Laboratory Evaluation of Differential White Blood Cell Count
Information from the Coulter S-Plus IV® and Technicon H-1® in
Patient Populations Requiring Rapid "Turnaround" Time
LYDIA NELSON, M.S., MT(ASCP), SH, SAMUEL CHARACHE, M.D., SANDRA WINGRELD, MT(ASCP), AND
EDWARD KEYSER, MT(ASCP)
Automated differential counts produced by the Coulter8 S-Plus
IV (S + 4) and the Technicon H-1® (H-1) were compared with
routine and reference manual differentials with the use of samples
from the adult emergency room (ER) and the neonatal intensive
care unit (NICU), populations in which rapid reporting of laboratory results is considered important. Error rates for routine
technologists were 9.3% in the ER and 15.3% in the NICU.
Error rates for the two instruments were higher than those for
technologists with samples from the ER but could be reduced to
4-5% if instrument flags and additional criteria were used to
signal the need for a conventional differential. Instrument error
rates were higher yet with NICU samples, and specificity was
very low (10% for each device). There were small differences
between the instruments in detection of immature neutrophils,
butflagsfrom the H-1 were more specific (except for detection
of nucleated red blood cells in samples from the ER). If either
instrument were used in an adult ER and flags and additional
criteria were used to signal the need for conventional differentials,
64-75% fewer manual counts would be performed with no decrease in accuracy and a considerable improvement in turnaround
time. (Key words: Differential leukocyte count; Automation and
flow cytometry) Am J Clin Pathol 1989;91:563-569
AUTOMATED HEMATOLOGIC INSTRUMENTS
such as the Coulter S-Plus IV® (S + 4), Technicon H-1®
(H-1), and others 2 8 1 0 " 1 5 1 8 are capable of generating
multiparameter blood counts and screening differentials
in approximately 1 minute or less. Because of inherent
analytic variability and cost of traditional eye-count differentials, many laboratories are relying on instrumentgenerated differential leukocyte counts to reduce operating
costs and provide rapid turnaround time.
Numerous studies have evaluated the reliability and
efficiency of automated electronic and cytochemical leukocyte differentials in adult populations,3'4-6'7-9'0"'516 but
few studies have evaluated such differentials in children.512
In this study we have investigated two patient populations
in which rapid reporting of test results is considered critical: the adult emergency room (ER) and the neonatal
intensive care unit (NICU). Our examination primarily
addresses the question of whether automated differentials
can be technically as accurate or contain the same infor-
Received June 23, 1988; received revised manuscript and accepted
for publication August 31, 1988.
Address reprint requests to Dr. Charache: The Johns Hopkins Hospital,
600 North Wolfe Street, Meyer B121D, Baltimore, Maryland 21205.
563
Departments of Laboratory Medicine and Medicine, The
Johns Hopkins Medical Institutions, Baltimore, Maryland
mation as manual examination of the traditional blood
film ("eye count"). If the latter is true, for the particular
patient populations studied, we asked if white blood cell
(WBC) "flags" associated with the automatic counters are
reliable guides to the need for traditional differentials. We
reassessed the method currently in use in our laboratory,
which combines a manual scan of the film with the use
of an automated microscope (see below). We also asked
if automated differentials based upon histochemical
staining and light scattering (five-part differentials performed by the H-1) are more useful than those based upon
electrical impedence (three-part differentials performed
by the S + 4). Because automated instruments "flag"
questionable results to indicate a need for further study,
are the flags reliable enough to safely curtail the use of
traditional differential methods, while providing the answers to questions considered important by clinicians?
To assess the clinical utility of electronic and cytochemical differential information, for a period of three
months, random blood samples from the ER and NICU
were run on both the S + 4 and the H-1. These samples
were compared with microscopic differentials, for correlation of results, instrument sensitivity and specificity, as
well as reliability and accuracy of instrument-generated
WBC flags (Coulter "R" flags and Technicon "NRBC,"
"blast," "left shift," "immature granulocyte," and "atypical lymphocyte" flags). Such a comparison of two hematology instruments, using different principles of operation,613 provides insight not only into problems of instrument operation, but also problems of unique patient
populations.
