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tga ple
qacs== 53,432
devert FL EGS
geet fia gs
Bags 5: soe
ie Eu
4235 2 t
5483 ¢ a
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sulficieotly low aperture current app~ar to be of impartance,

#pp .
factors appear to

La tly
eo of
rng hematocrit and/;

For consstent uetenaination of the Litter, the

sulficieatly low aperture curtent appears to be of

mean chan-

selectivn of a
importance,

for

EI
N. FOR P! ING
VOL! ECTRA by J. Mo Mo.
BP. J. Lyne, March 196.

by errors wm Beasur:

»
be Lmted

aed.

aE hdl
de Ban
Lite



ACKNOWLEDGMENTS

The authors are indebted to Dr. James Hagans of the Biomathematical Service
Staff for advice and aid in the analysis of data and to Dr, Wallace Friedberg, Radio-
biology Section, for radiological measurements,


.¥

ane

~

cA nO Ren A I 8

Spectes®

SD.
CY.(5)

Spectes®
x
SD,
CY, ($)

Species*
x

SD, ,

CV. (3)

sD.
cV. (3)

TABLE I

SUMMARY OF RESULTS OF THREE REPLICATIONS
OF MEASUREMENTS ON VARIOUS SPECIES

LA Erythrocyte Counts * 10° per mm’
Mt M2 M3 RI R2 Cl C2 C3 C4

526 456 S2l 477 440 838 406 805 869
0.03 0.09 0.24 0.04 00f 0.17 0.06 9.05 0.05
048 101 451 074 080 204 193 0.62 0.60

1B Moon Channel ( © ) Current = 52
Mal M2 M3 Rl R2
124 138 128 98 al
012 oki (0B
09 4s 09 48 08
1c Moen Channel (G) Current = 7

Cl ca C4 c3 Ra M3
1023 10.08 s«1096 = 1088S 4.03 17.00
uv
0208 | G23t SNL ONSS OSL TAL

f

ad 33 «oo, ue _3 43
1D | —_Hemehocrt (Per Cont) — Dye Method

Ml Ma RL. Gl, C8 C4

a9 so me 23 6 40

13 lot so at 20

29 a2 a4 138 al ar

Range for
All Species

20-110

22.188

M ce Many Roa Rabbit; C a cat; X mn enean, SD, = standard deviotion; CV, = coefficient
variation,

-35-


plasma were.placed in matched quartz erivets
and their optical densities measured in a Béck-
man model DU spectrophotometer at 600 me
(slit width = 0.073 mm.), In the-dye-conten-
trations employed, adherence to Beer's law was
demonstrated. :

The optical density determined In the plasma
standards was plotted so that the 5:1 ml. of
plasma:dye mixture represented 0 ml, of cells
and the 3:1 and 2:1 ml. mixtures 2 and 3 mi.
of cells, respectively, The values for the mean
optical density of the three plasma-dye mix-
tures obtained from the whole blood were then
superimposed on the established calibration
line, us, the total erythrocyte volume con-
tained in 5 ml, of the sample was determined,
The Evans blue hematocrit amounted to 100/5
of the value obtained.

RISA Hematocrit

In one experiment the Evans blue hematocrit
technique was evaluated by mixing portions cf
identical vhole blood <r plasma samples with
the dye and with 1 dilution of radio-lodinated
human serum albumin (RISA).' Sixty millf-
liters of venous blood were withdrawn from a
fasted human subject and mixed immediately
with O.4 ml. of heparin solution, The Evans
blue technique was performed on two 5.0 ml.
portions of this blood, and three portions of 3.0
ml, each were taken at the same time to be
thoroughly mixed with 100 microliters (A) each
of RISA solution (approximately 24 ¢e/ml.).
Plasma samples of 1.0 and 3.0 ml. were also
mixed with 100 A of the RISA solution, After
centrifuging the blood-RISA and blood-dye mix.
tures, the dye concentrations! were measured
as usual and: 500 A postions of the RISA-plasma
samples were counted for two minutes each in

“> @ scintillation counter with two facing thalliums

activated Nal crysta!s (each 4 inches in diam-
eter by 3 inches thick). ;

The calculations for RISA tocrit_ were
based on the relative concenftations of 1.
albumin in the plasma and whole blood samples,

P Abbot Laboretorien, Oak Ridge, Tenmenen |

* Fogente Lahoratory SpeciaRies Comp, Janiaica 35,

© Chictigo Surgical and Electrical Cumpany, Chica
Mints.

