Eight-parameter Automated Hematology Analyzers:
Comparison of Two Flow Cytometric Systems
BENJAMIN DREWINKO, M.D., PH.D., PAMELA BOLLINGER, MT(ASCP), MARY ROUNTREE, MT(ASCP),
DENNIS JOHNSTON, PH.D., GAIL CORRIGAN, B. S., WILLARD T. DALTON, M.D., AND JOSE M. TRUJILLO, M.D.
The performance of two high-speed 8-parameter automated
hematology analyzers (Coulter Counter S-plus and ELT-8)
were compared with that of reference instruments (Coulter
Counter S Sr and Technicon Autocounter). The precision, linearity, and lack of carry-over of both instruments were superior
over that of existing equipment. The especially noteworthy
feature of the instruments was their excellent performance in
the range of extreme values of both white blood cells and platelets. This enhanced performance, and the fact that all presently
known relevant hematologic parameters can be measured by
a single instrument on a single sample, make the contribution
of these two flow cytometric-based instruments a significant
advance in the field of automated laboratory medicine. (Key
words: Automated analyzer, cell counts; Platelet count; Hematology; Flow cytometry) Am J Clin Pathol 1982; 78:
738-747
DEVELOPMENT OF INSTRUMENTS for highspeed analysis of cells in a fluid suspension (flow cytometry) has revolutionized the operation of the modern
hematology laboratory. 3,5,81012 Improvements in design
have yielded instruments that provide greater resolution,
superior precision, enhanced accuracy, and a faster analysis rate than currently available automated hematology
analyzers. 8,10 "' 14 Additionally, such instruments have
the potential to provide increased information on hitherto unsuspected cellular parameters' 14 that may prove
of vital and relevant clinical importance in the evaluation of the disease state. 1 , 3 " 1 2
This report provides a side-by-side evaluation of two
currently available 8-parameter automated hematology
analyzers that employ flow cytometric technology: the
Coulter Counter® Model S-Plus, manufactured by Coulter Electronics, Inc. (Hialeah, FL), and the ELT-8,®
manufactured by Ortho Instruments (Westwood, MA).
Evaluation was conducted in a large oncologic hematology laboratory7 that provided ample opportunity to
analyze the performance of such instruments under the
usual conditions of either severe myelosuppression or
of marked increments of circulating white blood cells
and platelets.
Received January 27, 1982; received revised manuscript and accepted for publication March 23, 1982.
Address reprint requests to Dr. Drewinko: The University of Texas
System Cancer Center, M. D. Anderson Hospital and Tumor Institute,
Texas Medical Center, Houston, Texas 77030.
The University of Texas System Cancer Center, M. D.
Anderson Hospital and Tumor Institute, Departments of
Laboratory Medicine and Biomathematics, Houston, Texas
Materials and Methods
Description of Instrumentation
S-Plus. The Coulter® Model S-Plus (Coulter Electronics, Hialeah, FL) is an automated eight-parameter
hematology analyzer that utilizes either 1 mL of whole
blood or 44.7 ^L of capillary blood (diluted in 10 mL
of isotonic diluent) to determine in 35-55 seconds the
routine seven-parameter hematology count (white blood
cell count, WBC; red blood cell count, RBC; hemoglobin
concentration, HGB; hematocrit, HCT; mean corpuscular volume, MCV; mean corpuscular hemoglobin,
MCH; and mean corpuscular hemoglobin concentration, MCHC), and also an eighth parameter, the platelet
count (PLT). The instrument is also capable of analyzing
four additional parameters: the RBC volume distribution width; the platelet-crit; the mean platelet volume;
and the platelet volume distribution width. Whole blood
and predilute samples are introduced into the instrument through separate aspiration pathways but analyzed
in the same sensing chambers.
The basic Coulter principle of nonoptical one-by-one
electronic impedance counting and sizing of particles is
utilized to enumerate WBC, RBC, and PLT; the MCV
is determined directly from the electrical pulse heights.
