Platelet Counts

1. PGIMER, Chandigarh Tata memorial Hospital PD Hinduja Hospital Platelet Counts Dr Kunal Sehgal, M.D. Associate Consultant Hematology Laboratory Department of Lab Medicine PD Hinduja National Hospital and MRC drkunalsehgal@gmail.com

2. 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) Briggs et al. Continuing developments with the automated platelet count. Int. Jnl. Lab. Hem.2007,29,77-91.

3. 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.

4. International Flow Reference Method The New Gold Standard RBC/Platelet Ratio Method Dual Platform Method Absolute Platelet Count= Platelet events X RBC count RBC events (Automated Cell Analyzer) Platelets CD41/61 RBC ISLH Task Force, Am J Clin Pathol 115, 460-464.(2001)

5. 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

6. Problems of Peripheral Smear Platelet Check • Platelet Clumps • Platelet Satellitism on WBCs • Poor Smearing • Highly subjective

9. 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

12. ARTEFACTS

13. Automated CBC Analysers • Impedance principle • Optical Principle Counters count many more cells and hence more reproducible results Improved C.V. - typically less than 5%

14. 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

15. CBC Histograms

16. Normal Platelets histogram Giant Platelets histogram

17. Problems with Impedance Counts

18. Optical Principle Two dimensional Light Scatter Two angles of laser ight scatter are measured Light Scatter- 2-3°C- volume (plt size) Light Scatter- 5-15°C- refractive index (plt density) Rbc fragments have a different RI as compared to platelets and hence can be separated Optical Fluorescence platelet counting Size vs. Fluorescence plot (Polymethine Dye) RBC fragments do not contain RNA while giant platelets and immature forms contain RNA and are called reticulated platelets These are easily separated from microcytic RBCs and fragments

19. Advantages of Optical Platelet Counting

20. Giant PLT Optical Platelet Enumeration Microcytic RBC

21. CASE STUDIES

22. Case Study 1 Automated CBC -Platelet count – 1.05lacs PS- 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.

23. Case Study 2 Automated CBC -Platelet count – 2.35lacs PS- many platelet clumps What do you do? Peripheral Smear – comment – Platelets are adequate on smear. Platelets are also seen in clumps.

24. Case Study 3 - 31/F,Blood Donor, East Indian Origin, Normal Hb and WBC, Impedance Plt- 134, Platelet O –162, Morphologically- Many Giant platelets

25. Case Study 4- CBC Histogram

26. Case Study 4- continued…

27. Platelet Clumps in WBC Ghost Area Ghost area in a case of platelet clumps Ghost area in a normal CBC

28. 72 year old male Hemogram revealed thrombocytopenia (54,000/cmm) Case Study 5

31. Based on platelet histogram findings, a peripheral smear examination was done • Giant platelets were seen • Platelet clumps seen The sample contained adequate platelets, however we got spurious results on automated analyzer

32. EDTA induced Pseudothrombocytopenia Citrated PB Sample –Platelet count- 2.35 lacs Peripheral smear showing many platelet clumps (10x).

33. Case Study 6 • 55/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

34. Case study 7- Acceptable C.V. Case Scenario 1 • First run, platelet count- 200000 • Second run, platelet count – 192000 A difference of 8000. Is this Acceptable? Yes- the difference is only 4% Case Scenario 2 • First run platelet count- 24000 • Second run platelet count – 16000 A difference of 8000. Is this Acceptable? NO- the difference is of 33% and will have a huge clinical impact!

35. Any Questions ?