 aliquots of bovine albumin–dissolved arachidonic acid may be frozen for later
of platelet dense granule ADP use
9 at agonist ADP concentrations near 10um, there is simultaneous irreversible
shape change, secretion, and formation of aggregates, resulting in a PRE-ASSESSMENT
monophasic curve and full deflection of the tracing 1. It is termed as a decrease in number of circulating platelet.
9 ADP concentrations between 1 and 10um induce a biphasic curve: primary 9 THROMBOCYTOPENIA
aggregation followed by a brief flattening of the curve called the lag phase and
then secondary aggregation 2. What is the most common platelet agonist used in aggregometry?
9 reagent ADP is stored at –20°C, reconstituted with physiologic saline, and 9 ADP
used immediately after reconstitution
9 leftover reconstituted ADP may be aliquotted and frozen for later use 3. In what objective do we count platelets?
9 OIO
Epinephrine
9 binds platelet a2-adrenergic receptors, identical to muscle receptors, and 4. Platelet function tests are designed to detect QUALITATIVE platelet
activates the platelets through the same metabolic pathways as reagent ADP abnormalities.
9 the results of epinephrine-induced aggregation match those of ADP
o except that epinephrine cannot induce aggregation in storage pool 5. What instrument is designed to measure platelet aggregation?
disorder or eicosanoid synthesis pathway defects no matter how high 9 AGGREGOMETER
its concentration
9 does not work in whole-blood aggregometry 6. What is the phenomenon that may occur when EDTA anticoagulant is used in
9 stored at 1°C to 6°C and reconstituted with distilled water immediately before counting platelet?
it is used 9 PLATELET SATELLITISM
9 leftover reconstituted epinephrine may be aliquotted and frozen for later use
7. What is the other name of platelets?
Collagen 9 THROMBOCYTES
9 binds GP Ia/IIa and GP VI, but it induces no primary aggregation
9 after a lag of 30 to 60 seconds, aggregation begins, and a monophasic curve 8. What is the anticoagulant of choice in performing platelet aggregometry?
develops 9 SODIUM CITRATE
9 aggregation induced by collagen at 5mg/mL requires intact membrane
receptors, functional membrane G proteins, and normal eicosanoid pathway 9. What is the most accurate way of counting platelets?
function 9 DIRECT METHOD
9 loss of collagen-induced aggregation may indicate a membrane abnormality,
storage pool disorder, release defect, or the presence of aspirin 10. When activated, the shape of platelets changes from DISCOID to
9 most laboratory managers purchase lyophilized fibrillar collagen preparations SPHERICAL.
such as Chrono-Par Collagen (Chronolog Corp.)
9 stored at 1°C to 6°C and used without further dilution
9 may not be frozen

Arachidonic acid
9 assesses the viability of the eicosanoid synthesis pathway
9 free arachidonic acid agonist at 500um is added to induce a monophasic
aggregometry curve with virtually no lag phase
9 aggregation is independent of membrane integrity
9 deficiencies in eicosanoid pathway enzymes, including deficient or aspirin-
suppressed cyclooxygenase, result in reduced aggregation and secretion
9 arachidonic acid is readily oxidized and must be stored at –20°C in the dark

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 MLS 12Aa: Hematology 2 (Laboratory)
PLATELET REVIEW BAD FILMS
• Blood films should first be examined macroscopically to make sure that
PRECAUTIONS ON BLOOD FILM PREPARATION they are satisfactory quality for examination.

REMINDERS:
• Always have a clean surface glass slides
o spreader should have a smooth end for every specimen
o it should be thoroughly clean to avoid carry over of blood cells
from one specimen to another

• Always wipe out the first drop of blood

o only the drop of blood and slide should be touched

• Observe the factors to determine the thickness of a smear such as:

o Film a is unevenly spread and too thick at the tail.
1. Size of drop of blood o Film b and c are too long and b is also broad.
2. Angle between the slide and spreader o Film d is too short and thick.
3. Speed of the spreader o Film e is too close to one edge of the glass slide.
4. Pressure of the spreader on the slide o Film f is too short.