The hemoglobin concentration is measured by the colorimetric cyanmethemoglobin method. HCT, MCH,
and MCHC are not measured directly, but are computed
internally from the measured parameters. Different
types of cells are discriminated by volumes in two separate chambers (WBC and RBC/PLT) each containing
three aperture tubes. Pulses amplitudes generated from
cells passing through the apertures are directly proportional to particle volumes and are calibrated to measure
cell volumes from 0 to 360 fL. Under these conditions,
a leukocyte is any particle with a volume greater than
45 fL that remains in the suspension after the RBCs
have been lysed. All cells in the RBC/PLT bath with a
0002-9173/82/1100/0738 $01.30 © American Society of Clinical Pathologists
738
Vol. 78 • No. 5
EVALUATION OF AUTOMATED HEMATOLOGY ANALYZERS
volume in the range of 36 to 360 fL are classified as
RBCs, while those in the 2 to 20 fL range are classified
as PLTs. The raw data of the PLT count from each
aperture tube is distributed according to volume by differential gating in a channelyzer. It is then analyzed and
smoothed by a log-normal distribution algorithm to
cover the PLT range of 0 to 70 fL. If the PLT count is
decreased, the instrument wilj continue counting for
four additional count cycles until at least 400 particles
have been detected and evaluated. If there still is an
insufficient number of particles, or if there are statistically discrepant results among the distributions generated by each aperture tube, the result from the raw data
is printed along with a code describing the anomaly. If
the distribution is not log normally distributed, the result
is suppressed and the operator is alerted by a visual signal. For all particles (WBC, RBC and PLT), Poisson
coincidence errors in counting are corrected internally
by appropriate algorithms.
ELT-8. The ELT-8® (Ortho Instruments, Westwood,
MA) is an automated hematology analyzer capable of
measuring eight hematology parameters (WBC, RBC,
HGB, HCT, MCV, MCH, MCHC, PLT) on a 100-microliter sample of whole blood in 60 seconds. The principle utilized by this instrument is that of optical detection of cells in a liquid suspension that scatter light at
a low forward angle. The intensity of the scattered light
is detected by a photodetector for conversion to electrical pulses: The technology involved is hydrodynamic
focusing of a narrow stream of cells through a flow channel 250 j*m in diameter. Focusing is achieved by a liquid
laminar sheath flow that is coaxial to the sample flow.
This method isolates the cell suspension, confining the
particles to the central portion of a liquid jet. A heliumneon laser beam is focused upon this stream of cells
forming a minute, cylindrical sensing zone (20 microns
in diameter and 7 microns in height) in the center of
the sample stream, allowing for an almost single-file array of cells for sensing and detection. WBC are enumerated following lysis of the RBCs. Discrimination
between PLT and RBC depends upon three cell variables: volume, refractive index, and "time of flight"
through the sensing zone. Each cell generates a pulse
whose height and width is proportional to all three discriminating parameters of that cell. A voltage value is
assigned to that pulse and compared to an "integral"
voltage. A value greater than the integral voltage is classified as an RBC, and a lower value is classified as a
PLT: HGB concentration is measured by the colorimetric cyanmethemoglobin method. The HCT is determined from the area under the curve generated by the
RBC pulses and the MCV, MCH, and MCHC are computed internally from the measured parameters. The
operator is alerted to certain sample abnormalities or
739
instrument malfunctions by means of varied audible
alarms or displayed sensor messages.
Reagents and Controls
S-Plus. The reagents used on the S-Plus were those
recommended by the manufacturer. These included Isoton® II (Coulter Diagnostics, Hialeah, FL) a balanced
electrolyte solution; Lyse S® II, an agent that lyses red
blood cells, removes the cytoplasm from the white blood
cells, and is the reactive agent for the HGB concentration determination; and Isoterge® II, a cleaning agent.
Strict precautions were taken not to introduce bubbles
into the Isoton II in order to prevent high background
counts. Reagent consumption per test cycle was 55 mL
of Isoton II, and 0.77 mL of Lyse S II. The primary
control used during the evaluation was 4C® Plus normal
(Coulter Electronics, Hialeah, FL), an eight-parameter
control that included fixed human PLTs. Secondary
controls for the standard seven parameters consisted of
4C® normal, abnormal low, and abnormal high (Coulter
Electronics, Hialeah, FL), and Quanticel™ HP-Human
Platelet Reference-normal and low (BHP, Inc., Westchester, PA) for the PLT parameter.
ELT-8. The only reagents used during the evaluation
were those currently approved for operation of the system and are manufactured by Ortho Instruments (Westwood, MA). These included Salac™, a buffered saline
diluent; Lysac™, a red blood cell lysing agent and diluent for WBC; and Cyanac™, a diluent and reactive
agent for determination of hemoglobin concentration.
We found it advantageous to prefill extra reagent containers with Salac to let particulate matter settle out
before installing on the instrument in order to avoid
high background counts. It is also important to record
the date on the Cyanac reagent container, as the reconstituted stability is 30 days. Reagent consumption per
test cycle was 1.6 mL of Cyanac, 0.8 mL of Lysac, and
16.0 mL of Salac. Late during our evaluation, the Salac
diluent was replaced with Isolac™ diluent (see Retrofits). The primary controls were CBC-trolR normal
(Pfizer Diagnostics, New York, NY) for the seven routine hematology parameters, and Quanticel HP-Human
Platelet Reference-normal for the PLT parameter. Secondary seven-parameter controls were Haem-CR-normal and abnormal low (J. T. Baker Diagnostics, Bethlehem, PA) and Quanticel -HP-Human Platelet Reference-low for the PLT parameter.
