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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 aect the platelet count such as method of collecting blood specimen,
preparation of the platelet sample, the type of instrument and the calibration
method used. !ailure to recogni"e these factors has sometimes led to the
misdiagnosis.
Platelet count is more di#cult to do than $%& and '%& count because it is very
small, it may disintegrate easily (hen exposed to air, unevenly distributed in the
blood because of the tendency to stic) or clump together, adhere on to glass
surfaces or foreign bodies, di#cult to dierentiate from debris, bacteria and dirt
and is not (ell distributed in the circulation.
Specimen: *enous blood is collected (ith +D,- as the anticoagulant. %lood
from s)in puncture (ounds produces variation but is satisfactory if the blood is
.o(ing freely and if only the /rst fe( drops are used.
INDIRECT METHOD
Platelets are counted simultaneously in a blood smear. Platelets are counted
in relation to 0,111 $%&s in the blood smear. Platelet &ount 2P&3 /L or P&/L is
calculated based on $%& count obtained using hemacytometer. ,his method is not
so reliable since results depend on the distribution on platelets and on the $%&
count. ,he advantage, ho(ever is that it allo(s the study of platelet morphology.
I. Dameshek Method (Wet Method)
Diluting .uid: Rees-Ece! Di"#en$
-does not lyse $%& and '%&
a. %rilliant &resyl %lue 4 1.05 g 2dye3
b. Sodium &itrate 4 1.6 g 2prevents clumping of platelets3
c. Distilled 'ater 4 011 ml
d. !ormalin 4 7 drops in 0:01 dilution 2preservative3
e. Sucrose 4 8.1 g
Procedure:
1. Disinfect the site of puncture.
2. Place a drop of diluting .uid on the disinfected area. This is done to
avoid exposure to air and disintegration of platelets.
3. Puncture through the drop of diluting .uid to a depth of 7 mm.
4. ,ransfer a drop of blood and diluting .uid mixture on the center of a
coverslip. 2$atio 9 %:& : blood to diluent3 -nd invert over a glass
slide and allo( it to stand in a moist chamber for 05465 minutes.
(Meanwhile, do not forget to do the RBC ount on the patient.!
". +xamine the prepared smear under the oil immersion of a
microscope.
#. &ount ;51 $%&s per area in 6 areas to a total of 0,111 $%&s and
count all the platelets seen. Platelets are lilac colored cells, tiny and
glistening.
$. &omputation:
Normal Value: 511,1114<11,111= mm
7
II. Fonio (Dry Method)
Diluting .uid: %'( M)*nesi#m S#"+)$e
4does not lyse $%&s
Procedure:
0. Disinfect the site of puncture.
;. Place a drop of diluting .uid on the disinfected area.
7. Puncture through the drop of diluting .uid to a depth of 7 mm.
6. ,ransfer a drop of blood4>gS?6 mixture on a slide. 2$atio 9 %:,3
5. >a)e a smear, dry and stain (ith 'right@s stain. 2Meanwhile, do not
forget to do the RBC ount on the patient.!
A. Under ?I?, count ;51 $%&s per area in 6 areas to a total of 0,111
$%&s and count all the platelets seen.
B. &omputation:
Normal Value: ;51,1114511,111= mm
7
-dvantage:
0. +asier to count $%&s and platelets
;. Si"e and shape of platelets can be observed
III. Olefs Method
,he ?lef@s method is the best method in the indirect procedures but
some(hat cumbersome.
Normal Value: 67B,111458A,111=mm
7
ESTIMATE PLATELET COUNT
Ratio of Eletroni !latelet "ount to #latelets #er oil$immersion
%eld.
%efore platelet estimates can be performed, the laboratory must calculate
the ratio of electronic platelet count to the number of platelets per oil4immersion
/eld of vie(. ,he procedure for calculation of this ratio and estimation factor is
as follo(s:
0. Perform electronic platelet counts on 71 consecutive fresh patient blood
samples. >a)e sure that the platelet count is in control.
;. !or each /lm, under oil immersion microscopy, /nd an area (here 51C of
the red cells are overlapping in doublets or triplets. ,hen count the
number of platelets in 01 consecutive /elds.
7. Divide the total number of platelets found by 01 to obtain the average
number per single oil4immersion /eld.
6. Divide the electronic platelet count by the average number of platelets
per oil /eld.
5. -dd the numbers obtained in step 5 and divide by 71 2the number of
observations in this analysis3 to obtain the average ratio of platelet count:
platelets per oil4immersion /eld.