• Observe the properties of the ideal smear: BAD TECHNIQUE

1. Gradual transition from thick to thin
2. Smear must occupy one – half to two – thirds of the slide
3. Margin free, with feathered edge
4. No overlapping of cells
5. Smear surface must be without scratched and free from
ridges and holes

• The film of blood should be narrower than the glass slide

o It should not extend to the end of the slide
o It should be narrower than the coverslip that will be applied
Why do we present the precautions and reminders?
• So we know that platelets adhere easily to foreign surfaces and if indi
name ang pag smear it will result to erroneous platelet count
• The spreader has been inadequately cleaned between spreading films
from two different samples.
o blasts have been carried over from a sample with high blast
cell count to a normal sample.

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 MLS 12Aa: Hematology 2 (Laboratory)
MACROSCOPIC APPEARANCE OF RED CELL AGGLUTINATION
• A blood sample may contain cold
agglutinins that very large red cell agglutinates
form.
• If the blood is warmed before spreading, the
degree of abnormality is much less.
EXAMINING THE BLOOD FILM
• Examine the edge and the tail.
• Exclude artifacts that make the film unsuitable for assessment.
• Cells of various lineages should be assessed.
• The ideal part of the film to examine for red cells morphology is shown
o the area where red cells are touching but without much overlap
• Examining red cells in the tail of the film will give a false impression of
spherocytes
• Examining a part of the film that is too thick will make a recognition of
whole cells unreliable.

GIANT PLATELETS
CARCINOMA CELLS IN BLOOD FILM
• Very large platelets are often referred to as
• Rarely, carcinoma cells are present in giant platelets.
the blood in very large numbers.
• There is no strict definition if this term but its
• Other abnormalities that can produce use is appropriate when platelets are as large as
a similar macroscopic appearance normal red cells.
include the presence of
cryoglobulins or a very large
platelet aggregates.

FIBRIN STRANDS IN THE TAIL OF THE BLOOD FILM

PLATELET SATELLITISM

• Platelet satellitism describe the phenomenon of

• Once a blood film has been examined
adherence of platelets to white cells.
macroscopically, it should be examined using a LPO
such as x20, x40, x50.
• It is an in vitro phenomenon of no clinical
significance.
• The edge and the tail of the film should be
examined for large abnormal cells, platelet
aggregates, or fibrin strands. • It is important that it is detected since the
platelet count will be factitiously low.
• Fibrin strands are very pale blue, sometimes
almost colorless, and deform the red cells through which they pass. When
they are present, the platelet count is often falsely low.

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 MLS 12Aa: Hematology 2 (Laboratory)
MAY-HEGGLIN ANOMALY THROMBOCYTOPENIA AND A GIANT PLATELET IN AUTOIMMUNE
THROMBOCYTOPENIC PURPURA

• Blood film in MHA showing large platelets and • The patient’s spleen has been removed.
a May- Hegglin inclusion in the cytoplasm of the
neutrophil. • This condition was previously known as
Idiopathic thrombocytopenia purpura (ITP).

• A giant platelet and two red cells containing

Howell-Jolly bodies.
BERNARD-SOULIER SYNDROME

M7 AML
• Blood film shows giant platelets.
• The lineage of the blasts was identified by
immunocytochemistry with a CD61 monoclonal
antibody.

• Down’s syndrome is associated with a greatly

increased incidence of M7 AML.

GRAY PLATELET SYNDROME • Blood film from an infant with Down’s syndrome
with M7 AML or acute megakaryoblastic leukemia
• Blood film in the grey platelet syndrome showing showing megakaryoblasts and one NRBC.
thrombocytopenia and apparently agranular
(“gray”) platelets.
EMPERIPOLESIS

• A small proportion of normal megakaryocytes appear

to have engulfed other hematopoietic cells.

PLATELTS IN WASyndrome • These cells have not been phagocytosed but have
actively entered the surface-connected demarcation
• Blood film in Wiskott-Aldrich syndrome membrane of the system of the megakaryocyte.
showing thrombocytopenia and abnormally
small platelets. • They can emerge again as intact cells.

