Automated Platelet Counters
A Comparative Evaluation of Latest Instrumentation
KLAUS MAYER, M.D., BERNADETTE CHIN, M.T.(ASCP), JEFF MAGNES, B.S., H. TZVI THALER, PH.D.,
CHERYL LOTSPEICH, M.T.(ASCP), AND AMY BAISLEY, M.T.(ASCP)
Mayer, Klaus, Chin, Bernadette, Magnes, Jeff, Thaler, H.
Tzvi, Lotspeich, Cheryl, and Baisley, Amy: Automated platelet
counters. A comparative evaluation of latest instrumentation.
Am J Clin Pathol 74:135-150, 1980. An extensive evaluation of
performance characteristics and accuracy of clinical results for
two automated multiparameter whole-blood cell counters (the
Coulter Counter® Model S-Plus and the Ortho ELT-8®) and
two single-parameter semiautomated platelet counters (the
J. T. Baker MK-4/HC® and the Clay-Adams Ultra-Flo 100®) is
described. Results of comparative assays performed on more
than 1,200 clinical specimens are analyzed. These results are
compared with manual determinations where appropriate.
Particular attention is accorded to the accuracy of platelet
counts, especially at abnormal levels below 70 x 103/cu mm,
where falsely elevated platelet counts may lead to serious
clinical consequences. Both multiparameter instruments
yielded accurate results, with the exception of low values
reported by the ELT-8 for mean corpuscular volumes above
100 cu jum. Results for platelet counts were accurate for most
specimens on all four instruments; the ELT-8 was the most
reliable (P < 0.01), especially for the critically low counts.
Although no instrument is infallible in determining platelet
counts at all levels, the authors conclude that addition of platelet-counting capability represents a significant advancement
over existing instrumentation. (Key words: Automated blood
count; Automated platelet count.)
THE PHYSICIAN'S clinical decisions are frequently
dependent on blood counts. Erythrocyte, leukocyte,
and platelet counts are essential to the early detection
of occult lesions and are equally important in the
management of many diseases, including neoplasms,
infections, and toxic effects of drugs or chemical
exposure. More recently, with the advent of automated
equipment, much clinical information has been obtained
from a knowledge of cell size and size distribution. The
latest generation of blood cell counting instrumentation
has introduced significant advancements in plateletcounting technology and accuracy. It is therefore
important that those who carry responsibility for
Clinical Hematology Laboratory, Hematology-Lymphoma
Service of the Department of Medicine and Biostatistics
Laboratory, Memorial Sloan-Kettering Cancer Center,
New York, New York
clinical laboratories, as well as those who order and
interpret hematologic data, have a full understanding of
the capabilities and limitations of the instrumentation
currently available. To this purpose, to judge and
compare the suitability of blood cell counting
equipment, the authors have undertaken an extensive
evaluation of the following instrumentation:
(1) Coulter Counter Model S-Plus, Coulter Electronics, Hialeah, Florida (eight-parameter blood count,
including platelet count).
(2) ELT-8, Ortho Instruments, Westwood, Massachusetts (eight-parameter blood count, including
platelet count).
(3) MK-4/HC, J.T. Baker, Milford, Connecticut
(platelet count only).
(4) Ultra-Flo 100, Clay Adams, Parsippany, New
Jersey (platelet count only).
Principles of Instrumentation
To facilitate reference to the hematologic parameters
analyzed in this evaluation, the following abbreviations
will be employed throughout this paper: leukocyte
(WBC), erythrocyte (RBC), hemoglobin (HGB),
hematocrit (HCT), mean corpuscular volume (MCV),
and platelet (PLT).
A brief description of the capabilities and basic
operational principles of each of the four instruments
will aid the reader in understanding the results and
discussion.
The Coulter Counter Model S-Plus
Received October 23, 1979; received revised manuscript and
accepted for publication January 7, 1980.
Supported in part (biostatistics) by grant CA20194-03 from the
National Cancer Institute.
Address reprint requests to Dr. Mayer: Director, Hematology
Laboratory, Memorial Sloan-Kettering Cancer Center, 1275 York
Avenue, New York, New York 10021.
The S-Plus is a fully automated hematology instrument that utilizes either 1 ml undiluted venous blood
or 44.7 /xl capillary blood prediluted to 10 ml total
volume to determine the standard seven-parameter
blood count plus PLT count in 34 to 50 sec. In addition,
0002-9173/80/0800/0135 $01.30 © American Society of Clinical Pathologists
135
136
MAYER ET AL.
a ninth parameter, erythrocyte distribution width
(RDW), is also determined. The whole-blood sample is
automatically diluted within the instrument prior to
nine-parameter analysis. Cell counting is based on the
principle of one-by-one impedance electronic counting
and sizing of particles. In addition to the nine parameters currently printed out, provision already exists for
acquisition of additional PLT parameters. The S-Plus
flags WBC, RBC, MCV, and PLT counts via a coding
system printed directly onto the report ticket, including
the following categories: counts exceeding the printer
maximum, statistical voteout of counts, and "no fit"
for PLT size distribution (caused when a PLT count is
less than 20 x 103/cu mm, when the mode of the PLT
distribution is not between 3 to 15 /wn, creating a
negative size distribution curve, or when there is a voteout of the fitted distribution curve). In addition, interruptions in the operating cycle that may affect results
{e.g., recount, clear) are also printed onto the report
ticket.
The Ortho ELT-8
The ELT-8 is a fully automated hematology instrument that utilizes 100-ptl quantities of venous or capillary blood to determine the standard seven-parameter
blood count plus PLT count in 60 sec. The sample is
automatically diluted within the instrument prior to
eight-parameter analysis. Cell counting is based on the
principles of laminar flow hydrodynamic centering of
cells prior to one-by-one counting using a laser optics
system. The ELT-8 signals all values exceeding preset
operating ranges for WBC, RBC, HGB, and PLT
counts. In addition, the system informs the operator
if giant platelets (or microcytic RBCs) represent greater
than 4% of the total PLT count. The Data Handler
emits an audible signal to alert the operator, followed
by an appropriate written message on the CRT screen.