Calibration
Both the S-Plus and the ELT-8 were initially calibrated using blood collected in tripotassium EDTA (K3
EDTA) obtained from five hematologically normal donors. Determinations were performed on each whole
740
DREWINK0£7"/1L.
blood sample utilizing the following reference methods
for each parameter: (1) WBC and RBC: five replicate
dilutions measured on a Coulter Counter® Model ZBI
(Coulter Electronics, Hialeah, FL); (2) HGB: five replicate dilutions measured on a Gilford spectrophotometer Model 300-N; (3) HCT: five replicate aliquots centrifuged at 10,500 rpm for four minutes and read on a
Damon/IEC Micro-Capillary Reader without correction
for trapped plasma; and (4) PLT: eight phase microscopy
readings of two Unopette® #5855 (Becton-Dickinson,
Rutherford, NJ) dilutions (1:100 with 1% ammonium
oxalate) with both sides of each chamber read by two
technologists. Also at this time, all other automated hematology analyzers available in our clinical hematology
laboratory were recalibrated, if necessary, to these reference values. These instruments consisted of three
Coulter Counter® Model S Srs, and four AutoCounters™ (Technicon Instruments, Tarrytown, NY).
For the purpose of the evaluation, the instruments in
the clinical laboratory were used as reference.
Quality, Control
Before each daily operation, quality control measures
were performed for each instrument according to the
manufacturer's recommendation (for the test instruments) or to our established laboratory procedures (for
the reference instruments). The instruments were considered to be in control when background counts were
below recommended levels, and when values for the
control materials fell within two standard deviations
(SD) of the mean value established in our laboratory for
15 replicate assays over three consecutive days (for the
S-Sr, S-Plus and AutoCounter), and 10 replicate assays
processed immediately after calibration (for the ELT-8).
S-Plus. Checks on instrument function and electronic
components were performed daily prior to routine operation and background counts were done. The primary
and secondary controls were then processed in duplicate.
ELT-8. Quality control measures were performed
daily on the ELT-8 in accordance with the manufacturer's recommendations. These included a check on the
background count and the processing of replicate samples of the primary controls (or secondary calibrator
materials) and the secondary control products.
AutoCounter. The background was zeroed before each
run. Platelet Reference N® (Technicon Instruments,
Tarrytown, NY), a suspension of fixed human platelets
at a normal level, was assayed as a standard at the beginning of each run and after every ten patient samples.
Later in the study, this product was replaced with Quanticel HP-Human Platelet Reference-normal and low
levels.
S-Sr. Daily electronic and instrument function checks
A.J.C.P. • November 1982
were performed on all three S-Srs according to our established laboratory protocol. Background counts were
performed prior to the processing of the control materials. 4C normal was used as a primary control, and
Haem-C normal and abnormal low were used as secondary controls.
Precision
Within-run precision was evaluated for the directly
measured parameters, WBC, RBC, HGB, and PLT (and
HCT for the ELT-8) at three levels: decreased, (below
normal range), normal, and increased (above normal
range). On the S-Sr and S-Plus, MCV precision was evaluated at the normal level only, due to the difficulty of
obtaining adequate samples at abnormal levels. The SPlus, S-Sr, and AutoCounter were evaluated simultaneously in both the whole blood and predilute mode
after making the appropriate dilutions from the same
sample of whole blood. Large amounts of blood were
required for each study and were obtained from normal
donors free of any known hematologic disorders. The
exception to this were the leukapheresis blood specimens
used for the concentrated WBC studies obtained from
two patients who had chronic, myelogenous leukemia.
To obtain the values desired in the abnormal ranges,
it was necessary to artificially dilute or concentrate the
donor blood by separating it into its various components
via several centrifugation steps, and then combining the
proper volumes of packed RBCs, platelet rich plasma
(PRP), and platelet poor plasma (PPP). Normal ranges
were evaluated using the donor whole blood sample
without additional manipulations. After preparing the
blood specimens, 26 consecutive counts were performed
on all instruments, and the first count was discarded for
statistical analysis.
Day-to-day precision of the S-Plus, S-Sr, and
AutoCounter was evaluated for the directly measured
parameters by calculating the coefficient of variation
(CV) of the values obtained over a 30-day period using
Counter Check® Plus (Diagnostic Technology, Inc.