A. $ound the number calculated to the nearest (hole number to obtain the
estimation factor.

Estimate #latelet ount.
?nce the platelet estimation factor is calculated, the laboratory scientist may
simply calculate the average number of platelets per oil4immersion /eld on all
subseDuent specimens and multiply this number by the estimation factor to
obtain the platelet esti%ate.
P")$e"e$ Es$im)$e -+ Rep-!$ P")$e"e$ Es$im)$e )s
1 : 6<,111=uE Marked derease
51,111 : <<,111=uE Moderate derease
011,111 : 06<,111=uE &li'ht derease
051,111 : 0<<,111=uE (o) normal
;11,111 : 611,111=uE NORMAL
610,111 : 5<<,111=uE &li'ht inrease
A11,111 : 811,111=uE Moderate inrease
-bove 811,111=uE Marked inrease
DIRECT METHOD
,his method employs a dilution of blood using an $%& pipet (ith the use of a
hemacytometer.
I. Rees$Eker Method
Diluting .uid: Rees-Ece! Di"#en$
-does not lyse $%& and '%&
a. %rilliant &resyl %lue 4 1.05 g 2dye3
b. Sodium &itrate 4 1.6 g 2prevents clumping of platelets3
c. Distilled 'ater 4 011 ml
d. !ormalin 4 7 drops in 0:01 dilution 2preservative3
Procedure:
0. $inse $%& pipet /rst (ith $+ diluting .uid by suc)ing in and out the
diluting .uid (to prevent disintegration of platelets!.
;. &ount platelets in the ;5 sDuares in the large central sDuare on
each side of the hemocytometer.
7. &omputation:
!latelet "ount * Platelet &ounted x Dilution !actor x -rea
&orrection !actor
F 01 2Depth &orrection !actor3

Normal Value: 051,1114651,111= mm
7
II. +reker "ronkite
,he reference method that uses phase4contrast microscopy (ith green
2platelets appear dar)3 or gray /lter 2platelets appear pin) or purple3. It also
uses a .at4bottomed hemacytometer (hose focus can be easily adGusted and a
thin coverslip 2because thic) coverslip retards refraction of light.3.
Diluting .uid: %( Amm-ni#m -.)")$e
4lyses $%&
Procedure:
0. 'ell4mixed blood is diluted 0:011 in diluting .uid, and the vial
containing the suspension is rotated on a mechanical mixer for 01
to 05 minutes.
;. ,he hemacytometer is /lled in the usual fashion, using a separate
capillary tube for each side.
7. ,he chamber is covered (ith a Petri dish for 05 minutes to allo( the
platelets to settle in one optical plane. - piece of (et cotton or /lter
paper is left beneath the dish to prevent evaporation.
6. Using this type of microscope platelets are seen very clearly against
'%&s.
5. Platelets are seen as round to oval purple bodies sometimes (ith
dendritic processes. ,heir internal granular structure and a purple
sheen allo( the platelets to be distinguished from debris, (hich is
often refractile. Hhosts of the red cells that have been lysed by the
ammonium oxalate are seen in the bac)ground.
A. -llo(able dierence bet(een each chamber is I=401 platelets. If
greater than 01, repeat mixing and charging o counterchec) the
test.
B. Platelets are counted in 01 small sDuares, /ve on each side of the
chamber. If the total number of platelets counted is less than 011,
more small sDuares are counted until at least 011 platelets have
been recorded401 sDuares per side or all ;5 sDuares in the large
central sDuare on each side of the hemocytometer, if necessary. If
the total number of platelets in all 51 of these small sDuares is less
than 51, the count should be repeated (ith 0:;1 or 0:01 dilutions of
blood.
8. &omputation:
!latelet "ount * Platelet &ounted x Dilution !actor x -rea
&orrection !actor
F 01 2Depth &orrection !actor3
Normal Value: 051,1114651,111= mm
7
III. ,uy and (eake Method
Diluting .uid:
a. &rystal *iolet 4 1.15 g 2dye3
b. Sodium &itrate 4 0.A g 2prevents clumping of platelets3
c. Distilled 'ater 4 011 ml
d. 61C !ormalin 4 <6.1 ml 2preservative3
Procedure:
0. Platelets are counted in all the ;5 intermediate sDuares of the
central sDuare. Platelets appear as rounded, oval or coma4shaped
highly refractile bodies measuring from ;47u. ,he amount of light in
the microscope should be adGusted so that the platelets appear as
highly refractile bodies.