The J.T. Baker MK-4IHC
The MK-4 is a semiautomated PLT-counting instrument. It utilizes 3 fj\ platelet-rich plasma prepared
from whole blood and suspended in 12 ml diluent; it
requires 3 min for start-to-finish PLT enumeration. The
system uses an electronic counting principle based on
interruption of conductivity, and then utilizes a predetermined HCT to calculate the PLT count. The
test procedure requires manual specimen preparation
and prior determination of HCT by other methods.
The MK-4 employs a visual warning system, which
advises the operator to verify questionable PLT counts
detected by the instrument (due, for example, to
aperture clogging).
A.J.C.P. • August 1980
The Clay Adams Ultra-Flo 100
The Ultra-Flo 100 is a semiautomated whole-blood
PLT-counting instrument that utilizes a 10-jd specimen
of whole blood suspended in 9.1 ml diluent to determine
a PLT count in 60 sec. The system uses hydrodynamic
focusing prior to electronic counting of platelets.
The RBC count must be determined prior to running
each sample and is used to automatically calculate
the PLT count. The Ultra-Flo 100 employs a visual
error code system, which warns the operator of possible
errors in the PLT count; the system detects and signals
deviations from normal PLT distribution as follows:
greater than 4% giant platelets (or microcytic RBCs),
or greater than 12% small platelets (or debris).
Materials and Methods
Equipment
In all phases of the study where manual procedures
were performed, the greatest technical care was employed; however, the reader is reminded of inherent
imprecision in all technics, including pipetting, hemacytometer charging, and phase microscopy.
The venous blood specimens used throughout all
phases of this study consisted of blood collected in
Vacutainer tubes* containing tripotassium-EDTA
(1.5 mg/ml blood) as anticoagulant. Capillary blood
collecting systems will be described where applicable.
The equipment was installed by the manufacturers on
the premises of the authors' institution—The Memorial
Sloan-Kettering Cancer Center. The manufacturers
were given every opportunity to have these instruments
in the best working order both before and during the
evaluation period. All instruments used during this
evaluation were production models operated by hematology technologists at the authors' institution, with
two exceptions: (1) The ELT-8 used during Phases I
and II was a prototype model operated by an Ortho
technologist; (2) a second S-Plus production model
was used during parts of Phases II (operated by a
Coulter technologist) and III.
The instruments were calibrated and quality controlled as follows.
The S-Plus was initially calibrated using 20 normal
whole-blood specimens against reference methods,
for WBC, RBC, HGB, HCT, RDW, and PLT. Daily
electronics check and calibration verification were performed, the latter using 4C Plus Control (Normal).t
The ELT-8 was initially calibrated using CBC-trol
(Normal)* for WBC, RBC, HGB, and HCT, and
* Becton Dickinson, Rutherford, New Jersey.
t Coulter Diagnostics, Hialeah, Florida.
t Pfizer Diagnostics, New York, New York.
Vol. 74 • No. 2
AUTOMATED PLATELET COUNTERS
Quanticel-HP Human Platelet Reference (Normal)§
for PLT; the initial calibration was checked using five
normal whole-blood specimens against reference
methods. Daily calibration was performed using CBCtrol (Normal) and Quanticel-HP (Normal) and monitored throughout the day using one normal whole-blood
specimen.
The MK-4 was initially calibrated by the manufacturer.
Daily electronics check and calibration verification
were performed, the latter using Haem-PC (Normal
and Low).H
The Ultra-Flo 100 was initially calibrated by the
manufacturer. A daily calibration verification was performed using Ultra-Flo Platelet Reference Control
(Normal and Abnormal).**
Terminology
For the purposes of this evaluation, the following
definitions apply:
Precision was defined as the ability of an instrument to report reproducible values for replicate assays
of the same specimen, without regard to the absolute
value obtained.
Accuracy was defined as the ability of an instrument
to report a value in agreement with a predetermined
reference value.
Procedure
Phase 1 consisted of a comparative evaluation of
performance characteristics of the S-Plus and ELT-8
for WBC, RBC, HGB, HCT, MCV, and PLT, particularly with regard to precision (WBC, RBC, HGB,
HCT, MCV, PLT), linearity (WBC, RBC, HGB, PLT),
and carry-over (WBC, PLT).
Precision. Precision of measurements by the S-Plus
and ELT-8 was evaluated for 14 days. Coefficients of
variation were determined based upon ten replicate
assays on each instrument for both morning and afternoon runs. A comparison of mean morning and afternoon values was used to evaluate within-day drift for
each parameter. Two control specimens were used: (1)
Normal Control: normal donor whole blood was drawn
each morning, pooled, mixed, and divided into two
aliquots designated as A.M. and P.M. (2) Abnormal Control: vials of CH-60 Hematology Abnormal Control,tt
were similarly pooled, mixed, and divided into A.M. and
P.M. aliquots. Since this control contains no platelets,
only the remaining five parameters were evaluated.
For both controls, the A.M. aliquot was assayed im§ BHP, Inc., West Chester, Pennsylvania.
11 J.T. Baker Diagnostics, Bethlehem, Pennsylvania.
** Clay Adams, Parsippany, New Jersey.
t t Dade, Miami. Florida.
137
mediately, and the P.M. aliquot was refrigerated at 4 C
for four to six hours prior to assay.
Linearity. Linearity of measurements by the S-Plus
and ELT-8 was evaluated as follows. Blood specimens
containing abnormally high levels of WBCs and PLTs
were obtained and serially diluted with Isoton 11.t
Normal whole-blood specimens with concentrated
RBCs and HGB were also serially diluted with Isoton
II or autologous plasma. All samples were run on both
instruments within one hour of collection and dilution.
Carry-over. Carry-over studies for WBCs and PLTs
were performed usjng selected specimens with high
values for the respective parameters. Direct measurement of carry-over for RBCs and HGB was performed,
but was not included in these data owing to software
limitations in the ELT-8 for printing low RBC and HGB
values (below 0.5 x 106/cu mm and 3 g/dl, respectively).
For WBC carry-over, six specimens were evaluated;
for PLT carry-over, nine specimens were studied. Each
specimen was consecutively assayed five times, followed by two diluent rinses.
