From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

1957 12: 671-676

An Explanation of the Discrepancy between Direct and Indirect Platelet Counts

HARRY J. FITCH

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From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

An

Explanation
and

of
Indirect
By

the

Discrepancy
Platelet
J. FITCH

between
Counts

Direct

HARRY

HERE ARE the indirect.

t-iso For

standard the direct usually on an usual,

in

nuethods nuethod
a hemocytomet-er

for the the

counting blood
pipet,

platelets, is accurately and nuethod the the to

the direct and diluted with au platelets blood are is diluted amid prepratio is to thems
Whems

anst-icoagulamut

sohutiomu,

enumsueiated

ims a counting

chanuber.1 ordinary a cosership cells are

platelet-red

For

indirect

amid a drop is placed dried3 or, as is more arat-ion.2 established. deternsimse

derised by

microscope is placed then coumut-cd

slide. It may on the drop field by directly in The platelet

to this direct

then be spread make a wet and the chamber is

counut.

Platelets The their

referring

and

red

are

field

red cells concentrations

the

also counted in the blood.

cell ratio

a counting concentration
red cell

platelets suit-s per

kmsosvn

ins the are msot the ; by

the simsce

sanue same. the

sample indirect
of

of

blood count

are

coumsted by the
It

by direct This
has

these discrepancy
also been

tsso

methods is about has

kmsosvns that

the 250,000 beemu

re-

A isormal method
the

nuet-hod

cu.mnuu.

about 500,000.
century.6

begimsning

si-hens

the ptoportioms rat-jo is about an the fronu The imidirect platelet that

of platelets 1 :20, while count. count is carried of nornsal by between for speculatioms is to demonstrate the

to red betis-eeis

cells is established a slide amid coverslip

in

a counting it. is about

chamber 1 : 10.

the When smear3,

out its a counstimug chambers or on a dried humams blood is about 250,000, sihich is no differemst direct results nsethod. usually or debate, the reasoms
ANI) phase-contrast of dilinent and

obtaimsed

diffetemuee

obtailsed has for not the

by

the

tsvo

nucthods,

si-hihe

oftems a subject of this repont

beems explained.7 differemuce ins these

The purpose couist.s.

MATERIALS

I\IETH0DS microscopy using oxalate a small as the method diluent.1 of Indirect Brecher counnts

1)irect
and were accordinsg

couunts

were a one I),

l)erformed per cenut blood nuixinng

by

Cronkite performed

with
to the

solution

ammonium in

wax with cup a

wax
was human betweens

stirring
Coverslips prepared blood. the were

rod
with This two clone. stuidy methods

were
General involved and The

rinsge(l
Electric the whenever crucial

with

nuet-hod of Olef.5 vaseline to

l)ri-Film SC-87. several were of

Damesheks solution prevent drying. Siliconized

All earlier conspared counsts theories were of done the

was
on

use(l the as
glassware normal blind difference

dilunent.2

testinng the two experiment field counisted coverslip. were figuures

methods with
from divided The on

experimentally,

counts opposite the figures

the
one by counts a
an

indirect
edge tine of graph
average

met-hod
of each
ten

consisted
to to thens those ins were to the fields

of counting
derive average

the
edge.

red
The cells 3. the

cells
total per

and
mumfield each

platelets
ber across these
point

ins every cells the values

innthe

the

coverslip of field

of

was

number similar

compared svhich

the
2 simplifies

with average and

and
However, figures

plotted
represents

coissecutive fields,

without

chansginsg

their

essential

characteristics.

Ten
Reed

such
Arnuy

experiments
Instittnte

were
of

performed.
Walter

Fronsu

the

1)epartment

of

Hematology,

Walter

Research,

Reed

Center, Washington, 1). C. Submitted Oct. 5, 1956; accepted for publicat ions Nov. 26, 1956. The author records his thanks Lt. Col. to William H.Crosby to this insvestigation amid to Miss Thelma Johnson svho performed
671

Arnsy

Medical

for a

sunggestion blind platelet

that

le counnsts.

the

From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

672

DIRECT

ANt)

INDIRECT

PLATELET

COUNTS

,.

