263
ic micr microc ocyt ytic ic anem anemia ias. s. error co nt isdete isdeterm rmin ined ed li in
(%) th
elec electr tron onic ic cell cell coun counte ters rs
ta
io
RDW (%)
o::_:.f_::.M_::_C.::._V-,MeanMCV to
nd Fa or
co ce te
the follow following ing formula formula MC
(g/d (g/dL) L)
Pr au on Te hn aL a n G en e n e a L C om o m m en en t
MCH
io
al ti
at
to
ap ea
--
MCV
Hct Example:
Lan
12_
MCHC
thes thes ci
33.3 33.3 g/c1 g/c1
45
,previous ,previous example MCHC
15 0.45
Re
nc
Va ue
33.3 33.3 g/dL g/dL
fL (ran (range ge 80-9 80-96.1 6.1 fL)
ce to belo belo 32 g/dL g/dL indi indica cate te h y p o c h r o m a s i a .
An (MCH (MCHC) C)
MC fc
(>40 (>40 g/ L) also also coul coul indi indica cate te th pres presen ence ce
mean mean 34.4 34.4 g/dL g/dL (ran (ran
Ce
Di
bu on
lu
g/dL g/dL
OU ta
is
33.4 33.4-3 -35. 5.
(ran (range ge 11.5% 11.5%-1 -14.5 4.5%) %)
OO
entr entr
es
ed
in
at
lo
W id id t I'
.mS()C"It:osi~
te
mult mult
me
tr clini clinica ca
th
ea
labor laborat ator ory. y.
special special circum circumsta stances nces < I S : ll in to ie
ce
es
is
to
chapte ar
CHAPTE
resented in
1'
form
that isconsiste
G en e a l M e h od s U s e Co Bl Ce
NCCLS. This format follows:
,......,.,.....
1.
3. 4. 6. 7. 8. 9. 10
11
in iple an clinical re sons fo th test Specimen collecti an prep ration Reagents supplies an equipmen io Qualit contro Procedur Calculation eporti result (nor al values Procedur notes, includin source of error, clinical applications an limitation of th procedure References
Er ll th following: 1. Diluti th bloo sa pl qu ntitativel usin specia measurin device with appropriate diluents,
flui, enu
fluj
dilute
sample rt th ls te sa pl to th fi al result th nu be of cell
of already-smal sa pl
of
te io individual's bl od
follows: le
so
ti te anne
il
th ti destro cellular elements le tr
de ic in such it
re Intern ti Haematology (ICSH).
Uni
to
count. -to;
ic
Specimens
ti aythat th number ofcell lu
fo counting cell ar designed
tru
ree-flowin capillar lo obtained from skin puncture (see Chapte . " 3 ) or venous bloo preserve it anticoagulan ay be used he nticoagla ic Before usin an bloo sample th technologist
ta
tari
1.
iz ti
in
R e po r e d
good coll ctio techniqu as used ac sa pl ld ly is ll know asfibri clots assoon as it isreceived. Clotted bloo or sample with fibrin clot ar unacceptable fo cell counts ta dard Prec utio st al ay be used he an bloo specimen ishandle 4.
of cell actually counte
(platelets RBCs
BCs)
li lo sl ll reported asthe number of cell pe cubi millimeter
D i L ue n Re
Use
C eL L C ou n ra te
ic
th
lu
isotonicity.
hemocyto-
meter, an accurately rule chambe or device wher cell
er counte
!l
in area of square
millimeter However, on cubi millimeter is essentially eq al to on icroliter. This is'su arized as follows mm
sary characteristic
illi eters,
in
I-lL
Therefore: 1x
I-lL
conductivity.
is
of
diluen fo re
cell co nt
II'lf 265
iasic procedure
PRINCIPLE
me
wi ma
me
me mo
ma
me
fluids). SPECIMEN
DT
wh
wi
me
me R EA GE NT S
.iinut portions ividual' blood.
S UP PL IE S
Un
me AN
E QU IP ME N
Mi
BD wt
bacteria. Caution:
wh
Ne
mo me wi mo Ha un ~ . C o n ve n o n a m ic ro sc op e
olood preserved Th anticoag :ie technologist
Q UA L
C O N TR O
ma
me
! . . ,. "" . . .
. . . , . ". . ~ . . ., , ,
PROCEDURE
ee
pr pe
ac
,,
,1
Mi
samp
me
Us follows:
ceived Clotte unacceptable
ml handled.
b. Remove
mb wi
remove.
wi
wi
me
ws
Wi lost
removal
me
mp ta
nicity.
c.