Phase II consisted of a comparative evaluation of the
four instruments as to accuracy of results, with particular emphasis on PLT counts, in a selected population of 200 patient specimens with abnormal hematologic counts, especially low WBC and PLT counts.
It included two parts:
In the first part of Phase II, results of five parameters
(WBC, RBC, HGB, HCT, MCV) obtained on the S-Plus
and ELT-8 were compared with reference values.
Results from the Clinical Hematology Laboratory's
Coulter Counter Model Sit were used as reference for
WBC, RBC, and HGB (n = 200). The Model S used
was checked daily for accuracy using 4 C (Normal)
Control,t and for precision using a normal whole
blood. Manual procedures were employed for the following parameters:
(1) Manual WBC determinations were performed
for all WBC counts of less than 1.5 x 10'Vcu mm
(n = 43). Blood was diluted 1:100 with 1% ammonium
oxalate using Unopette Test 5855* and incubated for
10 min to hemolyze the RBCs. After a Neubauer hemacytometer was charged, it was placed on moistened
filter paper in a Petri dish and allowed to stand 10
min to permit the cells to settle. WBCs were enumerated
microscopically by counting all nine large squares on
both sides of the hemacytometer, adding 10% to the
average nine-square count, and multiplying this figure
by 100 to obtain the total count.
(2) Manual HGB determinations (cyanmethemoglobin
method 2 ) were performed for selected patients (n
= 150), using a Beckman Model 24 spectrophotomtt Coulter Electronics, Hialeah, Florida.
138
MAYER ETAL.
eter.§§ Absorbance readings at 540 nm were converted to
HGB values by referring to a standard curve established
using Cyanmethemoglobin Certified Standard.HH
(3) Microhematocrits were performed in duplicate
on all specimens (n = 200) using an Autocrit II centrifuge.** Samples were centrifuged at 13,700 RCF for
3 min and read directly without correction for plasma
trapping.
(4) Manual MCVs were calculated for all specimens
(n = 200) by dividing the manual hematocrit by the
mean RBC value obtained on the S-Plus, ELT-8, and
Model S.
In the second part of Phase II, results of PLT counts
obtained on four instruments were compared with
manual phase PLT counts used as reference for all
specimens (n = 200). Blood was diluted 1:100 with 1%
ammonium oxalate using Unopette Test 5855 (a modification of the reagents developed by Brecker and Cronkite 3 ), and was incubated for 10 min to hemolyze the
RBCs. After a Neubauer hemacytometer was charged,
it was placed on moistened filter paper in a Petri dish
and allowed to stand 10 min to permit the cells to settle.
Using phase microscopy, platelets were enumerated
by counting all 25 small squares of the large center
square of both sides of the hemacytometer and multiplying the average 25-square count by 1,000 to obtain
the total PLT count.
Since the accuracy of manual microscopic reference
methods (especially phase PLT counts) is critical to the
conclusions reached from the comparative evaluation,
the authors feel that the procedures employed deserve
special attention.
Each manual WBC count and phase PLT count was
performed using a clean Spencer "Bright Line" Neubauer hemacytometer AO Model 1475,*** with a No.
V/2 Cover Glass. The three microscopes used to perform manual WBC counts and phase PLT counts were
as follows: (1) AO Model 1036A***; (2) AO Model
1036A, "Phase Star"***; (3) Nikon Model A21404.ttt To ensure functional optical alignment of the
microscopes before and during the course of the evaluation, the following procedure was employed: Before
the evaluation, the two AO microscopes were properly
set and serviced in September, 1978.Mt The Nikon
microscope was inspected before rental.§§§ On a daily
basis, the phase alignment of each microscope was
checked and adjusted according to manufacturers'
specifications, and all lenses were cleaned with lens
paper. On a weekly basis, the optical alignment of
§§ Beckman Instruments, Fullerton, California.
1iH Hycel, Houston, Texas.
*** American Optical Corp. Buffalo, New York.
t t t Nikon Corp., Tokyo, Japan.
ttt August Waeldin, Inc., New York, New York.
§§§ Atlantex and Zieler Instrument Corp., Dedham, Massachusetts.
A.J.C.P. • August 1980
each microscope was checked and adjusted as necessary. WBC counts were performed under 100X total
magnification on all three microscopes. Phase PLT
counts were performed under 450x total magnification
on the two American Optical microscopes and under
400x total magnification on the Nikon microscope.
Four technologists participated in the WBC and
phase PLT counting process. Three of these were MT(ASCP) hematology technologists at the authors'
institution, who were selected because of their expertise in hematology laboratory work. The fourth was an
Ortho employee who was also experienced and well
qualified in hospital hematology technology. Phase
counts were performed on each specimen by three of
these technologists (chosen at random), and the mean
was used as reference. In addition, approximately
15 phase PLT counts were repeated and confirmed by
Coulter technologists (not included in the data). Specimens were selected primarily on the basis of abnormal
PLT counts and, as such, represented many of the
"worst" hematologic cases one would expect to encounter in clinical work. In this way, each system was
maximally challenged to perform and produce reliable data.
All manual reference procedures described above
were performed under "blind" experimental conditions; that is, the technologist(s) performing the manual
determination had no previous information regarding
either the specimen itself or any of the instrument
values obtained.
Phase III consisted of a comparative clinical evaluation of results obtained on the S-Plus and ELT-8 for
six parameters (WBC, RBC, HGB, HCT, MCV, PLT)
in a random population of 1,045 patient specimens received by the Clinical Hematology Laboratory at the
authors' institution. These data were obtained under
essentially routine clinical conditions. All specimens
were assayed within three hours of collection and run
through both instruments within one hour of each
other. Reference methods were employed where
discrepancies between the two instruments were felt
to be clinically significant for WBC (n = 38) and PLT
(n = 46). The methodologies employed were as described above, with the exception that the manual
counts were performed under routine clinical conditions by a hematology technologist at the authors'
institution.
Phase IV of the study consisted of a comparative
clinical evaluation of the capability of the S-Plus and
ELT-8 to perform hematologic determinations on
venous and capillary blood specimens from the same
patient.
