PGIMER, Chandigarh

Tata memorial 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

PD Hinduja Hospital

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 CountsThe 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 Standard

t
P la

RBC/Platelet Ratio Method

ts
ele

Dual Platform Method

CD
41/61

Absolute Platelet Count=

RBC

Platelet events X RBC count

RBC events
(Automated Cell
Analyzer)

ISLH Task Force, Am J Clin Pathol 115, 460-464.(2001)

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 results
Improved 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 Principle
Two dimensional Light Scatter
Two angles of laser ight scatter are measured
Light 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 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

Advantages of Optical
Platelet Counting

Optical Platelet
Enumeration

Microcytic RBC

Giant PLT

CASE STUDIES

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.

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.

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

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

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

EDTA induced Pseudothrombocytopenia

Citrated PB Sample Platelet count- 2.35 lacs

Peripheral smear showing many

platelet clumps (10x).

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

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!

Any Questions ?