03 platelet counts
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TMH proceedings 2010-2011,pdfTRANSCRIPT
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Platelet Counts
Dr Kunal Sehgal, M.D.Associate Consultant Hematology LaboratoryDepartment of Lab MedicinePD Hinduja National Hospital and MRC
PD Hinduja HospitalTata memorial HospitalPGIMER, Chandigarh
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Platelets Historical Perspective 1882- Platelets recognised as distinct corpuscles Italian pathologist Giulio Bizzozero
1953 - Manual Phase Contrast Microscopic Method using Neubauer chamber - ICSH - Gold Standard 1988-2001
1965 -72- Semi- Automated and Fully Automated Counters
1981- Hydrodynamically focused whole blood aperture IMPEDANCE counter
1985- OPTICAL Platelet Counts
Early 1990s- Flow cytometric methods based on CD41/61
2001- Flow Cytometric RBC platelet Ratio the new International reference Method (IRM)
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Manual Platelet Counts- The Old Gold Standard
Laborious Time Intensive Subjective High Inter- observer CVs of 10-25 %
Briggs et al. Continuing developments with the automated platelet count. Int. Jnl. Lab. Hem.2007,29,77-91.
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International Flow Reference Method
The New Gold StandardRBC/Platelet Ratio Method
Dual Platform Method
Absolute Platelet Count=
Platelet events X RBC count RBC events (Automated Cell Analyzer)
ISLH Task Force, Am J Clin Pathol 115, 460-464.(2001)
CD41/61
RBC
Platele
ts
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Peripheral Blood Smear (Platelet count check only)
Platelets to be counted in a region where RBCs and platelets are well dispersed.
Atleast 10 oil immersion fields to be counted (more in lower counts)
Average no. of platelets in a field multiplied by 10000 is the approximate platelet count
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Problems of Peripheral Smear Platelet Check
Platelet Clumps Platelet Satellitism on WBCs Poor Smearing Highly subjective
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Peripheral Blood Smear (Platelet count check only)
Eg :
a) 10 fields 45 platelets
Avg. plt per field is 4.5
Approximate Platelet count=4.5x10000=45000
b) 20 fields 40 platelets
Avg. plt per field is 2
Approximate Plt count=2x10000=20000
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ARTEFACTS
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Automated CBC Analysers
Impedance principle Optical Principle
Counters count many more cells and hence more reproducible resultsImproved C.V. - typically less than 5%
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Impedance Principle
Coulter Principle or Resistance detection method
Cells suspended in an elecrolyte solution
Change in electric impedance impedance signal
Impedance signal Directly proportional to the volume of the cell
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CBC Histograms
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Normal Platelets histogram
Giant Platelets histogram
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Problems with Impedance Counts
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Optical PrincipleTwo dimensional Light Scatter
Two angles of laser ight scatter are measuredLight Scatter- 2-3C- volume (plt size)Light Scatter- 5-15C- refractive index (plt density)
Rbc fragments have a different RI as compared to platelets and hence can be separated
Optical Fluorescence platelet countingSize vs. Fluorescence plot (Polymethine Dye)
RBC fragments do not contain RNA while giant platelets and immature forms contain RNA and are called reticulated plateletsThese are easily separated from microcytic RBCs and fragments
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Advantages of Optical Platelet Counting
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Microcytic RBCGiant PLT
Optical Platelet Enumeration
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CASE STUDIES
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Case Study 1
Automated CBC -Platelet count 1.05lacsPS- many large platelet clumps
What do you do?
Peripheral Smear comment Platelets are seen in many clumps. Platelets are adequate on smear (>1lac). Kindly repeat CBC for accurate platelet count if clinically indicated.
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Case Study 2
Automated CBC -Platelet count 2.35lacsPS- many platelet clumps
What do you do?
Peripheral Smear comment Platelets are adequate on smear. Platelets are also seen in clumps.
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Case Study 3 - 31/F,Blood Donor, East Indian Origin, Normal Hb and WBC, Impedance Plt- 134, Platelet O 162,
Morphologically- Many Giant platelets
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Case Study 4- CBC Histogram
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Case Study 4- continued
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Platelet Clumps in WBC Ghost Area
Ghost area in a case of platelet clumps
Ghost area in a normal CBC
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72 year old male
Hemogram revealed thrombocytopenia (54,000/cmm)
Case Study 5
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Based on platelet histogram findings, aperipheral smear examination was done
Giant platelets were seen Platelet clumps seen
The sample contained adequate platelets,however we got spurious results onautomated analyzer
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Peripheral smear showing manyplatelet clumps (10x).
EDTA induced Pseudothrombocytopenia
Citrated PB Sample Platelet count- 2.35 lacs
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Case Study 655/M A know case of Acute Leukemia
Hb -7.5g% WBC- 21.5 x103 /ul Platelet count- 18 x103 /ul
What do you do next?
Peripheral smear check-
Rule out micro clots in sample
Look for fibrin strands and platelet clumps on slide
Do a peripheral smear estimation of platelet counts
Be aware of the clinical decisions that depends on your result- i.e know the transfusion threshold levels
Discuss case with clinician if required
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Case study 7- Acceptable C.V.Case Scenario 1 First run, platelet count- 200000 Second run, platelet count 192000A difference of 8000. Is this Acceptable? Yes- the difference is only 4%
Case Scenario 2 First run platelet count- 24000 Second run platelet count 16000A difference of 8000. Is this Acceptable?NO- the difference is of 33% and will have a huge clinical impact!
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Any Questions ?
Slide 1Platelets Historical Perspective Manual Platelet Counts- The Old Gold StandardInternational Flow Reference Method The New Gold StandardPeripheral Blood Smear (Platelet count check only)Problems of Peripheral Smear Platelet CheckSlide 7Slide 8Peripheral Blood Smear (Platelet count check only)Slide 10Slide 11ARTEFACTSAutomated CBC AnalysersImpedance PrincipleCBC HistogramsSlide 16Problems with Impedance CountsOptical Principle
Slide 19Slide 20Slide 21Case Study 1Case Study 2Slide 24Slide 25Slide 26Platelet Clumps in WBC Ghost AreaSlide 28Slide 29Slide 30Slide 31Slide 32Slide 33Case study 7- Acceptable C.V.Any Questions ?