Because the study addressed differential WBC counts,
rather than examination of blood smears, assessment of
red blood cell (RBC) morphologic characteristics by either
technologists or automated devices was not examined.
Determinations of erythrocyte size, shape, and hemoglobin content are an essential part of patient management,
and omission of such data from this study should not be
construed as denial of their importance.
564
NELSON ET AL.
ER staff collects
K3 EDTA Vacutainer
or
NICU staff collects
2 K2 EDTA Microtainers
Run sample on H-1
Run sample on S + 4
Prepare blood film
Mlnlprep. Stain slide.
I
Perform 200-cell differential
on Hematrak 360 ("routine
differential"—see text)
Slide Review
One of eight reference technologists
does a 300-cell scanning review of
reported differentials.
I
Use 100-cell differential limits (14) as a
guide and perform 100-cell eye count
differential when discrepancies occur.
I
Reference technologists resolve
identification of questionable cells.
I
Use reviewed differential as "truth"
for instrument evaluation.
FlG. 1. A flow chart of the steps involved in analyzing differential data.
Materials and Methods
Blood Samples
Seven hundred seventy-six ER samples of venous blood
were collected in K3EDTA (Vacutainer®, Becton Dickinson Corporation), whereas 176 neonatal blood samples
were collected in K2EDTA (Microtainer®, Becton-Dickinson). Over a three-month period, the samples were collected from adult and neonatal patients (gestational age,
23-41 weeks) with a wide variety of diagnostic problems.
The adult samples were almost always collected by venipuncture, whereas pediatric samples were collected by
heel stick or from umbilical venous catheters. All samples
were collected only when requested by the physician caring
for the patient. The time between sample collection and
analysis was between 30 minutes and four hours for most
specimens.
Sample Handling
From the time of sample collection to testing, all samples were maintained at room temperature. Typically, two
A.J.C.P. «May 1989
Microtainers were collected from each child: one was used
for the S + 4 count and the other for the H-1, with the
blood film pulled at random from one of the Microtainers.
Both instruments were maintained according to manufacturers' guidelines. The S + 4 was calibrated against
fresh blood, and the H-1 was calibrated against the S
+ 4. The S + 4 calibration was checked every eight hours
against samples that had previously been assayed by reference methods, whereas quality control of the H-1 was
maintained with materials supplied by the manufacturer
as well as carryover samples previously analyzed on the
S + 4 counter.
In our laboratory, rather than routinely performing
traditional (or "eye count") differentials on all blood films,
differential leukocyte counts are routinely performed on
Wright-stained blood films with a Hematrak 360® (Geometric Data). Before starting the Hematrak count, a technologist scans the blood film for quality of preparation
and staining. If they are satisfactory, the instrument attempts to classify 200 WBCs. If the technologist's impression of the scanned film disagrees with the instrument
count, or if abnormal cells are present, a 100 or 200-cell
manual differential is done.
It became evident early in the study that incomplete
lysis of neonatal red blood cells in the peroxidase channel
of the H-1 produced inaccurate WBC data in more than
half of the samples (65 of 99). There were no problems
with red blood cell (RBC) lysis in the basophil channel,
and if initial WBC counts from the two channels disagreed,
and there was enough blood remaining, the sample was
diluted 1:1 with saline and reassayed. The diluted neonatal
samples generally generated reliable WBC count and differential results.
Data Analysis
Reference Method. To provide a basis ("truth") for
comparing electronic and cytochemical differentials with
traditional differentials, all routine differentials were reviewed by one of eight experienced technologists. At least
300 WBCs were scanned17; in cases of leukopenia, blood
smears were scanned for 5 minutes. If the reviewer's estimate for any cell line was outside the limits that might
be expected from sampling errors,17 a 100-cell eye-count
differential was performed: in cases of questionable cell
identification, a third technologist was used to settle disputes (Fig. 1). Errors on the original differential reports
were tabulated and the frequency of laboratory errors calculated.