;. &omputation:
IV. -NO!E..E &ystem
Principle: %lood is diluted in buered ammonium oxalate (hich hemoly"es
mature red cells and preserves, platelets, leu)ocytes and reticulocytes.
Platelets and Eeu)ocytes may to be counted in a standard
hemacytometer.
Diluting !luid: %( B#/e!e0 Amm-ni#m O.)")$e
Dilution: %:%11
Procedure:
0. -dd blood from capillary to diluent.
;. $inse capillary.
7. >ix by inversion. Diluted sample is stable for 7 hours.
6. Eet stand for 01 minutes to allo( red cells to hemoly"e.
5. >ix thoroughly by inversion.
A. &onvert to dropper and charge hemacytometer. - hemacytometer
(ith a Jeubauer ruling is recommended.
B. &over hemacytometer (ith Petri dish to prevent dehydration (hile
(aiting 01 minutes for platelets to settle.
8. &ount platelets in all ;5 small sDuares (ithin a large center sDuare
(ithin large center sDuare of the Jeubauer ruling.
<. >ultiply the total number of platelets counted by 0,111.
Jote: If leu)ocyte is also to be counted, one must perform the leu)ocyte
count before platelet determination.
V. .oantins
VI.Ny'ards
VII. Walker
VIII. Van /llens Method : reported in percent
PHYSIOLO2IC VARIATION OF PLATELET COUNT:
Platelet count is normally increased in high altitudes, administration of
epinephrine, after strenuous exercise, trauma and excitement and in (inter as
platelets are from the splenic pool into the peripheral blood.
Jormally decrease before menstruation and during pregnancy.
?ral contraceptives may cause slight increased platelet count.
Platelets are normally absent in lymph and other body .uids.
INCREASED P")$e"e$ C-#n$ DECREASED P")$e"e$ C-#n$
Polycythemia vera Pernicious -nemia
>yeloproliferative Syndrome -plastic -nemia
Splenic *ein ,hrombosis Infectious Disease
Post4splenectomy States Eesions involving the %one
>arro(
-cute %lood Eoss I,P
&arcinomatosis -cute Eeu)emia
Jephrotic Syndrome Kemorrhagic !ever
&hronic >yelogenous Eeu)emia
-cute -lcoholic Kepatitis
&irrhosis of the Eiver
Ulcerative &olitis
C-mmen$s )n0 S-#!ces -+ E!!-!:
,he method of collecting blood in.uences the platelet count. "a#illary 0lood
obtained by /nger4pric) generally has a lo(er platelet count than the blood
obtained by venipuncture. ,his is caused by the adhesion of some platelets at
the site of the (ound and the diluting eect of tissue Guices occurring (hile
sDuee"ing the /nger. ,he small amount of blood collected in capillary blood
does not allo( for a rechec) on the sample if the initial result is doubtful. In
practice a venous blood is preferable and reliable. &apillary blood maybe used
(hen there is poor venous access but (hen collection is indicated /ngertip and
heel is recommended but not the earlobe because the /ne hair favors adhesion
of platelets. !ree .o( of blood is ideal. It is especially di#cult to obtain accurate
platelet counts from heelstic) puncture on babies.
%lood in ED./ is satisfactory for /ve hours after collection at ;1& and for ;6
hours at 6&, provided that no di#culty (as encountered during collection.
+D,- is a convenient anticoagulant to use for routine platelet countsL
occasionally the presence of +D,- causes the platelets to clump and the count
to be falsely lo(. ,he platelet agglutinins responsible for this may be IgH and Ig>
antibodies active in the presence of +D,-. If #latelet lum#in' is observed, the
count is rediluted. If clumping is still present, obtain a fresh specimen. -n even
distribution of platelets through the counting area is critical, because of the
adhesive Duality of platelets, /ngerstic) specimens are least desirable. Poor
venipuncture techniDue or inadeDuate mixing of the specimen after collection
can cause platelet clumping.
,he technologist should be a(are of M#latelet satellitosisN (hen using +D,- as
an anticoagulant. Platelet satellitosis appear as neutrophils ringed (ith adhesive
platelets. If platelet clumps and satellitosis are observed, the only (ay to get an
accurate count in these patients is to collect blood into itrate 2blue4top tube3
anticoagulant 2remembering that the citrate (ill decrease the platelet count by
01C due to dilution eectsL this eect should be compensated for3. 'hen
sodium citrate is used as an anticoagulant, ma)e the correction for the dilution
by multiplying by 0.0. Platelet clumping increases (ith time, so platelet counts
should be done as soon as possible after collection to maintain accuracy.