+ Comming Glass Works, Coming, New York
°° Fisher Scientific Company, New York 14, New York,

Centrifuge Hematocrit

Small portlons of cach sample were taken
bato-heparin-coated capillary tubes? and centri-
foyed-tor four-minutes on a hematocrit “Elec-
trofuge."** The hematocrits were read directly
from thesw tubes with an International Micro-
Capillary Reader, Model CR,

Recording of Erythroryte Volume Spectra

Precision grade micro pipets* and a Machieit-
type auto pipet (25.0 ml.)** were used to
make all ‘liutions: Fifty \ of the sample were
mixed with 25.0 ml, of a Eltered 0.9% NaCl
solution; 250 of this dilution were then mixed
with another 25.0 ml. of the NaCl solution.
This final dilution of 1:50,000 was used for the
recording of volume spectra and for the cell
counting procedure, Three determinations were
made of cach sample.

The 1:50,000 dilution, in a glass vial of appro-
priate size, was quickly placed under the aper-
ture tube of the counting assembly and a
volume distribution spectrum was obtained: the
plotter mechanim was engaged, the manometer
stopcock was opened, and the “start” and
“record” switches were actuated, Previously,
blood samples from various species of interest
had heen studied to determine the optimal set-
tings for amplification, aperture current, and
plotter scale factor.

The aperture current gain control was set on
90, (Care must be taken that the gain control
setting ts recorded ant that this setting is not
changed during and after calibration!) Because
of shifts in volume spectra which occurred at
about 10 minutes following the second dilution,
recordings were always made within three min-
utes after dilution.

Sack
Erythrocyte Counts

After the volume spectrum had been re-
conted, the same dilution was used to obtain
the erythrocyte count, Examination of the vol-
ume spectrum revealed a suitable counting
threshold, i. ©, a “lower threshokt” setting mid-
way between the lowest channel which is free
from nolse and the first channel which shows

-~2-


_¥

CALIBRATION OF AN ELECTRONIC COUNTER
AND PULSE HEIGHT ANALYZER
FOR PLOTTING ERYTHROCYTE VOLUME SPECTRA

JESS M. McKENZIE, PH. D.
P. R. FOWLER, M. $.
P. J. LYNE, M. T. (ASCP)

The development of electronic instruments
for counting blood cells with unprecedented
accuracy (1) has revived interest in this hema-
tological variable, not only for clinical studies,
but also in other research areas requiring the
most exact measurements, A recent study (2)
has Indicated that electronic instruments of
certain design can be calibrated to yickd cell
tolupe ¢ Gate accurate enough for the detection

sul ifferences In erythrocyte populations,
However, the employment of expensive pulse.
height ig systems and computing equip-
ment (3) for calibrating tho instrument seems
to place such research outside the jeer of the
ordinary laboratory, The recent development
of a research model counter with a companion
pulse height analyzer males available a system
which can be casily calibrated, The principles
employed are’ simple (3, 4), but nu fletailed
descriptions or evaluatigns of calibration tech-
niques have appeared in the lite: This
report offers a technique for: calibration and
considers the limiting factors for obtaining pre.
cise calibration, -o

METHODS

The systom used in these studies was a
Coulter Counter Model B with its companion
Particle Size Distribution Plotter.® This sys-

* Coulter Electronics Co,, Hialeah, Moria.

tens has been described by Brecher, ef al (4).
The same aperture tube was used throughout
the experiment; its opening measured approxl-
mately 100 # In diameter by 75 # In length.
Forty to 50 ml. of venous or heart blood
were withdrawn through 20 gauge needles into
silicontzed syringes and immediately mixed in
a plastic centrifuge tube with 0.2 ml, of sodium
heparin solution, This mixture constituted the
“sample,” and portions of it were used for the
measurment of Evans blue hematocrits, centri-
fugo hematocrits, erythrocyte counts, and for
the recording of erythrocyte volume spectra.