The methods used to assess the automated differentials
differ from those recommended by the National Committee on Clinical Laboratory Standards.19 Those recommendations are not well-suited to conditions in our
laboratory, and alternative techniques were devised to
meet our physical andfinancialconstraints.
Vol.91 •No. 5
EVALUATION OF DIFFERENTIAL WHITE BLOOD CELL COUNT
565
Table 1. Criteria for the Evaluation of the
Correlation and Regression Analysis. Classic correlation
Reference Differential
and linear regression analyses were performed on WBCs,
RBCs, hemoglobin, and platelet counts as well as differThe manual differential was classified as abnormal if one of the
ential leukocyte data from all adult emergency room
following criteria were met (all absolute numbers are cells/mm3):
Any blasts are seen on the blood smear.
samples. The NICU data were not analyzed in this fashion
Promyelocyte absolute number a 100 cells.
because more than half the samples had to be diluted and
Unclassified young cell absolute number a 100 cells.
almost all samples had abnormal WBC distributions (i.e.,
Myelocyte absolute number a: 100 cells.
differential counts were flagged or not displayed).
Plasma cell absolute number a 100 cells.
Any combination of the above cells a 100 cells (i.e., 50
Overall Efficiency. Sensitivity, specificity, and efficiency
myelocytes plus 50 promyelocytes).
for detection of abnormal samples were determined for
Metamyelocytes a 3% or absolute number a 1,500 cells.
Atypical lymphs a 5% or absolute number a 500 cells.
the two automated systems. Reference differentials were
Bands S; 10% or absolute number S; 1,000 cells.
considered normal if they did not violate any of the criteria
NRBCs ^ 3% or absolute number a 200 cells.
in Table 1. S + 4 data were judged abnormal if the inEos ^ 8% or absolute number a 700 cells.
Basos i 3% or absolute number s 200 cells.
strument generated aflagor backlighted a parameter, and
H-1 data were considered abnormal if a WBCflagor star*
was generated. If no flags, backlighting, or stars were displayed, the differential was considered normal by instrutrophils) and lymphocytes enumerated by either counter
ment standards.
showed excellent correlation with reference differential
counts. H-l eosinophil counts showed a fairly good corReliability of Flags. The final instrument evaluation
involved a study of the reliability of individual WBC flags. relation (r = 0.811) with manual methods, but H-l and
Comparisons were made between the reference differenS + 4 mononuclear or monocyte counts and H-l basophil
tial, impedence differential, and the cytochemical differcounts did less well. These differences were undoubtedly
ential.
exaggerated by the inaccuracies of our reference differentials,17 but the calculations may overemphasize differEach instrument-generated WBCflag(or lack of one)
was classified as true or false positive or true or false neg- ences that are clinically not significant.
ative, based on the criteria listed in Table 2.
Routine Differential Count Error Rate
(Technologist Errors)
Results
Sample Handling
Preliminary studies showed that blood counts from
Vacutainers and carefully collected Microtainers were
stable from 30 minutes to six to eight hours. Conventional
differentials performed on films prepared from two Microtainers collected one after the other from the same
infant showed considerable differences, particularly in the
proportion of cells classified as lymphocytes. Discrepancies between the pairedfilmswere greater with some reference technologists than with others. The consensus
method used to establish "truth" minimized such differences but probably did not eliminate them.
Correlation and Regression Analyses
The two instruments showed good agreement (Table
3) in measurement of hemoglobin concentration and
WBC, RBC, and platelet counts. Granulocytes (or neu-
* Within the context of this study, "backlighting" of a result on the
S + 4 report indicates failure of WBC distribution criteria in addition
to those that evoke R-flags, or a mononuclear cell number in excess of
1,500/mm3. "Starred" results on the H-l indicate disagreement in the
WBC count between the peroxidase and basophil channels or failure of
other instrument "self-check" analyses.
When eight experienced reference technologists reviewed 776 routine emergency room differentials and 176
routine neonatal differentials, we found considerable differences in error rates. Samples from the ER had a 9.3%
(72 of 776) error rate, whereas those from the NICU had
a 15.3% (27 of 176) error rate. Both error rates are higher
than our earlier laboratory error rate of 7%,12 although
none of the errors were of sufficient magnitude to have
altered clinical management. The most common errors
with samples from both sites were in classification of band
and immature neutrophils and monocytes.
