Platelets count estimation on peripheral

smear : what should be an acceptable

‘Multiplication factor’?

Noer Hafni
INTRODUCTION
 A manual
Automated verification of
In an era of evidence- haematology one needs to keep platelet counts is
based medicine, analysers are done include
in mind that
essential to explore routinely being used flagging by
worldwide to platelet count is a
and document automated
standards required determine blood parameter which
analyser, in case of
for accurate results counts with invariably needs thrombocytopenia,
in laboratories acceptable accuracy manual platelet clumps
and precision. verification. and giant platelets.
 One such manual methods of platelet count

Platelet (PLT)/red blood cell Counting the average

(RBC) ratio, estimated on number of platelets
the Peripheral Smear by per oil immersion
counting the number of field (OIF) and then
platelets per 1000 RBCs and multiplying it with a
can back calculated from multiplication factor,
electronic blood cell either 15,000 or
counter 20,000

Different laboratories using a wide range of multiplication factors such as 10,000,

15,000 and 20,000.
This leads to a wide variation in the platelet counts estimated on peripheral
smear, resulting in high inter‑observer variability
• Multiplication factor required to obtain correct platelet
count has been questioned in the past, and a focussed
unbiased study may be helpful in resolving the dilemma
• This study aims to validate the ideal multiplication
factor for estimating manual platelet counts on
peripheral smear, and to identify a manual method
having comparable results with the haematology
analyser
METHODS
• An observational study conducted in a tertiary care hospital in Uttarakhand, India

Peripheral smears were prepared from ethylenediaminetetraacetic

acid‑anticoagulated blood samples of 100 patients.

The peripheral smears were made by the standard ‘wedge’ procedure and
stained with Leishman stain.

Automated counts by a calibrated and adequately quality-controlled

Beckman Coulter LH750 analyser

The manual platelets counts using a magnus microscope (MLXi plus) with
0,2 mm diameter
• Automated analyser used as the reference methods  samples with inaccurate platelets
count (platelet clumps, giant platelet was excluded.
• Estimate of the total platelet count by the first method (PLT/RBC ratio method) was made
by counting the number of platelets/1000 RBCs on OIF examination.
• The number of RBCs observed in a quarter of OIF was multiplied by four instead of
counting all the RBCs in the field.
• The total platelet counts were estimated by multiplying the ratio of platelets to the RBCs
by the RBC counts derived from the analyser
Estimate of the platelet count by the second
method(PLT×15,000 method) was made by counting the
number of platelets in at least 10 OIFs in an appropriate
area (junction of body and tail).

The average number of platelets per OIF was calculated

and the total platelet count was estimated by multiplying
the average number of platelets per OIF by 15,000 to give
an approximate count per mm3

Total platelet counts by the third method (PLT ×20,000

method) and fourth method (PLT ×10,000 method),
respectively, were done in a similar manner.
 Statistical analysis

The differences  peripheral smear

estimate of platelet count and analyser

count were calculated.

The number and proportion of peripheral smear
.Anacceptable difference in results between
estimates of platelet count having acceptable
the peripheral smear‑based estimates
difference from the analyser count were
and the analyser counts was deemed to be
calculated.
25% as suggested by Clinical Laboratory

Improvement Amendments (CLIA)

Statistical analysis

Same analysis was then repeated :

- platelet counts <100,000/mm3 (low platelet count)

RBC counts <4 million/mm3 (low RBC count)

- Both platelet counts <100,000/mm3 and RBC counts <4

million/mm3

- RBC counts between 4 and 6 million/mm3

. The analysis was done using R

statistical environment with R commander package

RESULTS
 The percentage of acceptable error
• Low by the PLT × 100,00
method (25%).
All cases
• The lowest by the PLT ×
20,000 method (18%).

• Both PLT/RBC and PLT×15,000

methods was equal (72.4%)
Platelet count
• PLT ×10,000 method (58.6%) group (<
• PLT ×20,000 method (6.9%) 100,000/mm3)
 The percentage of acceptable error
• PLT/ RBC method was the highest (83.9%
and 80%),
• PLT ×15,000 method (80.6% and 73.3%),
• PLT ×10,000 method (29%, 53.3%),
Low RBC and and
• and lowest in PLT ×20,000 method (9.7%, Low platelet
0%). counts

• PLT ×15,000 method has the

highest accuracy (88.1%),
• PLT/RBC method (82.1%), Normal RBC
• PLT×10,000 and PLT ×20,000 counts
methods (23.9% and 20.9%).
DISCUSSIONS
A result of thrombocytopenia  automated analysers  re‑checked and
confirmed  peripheral smear examination.

Because there are many reasons of pseudo‑thrombocytopenia  analytical or

pre‑analytical factors.

Such as
• Platelet clumping,
• satellitism,
• giant platelets,
• RBC microcytosis
• and bacterial contamination
 • Accurate platelet count and
• To ensure uniformity
Standard
multiplication
• Objectivity in assessing platelet counts
factor

• PLT ×15,000 method

• PLT/RBC ratio method
The present study
• the best amongst the various peripheral smear‑based methods

• Abbey and Nosanchuk suggested a factor of approximately 20,000 for platelet counts
• Webb et al. suggested a multiplication factor of 15,000.
• Moreno and Menko suggested a factor between 15,000 and 20,000, without
Different
researchers committing to either .
PLT/RBC ratio method

Brahimi et al. also

stands validated by this
study

Require a haematology
analyser

Determining RBC count

 PLT
×15,000 • Accurate estimates for all subgroups

and • Confirming their utility in laboratory practice

PLT/RBC

PLT • Prove useful in validating the peripheral smear review

• Not require an analyser and can independently assess

×15,000 platelet counts

• Further utilised in resource‑poor settings

method
• Without the need of analyser
• Reliable and dependable results
PLT × 15,000 method used in
this study

Agreeable rate of ± 25%

CLIA recommendations
 Agreeable rate
of ± 40%,
Cumbersome
much
process
higher/lower
Influenced by than the actual
the field platelet count
diameter of the
Depends on oil immersion
the lens
determination
of a
Another conversion
manual factor
method of
platelet
estimation
Needs to
re‑emphasised

Importance of manual method

cannot be undermined

Needs to pay attention

Which method to adopt to

ensure accurate results.
CONCLUSIONS
 Manual platelet count Conclusions

Estimation under OIF Peripheral smear using a

multiplication factor of
15,000

Comparable accuracy
to the analyser
PLT/RBC ratio method
counts.

This method is simple and easy

Laboratory setting and Platelet counts on

provides a rapid and accurate peripheral smear.
estimation