Evans Blue Hematocrit

The method used was essentially that of
Shohl and Hunter (5), Three 5.0 ml, portions
bel na erred drawn a, volumetric

pets (calibes contain” transferred
to plastic centrifuge tubes containing 1.0 ml.
of a solution of Evans blue dye in 0.9% NaCl.
After thorough mixing, each 5.0 ml, transfer
pipet was rinsed in the dye-blood mixture, and
after a second mizing the tubes were centri-
fuged at approximately 2500 x G for 18 minutes
at 20°C, fAnother portion of the sample was
also centrifuged and the plasma was transferred
with calibrated 20, 20, and 5.0 ml. pipets
into 1.0 ml. portions of the dye solution, The
plasma-dye mixtures obtained from whole blood
and those obtained from known quantities of


*

Mean Cozpuscular Volume (MCV):

Het
Ce

(B) MCV = X10, eg.

50
—— * ]0'= "Hee!
3x1 10° = 100 p’/cell

When Het {s the hematos sit i» Vol. % and Ct
is the erythrocyte count per iam’,

Mean Channel (C):
4925 i928
(C) C# FC;U;/ TU;
iT eT
(925
ru;

When /#T Is the product of the channel
number and the units of pen deflection for any
given (I*) channel and the denominator is the
same denominator used in equation (A).

Volume Calibration Factor (a):
uy
t i
‘MCV
D ‘ a —g— = p’/channel
(D) a rom wy

Using the raw date from the plotter (Fig. 1)
(and) the hematocrit and the fe count
a volume calibration factor (a) /is calculated.
This factor may be used to’ express any channel
number in volumetric dimensions,

For cxample, once @ is obtained for given
settings of aperture current and amplifications
It may Be used to obtain the MCV of an un-
known sample by employing the formula

(E) “MCV aaG

When volume spectra are graphically pro-
jected it is advantageous to cunsider the cells
recorded in a given channel as having a volume
corresponding to the channel mid-point (4).

The channel mid-point is derived by subtract-
ing 0.5 from the channel number; e.g. channel
number 1 fs considered to be channel number
0.5, number 2 is then L5, ete.

Hence, the mean corpuscular volume at mid-
channel will be

(F) (a) (7-05) = MGV at mid-channel

RESULTS

Hematocrits — Replication of the Evans blue
method for estimation of hematocrits gave
satisfactory results; the coefficient of variation
ranged from 22% to 128% (Table I-D). The
samples exhibited little hemolysis or lipid tur-
bidity, There was a slight tendency for centri-
fuge hematocrits to be lower then those
obtained with the dye technique, but there was
no evidence of a statistically significant dif-
ference (Table II1-A), In a single experiment
(Man-3) the RISA hematocrit of 47.38 com-
pared favorably with the dye hematocrit of
48.0%,

Erythrocyte Counts are summarized in
Tahle I-A. These results were typical for those
obtained with the electronic counter (1); the
pooled standard error of the measurement was
only + 44,000 counts and the coefficient of
variatich ranged between 0.5 and 453,

_ MCV-Table. III-B presents the mean cor
puscular volumes as calculated from the elec
tronie counts and the centrifuge and dye
hematocrits, No statistically significant dif.
“ference was found.

Volume Spectra and Volume Celibretion
Factors — In Fig. 2 volume spectra are plotted
as per cent of total count vs, channel, Volume
spectra were obtained from triplicate samples
at two aperture currents (1/.707 and 1/5);
thus, the mean channels as expressed in
Table I-B and LC represent the average of

-4-


i
i

|

CALIBRATION OF AN
ELECTRONIC COUNTER
AND
PULSE HEIGHT ANALYZER

CATALOGED By

AVIATION MEOICAL SERVICE
AEROMEDICAL RESEARCH DIVISION
CIVIL ARROMEDICAL RESEARCH INSTITUTE ‘

OKLAHOMA CITY, OKLAHOMA j

MARCH 1963



TABLE Ill — B

COMPARISON OF ESTIMAVES OF MEAN
CORPUSCULAR VOLUME

A. B.
MCY from MCV from
Subject Centrif, Hct. Dye Mect. Diff = 9-A
Dog 15.8 855 +97
M-1 90.3 83.7 -66
M-2 09.3 08.7 ~06
M3 87.1 O15 +44
RA 68.1 70.0 +19
R-2 60.3 69.5 40.2
Cl AL3 45.3 +35
C2 44.0 61.7 +177
c3 473 48 -35
C4 48.7 50.6 +19

a= 429
"(paired control experiment, df # 9) = 1.323; p > 0.10

SEERA,

» CELL VOLUME ww) AT CHANNEL MIDPOINT
Prouna 2 Volume spectrum taken from a s of cat blood (see Fig. Land equatic
on oad unt weet caked