For the S-Plus, two systems were used to collect and
predilute the capillary blood: (1) Unopette Test 5925*
using a 44.7-jLtl "flagtype" pipette and 10 ml Isoton
II already contained in the sealed reservoir; (2) Ac-
AUTOMATED PLATELET COUNTERS
Vol. 74 • No. 2
o
c
139
I2h
8
4
•04-0 3 02 -01
0 01 02 03
WBC DriftUK&uinm)
-006
0
006
RBC Drift dicfitumm)
dL
0.13 -0.4-0.3 -Q2 -01
128
4
* £ 12"
84 F I G . 1. Precision: wilhinday drift (afternoon value
minus morning value) for
normal blood on S-Plus M and
E L T - 8 * for leukocyte count
( W B C ) , erythrocyte count
(RBC), hemoglobin ( H G B ) ,
hematrocrit ( H C T ) . mean
corpuscular volume ( M C V ) ,
platelet count ( P L T ) .
,Etb, W i n
-0.4 -03-02-01
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WBC Drift (.lO^cumm)
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HCT Drift(%)
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12-
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-50-40-30-20-10
0
cuvette 11 vialt using Coulter's "end-to-end" pipette
and 10 ml lsoton 11 dispensed from the S-Plus.
For the ELT-8, the Microtainer Capillary Whole
Blood Collector* was used to collect and anticoagulate
200-300 yA capillary blood; the system utilizes disodium-EDTA and two inert plastic mixing beads to
ensure complete anticoagulation (A second system,
the Hemette Capillary Blood Collector,H1l1l was also
tried initially, but proved technically too cumbersome
and was not used in the evaluation.).
Owing to the difficulty in coordinating the drawing
of venous and multiple-system capillary specimens
from the same patient, specimens from 35 different
random patients were used to evaluate each instrument.
Both venous and capillary specimens were drawn from
each patient, using the appropriate collecting systems.
For the patients from whom both Accuvette and Unopette capillary samples were drawn, the order of capillary specimen collection was randomized. Additionally,
results for any patient for whom either specimen was
technically suspect (i.e., clots observed in assaying
specimen) were not included in the data.
In all cases, venous and capillary specimens were
collected and assayed within three hours of each other.
All capillary specimens were assayed within one hour
of collection. For this portion of the study, no manual
i ' i * T * * 'r-L-r-330 -120 -9.0 -6.0-30 0 30 60 90
MCV Drift ( M J )
PLT Drift (»!0\umm)
12
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4U
1.0 2.0 30 40
HCT Drift (%)
111111 Ortho Instruments, Westwood, Massachusetts.
0 01 02 03 04
HGB Drift (g /di)
RBC Drift (nicftcumm)
•o
0 0.1 0.2 03 Q4
HGB Drift (g/dl)
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4-
-90-6.0-3.0 0 3.0 60 9 0
MCV Drift (/i.3)
L.
•«0 -120 -90 -60 -10 0 3.0 60 9.0
PLT Drift (il03/cu«nm)
reference methods were employed; for each patient,
venous values were taken as reference for comparison
with capillary values obtained on the same instrument.
Results
Performance
Characteristics
Precision. Table 1 shows a comparison of mean
morning and afternoon coefficients of variation in reproducibility studies performed on both instruments for
Table I. Comparison of Mean Coefficients of
Variation (%) Over 14 Days
ELT-8*'
S-Plus®
Normal
Blood
Test Cellst
Normal
Blood
Test Cellst
Parameter*
A.M.
P.M.
A.M.
P.M.
A.M.
P.M.
A.M.
P.M.
WBC
RBC
HGB
HCT
MCV
PLT
1.4
0.6
0.6
1.2
1.1
2.4
1.6
0.7
0.5
0.9
0.7
2.3
1.1
0.8
0.7
1.2
1.1
1.0
0.7
0.8
1.3
1.1
1.3
0.9
1.6
1.8
0.7
—
2.3
0.8
1.1
1.1
0.7
1.4
1.4
0.9
1.5
1.1
0.6
—
2.2
0.7
1.4
0.9
0.7
1.2
—
—
* WCB = leukocyte count; RBC - erythrocyte count; HGB hemoglobin; HCT
= hematocrit, MCV = mean corpuscular volume; PLT platelet count.
t Dade Abnormal.
140
MAYER
1:64
A.J.C.P. • August 1980
ETAL.
1:16
SERIAL DILUTION
SERIAL DILUTION
3
St
10-0
1:64
1:16
SERIAL OILUTION
SERIAL DILUTION
ffYTOSYHOlS
o
S-PLUS
•
ELT-8
FIG. 2. Linearity of measurements on S-Plus® and ELT-8® for leukocyte count (WBC), erythrocyte count (RBC),
hemoglobin (HGB), platelet count (PLT).
six parameters. Daily coefficient of variation (%) was
calculated as:
Standard deviation for ten
replicate assays
x 100%
Mean value of ten replicate assays
and was averaged for the 14-day evaluation period.
Figure 1 shows the within-day drift of morning versus
afternoon values for six parameters of normal blood
assayed on both instruments for 14 days. The difference
in morning and afternoon values (drift) for each parameter was calculated as the afternoon value minus the
morning value.
Linearity. Data on linearity are presented graphically. All graphs are logarithms of the raw counts,
due to the great range of values.
Combined data for two linearity studies performed
on WBC, RBC, HGB, and PLT are shown in Figure 2.
In each graph, the line shown was calculated using a
regression formula applied to several of the midrange
points, where the instruments were the most linear.
Carry-over. Table 2 shows mean and range of carryover for WBC and PLT counts on both instruments.
Carry-over (%) was calculated for each specimen as:
1st diluent rinse - 2nd diluent rinse
x 100%.
Mean value of five replicate assays
Clinical Specimens
The data for Phases II and III are presented graphically.