Great Neck, NY) for the S-Plus, 4C normal for the SSr, and Quanticel-HP normal for the AutoCounter. For
the ELT-8, day-to-day precision was evaluated by calculating the CV of values obtained on CBC-trol normal
and Quanticel-HP normal over a 20-day period. There
were 58 separate values used for the WBC, RBC, HGB,
HCT, and MCV parameters, and 48 were included in
the calculations of the PLT parameter.
Linearity
Linearity was evaluated on eleven successive percentile dilutions (5 to 100%) of a given value of each pa-
Vol. 78 • No. 5
EVALUATION OF AUTOMATED HEMATOLOGY ANALYZERS
rameter at decreased, normal, and elevated levels in the
whole blood mode (and in the predilute mode for the
S-Sr, S-Plus, and AutoCounter)! Blood for each study
was obtained from a single donor, separated into its
components through several centrifugation steps, and
combined in specific volumes to yield the appropriate
dilutions. The only exception was the linearity study at
an elevated WBC level, which was performed using a
WBC concentrate from a patient with chronic myelogenous leukemia diluted with Isoton II (for the S-Sr, SPlus, and AutoCounter), and from a patient with lymphoma diluted with saline (for the ELT-8). Each dilution
was processed in quadruplicate for each parameter, and
the average value was plotted against the expected value
from the dilution percentile.
Carryover
Two separate whole blood samples were used in the
carry-over studies; one with high parameter values and
a second with low parameter values. These samples were
prepared utilizing normal donor components and the
original samples were either concentrated or diluted to
obtain the desired values. Predilute samples were prepared from the high and low specimens for the S-Sr, SPlus, and AutoCounter. A mean value for each counted
parameter was obtained for both specimens by running
ten replicates. The high and low value specimens were
then run interspersed ten times, and these mean values
were calculated. Per cent carry-over for each parameter
was calculated from this formula:
where: LA = x of alternated low value sample; Ls = x of
straight replicates of low value sample; H s = x of straight
replicates of high value sample; and HA = x of alternated
high value sample.
RBC Interference on PLT Enumeration
Because the AutoCounter, S-Plus, and ELT-8 analyze
platelets in the presence of intact or lysed RBCs, it was
necessary to evaluate the effect of the RBC concentration on the PLT count. Assays at normal and low platelet
levels were performed utilizing blood obtained from a
single hematologically normal donor and separated into
its various components by several centrifugation and
filtration steps: platelet poor RBCs (4 X 103 platelets/ML), PRP, and PPP. Platelet free plasma (PFP) was
produced by passing the PPP through a 0.2-fim filter.
Eleven tubes were prepared for each trial by varying the
amount of RBCs and PFP and adding the same amount
of PRP to each tube. The tubes were weighed before and
741
after the addition of each component in order to verify
the accuracy of pipetting. Each sample dilution was
counted in triplicate; the least-squares fit method was
used to plot the platelet count versus the RBC concentration.
WBC Interference on HGB Determination
To determine the effect of an increased WBC count
on the colorimetric determination of HGB, and WBC
concentrate was obtained during a therapeutic leukapheresis of a patient with acute lymphocytic leukemia. The
concentrate was centrifuged and only the uppermost
layer of leukocytes was aspirated to prevent contamination with RBCs. Type specific PPP and washed RBCs
obtained from the Blood Bank were used to prepare
eleven successive dilutions in which the amount of RBCs
was constant and the amount of WBCs and PPP varied.
Each dilution was anajyzed in quadruplicate on the SSr, S-Plus, ELT-8, and also on a Coulter® Hemoglobinometer (Coulter Electronics, Hialeah, FL) after
centrifugation to remove the leukocytes. The hemoglor
bin concentration versus the WBC count was plotted
using the least-squares fit method.
Retrofits
A few inherent problems became apparent during the
evaluation of both instruments. For the S-Plus, the RBC
concentration artificially decreased the displayed PLT
count by an average of 5.2% for every additional 106
RBCs//iL.6 In addition, the PLT counts obtained in the
predilute mode were, on the average, 15% lower than
those of the whole blood mode. In the case of the ELT8, a continuous downward trend was noticed for the
MCV, HCT, and PLT parameters on the control materials. Neither the degree nor the occurrence of the
downward trend could be predicted for a routine eighthour work period, let alone after overnight shut-down
periods. Patient correlations were run whenever it appeared that a trend had occurred, and reference determinations were performed on all discrepancies. Results
confirmed that the calibration of the ELT-8 was continuously drifting downward.