.,(

Fma.
of red centration accunracy

1.-A
cells of of at

snsall
the red cells

drop
edge at

of
of the

blood
the drop edge

1)enseath
is occunrs

a thins, snncoated
also with

coverslip. is fluid platelet-counting

The due and to

councenstration rouleaunx. destroys the Tue con-

apparent..

Granstnlarity

the

indirect

Platelet

cotnnt. H
ESULTS

It
neath Two

si-as
the thirds

found
cos-ership, of

ins f-he
the the all

indirect
red red cells cells

method
becomnc nsay be

that
foumid

is-hems
conscemstrated

the

drop
atthe the

of
outer

fluid
periphery 25

spreads
(fig. per

be1). of cemst

bemucath

the covershps area (figs. 2, 3). dispersed beneath the coscrship

related
sanse

Platelets, (figs. too

to

the

edge

of

the

covershp

thing

o(curred Both amid Whems

si-it-h

drops

oms the other hand, are almost randonuly 2, The 4) conscemutratioms of red cells is muot coimsciding the ivith edge of the drop. The small to reach the edge of f-he coserslip
.

(fig. 4). covership (fig. 5). ample:

count

u-ed cells amid platelets si-crc almost ramidomly dispersed whems the slide were coat-ed with silicone to issake the glass muons wettable this si-as done the imsdireet count alnsost equaled the direct. Excount (as-erage
platelets

Direct

of

foitr
per2700

chamushers)

199,000

per

cu.numsu.

Indirect

(bhimud counst): 96 184,000 platelets; (author

=

count)

red cells X 101 platelets per 2727

5.17 mssillioms RBC = red cells X 5.17 musilhioms

RBC

191,000

platelets

per cu.nuns.
Discussiox

The indirectplatelet coumut-, as it. is usually red cells are muot ransdomly distributed hemieat.h the t.ion of platelets ratio and to red cells is established high final because platelet, by the count edge, the reseuuted, ficially is artificially therefore the

domue, is covership. coumstinsg

imsaccurat-e When f-he ims away areas

from

because proporthe rep-

the

red cellsnot are adequately by the imudirect.

nsethocl

is arti-

high.

From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

HABRY

J.

FITCH

673

INDIRECT

PLATELET

COUNT

RED w
0 uIZ

CELLS

#{149}---.
#{176} #{176}

PLATELETS 300%%
I 8

I I 1 I

cr

LU >

I I I I

200%
I-

z
lOO%

LI-

EDGE
I

CENTER

EDGE

2345

6789101112131415

POSITION
FIG.

ON
platelets in

COVERSLIP
153 of couustof fields, frons edge to lut edge frons counsts
775,000; fields

2.-Distribution

of

red cells

cells

and

of point is

tine

coverslip.
sents would ten

Red
fields.

cells
The

averaged
red be matte.

11.6
are The

per

field,

the
at

platelets
the edge

0.75.
tlse

Each
coverslip,

point-

graph in

therepre-

concenstrated insclirect

3 to poinst the indirectwifh perisaps

13 the
counst

distribution
1)ased

is fairly

unniforusi.

It- is ins this that area indirect

i)ased tine ons platelets

platelet
tisese

ordinarily

platelet

on all fields is 359,01)0. l)istribiutionu ;s tendency to concenstrate ins tine cenntral

is fairly

random

area,

PERCENTAGE OF AREA IN EACH BAND

ALL
ON

FIELDS
THIS LINE

PERCENTAGE RED CELLS BAND

OF IN EACH

WERE

COUNTED POSITION FIGURE IN 2

FIG.