Computer-generated scattergrams of RBC, HGB,
HCT, and MCV use raw counts. Less information is
AUTOMATED PLATELET COUNTERS
Vol. 74 • No. 2
141
Table 2. Comparison of Carry-over (%)
for WBC and PLT*
S-Plus
ELT-8
Mean
Range
Mean/Range
WBC
(six specimens:
27 x 103 to
143 x 103/cu mm)
0.6
0.5-0.7
0.0
PLT
(nine specimens:
349 x 103 to
2,402 x 103/cu mm)
0.3
0.0-1.3
0.0
Parameter
* WBC = leukocyte count; PLT = platelet count.
gained from WBC and PLT scattergrams displayed
either as raw counts (due to overemphasis on high
values) or as logarithms of raw counts (due to exaggeration of variability at low values). The square
root of raw counts, although less intuitive, is a proper
compromise and is justifiable on mathematical grounds.
The square-root transformation tends to equalize the
variability independent of the overall magnitude of the
counts, as well as to make the distribution more nearly
Gaussian (and symmetric).
Figures 3, 4, and 5 pertain to 200 selected clinical
specimens (Phase II). Figure 3 shows the results of
values obtained on the S-Plus and ELT-8 versus Model
S determinations (n = 200) for RBC, WBC, and HGB.
Figure 4 shows the results of values obtained on the
S-Plus and ELT-8 versus "manual" determinations for
WBC (n = 43, hemacytometer), HGB (n = 150, cyanmethemoglobin method), HCT (n = 200, microhematocrit), and MCV (n = 200, manual method).
Figure 5 shows the results of PLT counts performed
by the S-Plus, ELT-8, MK-4, and Ultra-Flo 100 versus
phase counts (n = 200). At the manufacturer's request,
the Ultra-Flo 100 data were plotted with different
symbols for flagged and unflagged specimens. The
statistical significance of the relative accuracy of PLT
counts on the four instruments was analyzed by comparing the mean differences between the square-root
transformations of the raw instrument and of the raw
phase counts, so as not to overemphasize discrepancies
in specimens with genuine high counts.
Figure 6 shows the results of WBC, RBC, HGB,
HCT, MCV, and PLT determinations performed by
the S-Plus versus the ELT-8 for 1,045 random clinical
specimens (Phase III). For the seven visually obvious
discrepant WBC counts, the manually performed
counts are indicated. For the six visually obvious
discrepant HGB values, the patients' disease states
10.0
40.0
REFERENCE WBC (MODEL S) XIOVCUMM
REFERENCE RBC (MODEL S)
Xlrf/CUMM
B.O
10.0
REFERENCE HCB <M00L'
FIG. 3. S-Plus® and ELT-8® results vs. Model S® results on 200
selected clinical specimens for leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB).
12.0
S I G/J'
ffY Tbsrmm
o
S-PLUS
•
ELT-8
A.J.C.P. • August 1980
MAYER ETAL.
142
or any pertinent characteristics of the specimens
are indicated.
For all manual WBC and phase PLT counts performed during Phases II and III, mathematical formulas
were derived to define "clinical outliers" {i.e., "incorrect" instrument values) according to clinical
experience regarding acceptable ranges of discrepancies
between instrument and manual reference values. For
WBC, the range of clinically acceptable instrument
values was defined as:
(Manual WBC count) ± (20% of manual WBC count
+ 0.3 x 103/cu mm).
For PLT, the range of clinically acceptable instrument
values was defined as:
(Phase PLT count) ± (20% of phase PLT count
+ 10 x 107cu mm).
Table 3 shows the number of clinical outliers reported by each instrument when these formulas are
applied to the manual values obtained during Phases
II (WBC, n = 43; PLT, n = 200) and III (WBC,
n = 38; PLT, n = 46).
Figure 7 summarizes the differences in WBC, RBC,
HGB, and PLT values obtained on venous and capillary blood specimens drawn from 35 patients for each
capillary blood collection system (Phase IV). For each
set of venous/capillary specimens, the difference in
value for each of the four parameters was calculated
as the capillary value minus the venous value.
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FIG. 4. S-Plus® and ELT-8® results vs. manual reference methods for leukocyte count (WBC), n = 43; hemoglobin (HGB), n = 150;
hematocrit (HCT), n = 200; mean corpuscular volume (MCV), n = 200.
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FIG. 6. S-Plus® vs. ELT-8® results on 1,045 random clinical specimens for leukocyte count (WBC), erythrocyte count (RBC),
hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), platelet count (PLT).
AUTOMATED PLATELET COUNTERS
Vol. 74 • No. 2
Operation
Table 4 is a comparison of reagent consumption
per cycle on the S-Plus, ELT-8, MK-4, and UltraFlo 100.
Discussion
Performance
Characteristics
Selected Clinical
Precision. Both the S-Plus and ELT-8 performed
excellently in reproducibility studies. The coefficients
of variation for the six parameters analyzed were
superior to manufacturers' specifications for both
instruments and were well within the limits of clinical
usefulness and acceptance. 5 The coefficients of variation were somewhat tighter for WBC and HGB on the
S-Plus, and for PLT on the ELT-8. Although the coefficients of variation showed no significant difference
between morning and afternoon results, the mean
afternoon recovery value was superior with the S-Plus
for all parameters except RBC and PLT, for which
both instruments performed equally well. In particular,
the ELT-8 used during this portion of the study exhibited
a within-day drift for HCT exceeding ±1.5% on five
of 14 days compared with one of 14 days on the S-Plus);
in these instances, a 10 min operator recalibration
procedure was necessary on the ELT-8.
Linearity. Deviations from linearity that occurred on
the S-Plus and ELT-8 at extremely high and low counts
for WBC and PLT are not felt to be clinically significant.
In Phases II and III, no consistent instrument bias was
found at either excessively high or low WBC levels in
those specimens for which manual counts were performed; furthermore, comparison of instrument and
phase PLT counts at low levels generally showed the
145
opposite instrument bias from that predicted by the
linearity data.
Carry-over. For both the S-Plus and ELT-8, mean
carry-over effects for WBC and PLT were superior
to manufacturers' specifications. The ELT-8 showed
no carry-over for WBC and PLT on the first rinse
following any sample.
Specimens
WBC, RBC, HCT, HGB, AND MCV. Referring to
Figure 3, the comparative data in the selected population
of 200 patient specimens showed excellent correlation
of the S-Plus and ELT-8 values with Model S values
for WBC (r = 0.923, 0.921), RBC {r = 0.985, 0.985),
and HGB (r = 0.979, 0.974). As seen in Table 3, the
S-Plus and ELT-8 showed good clinical agreement
with hemacytometer WBC counts for counts below 1.5
x 103/cu mm. Figure 4 demonstrates that the S-Plus
and ELT-8 values showed excellent correlation with
microhematocrit (r = 0.971, 0.969) and manual HGB
(r = 0.987, 0.990) values.