Sensitivity and Specificity Studies
Overall, 224 blood films from adults (29%) and 135
from neonates (77%) were considered abnormal, with 1.7
and 2.3 abnormalities per film, respectively. Among the
ER samples, 276 were flagged by the S + 4 and 192 by
the H-1. Sensitivity and specificity data from samples from
both sites (Table 4) showed only slight differences between
the two instruments, when their ability to detect abnormal
samples (regardless of type of abnormality) was compared.
For ER patients, the S + 4 had a slightly better sensitivity
rating than the H-l, whereas the H-l had a better predictive value for abnormal specimens and a slightly better
efficiency. Data from newborns showed little or no dif-
566
N E L S O N ET AL.
AJ.C.P. • May 1989
Table 2. Criteria for the Evaluation of Flags for the Coulter S-Plus IV and Technicon H-l*
Coulter
Flag
Technicon
Flag
Blood Smear
Criteria
R-1
N
True positive
False positive
False negative
Theflagis present and NRBCs are noted on the blood smear.
Theflagis present and no NRBCs are noted on the blood smear.
There is noflagand there are a3% NRBCs or a300 cells.
R-2
Blast
Atyp
IG
True positive
True positive
True positive
There is aflagand at least one blast is seen on the blood smear.
There is aflagand atypical lymphs are i 5 % or &500 cells.
There is aflagpresent and young cells, myelocytes, and
promyelocytes are a 100 cells or metamyelocytes &3% or 200
cells.
R-3
L-shift
True positive
There are a 15% bands or & 1,500 immature granulocytes.
R-M
—
True positive
Theflagis present and at least one or more of the R-1 through
R-3 criteria are met.
True positive
Theflagis present and any abnormal diff criteria are met.
' The criteria for false positive and false negative flags are similar to those for R-! or N flags.
ference in overall performance between the two instruments.
Studies by Payne and Pierre14 suggested that instruments could vastly improve their ability to detect samples
with abnormal differential counts by combining instrumentflagswith "check and review" limits. When all but
two (scaled histograms and y-axis take ofF>2 mm) of the
check and review limits of Payne and Pierre14 were applied
to the unflagged abnormal ER samples, we found the S
+ 4 missed 39 samples or 5%. Missed abnormalities included increased band counts (24), eosinophils (8), atypical lymphocytes (3), basophils (3), and monocytes (1).
The H-l missed 34 (4.4%) of the abnormal samples. These
abnormalities included increased band counts (27), atypical lymphocytes (3), nucleated RBCs (2), and monocytes
(2). Based on thesefigures,if we had used only instrument
Table 3. Correlation Between Automated and
Reference Differentials (specimens
from adult emergency room)
Measurement
flags and check and review limits, the Coulter would have
safely reduced manual differentials by 64% and the H-l
would have reduced them by 75%.
Reliability of White Blood Cell Flags
The accuracy of individual WBC flags was evaluated
with the use of manufacturer's criteria (Table 1). For example, an increased number of band neutrophils should
generate an "R-3" flag on the S + 4 (or an RMflagif
multiple abnormalities are present) and a "left shift" flag
on the H-1; more immature neutrophils should generate
"R-2" and "immature granulocyte"flags;and nucleated
RBCs should generate "R-1" (S + 4) or "N" (H-l) flags.