Both manufacturers were alerted to take remedial
action. For the RBC interference on the S-Plus, improvements consisted of software changes with the installation of a program revision to compensate electronically for the decrease in PLT count as the RBC
concentration increased from 0 to 6 X 106/ML. TO correct for the discrepancy between PLT values determined
by both aspiration modes, the changes included a new
blood sampling valve (BSV), shortening of the bloodcarrying lines from the BSV to the RBC/PLT chamber,
742
Table I. Within-run Precision at Varying Levels
on 25 Replicate Counts
Reference
Instruments
Parameter
WBC
Level
HGB
HCT
PLT
Predilute
Mode
Whole
Blood
Mode
Predilute
Mode
ELT-8
11.1*
9.2
4.9
2.7
1.8
1.2
1.2
1.6
12.1
J6.7
5.7
ND
3.0
2.7
2.5
2.6
6.3
6.2
3.6
1.9
1.2
1.1
1.8
5.8
5.6
6.8
1.3
2.0
2.1
2.7
2.2
5.3
NDf
3.2
2.0
2.3
1.1
0.9
1.5
1.2
0.9
0.5
'•4
1.8
1.0
1.3
0.7
0.6
0.8
0.7
0.6
1.2
1.5
0.7
1.3
0.8
0.7
0.7
0.8
0.8
0.7
0.6
0.6
0.4
1.8
ND
1.1
1.0
0.9'
0.7
0.4
0.4
0.5
0.5
1.6
2.2
0.8
0.7
0.9
1.0
2.1
1.4
0.4
0.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.8
1.0
0.6
After retrofitting, correlations among results from all
instruments were performed on random routine patient
specimens processed by the Section of Hematology over
an 18-month period. The S-Plus was compared with the
S-Sr and the AutoCounter for 429 specimens in the
whole blood mode, and for 205 specimens in the predilute mode. The ELT-8 was compared with the S-Plus,
the S-Sr, and the AutoCounter for 373 samples. Results
from each instrument, considered on a mode pair basis,
were plotted as linear regression curves and correlation
coefficients were calculated by the Pearson Method. For
detailed analysis of the WBC and PLT counts, values
were plotted separately at low, normal, and ejevated
levels.
A correlation analysis between results of specimens
obtained from capillary blood (fingerstick) and those
from venipuncture were conducted for 25 normal donors for the S-Plus and ELT-8, and also for 25 patient
donors for the S-Plus only. Both venous and capillary
samples were obtained from each donor, and predilute
samples were prepared from the venous specimens for
the S-Plus. Duplicate fingerstick specimens were collected utilizing the Hemette II® (Ortho Instruments,
0.9
0.9
0.6
0.6
0.5
0.4
0.8
0.5
ND
ND
Table 2. Day-to-day Precision at Normal Levels
7.9
7.4
2.6
3.0
1.4
1.9
3.2
2.5
11.9
7.8
5.2
3.3
2.2
2.4
2.2
2.4
7.4
5.0
3.0
2.0
3.1
2.2
2.2
2.1
8.4
6.6
3.4
3.3
2.2
2.7
2.4
1.8
9.0
ND
2.4
3.0
2.2
1.7
ND
1.5
i.3
(X106/ML)
(g/dL)
6.3
9.2
13.0
14.8
18.9
(%)
26.9
34.6
43.1
MCV
Whole
Blood
Mode
(XIO /ML)
1.98
3.04
4.50
6.10
(fL)
87
94
and carry-over were evaluated again, and the values
compared to those obtained before corrections.
Correlations on Clinical Specimens
S-Plus
3
0.9
1.2
1.8
5.0
8.0
11.0
25.0
43.0
RBC
A.J.C.P. • November 1982
DREWINK.O£7"/lL.
3
(X10 A*L)
12
23
43
69
112
326
401
562
Parameters
and an adjustment to the RBC diluent dispenser. These
improvements brought the predilute PLT count to
within 5% or less of the PLT count determined in the
whole blood mode. For the ELT-8, the retrofit consisted
of certain software changes in the Data Handler module
as well as physical product improvements. The physical
changes included a new reagent plumbing system, a
smaller blocker bar, a V-2 pressure relief valve, and replacement of the photomultiplier tube with a solid state
scatter detector. In addition, a new saline diluent, Isolac,
that contained a surfactant expressly designed for optical
systems replaced the original Salac. Upon retrofitting of
both instruments, the calibration, precision, linearity,
S-Plus
ELT-8
8.9
0.2
2.7
6.9
0.2
2.4
7.2
0.2
2.6
5.00
0.06
1.2
4.63
0.04
0.9
4.52
0.03
0.7
WBC
Mean ( 1 0 3 / M U
SD
CV
RBC
Mean (10 6 /ML)
* Coefficient of variation (%).
t ND = not done.