The S-Plus and ELT-8 MCV values showed relatively
weaker correlation with the "manually" determined
reference MCVs {r = 0.854, 0.809). This may be partially explained by the fact that the "manual" MCVs
were calculated from two independent variables (microhematocrit and mean instrument RBC) and may be
expected to show more variability than reference
values used for the other four parameters. For MCV
values above 100 cu ;u,m, the ELT-8 reported lower
values than either the S-Plus or "manual" MCVs. This
may be due in part to the Ortho system's optical
methodology. In the authors' opinion, the MCVs reported by the ELT-8 do not mislead the clinician, as
macrocytic anemias are still identifiable.
Table 3. Clinical Outliers for WBC and PLT in Phases II and III: Comparison of Instrument Values
with Manual Reference Values*
Number of Clinical Outliers
Parameter
Instrument
All Other Instrument
Valiue(s) "Correct"
One or More Other
Instrument Value(s)
Also ' 'Incorrect"
2
3
0
0
2
3
Total
Phase 11 WBC
(comparison with 43
manually counted specimens)
S-Plus®
ELT-8®
Phase II PLT
(comparison with 200
manually counted specimens)
S-Plus
ELT-8
MK-4®
Ultra-Flo 100®
16
2
3
15
18
12
16
24
34
14
19
39
Phase III WBC
(comparison with 38
manually counted specimens)
S-Plus
ELT-8
5
11
7
7
12
18
Phase III PLT
(comparison with 46
manually counted specimens)
S-Plus
ELT-8
19
6
0
0
19
6
' For clinical outlier formulas, refer to text. WBC = leukocyte count; PLT = platelet count.
146
MAYER ETAL.
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A.J.C.P. • August 1980
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4
FIG. 7. Comparison of differences between venous and
capillary results (capillary value
minus venous value) on S-Plusot
and ELT-S* for leukocyte count
(WBC),n = 35; erythrocyte count
(RBC), n = 35; hemoglobin (HGB),
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PLT. Attention is directed to the PLT count because
this marks the major innovation of the S-Plus and ELT-8
compared with existing multiparameter blood-counting
instrumentation; in fact, it is the raison d' etre for
acquiring these two instruments. In addition, the two
single-parameter instruments were evaluated in this
portion of the study.
Figure 5 shows the correlation between PLT counts
obtained on four instruments and manually performed
phase PLT counts. It is generally accepted that phase
PLT counts are far less precise than counts obtained
on any of the instruments tested. 34 However, it has
been documented that automated equipment may at
times detect and count as platelets other particles
having a size, refractive index, or other characteristic
that is seen and recognized by the instrumentation
as platelets.1,6-8 These particles include cytoplasmic
fragments in leukemia patients, Howell-Jolly bodies,
nucleated erythrocytes, leukocytes, malarial parasites,
and aggregated erythrocytic stroma secondary to
erythrocyte antibodies or agglutinating paraproteins.6
It has been further suggested that in these instances
AUTOMATED PLATELET COUNTERS
Vol. 74 • No. 2
phase PLT counts, in conjunction with careful scrutiny
of the stained slide for cytoplasmic debris, are a more
accurate method of platelet counting. 78
The overall correlation of all four instruments with
phase PLT counts was good: S-Plus (r = 0.973),
ELT-8 (r = 0.981), MK-4 (r = 0.980), and UltraFlo 100 (r = 0.948). Nevertheless, a number of
clinically meaningful discrepancies occurred. The vast
majority of discrepancies occurred for PLT counts
below 70 x 103/cu mm. Rarely, the phase count
exceeded instrument counts. However, all too often
the instruments yielded a higher PLT count than
that obtained either quantitatively by phase count or
by estimation from the stained slide.
Two methods may be used to evaluate PLT-counting
performance by the four instruments: (1) Comparison
of the mean difference between the square-root transformations of the raw instrument and of the raw phase
PLT counts. The ELT-8 performed significantly (P
< 0.01) better than the other three instruments. (2)
Comparison of the number of clinical outliers, as
indicated in Table 3. The ELT-8 showed the least
number of these clinical outliers for the PLT parameter,
followed by the MK-4, S-Plus, and Ultra-Flo 100. Of
particlar interest are those cases in which three of
the four instruments tested were in clinical agreement
with phase PLT counts.
All clinical outliers identified were falsely elevated
PLT counts. Such errors are potentially dangerous in
that they may convey a false sense of security to the clinician, especially the oncologist who might administer
more radiation or chemotherapy than would be prudent
if the true PLT count were known. At the critical levels
below 70 x 10;Vcu mm, sufficiently accurate PLT
counts must be reported to permit the oncologist to
evaluate hematologic trends in response to cytotoxic
agents, to alert the clinician of the danger of bleeding
or necessity for platelet transfusion, and to assist in
subsequent monitoring of the patient to determine the
effectiveness of transfusion.
After approximately 80 specimens had been analyzed
in our selected 200-patient study (Phase II), the S-Plus
147
developed a problem with the sweep-flow system
(designed to eliminate artificial pulses in the RBC/
PLT aperture bath by sweeping particles away from the
sensing zone of the apertures). Correction of this
problem preceded the second 100 specimens. No
significant change in performance characteristics with
regard to PLT count on the S-Plus was seen in the
second 100 specimens.
To ensure that some or all of the falsely elevated
PLT counts obtained on the S-Plus were not due either
to faults in the S-Plus instrument selected for this study
or to electrical current fluctuations in the authors'
laboratory, a second S-Plus unit was installed in the
Clinical Hematology Laboratory (located in another
building); the second 100 specimens were assayed on
both instruments. Aside from the expected statistical
fluctuations obtained with both instruments on low
PLT counts, the two instruments showed excellent
agreement in the determination of all hematologic
parameters, and no clinically significant difference
was seen between values obtained on the two instruments.