3.-Diagram
corresponds represents
a differenst

of

a coverslip
to the two poinits
of the

representing
in figunre 2 and of the conscennt red
area.

tine value

diagram which was

the the
in Tue

experimentin figure 2. average of the two

the red particuilar
of red

Each
poinsts bausd.
cells

bannd ins
gives Each
in each

a baund

concentrations

cells

represents band two-thirds

amounst

coverslips

percentage of cells

established of the red

by

mttltiplvinsg cells are

concenstrationn rated the mu peripheral

by cernt of

tise tise

area. area.

Almost

25 per

From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

674

DIRECT

AND

INDIRECT

PLATELET

COUNTS

INDIRECT

PLATELET SMALL DROP

COUNT

RED
LU

CELLS

#{149}---.
#{176}#{176}

PLATELETS
300%

LU >

200% I-

0
0 0 -J

I 00%

LU LL

10

II

12

13

14

15

POSiTION
FIG.

ON
17.1
at

COVERSLIP
so snuall are that it (ounld the Tine platelet nnot edge indirect reach

the
fields

4.-Distribution of red cells and platelets edge of tine coverslip. Red cells averaged
counted. drop even Each though l)oinst the represents edge that of the 10 fields. drop not is

ins a drop

per
Tine the

field,
red edge of

the
cells the

platelets
coverslip.

0.88.

Sevenst.y-eight

concentrated at

of the platelet
points indirect

coumnt based on all fields 4 to 12 inclusive (areas l)latelet count is 420,000.

is 290,000

per cuu. nnm.; wouuld be scrut

based
insize(l

on

the

fields

rel)resennted
counut

i)y
mug) the

inn ordinsary

The bilities siett-able

rcasomu

for

this

phenonssenoms
cells

has
nsay

muot- beems
be inupelled

established, 1)ut
toward

ses-en-al

possi-

conic to Isuind. rfhe red glass is hydrophihic

the

edge

i)ecause

si-hue

f-he

red

cells

arc

hydrophobic

ansd,

tepelled

by the glass, f-hey temud to stream ins f-he directions of t-hue air-ssater insterface. There musay also h)e sonic slight warping of the flexible coi-erslip : if a smusall chtop of si-hole blood is placed betis-eems a covership amid slide amid examimued imuumssediat-ely, the comsccntrat-ed thatthe f-he few because red tsio red cells glass cells they at the periphery forns rouleaux alud apart. about i)eing
stamsd

on

edge

in-

dicatimsg the drop presunual)hy platelets prescisce

surfaces are at least 8 lie flat amid as they nuose canmsot, the slide amid slip

iii But the

they too
together. close

(enter of do msot tunnu os-cr The

by the

are smssahl amsd f-hey are probably pmot-ected from squeeze the of larger red cells.rFhe red cells t.heiussehs-es cams h)e so protected the covership floats abose wit-h silicomsc reduces the their

by

usimug sun-faces of for one

a drop so large that the slide amid coscrship

slide. Coating the capillary at-fractions ivould 150

longer

amsot-her, amid also nuakes the surfaces hydrophobic to repel the red cells. The lack of ramudonuness of red cell distribution nuaked the drop eye by placimug f-he a smssall red drop of is-hole blood spreads (ehis immediately

so they

t-emud
to

cams denuomsstmatecl he

f-he As edge of

l)emseath

a thims

covenship. at- flue

hecomuse

comscentrat-ed

From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

HARRY

J.

FITCH

675

INDIRECT

PLATELET SILiCONE

COUNT

RED 300%
LU

CELLS

#{149}--

PLATELETS

o----o

LU

>
I-

200%

0
0
-j
LU Ln

100%

0 EDGE
I 2 3 4

CENTER
5 6 7 8

EDGE
9 10 II

POSITION
FIG.
surfaces.