%lood preserved (ith he#arin is J?, ideal for blood smear preparation using
'right@s stain due to blue discoloration of the bac)ground.
-ccurate #latelet estimates are possible only (hen there are no platelet
clumps, or at most, rare clumps of t(o to three platelets. Earger 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 +D,- should be suspected and noted on the
specimen report. Platelet estimates reports and leu)ocyte dierential reports
should indicate the presence of large or morphologically abnormal platelets.
%ecause of their si"e they may be counted by automated counting instruments
as erythrocytes or even lymphocytes, and the result (ould be an inaccurate
platelet total.
,he platelet determination should be compared (ith a revie( of the blood /lm
for con/rmation of count and morphology.
In diret ountin' method, overcharging (ill lead to decrease in cell count
since cells (ith fall in the moat of the counting chamber. It is also important to
discard the /rst 645 drops of mixture from the pipet before charging the chamber
because these drops contain drops of pure diluting .uid and may contain foreign
materials li)e dead epidermal cells.
It is preferable to use #hase$ontrast miroso#e as opposed to a light
microscope (hen counting platelets as it allo(s for better dierentiation from
debris and $%& inclusions. In phase contrast microscope using the %rec)er4
&ron)ite metod, platelets appear as dense, dar) bodies and can be round, oval,
or rodli)e, sometimes sho(ing dendritic processes 2hairy proGections3. ,heir
internal granular structure and pearlescent sheen allo( platelets to be
distinguished from debris, (hich is often refractile. $%&@s appear as ghost cells.
Use caution (hen $%&s have inclusion present, so as not to confuse their
inclusion (ith platelets.
,he capillary pipette 2-NO!E..E &ystem3 measures ;1 uE of blood diluted (ith
0.<8 mE ?! 0 g=dE ammonium oxalate.
Platelet counts (ill also decrease (ith stora'e 2and >P*s increase3, therefore
counts should be performed as soon as possible after blood collection. Jote that
clotting of the sample (ill totally invalidate the platelet count.
ELECTRONIC COUNTIN2 3A#$-m)$e0 P")$e"e$ C-#n$s4
-utomated counters employ either optical methods 2detection of the degree
of light scattering3 or electrical methods 2change in the electrical resistance or
capacitance across a circuit3 to detect particles as they stream through an aperture
tube or .o( cell. %rie.y, various instruments exist for counting platelets, including
multiparameter instruments and a fe( instruments dedicated solely to platelet
counts. -utomatic platelet counters may sample (hole blood and discriminate
platelets from red cells on the basis of their si"e, or sample platelet4rich plasma and
calculate platelet counts on the basis of sample dilution, volume counted and
hematocrit of the original sample.
5#)"i$6 C-n$!-":
Instruments must be standardi"ed against reference methods such as the
phase4contrast method or against a single4channel instrument that has been
calibrated by means of platelet threshold curves. ?nce an instrument has been
standardi"ed, suspensions of /xed human platelets (hich are available from several
suppliers may be used as day4to4day controls. &ontrols are usually run at the
beginning of each batch of platelet counts and may be interspersed bet(een
patient samples (hen batches of more than 05 samples are run. $ecords of O& data
must be examined periodically to detect subtle instrumental or regeant problems.
Samples from each batch should be screened for accuracy by Duic) estimates
from the peripheral blood /lm. 'here the estimates and automated counts do not
agree, results should be obtained by phase4contrast method. Kistograms of platelet
populations provide a further means of Duality control.
C-mmen$s )n0 S-#!ces -+ E!!-!:
!alsely high platelet counts may result from contamination of reagents (ith
particles or bacteriaL carry over from a prior sample (ith a high platelet countL or
particles that should not be counted but are registered as platelets, such as
fragments of red or (hite blood cells, insu#ciently lysed red cells, or red cells
(ith inclusion bodies.
!alsely lo( counts may result from platelet agglutinins or platelet satellitism.
+xceptionally large platelets may not be counted if upper as (ell as lo(er
thresholds are used. +ach of these sources of error can be eliminated (ith a
good Duality control regime, including careful revie( of histograms generated by
automated instruments and revie( of the peripheral blood /lm.