LABORATORY EVALUATION OF PLATELETS

Prepared by: Kenneth S. Destura

PLATELET COUNT
 Platelet count is an integral part of the complete blood count. Unexpectedly, abnormal platelet
counts are sometimes obtained in clinical practice. It is important to understand the
physiological and various technical factors that could affect the platelet count such as method of
collecting blood specimen, preparation of the platelet sample, the type of instrument and the
calibration method used. Failure to recognize these factors has sometimes led to the
misdiagnosis.
 Platelet count is more difficult to do than RBC and WBC count because it is very small, it may
disintegrate easily when exposed to air, unevenly distributed in the blood because of the
tendency to stick or clump together, adhere on to glass surfaces or foreign bodies, difficult to
differentiate from debris, bacteria and dirt and is not well distributed in the circulation.
 Specimen: Venous blood is collected with EDTA as the anticoagulant. Blood from skin puncture
wounds produces variation but is satisfactory if the blood is flowing freely and if only the first
few drops are used.

INDIRECT METHOD
Platelets are counted simultaneously in a blood smear. Platelets are counted in relation to
1,000 RBCs in the blood smear. Platelet Count (PC) /µL or PC/L is calculated based on RBC count
obtained using hemacytometer. This method is not so reliable since results depend on the
distribution on platelets and on the RBC count. The advantage, however is that it allows the study of
platelet morphology.

I. Dameshek Method (Wet Method)

 Diluting fluid: Rees-Ecker Diluent
-does not lyse RBC and WBC
a. Brilliant Cresyl Blue - 0.15 g (dye)
b. Sodium Citrate - 0.4 g (prevents clumping of platelets)
c. Distilled Water - 100 ml
d. Formalin - 3 drops in 1:10 dilution (preservative)
e. Sucrose - 8.0 g

 Procedure:
1. Disinfect the site of puncture.
2. Place a drop of diluting fluid on the disinfected area. This is done to avoid
exposure to air and disintegration of platelets.
3. Puncture through the drop of diluting fluid to a depth of 3 mm.
4. Transfer a drop of blood and diluting fluid mixture on the center of a coverslip.
(Ratio = 1:5 – blood to diluent) And invert over a glass slide and allow it to stand
in a moist chamber for 15-45 minutes. (Meanwhile, do not forget to do the RBC
count on the patient.)
5. Examine the prepared smear under the oil immersion of a microscope.
6. Count 250 RBCs per area in 4 areas to a total of 1,000 RBCs and count all the
platelets seen. Platelets are lilac colored cells, tiny and glistening.
7. Computation:

Normal Value: 500,000-900,000/ mm3

II. Fonio (Dry Method)

 Diluting fluid: 14% Magnesium Sulfate
-does not lyse RBCs
 Procedure:
1. Disinfect the site of puncture.
2. Place a drop of diluting fluid on the disinfected area.
3. Puncture through the drop of diluting fluid to a depth of 3 mm.
4. Transfer a drop of blood-MgSO4 mixture on a slide. (Ratio = 1:3)
 5. Make a smear, dry and stain with Wright’s stain. (Meanwhile, do not forget to do
the RBC count on the patient.)
6. Under OIO, count 250 RBCs per area in 4 areas to a total of 1,000 RBCs and count
all the platelets seen.
7. Computation:

Normal Value: 250,000-500,000/ mm3

 Advantage:
1. Easier to count RBCs and platelets
2. Size and shape of platelets can be observed

III. Olef’s Method

 The Olef’s method is the best method in the indirect procedures but somewhat
cumbersome.
Normal Value: 437,000-586,000/mm3

ESTIMATE PLATELET COUNT

 Ratio of Electronic Platelet Count to platelets per oil-immersion field.
Before platelet estimates can be performed, the laboratory must calculate the ratio of
electronic platelet count to the number of platelets per oil-immersion field of view. The
procedure for calculation of this ratio and estimation factor is as follows:
1. Perform electronic platelet counts on 30 consecutive fresh patient blood samples. Make
sure that the platelet count is in control.
2. For each film, under oil immersion microscopy, find an area where 50% of the red cells
are overlapping in doublets or triplets. Then count the number of platelets in 10
consecutive fields.
3. Divide the total number of platelets found by 10 to obtain the average number per single
oil-immersion field.
4. Divide the electronic platelet count by the average number of platelets per oil field.
5. Add the numbers obtained in step 5 and divide by 30 (the number of observations in
this analysis) to obtain the average ratio of platelet count: platelets per oil-immersion
field.
6. Round the number calculated to the nearest whole number to obtain the estimation
factor.