ON
small 441

COVERSLIP
suspenn(lcd
Forty-fouir (-ells :nmnd 26

5.-l)istribuitioun

of

red
was

cells
perfornsued fields.

aun(1 platelets
oun a were Tisere

inn a drop
drop. red

i)et ceui
fields platelets.

silicouuized
were Thnere counted; is a fairly

Tine poiunt

experimenst represents

each

founr

ranndomsu

(histrih)utioms

of tine

red

cells.

the
pateust

drop.

But

if thesumfaces the red (d)s-emshil)

are

coat-ed
es-emily

ssit-h

silicomse,
Tise

or

if the

(In-op is

of less

l)loodI ap-

is

a large

onse, if the

cellsappear is t-hsick

IIts(l
S

diipersecl. nigid, of the

phenionsenions sont- used a counst.imng omn

chnanssher.

IJ.SIMAIIY

ihse cehls,

inschitect
sVhui(h tune

l)lItt(l(tused as

(ounni
a point-

is

highen
of

tisani

time
ins tise

dinert. (ounitinl(hitect-

hecautse

time

neferensce

nnnct.hod, nnot ane

red mann-

domssly chist-rihuiteci i)enn(at-ls I he (osenslip. Vise ne(l ((115 (ons((unt n-muted at tine ane (Nhge of f-hue (OVenShil) t hIlt tise tine nat-ioof ned (-ells to l)lItt-clct -i cannnoth)e
accumately est.ablishe(l
.

areas

of

the

(o\-(tsIiI)

I mI(hine(t l)lat-elet are tooigh. h

Si-snsn.smuo IX

(outnsts

h)aSed

oil I hse

n-tntio

in

t Inc((lit tal

INTEItLINGIJA

Le proque directe,
cytos

numsuciationi

indinecte

tie l)1IUlsettas

es

plus
base

alte
die

que

Icnnunnsematiomn

chinecte in-

he erythroeytos, es

clue

es

usate01150 (

neferennt-ia

ins he nsethsocio

iion es chist-ribuit e ah hasancho innfm-a he lamusehla (open-i-oh os. ecte en-yt hsro)j I eomsccnsttate al mssaugine del lansseila die mssaniena que le \ei l)noPontioni
chcternu;nabile. al cenntro (Id Numusem-at anea del ion nes

inter erythrocyt Os plachet C f-as muonns e plachet-tas, basate supen he proportions

inch i net-fe

de

lamehia tmoppo alte. (5

From bloodjournal.hematologylibrary.org by guest on January 21, 2012. For personal use only.

676

DIRECT

AND

INDIRECT

PLATELET

COUNTS

REFERENCES
BRECHER,

G.

AND

CRONKITE,

E.

lets.
2 DAMESHEK,

J.

Appl.

Physiol.

3:365,
Med.

P.: 1950.

Morphology

and

enumeration blood of

of human
platelets

l)lood and
reticu-

l)late-

W.:
Arch.

A method
Int.

for the simultaneous 50: 579,

Verfahren
J.A.M.A.

enutmeration

locytes.

1932. der Blutpl#{228}ttchenzahlung.

4(3:1022 and

FoNlo, 176,
KEMP, OLEF,

A.: 1912.

IJeber
The 585,

em
blood

neues

Deut.

Ztschr.

117: Chir.

G. T.:
I.:
Med.

plates.

1091,

1906.

Blood
46:

platelets:
1930.

an

improved various

indirect methods

method employed

for

their for

enumeration. enumerating

Arch. blood
&

Int.
6 PRATT,

J. H.: A critical platelets. J.A.M.A. 45:

Stratton,

study of the 1999, 1905.

L. M. : The Coagulation of Blood. Methods of Stuidy. p. 34. New York, Grune 1955. VAN EMDEN, J. E. G.: Klinische Untersuchungen umber die Blut-plattchen. I. 1)a.s Zfihlen der Blutpl#{228}ttchen. Fortschr. d. Med.16: 241, 1898. WRIGHT, J. H. AND ItINNICUTT, R.: A new method for counting blood platelets for clinical purposes. J.A.M.A. 56: 1457, 1911.
TOCANTINS,