:J
d. Cover
pecifi ai
me
Do wi
overflow reservoir neck. reservoir,
gravity,
then remove
Ne
wi reservoir overflow
isot ic -3
me
mb mi
me me
mi
wi C on t n u
..
M4
,_
reservoir.
w in g
pa
PTER
--·--··-""--~·--"----"l
C E D U R E 12-3 (Continued)
I---'-----'''---''--'"'"''~""---"---~ ..-""""""·"··"---'-'·"--""--""-,,.~"'-'''---,-,--,-. ma
not:
r ev er s
p os it io n
d ro ps . mo mo
me
No
squeezinq
me Do no
mp mo
me
lOx (low-power)
Oft
7. s.
S.
vo
ha
CALCULATION
N o o f e ry th ro cy te s
squares
Di ut on co ec on
ac or
Vo me
on
me
2.
.T 0 .0 2
)b
J .L )
Example: 50
3.
10 12/L
1 06 /! J L , o r 3 . Example:
35
he ca cu at on R EP OR T N G
R ef er en c
10,000
3.
10 12/L
o rm u a .
R E S UL T
v a lu e Males Females
12
4.2-5.4
/L 10 12/L
2"67
12-3
(Continued)
mp 2. rr
Clinical
REFERENCES
Turgeon
M: C l i n ic o l h e m a t o l og y ,
e d 4 ,P h il ad e lp h ia ,
365850
2005, L ip pi nc ot t
W il li am s
Wilkins.
1998
ng .le, ~r
Whi
C e l C ou n diluting
self-filling pipette availabl in various sizesdependin on th procedur to be performed. Each pipett
us destro th more numerous BC that th WBCs ma be counte more readily. (WBC need no be eliminated when RBCs ar bein counted. The principle of osmotic pressure is again employed, iffere tt il tains ic
capacity Th en opposite th pipett ti isterme the v e o w c ha m b e (Fig ). ie th ip te ti rote ts th ip tt isal to tu th ia th rvoir before use. The reservoir
In th ln
of
~s
th ds fo le kocyte ounts, th
see. \V en th acetic acid he olvzes th cell into acid hemati solution br olor gl bi
rese
in th
Cs it
whic gi es th re ulting he inte sity th rown
rn
ia th
re it
lu
il
ri
of plastic, th
ra th ip tt shield before actu
tain diaj)lnagm, tu
th
se
BCs.
Wh te se
Code labele wit capacity
th rythro te is ls id routin laborator examination Before the advent auto atic cell-c unti evices th re ount rt ll li in te ro ti laboratory test becaus of th larg erro (±20 in th nual thod ountin BCs.
I,
Self-fillin
pipe
ll la ra ie refu ly this is an ccurat asurement; Pr cedure 12-4 describe manual whit counts usin th Unopette Shield disengage
in certai
Reservoi
circumstance an ar th usua method
cerebrospina flui or synovial fluid, whic do no lend themselves to electroni cell counts Th basi principles ofelectroni cell counts ar describe in hapter 0.
13 pipett
an
diluen
reservoir.
with diluen
PTER
PRINCIPLE
me
me
a te le t
me
mo
s pe c a l c i c um s a nc es .
SPECIMEN
O TA a mp AG NT
UP
AN
me
Q U M EN T
mm
me
wa Caution:
no
ma
c le a mo
Ha Co
me mo
mi
QUALITY
CONTROL
1---------- ..·'"·-···--· PROCEDURE
·--
.- .-
~.
.-
------·-----·---i
1.
follows:
mo
follows: me Wi
o n r es er vo ir .
PRINCIPLES AN
C E D U R E
12-4
to....
PRACTICE OF CLINICAL HEMATOLOGY
269
(Continued) m e to
e tu r
m ix tu r
e se rv o
5. L o a
h e m ac y to m et er . e su sp en d c e not:
of or
mo
sed.
voir l,
me
No
Do no
10
lOx (low-power)
6.
t-rnm"
is
drops.
oi O UN T
bi ha AN
Using 100x m ag n
o n a in e
A L U LA T
at
areas.
KO YT
wi
br ht gh in
m ic r
py
k oc y
a pp ea r
ct e.
specimen.