Reference
Instrument
SD
CV
HGB
Mean (g/dL)
SD
CV
15.0
0.2
1.1
13.1
0.1
0.9
13.7
0.1
0.9
HCT
Mean (%)
SD
CV
41.7
0.7
1.7
40.8
0.4
1.0
42.2
0.4
1.0
1.3
0.8
0.9
93
0.8
0.9
145
15.0
4.4
265
5.9
2.2
355
7.3
2.1
MCV
Mean (fl)
SD
CV
PLT
Mean(10 3 /ML)
SD
CV
83
EVALUATION OF AUTOMATED HEMATOLOGY ANALYZERS
Vol. 78 • No. 5
743
WBC
100 r
20
40
60
80
0
2
4
6
8
10
3
Expected Value ( X 1 0 / M L )
FIG. 1. Relation between expected and observed values of WBC (at high and low ranges) measured by reference and test instruments.
Westwood, MA) for use on the ELT-8 and the Unopette® #5925 (Becton-Dickinson, Rutherford, NJ) for
use on the S-Plus. All specimens were analyzed in du-
2
4
6
8
6
Expected Value (x10 //xL)
plicate within 4 hours. The capillary specimens collected
for the ELT-8 were run in duplicate again after 24 hours.
The mean values for venous, capillary, and predilute
4
8
12
16
Expected Value (g/dL)
FIG. 2. Relation between expected and observed values of RBC and HGB measured by reference and test instruments.
DREWINKO£r/lL.
744
A.J.C.P. • November 1982
PLT
High Range
1000 -
o 800
•
160
Low Range
• S-Plua
o ELT-8
• AutoCounter
/ *
ji
y
t£
*_
co
o
- 120
/*
^
jio
M
So
~ 600
«
3
o
Jf
80
«
>
•o 4 0 0
o
>
e
/
|
>
•D
>
Q>
- 40
O 200
1
1
200
400
1
1
600
800
0
Expected Value
1
1
1
40
80
120
(0
a
O
160
(X103/ML)
FIG. 3. Relation of expected and observed values of PLT (at high and low ranges) measured by reference and test instruments.
specimens were plotted as described above, and correlation coefficients were calculated.
Results
Within-run Precision
Table l shows the within-run precision (expressed as
CV) at varying levels for each of the directly derived
parameters measured by all instruments. At normal levels, both the ELT-8 and the S-PLUS exhibit CVs lower
than those of the manufacturer's stated specifications for
every parameter. At abnormal levels, the instruments
routinely display CVs below 1.5%. Noteworthy are improvements in the precision over that of reference instruments occurring at low concentrations of WBC man-
ifested by a 50% decrease in CV. For the PLT parameter,
the CV of all instruments were similar, but most important, they were less than 5% for PLT counts down
to 50 X 103/>L, and less than 10% for counts below that
value. For the S-Plus, a higher CV was exhibited by all
parameters when measured in the predilute mode than
in the whole blood mode.
Day-to-day Precision
The ELT-8 and the S-Plus exhibited comparably excellent day-to-day precision for all parameters (Table 2).
The most noteworthy difference between these two instruments with respect to the reference instrument occurred for the PLT count. The day-to-day CV for the
AutoCounter (4.4%) was twice that of both the ELT-8
(2.1%) and the S-Plus (2.2%).
Table 3. Per Cent Carry-over for Direct Parameters Measured by Evaluation and Reference Instruments
Reference Instruments
S-Plus
Mean Value of Specimen
Parameter
High
WBC
RBC
HGB
PLT
30 X 10 3 /ML
4.30 X 10 6 /ML
* Per cent.
16 g/dL
950 X 10 3 /ML
Low
Whole Blood
Mode
Predilute
Mode
Whole Blood
Mode
Predilute
Mode
ELT-8
0.2 x 103AiL
1.2 X 1 0 % L
4.5 g/dL
5 X 107^L
1.67*
1.62
1.25
0.65
4.22
2.26
1.90
0.66
0.51
0.12
0.32
0.30
1.17
0.71
1.33
0.32
0.0
0.0
0.0
0.0
EVALUATION OF AUTOMATED HEMATOLOGY ANALYZERS
Vol. 78 • No. 5
Linearity
745
450 -
Figures 1, 2, and 3 display the linear relationship between expected and observed values of the directly derived parameters measured by all instruments. Of particular significance is the enhanced linearity achieved by
both newflowinstruments at the low range of WBC and
PLT counts rendering them of great value in the routine
follow-up of myelosuppressed patients.
350
^
250 -
5.
m-^
O
Carry-over
z
o
o
No detectable carry-over was noted for all direct parameters measured by the ELT-8, and a negligible
amount was observed for the S-Plus (Table 3). In this
respect, it is interesting to note the marked improvement
of the S-Plus over the S-Sr in regards to the carry-over
in both the whole blood and predilute mode.
200 180 160 140
120
a
LU
><
_l
0.
