Platelet Counts

Dr Kunal Sehgal, M.D.Associate Consultant Hematology LaboratoryDepartment of Lab MedicinePD Hinduja National Hospital and MRC

drkunalsehgal@gmail.com

PD Hinduja HospitalTata memorial HospitalPGIMER, Chandigarh

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)

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.

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

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

Problems of Peripheral Smear Platelet Check

Platelet Clumps Platelet Satellitism on WBCs Poor Smearing Highly subjective

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

ARTEFACTS

Automated CBC Analysers

Impedance principle Optical Principle

Counters count many more cells and hence more reproducible resultsImproved C.V. - typically less than 5%

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

CBC Histograms

Normal Platelets histogram

Giant Platelets histogram

Problems with Impedance Counts

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

Advantages of Optical Platelet Counting

Microcytic RBCGiant PLT

Optical Platelet Enumeration

CASE STUDIES

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.

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.

Case Study 3 - 31/F,Blood Donor, East Indian Origin, Normal Hb and WBC, Impedance Plt- 134, Platelet O 162,

Morphologically- Many Giant platelets

Case Study 4- CBC Histogram

Case Study 4- continued

Platelet Clumps in WBC Ghost Area

Ghost area in a case of platelet clumps

Ghost area in a normal CBC

72 year old male

Hemogram revealed thrombocytopenia (54,000/cmm)

Case Study 5

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

Peripheral smear showing manyplatelet clumps (10x).

EDTA induced Pseudothrombocytopenia

Citrated PB Sample Platelet count- 2.35 lacs

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

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!

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 ?