Re 4) cell columee at channel mkt-point and %
Fite the raw data givea tn Fig, 1

a -t=


.*

TABLE I

SUMMARY OF THE THREE ESTIMATES OF a
FROM THE APPROPRIATE THREE REPLICATIONS OF
ERYTHROCYTE COUNT, MEAN CHANNEL, AND DYE-HEMATOCRIT

Range for
All Species
1
CURRENT o707
Spectes® M-L R-1 R-2 M2 M3 _
x 6.70 731 717 1.27. 7.06 _
SD, 0.27 0.49 0.14 0.34 0.37 _
CV. (%) 4.00 6.74 0.20 4.72 5.23 0.2-6.7
. 1
CURRENT = FY)
Spectes® Cl R-2 C2 C4 M3 c3 _
x 457 Sauk 5.99 440 5.27 4.13 _
SD, 0.44 0.06 87 0.6L 0.36 0.06 _

CY. (%) 9.63 aE 14.46 13.92 684 LSS LL. 14S
© OM Many Asx Rabba; Cas cat; X or mean; SD, = tandard Jevistlon; C.V, = covifictont
variation,

TABLE I-A
COMPARISON OF TECHNIQUES FOR
ESTIMATION OF HEMATOCRIT
Si RISA Hick, A B Diff = B-A
weet ; Contrif, Het. Dye Het. t
. i (Afeen of two) (Mean of three)
Dog |, 523 » $0 +07
ML; a8 40 ~35
M2 483 48.0 ~03
iM3 a3, 458 480° +23
RI, f° ~ $95 art | : +09
R2 v DS. ' 306 +0.1
C1 350 380 +30
ca _ 8 25.0 +728
C3 1 380 ' 382 28
C4 423 440 +17
. d= +132

linet mat rns ats ® 1,367; p > 0.10
M = mem, Ro rebbm C 22 cat
“Mean of two

~6-—


_¥

CHANNEL

PVIFSCTOMMNA HHS BT BNDIRD
[a

loo r 7

UNITS

"tT" Cqe 16 CIVISIONS

Piyas 8. Plotter Reed-Out from a sample of rat bloud: Note selection of lower counting,
threshold (17), The mean channel (T) is cakulated from the equation (C)

pegs 4.

@ pen excursion ter than the background
(See Fig. 1). mee

The “upper threshold” setting was completely
locked out and the plotter was disengaged. A
count was then recorded in the usyal manner:
the manometer stopeock: was opentd, the reset
switch was sapped in Wako? dire direction, and
the manometer cock closed, The first three
digits of the count were recorded and corrected
poi wp Auaglik Se eeaicinaticed

counting” procedure was

formed oa each dilution sod the mean of the
three counts was obtained, = /

Calculations
Per Cent of Total Count ,
pa se Rreleet volume spectra on accom:

were plotted as per cent of
Beal cell count (above ‘thesboldy vs, cell

® Coulter Electronics Co, Hiskeah, Florida,

volume at channel mid-point (4). The former
was calculated for cach channel:

(A) uy

% Total Ct. » Sor

iT

When U, is the number of units of pen ex-
cursion for adygiven (1) channel; and

425
ICU;
eT

is the sum of all units in all channels from the
threshold setting (T) through Channel No, 25,
including the i* channel,

=3-


_¥

FOREWORD

Certain studies of blood samples drawn from large numbers of airmen,
are hampered by the lack of rapid and efficient means of obtaining
quantitative cata on red cells, widely used optical techniques are
slow and fraught with opportunities for error, This report presents infor-
mation concerning a new technique for calibrating electronic sizing
instruments, These instruments measure cell volume spectra with a greater

degree of accuracy and speed than conventional techniques,


.*

a

CALIBRATION OF AN ELECTRONIC COUNTER
AND PULSE HEICHT ANALYZER
FOR PLOTTING ERYTHROCYTE VOLUME SPECTRA

JESS M. McKENZIE, PH. D.
P. R. FOWLER, M. S.
P, J. LYNE, M. T. (ASCP)