RBC Interference on PLT Count
w
a
The RBC concentration of the blood sample had virtually no effect on the displayed PLT counts of the ELT8 and a neglible effect on the PLT counts of the
AutoCounter. However, there was a notable effect on
50 45 40 35 30
25
• - S-PLUS
° - ELT-8
• -AUTOCOUNTER
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
6
RBC COUNT ( X 1 0 / M L )
FIG. 5. Effect of RBC concentration on displayed PLT count by the
S-Plus after retrofit. Points represent the mean of values obtained from
six separate S-Plus units.
•
S-Plus
S-Sr
o ELT-8
a Hemoglobinometer
10.4
c
3
O
u
35
£
a
e
•o
•
30
„•
a>
>,
«
a
CD
'••"it
0
25
0
o"
0
A ,
**•
"-'^
9.6
.
CO
0
r
I
—•-
9.2
'
1.0
1
1
2.0
3.0
RBC count (X
!
4.0
5.0
106/ML)
FlG. 4. Effect of RBC concentration on displayed PLT count by
three different analyzers.
I
e.o
A
_o
p
Z-*-
o
6"
O
i
a
a
20
0
A
5"
^ -
A
(3
o
•
A
>-
10.0
•
•o
•
A
o
—o—
a
20
40
60
80
•
100
3
WBC Count ( X 1 0 / M L )
FIG. 6. Effect of WBC concentration on displayed HGB measured
by different instruments.
DREWINKO ETAL.
746
Table 4. Correlation of S-Plus vs.
Reference Instruments
Parameter
Whole Blood Mode
Predilute Mode
WBC
RBC
HGB
HCT
MCV
PLT
0.99*
0.99
0.99
0.99
0.97
0.99
0.99
0.99
0.99
0.98
0.97
0.95
the counts were made in the whole blood or in the predilute mode. Upon retrofitting, this effect was slightly
reversed to the extent that the PLT count increased an
average of 3.5% for each additional 106 RBC/^L
(Fig. 5).
WBC Interference on HGB Determination
Increments of WBC had no effect on HGB determinations performed by the Coulter Hemoglobinometer,
and virtually no effect on the values determined by the
ELT-8 (Fig. 6). For the S-Sr, displayed HGB values increased at the rate of 0.03 g/dL for each 5 X 103 WBC,
and for the S-Plus, at the rate of 0.05 g/dL per 5
X 103 WBC.
' Pearson correlation coefficients.
Table 5. Correlations among S-Plus, ELT-8, and
Reference Instruments
Parameter
S-Plus vs.
Reference
Instruments
ELT-8 vs.
Reference
Instruments
S-Plus vs.
ELT-8
WBC
RBC
HGB
HCT
MCV
PLT
0.99*
0.99
0.99
0.98
0.97
0.98
0.99
0.99
0.99
0.98
0.92
0.98
0.99
0.99
0.98
0.98
0.95
0.98
* Pearson correlation coefficient.
the PLT counts of the S-Plus (Fig. 4). The displayed
PLT count on the S-Plus decreased an average of 5.2%
for every additional 106 RBC/jtL, regardless of whether
Table 6. Instrument Correlations of Separate Ranges
S-Plus vs.
Reference
Instruments
ELT-8 vs.
Reference
Instruments
S-Plus vs.
ELT-8
n
n
n
WBC
(XIO 3 /ML)
Level
<4.0
4.0-10.0
>10.0
0.98*
0.96
0.99
165
158
37
0.98
0.91
0.99
166
157
35
0.99
0.96
0.99
164
161
38
0.90
0.95
0.87
44
236
54
0.91
0.95
0.85
44
237
47
0.86
0.95
0.85
42
258
46
PLT
(XIOVML)
Level
<50
50-400
>400
A.J.C.P. • November 1982
' Pearson correlation coefficient.
Retrofits
No significant difference between values of precision,
linearity, and carry-over of all directly measured parameters were noted for either instrument after retrofitting.
For the S-Plus, corrections resulted in less interference
of the RBC on the PLT count and good correlation of
PLT counts between the two modes of aspiration. For
the ELT-8, improvements gave a superior day-to-day
precision of the MCV, HCT, and PLT parameters.