Calculation dd 10%
100 to
Example:
90 (90
CO
NG
8)
100
9.8
l09jL
N D CALCULATING PLATELETS 43
in
or 44>< m a n i
on
us
gh
ht
or
ha e- nt
m ic r c op y
25
mi Using 43 or 44x
ma me ma
mi
mi
Calculation Mu re
1000
Example:
300
jL
12
Note:
II
C on t n u
w in g
pa
PTER
U R E
12-4
(Continued)
r-·-·~---------·-"-'-"-·-·-----------'-·-----·------·---'--------------.----., R EP OR T N G
R E U LT S
10 jL
n f r n mo
me ws Co ec ed WBC
A_ve_r_a=-ge_to_t_a_l_le_u_k_o_:cy~t_e_c_o;_u_n_t_x_1 _O 10
a te le t
e fe re nc e
WB 1012jL
v a u es :
R OC ED U
mi
ma my
.2
Ot mb
Clinical
Applications
al
ba
nf ct mb
ma
ma
REFERENCES
C l in i c a l h e m a t o io g y t
L - . . . . ~ _. _~ ~~ ~
Th
. . . .. ._ ~
i
t
N
~ ~ = ~ _ ~ _ ~ ~...... ._
,~ ~ ~ _ ,
••
Unopette system is
Li K ~ ' < - o ' ~ ~ _ . _ _ . _ .~. ~~ ~
self-filling disposable
manual cell counts must be performed: manual cell ts or eo in il pl tele ts hr bocytopeni patients an leukocyt counts in neutropenic patients."
Wi
diluting pipettes Th hemocytometer
id
M B
le
nc
tr te
an in luti
lo th
sa
hemocytom-
term counting chambe
and
with Neubauer ruling
eryth-
Ma Wh L eu ko c e ) C ou n ro
_!
' - _
called hemocytometer. Technically eter consists of
is
ft
Wilkins.
~ .~ ~
hemocytomete reticulocyte counts eosinophil counts an rocyt fragility tests.
ms
flat ur ac rd tu
to
al
ts
ta
it ea
at
0.
side
in de h.
an ll in
mine by couritin th number of cell in specia counting chamber. Th counting chambe is ofte
(F
re
14
sp
tw
to
111'jl 271
0.1-mm dept fo counting chambe
I'
Cove glas
!'
~I I'
(9 square millimeters)
of
,!
ocvtom-
special ~ber and 1,2
ounting Hausser
(80/400 s q
ich side. mm
epth. .d from .verglass
supports ittom of ounting
hamber
cytometers used in th hematology laboratory must an
e.
OiI-mm slice ar
cham Each counting chambe ha tw precision-rule
of Standards
in
le
ea
o ve r s li p
Z- 16
( Fr o
bI,
R o a k B F H em a t ol og y
c li n c a l p r n ci pl e
a n d a p p li ca ti on s
ed 2, Philadelphia
2002 Saunders.)
Cc into
16
25 equa
12-16). The with dilut:
In
portions
16
ro
10
l/
0.02: and
1/16-1n111,
l4
of
'.calcu
l/2s-mm, l/eo-mm, an r e 0.1 nu in depth.
tion
Tl M I C EL L M I
M O
W H
C OU NT S
M P
H EM O C YT OM E TE R
5).
is
soluti For tl are 0.1 rn is 0.4
unitvolume To the
be considered:
Lrnm square
W H C EL L
2.
C OU NT S
11\
by l/C
1. (Figur
12-17)
FO
four
/4
12-4), formula:
forrnu
273
on
corner
40Counted No counte
I-rnm/ areas nt ut of Area counte ep of hamb
blood
Volume Whit
rnrrr')
\X/her volume /L
with
the .Unopette 475
area
cellsjul
depth.
ou 140
ul,
20
7000/VL, or
White tion factor .T
liter:
po
hi
vo
is 0. rnrrr', mm
0. fis ou
ho tain d,
ft
ou whic
dupl cate de rm na ions
ul
1/
counts of seriou clinical importance).
ur
32
40
35
140. /[lL,
0/ formul
is
qu to
APTE
th
ea
el
ca
ts
ed to ea
calculated as 8050
ould be ep rted as 8100/I-l
be reported as 8000/I-lLor 8.
10
Th
genera te
cant figure isused.