Flagging. Identification of falsely elevated PLT
counts was only partially accomplished by flagging of
questionable results by the four instruments evaluated.
The S-Plus flagged approximately two thirds of the PLT
counts it reported for the 200 selected clinical specimens
(including 113 clinically correct PLT counts), thus
rendering it difficult for the technologist to differentiate
between accurate and falsely elevated counts at these
levels. In addition, all instruments failed to flag a number of spuriously elevated PLT counts; this, of course,
is the most serious flagging deficiency.
Manual Verification. None of the instrumentation
evaluated completely eliminated the necessity of slide
check or phase count for low PLT counts. In the
authors' institution, with a large population of patients
including leukemia patients and those receiving chemotherapy, this represents an especially troublesome
drawback. In those laboratories that encounter relatively few low PLT counts, it may be feasible to
check all instrument counts below a fixed level (e.g.,
Table 4. Comparison of Reagent Consumption per Cycle
Reagent
Diluent
RBC lysing
S-Plus«
Isoton 11*
Lyse S 11*
HGB determination
* Coulter Diagnostics, Hialeah, Florida.
t Ortho Diagnostics, Westwood, Massachusetts.
MK-4«
ELT-!
55 ml
0.77 ml
Salact
16 ml
Lysact
.08 ml
Cyanact
1.6 ml
Haema-line 0 $
Ultra-Flo 100®
12 ml
Ultra-Flo Diluent and
sheath fluid§
t J. T. Baker Diagnostics, Milford, Connecticut.
§ Clay Adams, Parsippany. New Jersey.
9.5 ml
148
MAYER ETAL.
100 x 103/cu mm) by phase and slide. For the oncologist, it may be appropriate to verify instrument
counts at least once per week on every patient with
a PLT count below 100 x 103/cu mm by slide check
or phase count to ascertain that particles counted as
platelets are not in fact cytoplasmic or other debris.
Manufacturers' Improvements. The manufacturers
of both multiparameter instruments are fully aware of
the above deficiencies and have stated that these are
amenable to correction. It is hoped that this will have
been accomplished by the time of publication of these
studies. Potential users and purchasers should assure
themselves of this.
Routine Clinical
Specimens
The S-Plus and ELT-8 performed adequately in the
clinical trials conducted in several hematology laboratories in the authors' institution (Phase III). Problems
were encountered by both instruments with specimens
from rare patients with cold agglutinin disease (n = 1;
all parameters) and Hemoglobin H disease (n = 1;
PLT). In addition, the S-Plus experienced difficulty in
several specimens from patients with macroglobulinemia
(n = 3; HGB), dysproteinemia (n = 1; WBC), and
hyperlipidemia (n = 1; HGB). These specimens are
usually recognizable by the bizarre results. In the above
instances, manual methods must be employed.
The results on 1,045 random specimens tested were
similar to those obtained on the 200 selected specimens.
As seen in Figure 6, the S-Plus and ELT-8 showed
excellent correlation with each other for five of the six
parameters evaluated: WBC (r = 0.988), RBC (r
= 0.994), HGB (r = 0.985), HCT (r = 0.983), and
PLT (r = 0.982). The MCV values showed good, but
somewhat weaker, correlation between the two instruments (r = 0.920). Table 3 shows that for those specimens where manual WBC counts were performed, the
ELT-8 reported one and a half times as many clinical
outliers for WBC as did the S-Plus. The majority of the
WBC outliers for both instruments occurred on specimens where examination of the peripheral smear revealed the presence of nucleated RBCs. The S-Plus
reported approximately three times as many clinical
outliers for PLT as did the ELT-8. Again, the ELT-8
reported lower MCV values than the S-Plus for MCVs
of greater than 100 cu (im. On specimens with a WBC
count of greater than 100 x 103/cu mm, the ELT-8
showed better agreement with manual HGB determinations than did the S-Plus, indicating an absence of WBC
interference in the HGB channel of the ELT-8.
Capillary
Specimens
For patient populations that include large numbers of
infants or persons whose veins have been traumatized
A.J.C.P. • August 1980
by repeated chemotherapy or transfusion, it may become necessary to depend on capillary blood specimens for daily follow-up of hematologic values. The
authors' institution falls in this category, and it therefore became essential to evaluate performance on
microquantities of blood obtained by fingerstick.
Figure 7 clearly indicates that results obtained on the
S-Plus using the Unopette system for capillary blood
collection showed better agreement with results on
venous blood for WBC, RBC, HGB, and PLT than
did results obtained using the Accuvette collection
system, although the latter system is recommended
by Coulter for use with the S-Plus. Large discrepancies
in venous/capillary PLT values were evident in specimens collected with the Accuvette system. Venous
PLT counts below 150 x 103/cu mm were consistently
reported higher on capillary specimens collected using
the Accuvette system, as compared both with capillary
specimens collected using the Unopette system and
with the venous specimens. These discrepancies in
PLT count using the Accuvette system were found to
be independent of minor discrepancies in HGB or
RBC values.
Both the S-Plus (using the Unopette system) and
the ELT-8 (using the Microtainer system) performed
acceptably on the majority of capillary blood specimens.
Since the venous values serve as reference, capillary
values for comparison measure within-system accuracy only, for each instrument. Absolute accuracy
of either venous or capillary values should not be
inferred from these data.
Mechanics of Capillary Blood Collection. Preliminary
fears that it would prove difficult to draw the 200-300
fjd of capillary blood required by the Microtainer
for use on the ELT-8 (in contrast to the 44.7 fi\ required for either of the two capillary collection systems
utilized on the S-Plus) were not borne out by our
clinical experience. Technologists were able to adapt
to the new system with a few days of practice, and
the extra volume required did not result in additional
discomfort for the patient. A single Microtainer specimen contained sufficient blood for two assays on
the ELT-8.
Aperture Clogging. In assaying capillary blood specimens with the S-Plus, we found that occasional aperture clogging occurred using the Unopette (in approximately 2 - 3 % of the specimens). In these instances,
corrective action was undertaken by the technologist
via a bleaching procedure to dissolve the debris. The
WBC apertures clogged more frequently than the
RBC/PLT apertures.