Correlations on Clinical Specimens
Table 4 presents the correlation coefficient of results
for the S-Plus versus the reference instruments for 429
whole blood and 205 predilute specimens. Correlation
values for all parameters were excellent with a slight
decrease in correlation for PLT seen in the predilute
mode (r = 0.95). Correlation of results obtained on the
S-Plus, ELT-8, and reference instruments is shown in
Table 5 for 373 clinical specimens. Again, all correlation
values were extremely good between the instruments,
except for a weaker correlation seen for the MCV parameter on the ELT-8 when measured against the S-Sr
(r = 0.92). A detailed correlation analysis for the WBC
and PLT parameters of the same 373 specimens at low,
normal, and elevated levels is expressed in Table 6. Particularly significant is the excellent correlation achieved
for WBC at both the low and elevated levels between
all instruments; the correlation values for the PLT pa-
Table 7. Correlation of Results between Different Collection and Aspiration Technic
Patient Specimens
Normal Donor Specimens
S-Plus
ELT-8
S-Plus
Parameter
Whole Blood
vs. Capillary
Whole Blood
vs. Predilute
Whole Blood
vs. Capillary
Whole Blood
vs. Capillary 24 hr
Whole Blood
vs. Capillary
Whole Blood
vs. Predilute
WBC
RBC
HGB
MCV
PLT
0.96*
0.98
0.97
0.97
0.96
0.99
0.97
0.98
0.98
0.97
0.98
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.99
0.95
0.94
0.99
0.99
0.98
0.96
0.95
0.98
0.96
• Pearson correlation coefficient.
vol. 78 • No. 5
EVALUATION OF AUTOMATED HEMATOLOGY ANALYZERS
rameter among all instruments decreased slightly at the
low and elevated levels.
Results of the correlation analysis between the mean
of capillary specimens and whole blood specimens for
both the S-Plus and ELT-8 is shown in Table 7. Excellent
correlation values were obtained by both instruments
between these two types of specimen collection technics,
indicating that the method of obtaining the blood will
not affect the accuracy of either instrument. The correlation values between the two aspiration modes on the
S-Plus were also very good. Particularly noteworthy were
the excellent correlation results obtained by the ELT-8
on the capillary specimens run 24 hours after having
been drawn.
Discussion
The ever increasing demand by clinicians for an expanded range of hematology laboratory tests reported
in a timely manner has been met by the development
and implementation of sophisticated high-speed multiparameter blood analyzers. However, to be useful in
routine practice, results provided by these instruments
must be accurate and reproducible over the wide dynamic range of hematology values encountered in clinical practice. This is especially critical at the low range
of both WBC and PLT counts where therapeutic decisions are based on relatively small changes of these parameters (i.e., transfusion, cessation, or resumption of
chemotherapy, etc.). Also, cost efficiency and timeliness
requires that the relevant parameters be analyzed with
the least possible number of instruments and with the
least number of sample preparative steps. These objectives have been achieved in both flow cytometric systems
evaluated in this report, where a breakthrough improvement was achieved in the capability of counting PLTs
along with the other seven parameters assayed in the
now obsolescent multiphasic hematology analyzers.
As determined in our laboratory, precision, Hnearity,
and lack of carry-over of both the ELT-8 and S-Plus
were superior to that of existing instruments, and slightly
better than that reported by both manufacturers and by
other investigators evaluating similar prototypes.1,1314
In particular, the precision of the PLT count was markedly superior to that performed manually or with other
technology.4,615 This was especially noteworthy for values below 50 X 103/|tL where both precision arid correlation coefficients with existing methods were better
than the results reported by other investigators.13 In a
study reported elsewhere, we showed that even at levels
of 20 X 103//iL, the accuracy of PLT counts determined
by the S-Plus were confirmed by counts performed under phase microscopy.2 Thus, in contrast to the suggestions of others,113 it is our practice to report results from
the automated count unless additional criteria established in our laboratory to prevent errors in PLT counts2
militate against this procedure.
747
For both instruments, results obtained on whole
blood showed an excellent correlation with those determined on capillary blood specimens. This observation
was remarkably useful in the case of the PLT count since
previous reports4,9 had indicated a highly significant discrepancy between the two sources. The interference of
the RBC concentration on the PLT count measured by
the S-Plus previously reported by us6 has been corrected
mathematically. Unfortunately, this correction has resulted in a slight bias in the opposite direction, creating
a constant increase of about 3% more platelets per million RBC. However, this error is considerably less than
that inherent to the precision (CV) at any level of the
dynamic range of the PLT count as to make it clinically
irrelevant.
In summary, both flow cytometric hematology analyzers have performed equally well under extreme conditions of hematologic disease. Routine utilization of
such instrumentation should have great impact on current large volume clinical hematology laboratory practice.
Acknowledgement. Coulter Electronics, Inc. and Ortho Instruments
provided instrumentation and reagents to perform this study. The
assistance of James J. Conroy, Ortho Instruments is gratefully acknowledged.
References
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Counter Model S-Plus. Lab World 1981; 32:64-67
3. Brecher G: The future of automation in hematology. Blood Cells
1980;6:111-114
4. Brecher GB, Scheiderman M, Cronkite EP: The reproducibility
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