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 MLS 12Aa: Hematology 2 (Laboratory)
MEGAKARYOCYTE IN THE PERIPHERAL BLOOD PETECHIAE IN Petechiae are pinpoint hemorrhage caused by
AUTOIMMUNE thrombocytopenia.
• Megakaryocytes are only occasionally seen in the THROMBOCYTOPENIC
PURPURA
circulating blood.
9 The brain of a child with autoimmune
thrombocytopenic purpura who suffered a fatal
• Peripheral blood megakaryocytes in FETAL CEREBRAL
HEMORRHAGE IN cerebral hemorrhage.
hematologically normal subjects are usually almost
bare nuclei with only a thin rim of residual cytoplasm. AUTOIMMUNE
THROMBOCYTOPENIC 9 This is a very rare complication of autoimmune
PURPURA thrombocytopenia.

HYPOLOBULATED MEGAKARYOCYTE IN BONE MARROW ASPIRATE ORAL HEMORRHAGE IN The mouth of a child with post-viral
POST-VIRAL thrombocytopenia showing hemorrhagic blisters.
• Bone marrow aspirate in the 5q – syndrome, THROMBOCYTOPENIA
type of myelodysplastic syndrome, showing a A child with ALL showing pallor and bruising
hypolobulated megakaryocyte. BRUISING IN ALL caused by thrombocytopenia.

ORAL BLEEDING IN The mouth of a child with quinine-induced

DRUG-INDUCED thrombocytopenia showing a hemorrhagic blister.
THROMBOCYTOPENIA
The legs of a patient with Septrin-induced
PURPURA IN DRUG- thrombocytopenia showing purpura (drug-induced
BM ASPIRATE SHOWING MICROMEGAKARYOCYTE
INDUCED
thrombocytopenia).
THROMBOCYTOPENIA

• Bone marrow aspirate in juvenile chronic myeloid

leukemia showing a binucleated micromegakaryocyte. FAILURE TO CLOT RETRACTION IN DRUG-INDUCED THROMBOCYTOPENIA

• Drug-dependent antibodies have interfered

with platelet function and clot retraction has
therefore not occurred.

NORMAL PLATELETS (AND RED CELLS) • The test samples are flanked by two normal
control samples
• Platelets are small cytoplasmic fragments which
can be seen between the red cells in a blood film.
HYPHAEMA
• They are composed of a central granulomere • Bleeding into the anterior chamber of the eye, referred to as
containing azurophilic granules and a peripheral hyphaema.
hyalomere that is agranular. • Such bleeding can occur as a complication of thrombocytopenia in acute
leukemia

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 MLS 12Aa: Hematology 2 (Laboratory)
THROMBOCYTOPENIA CAUSED BY DIC THROMBOCYTOSIS IN INFECTION

• Thrombocytosis as a response to bacterial

• Blood film showing thrombocytopenia caused infection
by DIC in variant form of hypergranular
promyelocytic leukemia. • There is also neutrophilia and monocytosis

• Platelets produced as a response to infection

THROMBOTIC THROMBOCYTOPENIC PURPURA
or inflammation are usually small, in contrast to
those produced in myeloproliferative disorders.
• Blood film in thrombotic thrombocytopenic
purpura showing thrombocytopenia, anemia, THROMBOCYTOSIS POST-SPLENECTOMY
and red cell fragmentation.
• Post-splenectomy thrombocytosis. The
patient also has post-splenectomy
lymphocytosis.

THROMBOCYTOPENIA CAUSED BY BONE MARROW REPLACEMENT • Following splenectomy there are usually
some platelets that are larger than normal
• Thrombocytopenia caused by bone marrow
replacement in acute lymphoblastic leukemia of
L2 category
ESSENTIAL THROMBOCYTHEMIA

• Blood film in essential thrombocythemia showing

increased numbers of platelets

THROMBOCYTOPENIA CAUSED BY INEFFECTIVE THROMBOPOIESIS • There is also increased variation in platelet size,
referred to as platelet anisocytosis
• Thrombocytopenia caused by ineffective
thrombopoiesis in the RAE category of • Anisocytosis = variation in size
MDS.
• Kung damo platelet in the film but lain size and
• In the myelodysplastic syndromes there staining = Essential Thrombocythemia, related with
is often thrombocytopenia despite normal MPNs
or increased number of megakaryocytes in
the bone marrow, providing evidence of
ineffective thrombopoiesis.