100
100
P re ca u o n Ma
nd Te hn Ce Co
th
ele die
ec
bO
te correcte
natu
to fol.
cl te
Fa or ll ma
hq
count.
v:
sample faulty laboratory equipment,
io
co ti
wh 10'
leu
Cl ni
cham
Si ni
an
E D B LO O
CE
of Ce
C ou n
var
C OU N
ll
Anemia
ec
An val' hig Th stre
au rapidl
to preven
ec PIa
coagulation.
lati wit: mel
of erythrocytes is increased. covergla
immediatel
be or each us
fl odin
WH
BL
CEll
the:
COUNTS
dfff:
.4
of
11.3
lt
10
An increase in th WB (leukocyte upperTimit ed
st Le en
ce en
er
anemia
anti
pe ti
.g.,
ti in an carcinomas an in leukemia ia is te ed liferation ofone or more of th variou hematopoieti
ia
If 10 10
ca
at
ma er
ti lc
ti
circulatin
il
cell of th
blood.
lood film prepared
ex erienced clinical laboratory scientist. Ther ar tw la ic io a, lymphocytic and myelocytic,
ma
ar ma the a c u t e co dition th
es
hay to lu i( disc nan oth. of
in pe niciou
in
ai 30
it
ch iq drugs, an
trib
en
plat
lo
ll
chronic no as marked
diseas
progresses apidly
Car: but tory gem
bee: pun cho: tene
Wid be svste oxal The cerU
IN PLES AN chamber. lUcleated
10
10
10 to 30 10
TI
LI
EM TOLO
.. ..
275
he la elets. Preven coagulation. 3. Preven hemolysis.
4. to
3C coun
It
decrease
10). Question-
cussion).
.d Anemias
P L a e le t C o un t
marrow anemia. ~number
hemostatic,
Manual Platelet Counts Usin U no pe t S ys te m 12-4),
'J
20
4. to ukocyte) termed ra ower
containing
ral infec:e radiace ta
I-g/dl, am on
lO specimen fo side of wo hernocytometers.
anticoagulant. fecsevere egnancy,
1.98
.t
Specimens same foca
plane.
illed proitopoietic
C o u n n g P La te L e
es prepared xamined
power (lOx)
.st
nphocytic iant type divide rapidly,
Me
ds
se
C ou n
La eL
I-g/dl, ammonium
H em o cy to m et e
l,
of
oxalate.
APTE
le
io
le
nu erou othe cell stil presen (e.g.,
th
scopically to chec le
small round, refractile bodies
sa (1 rnrrr') on th
he acytometer
anua
ns
must be calculated Th
acce t-
importan factor follow tele in
mrn/.
2. system), 3. Th volu ofdiluted bloo counted, hich iseq al to th epth ofth chambe (0.1 time th area in hich th cell re counte (1 mm+), or 0. Il-L Th following genera formul applies Unopette
squares
mm
in
100
0.1 mm tele In this case
Pr au on
/L
'constan fa to
an
draw
Te hn
counts Th
finger
us
into th pipette.
ni
nc
Pl
C ou n
Th normal
C ou n
Averag number of platelet
il be sed, it isnecessar to
th re aini
Cl ni
o r M an u
ti
platelet
neares 1000/1l-L.
Ca ul
each platelet count. in
fi ge pu ct re
perfor in agin th replicates if they 'arewithin th
Cs).
viewed micro-
th
umbe ofplatelets, epending in part lo io 10 /L whol blood. lo ss ia it le in lo bleedi ti e. coun higher than normal ay be associated with tendency toward thrombosis or lo platelet ount ca result
Thrombocytopenia, infe ti in te some anemia (aplasti an pernicious), an when th patien is undergoing radiatio treatmen chemotherapy, Thrombocytosis, or an increase in platelet's can be foun in rheumati fever, in asphyxiation fter surgical treatm nt afte splenectomy, with acut bloo loss an it so type of chemotherapy used in th tr at en of leukemia
Automated Celt-Counting
Fa or
Methods
ti ib ly co ntin erythrocytes an leukoc te also appl la ts In late is tive that peripheral bl od be freely flowin he
hematologi para eters. Th most useful instru me ts ar th se fo counting cells. Auto ated cell
an debris Rapi dilution of th bloo
throug
ma
clot If
repeated tech iq
isessential
th instrument's aperture in
fe
seconds.
la
Clumping
ay result from inadequate it th lu in obtainin th bloo sa ple.
co nts, duplicates ar al ay prepared an
du li
counted.
utomated counting method have been
usin focuse lase beam an
impedanc
in
agre within 10 to be acceptable onstan focusi of th icroscop is eces saryto identify th platelet amon th larger more
lt ls in in th s, used so extensively, it is referred to as th principle.
method
Coulte