Platelet Clumping. In assaying capillary blood specimens with the ELT-8, we found that occasional PLT
clumping occurred in the Microtainer (in approxi-
Vol. 74 • No. 2
AUTOMATED PLATELET COUNTERS
mately 2 - 3 % of the specimens). Usually, this phenomenon was detected in the WBC histogram, which
for these cases appeared to show a " d u a l " population
of WBCs (Fig. 8); the "second" population, residing
in the lower threshold zone of the WBC histogram,
represents groups of clumped platelets, each " r e a d " by
the instrument as a small WBC. The net result was a
spuriously high WBC count and low PLT count in the
capillary specimens, compared with their venous
counterparts. These errors were screened out by observing the WBC histogram, and the specimens were
redrawn. With further experience, we found that almost
all of these WBC/PLT discrepancies were eliminated
by waiting five min between specimen collection and
testing.
Despite the technical care employed in the collection of the capillary specimens, some clinically significant discrepancies between venous and capillary
results did occur with all three capillary collection
systems. As seen in Figure 7, WBC and PLT values
showed the largest venous/capillary discrepancies.
These may make it difficult for the physician to monitor
and interpret small daily changes in the values of these
parameters. Therefore, the authors recommend that
venous specimens be run whenever possible on either
instrument to obtain the most accurate results.
Operation
The four technologists participating in this evaluation
experienced no difficulty in learning the routine
operating and maintenance procedures for all four
instruments. Because of the technologists' previous
familiarity with the Coulter S system, the staff initially
found the S-Plus easiest to operate; however, once
the operator became practiced in using the other three
instruments, routine operation was simple and uncomplicated.
The daily start-up and calibration procedures for all
four instruments presented no problem. The S-Plus
required approximately 30 min for daily start-up and
calibration check. The ELT-8 required approximately
15 min for daily start-up and calibration. Both the
MK-4 and Ultra-Flo 100 required approximately 20
min for daily start-up and calibration check.
Routine maintenance downtime was as follows: for
the S-Plus, 30 min for bleaching apertures, flushing,
and sandwich cleaning after every 250 cycles; for the
ELT-8, 15 min for flushing, reagent change, and PLT
control verification after every 100 cycles; for the MK-4,
15 min daily for flushing and air purging; for the UltraFlo 100, 5 min daily for flushing.
Reagent Use. As seen in Table 4, for multiparameter instruments, the S-Plus uses significantly more
149
FIG. 8. Leukocyte count (WBC) histogram with "'dual population"
obtained on ELT-8.11
diluent per test cycle than does the ELT-8; however,
the HGB reaction in the ELT-8 requires an extra
reagent. Both single-parameter instruments use comparable amounts of diluent per test cycle. In addition,
the Ultra-Flo 100 uses a constant 2 ml per min of sheath
fluid when the instrument is on, regardless of whether a
sample is being processed.
Test Time Per Cycle. With regard to the rate of sample
throughput, under optimal clinical conditions {i.e.,
continuous cycling, no instrument downtime, and
specimens with PLT counts in the normal ranges),
the S-Plus performs at a faster rate (34 sec/cycle)
than the ELT-8 (60 sec/cycle). For specimens with
low PLT counts (less than 50 x 103/cu mm), counting
time is extended on the S-Plus (50 sec/cycle). All
specimens with "no-fit" PLT size distribution on the
S-Plus require an additional 34 sec to obtain a hard
copy print.
The two single-parameter instruments required
varied amounts of specimen preparation prior to assay.
The total time required to prepare and assay a specimen is approximately 3 min on the MK-4 and 1 min on
the Ultra-Flo 100.
Downtime and Repair. The clinical portion of this
evaluation was not of sufficient duration to justify a
quantitative comparison of instrument downtime;
furthermore, any downtime data generated with the
instruments used in this evaluation would not be
representative of downtime found in other instruments.
Conclusions
It has been confirmed that the manufacturers of the
latest generation of automated blood cell counting
instrumentation have, by adding the capacity to perform
automated platelet counts, effected a major accomplishment that will contribute significantly to the clinician's
ability to manage patients with existing or potential
150
MAYER ETAL.
hematologic disease. With respect to the standard
multiparameter automated blood count, the S-Plus and
ELT-8 performed at least as well as currently existing
instrumentation, with the notable exceptions of slight
hematocrit drift in the ELT-8 and low MCV values
above 100 cu /i,m reported by the ELT-8.
Because the newest feature of the instrumentation
tested was ability to perform platelet counts, the data
and discussion presented in this paper concentrate
largely on the accuracy of these results, the major
rationale for acquiring these new instruments. In
general, platelet counts were performed reliably on all
instruments tested, but at times all instruments counted
particles and/or debris other than platelets (resulting in
spuriously high platelet counts) without adequately
flagging these specimens to alert the technologist to
possible error. In the experience of the authors' institution, with a large patient population including leukemia
patients and those receiving chemotherapy, the reporting of falsely elevated platelet counts represents
a serious deficiency, especially for counts below 70
x 103/cu mm. At these critical levels, sufficiently
accurate platelet counts must be generated to permit
the oncologist to evaluate hematologic trends in response to cytotoxic agents, to alert the clinician of
the danger of bleeding or necessity for platelet transfusion, and to assist in subsequent monitoring of the
patient to determine the effectiveness of transfusion.
The ELT-8 was the most reliable instrument for
platelet enumeration (P < 0.01) and reported the least
number of falsely elevated platelet counts. The MK-4,
S-Plus, and Ultra-Flo 100 required closer scrutiny to
A.J.C.P. • August 1980
avoid the reporting of falsely elevated counts at critical
levels. With all instruments tested, the role of the
hematology technologist in monitoring results remains
of paramount importance. In particular, for low platelet counts, slide check or phase count will continue to
be necessary. Despite these limitations, the availability
of platelet counts with every complete blood count in
one minute or less represents a major advancement
over existing instrumentation.
Acknowledgments. Technical assistance was provided by Alfred
Pukite, C.L.S., Duane Sadula, M.T.(ASCP), and other members of
the Hematology Department; also by Suzanne Hecht, M.P.H., and
Elana Friedman, B.S., of the Biostatistics Department.
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