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 MLS 12Aa: Hematology 2 (Laboratory)
BONE MARROW ASPIRATE IN ESSENTIAL THROMBOCYTHEMIA • Cannot control the function of thrombopoietin

• No or less iron to regulate the TPO

• Bone marrow aspirate in essential
thrombocythemia showing increased • Continuous production of platelets resulting to thrombocytosis
pleomorphic megakaryocyte
GIANT PLATELET IN IDIOPATHIC MYELOFIBROSIS

ABNORMAL PLATELETS IN CG • Blood film in idiopathic myelofibrosis showing

a normal-sized platelet and a giant platelet
• Blood film in CGL showing increased
numbers of platelets which show increased • The leukocyte is a basophil
variation in size (Platelet anisocytosis)

• There is a mixture of normally granulated

and agranular platelets MICROMEGAKARYOCYTE IN MYELOFIBROSIS

BONE MARROW ASPIRATE IN ATYPICAL CML SHOWING A MULTINUCLEATED • They are cells less than 30 mm in diameter
MEGAKARYOCYTE (about the same size as a myeloblast or
promyelocyte.
• Most megakaryocytes have a single nucleus
although is polylobulated. • They are almost always indicative of a
hemopoietic neoplasm.
• This bone marrow aspirate from a patient with
atypical chronic myeloid leukemia shows a • Micromegakaryocytes are more often seen in the bone marrow.
multinucleated megakaryocyte.
REVIEW
BONE MARROW ASPIRATE IN CGL • ___25___ platelets per oil immersion field
• __3-10__ platelets per 100 RBCs
• BM aspirate in CGL showing increased
numbers of megakaryocytes • _____/OIF – few
• _____/OIF - moderate
• Their average size is less than normal • _____/OIF – markedly many

THROMBOCYTOSIS IN IRON-DEFICIENT POLYCYTHEMIA • Significant platelet levels:

à _______uL – abnormally low
• In this case, thrombocytosis is likely to have à _______uL – bleeding possible with trauma
been caused by the underlying à _______uL – spontaneous bleeding possible
myeloproliferative disorder but it may have à _______uL – severe spontaneous bleeding
been aggravated by iron deficiency

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 MLS 12Aa: Hematology 2 (Laboratory)
DETERMINE WHETHER THE FOLLOWING WILL RESULT TO FALSELY
INCREASE OF FALSELY DECREASE PLATELET COUNT
FALSELY INCREASE
Normal platelets
• carry over from a prior sample with a high platelet count
• contamination of reagents with particles or bacteria Normal blood smear and patient condition
• traumatic venipuncture (may result in activation of the clotting
process)
• insufficiently lysed red cells
• white cell cytoplasmic fragments

FALSELY DECREASE Thrombocytopenia with giant platelets

• abnormal amount of plasma proteins in various paraproteinemias
• EDTA-induced platelet clumping
• large platelets/giant platelets
• lipemic or clotted sample
• platelet agglutinins Giant platelets (right)
• platelet satellitism
Platelet aggregates (left)
• previous contacts of platelets with foreign surface such as dialysis
membrane
• red cell fragments or small red cells
• red cells with inclusion bodies
• patients receiving chemotherapy for acute leukemias and
lymphomas

DESCRIBE THE FOLLOWING BLOOD PICTURE AND CORRELATE WITH THE Essential Thrombocythemia (left
RESULT OF PLATELET COUNT AND PLATELET DISORDER bone marrow picture)

Platelet aggregates over the film

Platelet aggregate (left)

Platelet satellitism (right)

Has giant platelets

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 27

 MLS 12Aa: Hematology 2 (Laboratory)
ADDITIONAL PRECAUTIONS AND KEY POINTS IN PLATELET COUNTING • Exclude red cells and granulocytes from PRP: interfere with the light
• Erythrocyte with overlying platelet transmittance and cause reduced response heights, which can be
9 Platelets may adhere to or overlap red blood cells mistaken for abnormal aggregation
9 Suggesting a red blood cell inclusion or parasite 9 May be removed further centrifugation of PRP at 150g for 2
min or after settling has occurred
• Platelet is surrounded by a thin clear zone or halo
9 not a feature of genuine red blood cell inclusions
• Centrifugation – cause cellular release of ADP and platelet
refractoriness to aggregation
• Carefully examine the morphology of the platelet
9 Should not be started within 30 min of preparing the PRP
9 comparing the size, staining characteristics, and granularity
tests should
with known platelets in the same field
9 After centrifugation, settle PRP before analysis
9 as well as determining if the platelet is in the same plane of
focus as the red blood cell
• Presence of cryoglobulins – cause changes in transmittance which
• Stress platelets resemble the appearance of spontaneous aggregation
9 Larger (6um) than ordinary mature circulating platelets 9 Warming PRP to 37C for 5 min
9 If patient have cryoglobulins, warm PRP at 37 degrees
9 Round up in EDTA, but are cylindrical and beaded in citrated
celcius for 5 mins because ma falsely increase ang
tubes
aggregation
• Platelet aggregation and satellitism
• Lipemic plasma – cause problems in adjusting the aggregometer
9 EDTA-induced pseudothrombocytopenia
and the responses may be compressed owing to the small difference
9 Does not occur when other coagulants are used
in transmitted light between PRP and PPP
§ citrate
9 Kung ga use spectrophotometer, do not use LIPEMIC
§ heparin
PLASMA
9 because it requires a calcium-poor medium