 Estimate platelet count.

Once the platelet estimation factor is calculated, the laboratory scientist may simply
calculate the average number of platelets per oil-immersion field on all subsequent specimens
and multiply this number by the estimation factor to obtain the platelet estimate.

Platelet Estimate of Report Platelet Estimate as

0 – 49,000/uL Marked decrease
50,000 – 99,000/uL Moderate decrease
100,000 – 149,000/uL Slight decrease
150,000 – 199,000/uL Low normal
200,000 – 400,000/uL NORMAL
401,000 – 599,000/uL Slight increase
600,000 – 800,000/uL Moderate increase
Above 800,000/uL Marked increase

DIRECT METHOD
This method employs a dilution of blood using an RBC pipet with the use of a
hemacytometer.

I. Rees-Ecker Method
 Diluting fluid: Rees-Ecker Diluent
-does not lyse RBC and WBC
a. Brilliant Cresyl Blue - 0.15 g (dye)
b. Sodium Citrate - 0.4 g (prevents clumping of platelets)
 c. Distilled Water - 100 ml
d. Formalin - 3 drops in 1:10 dilution (preservative)

 Procedure:
1. Rinse RBC pipet first with RE diluting fluid by sucking in and out the diluting
fluid (to prevent disintegration of platelets).
2. Count platelets in the 25 squares in the large central square on each side of the
hemocytometer.
3. Computation:
Platelet Count = Platelet Counted x Dilution Factor x Area Correction Factor
X 10 (Depth Correction Factor)

Normal Value: 150,000-450,000/ mm3

II. Brecker Cronkite
The reference method that uses phase-contrast microscopy with green (platelets appear
dark) or gray filter (platelets appear pink or purple). It also uses a flat-bottomed
hemacytometer whose focus can be easily adjusted and a thin coverslip (because thick coverslip
retards refraction of light.).

 Diluting fluid: 1% Ammonium oxalate

-lyses RBC

 Procedure:
1. Well-mixed blood is diluted 1:100 in diluting fluid, and the vial containing the
suspension is rotated on a mechanical mixer for 10 to 15 minutes.
2. The hemacytometer is filled in the usual fashion, using a separate capillary tube
for each side.
3. The chamber is covered with a Petri dish for 15 minutes to allow the platelets to
settle in one optical plane. A piece of wet cotton or filter paper is left beneath the
dish to prevent evaporation.
4. Using this type of microscope platelets are seen very clearly against WBCs.
5. Platelets are seen as round to oval purple bodies sometimes with dendritic
processes. Their internal granular structure and a purple sheen allow the
platelets to be distinguished from debris, which is often refractile. Ghosts of the
red cells that have been lysed by the ammonium oxalate are seen in the
background.
6. Allowable difference between each chamber is +/-10 platelets. If greater than
10, repeat mixing and charging o countercheck the test.
7. Platelets are counted in 10 small squares, five on each side of the chamber. If the
total number of platelets counted is less than 100, more small squares are
counted until at least 100 platelets have been recorded-10 squares per side or
all 25 squares in the large central square on each side of the hemocytometer, if
necessary. If the total number of platelets in all 50 of these small squares is less
than 50, the count should be repeated with 1:20 or 1:10 dilutions of blood.
8. Computation:
Platelet Count = Platelet Counted x Dilution Factor x Area Correction Factor
X 10 (Depth Correction Factor)

Normal Value: 150,000-450,000/ mm3

III. Guy and Leake Method
 Diluting fluid:
a. Crystal Violet - 0.05 g (dye)
b. Sodium Citrate - 1.6 g (prevents clumping of platelets)
c. Distilled Water - 100 ml
d. 40% Formalin - 94.0 ml (preservative)

 Procedure:
1. Platelets are counted in all the 25 intermediate squares of the central square.
Platelets appear as rounded, oval or coma-shaped highly refractile bodies
measuring from 2-3u. The amount of light in the microscope should be adjusted
so that the platelets appear as highly refractile bodies.
2. Computation:

IV. UNOPETTE System

  Principle: Blood is diluted in buffered ammonium oxalate which hemolyzes mature red
cells and preserves, platelets, leukocytes and reticulocytes. Platelets and Leukocytes
may to be counted in a standard hemacytometer.