Hametology Section
Broorwamice Baance

63:8

FEDERAL AVIATION AGENCY
AVIATION MEDICAL SERVICK
* AEROMEDICAL RESEARCH DIVISION
CIVIL AKEROMEDICAL RESEARCH INSTITUTE
OKLAHOMA CITY, OKLAHOMA

MARCH 1963 -


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triplicate measurements, The range of coefli-
cients of variation was greater when the higher
current was used, and in general there was less
reproducibility with the higher current, Con-
sistent with these results, the estimation of the
volume calibration factor (a) was more repro-
ducible at the lower current (Table II),

DISCUSSION

The resulis of this study indicate that eryth-
tes may be counted by: eiectronic. means
with an impressive reproducibility. The eryth-
rocyte counts presented in Table I-A indicate
that populations as small as 4.04 X 10° cells/mm!
and as large as 8,69 X 10° cells/mm’ may be
estimated within a reasonable error of measure-
ment,

One might suspect that the limiting factor
in estimating the volume calibration factor (a)
{s the measurement of hematocrit, Examination
of the cocfficients of variation suggests that the
mean channel determinations at the higher
current may introduce almost as much error
as the hematocrit (Tables 1-C and I-D), This
is consistent with the report of Brecher, et al,
(4) that high aperture currents may alter the
size of mammalian erythrocytes; the coefficients
of variation obtained when a was calculated
from the cell count, dye hematocrit, and C
support this statement,

The characteristics of the size distribution
plotter used in these experiments require that
the pulse heights fali within a certain voltage
range for proper analysis, It is not necessary,

» to use hi; aperture currents in
order to satisfy this criterion! Asa rule, changes
in pulse |height ‘amplification serve to bring
the pulse pattern within the range of the
plotter; too high an amplification (e.g. 1/.4 or
al 5 May result in an excess of
electronic noise (4), A good balance between

tion and aperture gdrrent intensity
is best obtained by trials with the erythrocytes
to be used in a particular experiment.

Ponder (8) stated that tho Evans blue dye
method for hematocrit estimations is, almost

»

ideal in the sense that the dye is neither ad-
sorbed to the erythrocyte surface nor taken
into the cell. Ta one experiment the Evans
blue hematocrit of 48.0% agreed more clovely
with a RISA hematocrit of 47.36 than the esti-
mation obtained by centrifugation (45.8%), The
latter technique, however, yields estimates of
the mean erythrocyte volume which compare
satisfactorily to those obtained by the more
elegant dye technique (Table 11).

The calibration factors obtained in these
experiments have been used for two months in
our laboratories for the plotting of erythrocyte
volumes in rats and we fave ol ed no drift
in the spectra obtained from control animals.

SUMMARY

A simple technique is presented [0, calibrat-
ing an clectronic system used in the plotting
of erythrocyte volume spectra, The calibration
factors, once obtained, apparently remain ap-
plicable for some time, Precise estimates of
calibration factors appear to be limited by
errors in measuring hematocrit and/or mean
channel, For consistent determination of the
latter, the selection of a sufficiently low aper-
ture current appears to be of importance.

REFERENCES

1. Brecher, G, Schaeiderman, M., snd William, G. Z.
Evaluation of Electronic ret blood cell counter, Am. J.
Clin, Path, 26:1499 (1956).

2. Lushbeugs, C C, Bamnan, XN. J, and Clascock, B,
Electronic measurement of celhilar volumes, 11. Fre
doze (1988) ten of ot

3 Lushbaugh, CC, Maddy, BAe ged Basmane, 3
Electronic measerement of cellular volumes. 1.
bration of the spparatus, Blood 20:233 (1942).

Brecher, C., RF
Williams, G. Z, and Schmidt, P. J. Sire
erythrocytes, Aan. N.Y. Aced. Sei, 00:242 (1988).

& Shahi A; Fst Honte. Tutte met of ool
vuhupe Wood u Evens met] Sy
Clin Mek $6:1839 (1941),

& Pooder, K Red blood cell Uinwasions: Measurement.
In Medical O, Classe (Ed), Year Book Pub-
Ushers, Chicago, pp. 120-1214 (1944),