• Platelets left standing at room temperature become increasingly

ADDITIONAL PRECAUTIONS AND KEY POINTS IN PLATELET
AGGREGOMETRY reactive to andrenaline and in some cases to collagen; the rate of
• Blood is collected in a 3.2% sodium citrate tube and the test should change increases after 3h.
be performed within 30 minutes of blood collection.
ADVANTAGES OF PLATELET LUMIAGGREGOMETRY
• The platelet count of platelet-rich plasma should be approximately • Uses electrical impedance in whole blood or optical density in platelet
40% aggregated rich plasma.
9 If the platelet count is higher, platelets are normal
• Whole blood-aggregation measures platelet function in
anticoagulated blood without the need to isolate them from other
• If the original platelet count is ____________ < /uL, then the test might
blood components.
be invalid.
9 If the test needs to be performed, then then the platelet count
of the control should also be _____________.

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 MLS 12Aa: Hematology 2 (Laboratory)
• Without the necessity for centrifugation, the entire platelet population MODULE 1 QUIZ (FORTUNO)
is tested and labile factors in the blood (e.g., prostacyclin and What is the specimen of choice for platelet count?
thromboxane A2) that may influence platelet function are preserved. - EDTA anticoagulated blood

Which of the platelet count methods below utilizes a counting chamber?

• Impedance aggregation in blood is not dependent on optical - Reese-Ecker
characteristic of the sample, so tests can be performed on lipemic and
thrombocytopenic samples. In which part of the counting chamber do we count the platelets?
- central large square

• The method is also useful in situation where sample volume is critical. Let’s say you did the routine method for platelet count. What should be the area of the
square in the formula?
2
• Whole blood is enough - 1 mm
• Measure low samples if critical ang sample volume
Compute for the platelet count:
• Not affected by lipemia or nubo platelet sample sample platelets in upper chamber: 53
platelets in lower chamber: 57
blood aspirated to 0.2 mark
RBC pipet used
number of squares counted: 1

answer: 275,000/uL
‘ What should be the dilution factor if you aspirated blood up to the 0.5 mark and you
used an RBC pipette?
- 200

You are doing modified Fonio’s method of platelet count. Below are your results:
field 1 -21
2- 14
3 – 14
4 – 12
5 – 18
6 – 20
7 – 16
8 – 16
9 – 16
10 – 10

What is the platelet count given that you are reading your results in the
correct area of the smear?
- 314,000/uL

Match the description to the name of the method.

- Wet method of indirect platelet count : Dameshek
- Dry method of indirect platelet count : Fonio’s

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 MLS 12Aa: Hematology 2 (Laboratory)
Match the aggregometer to its principle.
- PAP 8E (PRP) : light transmitance
- Model 700 (whole blood) : electrical impedance
- Chrono-Log : chemiluminescence

Match the agonist to its receptor on platelets:

- thrombin - PAR
- ADP – P2Y1 and P2Y12
- epinephrine – adrenergic receptor
- collagen – GP Ia/IIa and GP VI

When doing platelet count, you noticed that there are platelet clumps in the sample
even after dilution. You recollected a sample using sodium citrate and your result was
316000/uL. What should be the reported result?
- 347,600/uL

In direct method of platelet count, what microscope objective should you use?
- HPO

PADAYON, RMT !!! QUITAG,BULQUERIN, DEALA 30