 Diluting Fluid: 1% Buffered Ammonium Oxalate

 Dilution: 1:100

 Procedure:
1. Add blood from capillary to diluent.
2. Rinse capillary.
3. Mix by inversion. Diluted sample is stable for 3 hours.
4. Let stand for 10 minutes to allow red cells to hemolyze.
5. Mix thoroughly by inversion.
6. Convert to dropper and charge hemacytometer. A hemacytometer with a
Neubauer ruling is recommended.
7. Cover hemacytometer with Petri dish to prevent dehydration while waiting 10
minutes for platelets to settle.
8. Count platelets in all 25 small squares within a large center square within large
center square of the Neubauer ruling.
9. Multiply the total number of platelets counted by 1,000.

 Note: If leukocyte is also to be counted, one must perform the leukocyte count before
platelet determination.

V. Tocantin’s
VI. Nygard’s
VII. Walker
VIII.Van Allen’s Method – reported in percent

PHYSIOLOGIC VARIATION OF PLATELET COUNT:

 Platelet count is normally increased in high altitudes, administration of epinephrine, after
strenuous exercise, trauma and excitement and in winter as platelets are from the splenic pool
into the peripheral blood.
 Normally decrease before menstruation and during pregnancy.
 Oral contraceptives may cause slight increased platelet count.
 Platelets are normally absent in lymph and other body fluids.

INCREASED Platelet Count DECREASED Platelet Count

Polycythemia vera Pernicious Anemia
Myeloproliferative Syndrome Aplastic Anemia
Splenic Vein Thrombosis Infectious Disease
Post-splenectomy States Lesions involving the Bone Marrow
Acute Blood Loss ITP
Carcinomatosis Acute Leukemia
Nephrotic Syndrome Hemorrhagic Fever
Chronic Myelogenous Leukemia
Acute Alcoholic Hepatitis
Cirrhosis of the Liver
Ulcerative Colitis

Comments and Sources of Error:

 The method of collecting blood influences the platelet count. Capillary blood obtained by
finger-prick generally has a lower platelet count than the blood obtained by venipuncture. This
is caused by the adhesion of some platelets at the site of the wound and the diluting effect of
tissue juices occurring while squeezing the finger. The small amount of blood collected in
capillary blood does not allow for a recheck on the sample if the initial result is doubtful. In
practice a venous blood is preferable and reliable. Capillary blood maybe used when there is
poor venous access but when collection is indicated fingertip and heel is recommended but not
the earlobe because the fine hair favors adhesion of platelets. Free flow of blood is ideal. It is
especially difficult to obtain accurate platelet counts from heelstick puncture on babies.
 Blood in EDTA is satisfactory for five hours after collection at 20C and for 24 hours at 4C,
provided that no difficulty was encountered during collection.
 EDTA is a convenient anticoagulant to use for routine platelet counts; occasionally the presence
of EDTA causes the platelets to clump and the count to be falsely low. The platelet agglutinins
responsible for this may be IgG and IgM antibodies active in the presence of EDTA. If platelet
clumping is observed, the count is rediluted. If clumping is still present, obtain a fresh
specimen. An even distribution of platelets through the counting area is critical, because of the
adhesive quality of platelets, fingerstick specimens are least desirable. Poor venipuncture
technique or inadequate mixing of the specimen after collection can cause platelet clumping.
 The technologist should be aware of “platelet satellitosis” when using EDTA as an
anticoagulant. Platelet satellitosis appear as neutrophils ringed with adhesive platelets. If
platelet clumps and satellitosis are observed, the only way to get an accurate count in these
patients is to collect blood into citrate (blue-top tube) anticoagulant (remembering that the
citrate will decrease the platelet count by 10% due to dilution effects; this effect should be
compensated for). When sodium citrate is used as an anticoagulant, make the correction for the
dilution by multiplying by 1.1. Platelet clumping increases with time, so platelet counts should
be done as soon as possible after collection to maintain accuracy.
 Blood preserved with heparin is NOT ideal for blood smear preparation using Wright’s stain
due to blue discoloration of the background.
 Accurate platelet estimates are possible only when there are no platelet clumps, or at most,
rare clumps of two to three platelets. Larger or more numerous platelet clumps cause
inaccurate estimates. If clumps are noted, the original blood specimen should be reexamined for
clots. If clots are not found, platelet clumping in the presence of EDTA should be suspected and
noted on the specimen report. Platelet estimates reports and leukocyte differential reports
should indicate the presence of large or morphologically abnormal platelets. Because of their
size they may be counted by automated counting instruments as erythrocytes or even
lymphocytes, and the result would be an inaccurate platelet total.
 The platelet determination should be compared with a review of the blood film for confirmation
of count and morphology.
 In direct counting method, overcharging will lead to decrease in cell count since cells with fall
in the moat of the counting chamber. It is also important to discard the first 4-5 drops of mixture
from the pipet before charging the chamber because these drops contain drops of pure diluting
fluid and may contain foreign materials like dead epidermal cells.
 It is preferable to use phase-contrast microscope as opposed to a light microscope when
counting platelets as it allows for better differentiation from debris and RBC inclusions. In phase
contrast microscope using the Brecker-Cronkite metod, platelets appear as dense, dark bodies
and can be round, oval, or rodlike, sometimes showing dendritic processes (hairy projections).
Their internal granular structure and pearlescent sheen allow platelets to be distinguished from
debris, which is often refractile. RBC’s appear as ghost cells. Use caution when RBCs have
inclusion present, so as not to confuse their inclusion with platelets.
 The capillary pipette (UNOPETTE System) measures 20 uL of blood diluted with 1.98 mL OF 1
g/dL ammonium oxalate.
 Platelet counts will also decrease with storage (and MPVs increase), therefore counts should be
performed as soon as possible after blood collection. Note that clotting of the sample will totally
invalidate the platelet count.

ELECTRONIC COUNTING (Automated Platelet Counts)

Automated counters employ either optical methods (detection of the degree of light
scattering) or electrical methods (change in the electrical resistance or capacitance across a circuit)
to detect particles as they stream through an aperture tube or flow cell. Briefly, various instruments
exist for counting platelets, including multiparameter instruments and a few instruments dedicated
solely to platelet counts. Automatic platelet counters may sample whole blood and discriminate
platelets from red cells on the basis of their size, or sample platelet-rich plasma and calculate
platelet counts on the basis of sample dilution, volume counted and hematocrit of the original
sample.

Quality Control:
Instruments must be standardized against reference methods such as the phase-contrast
method or against a single-channel instrument that has been calibrated by means of platelet
threshold curves. Once an instrument has been standardized, suspensions of fixed human platelets
which are available from several suppliers may be used as day-to-day controls. Controls are usually
run at the beginning of each batch of platelet counts and may be interspersed between patient
samples when batches of more than 15 samples are run. Records of QC data must be examined
periodically to detect subtle instrumental or regeant problems.
 Samples from each batch should be screened for accuracy by quick estimates from the
peripheral blood film. Where the estimates and automated counts do not agree, results should be
obtained by phase-contrast method. Histograms of platelet populations provide a further means of
quality control.

Comments and Sources of Error:

 Falsely high platelet counts may result from contamination of reagents with particles or
bacteria; carry over from a prior sample with a high platelet count; or particles that should not
be counted but are registered as platelets, such as fragments of red or white blood cells,
insufficiently lysed red cells, or red cells with inclusion bodies.
 Falsely low counts may result from platelet agglutinins or platelet satellitism. Exceptionally
large platelets may not be counted if upper as well as lower thresholds are used. Each of these
sources of error can be eliminated with a good quality control regime, including careful review
of histograms generated by automated instruments and review of the peripheral blood film.