In the adult ER population, the S + 4 generated more
true positive and fewer false positive flags than did the
Table 4. A Comparison of Electronic and Cytochemical
Screening Differentials for Adult and Pediatric Patients
ER
r
Test Method
H-l versus S-Plus IV
WBC
RBC
HGB
Pit
0.996
0.989
0.997
0.984
H-l versus reference differential
Neutrophils
Lymphocytes
Eosinophils
Monocytes
Basophils
0.941
0.935
0.811
0.634
0.242
S-Plus IV versus reference differential
Granulocytes
Lymphocytes
Mononuclear cells
0.938
0.937
0.488
Sensitivity—
% true positive
Specificity—
% true negatives
Predictive value of
normal
Predictive value of
abnormal
Efficiency—
% of samples
correctly
classified
NICU
Coulter
Technicon
Coulter
Technicon
57
50
97.0
97.8
71
81
9.8
9.8
84
80
50.0
57.1
38
51
77.9
78.1
68
72
76.7
77.2
Note: These data reflect only the ability of flags to detect abnormal samples (i.e., those that
require conventional differentials). They do not combine the use of "check and review limits"
with instrument flags, as recommended by Payne and Pierre."
Vol.91 'No. 5
567
EVALUATION OF DIFFERENTIAL WHITE BLOOD CELL COUNT
Table 5. Reliability and Frequency of Individual Flags as Detectors of Specific Abnormalities
in the WBC Differentials of Adult ER Patients
Emergency Room Population (N = 776)
Coulter
Flag
Technicon
Hag
Total
T+
F+
Total
N
8
(14%)
49
(86%)
57
16
IG*
7
(78%)
21
(57%)
4
(24%)
2
(22%)
16
(43%)
13
(76%)
9
3
37
5
17
0
Lt Shift* Bands £ 15%
25
(49%)
26
(51%)
51
76
106
171
100
F-
T+
F+
R-1
11
(73%)
4
(27%)
15
4
R-2
15
(41%)
22
(59%)
37
12
ATL
Blast
R-3* Bands ;> 15%
20
(37%)
34
(63%)
54
57
R-M*
35
(36%)
62
(64%)
97
8
—
30
(50%)
30
(50%)
60
NA
Totals
111
152
263
81
• Discrepancies between numbers of flags result from cross-flagging (i.e.. increased band counts
may be flagged by "R3" or "RM" or by "Left Shift" or "IG").
H-1 (Table 5), whereas in the neonatal population there
were more false positive and negativeflagswith the H-1.
Overall, the reliability of the two instruments was similar
with samples from the adult ER, with only 42% (Table
65
F-
T+ = true positive: F+ « false positive: F - <= false negative.
5) of the S + 4 and 38% of the H-1 WBCflagscorresponding to abnormalities seen on the blood smear. In the NICU
population, the S + 4 WBCflagscorresponded to blood
smear abnormalities 71% (Table 6) of the time, whereas
Table 6. Reliability and Frequency of Individual WBC Flags as Detectors of Specific
Abnormalities in the WBC Differential
NICU (N = 176)
Coulter
Flag
Flag
Total
F-
Technicon
T+
F+
R-1
20
(77%)
6
(23%)
26
5
N
19
(86%)
3
(14%)
22
15
R-2
63
(63%)
37
(37%)
100
3
IG*
19
(86%)
18
(56%)
0
(0%)
3
(14%)
14
(44%)
150
(100%)
22
4
32
4
150
0
12
(29%)
42
10
182
268
33
T+
ATL
Blast
R-3* Bands £ 15% or 1,500
11
(85%)
2
(15%)
13
8
R-M*
71
(76%)
23
(24%)
94
11
—
9
(75%)
3
(25%)
12
0
Totals
174
71
245
27
' Sec footnote to Tahlc 5. T+ •= true positive: F+ = false positive; F - = false negative.
Lt Shift* Bands 2: 15% or 1,500
30
(71%)
86
F+
Total
F-
NELSON ET AL.
568
the H-1 corresponded 32% of the time. If false positive
blast flags were ignored, performance of the two instruments was very similar (71 vs. 73% "correct" flags).
Discussion
Electronic and cytochemical differential counters have
made tremendous advances over the last ten years. By
decreasing the number of conventional differentials, their
use can reduce laboratory operating costs, decrease turnaround time, and yield a greater precision and accuracy
than most manual differential counts. Used for that purpose, the questions of interest are the numbers of truly
abnormal samples detected or missed and the number of
false positive results. In the present study, "flags" generated by both instruments did not do particularly well in
identifying abnormal samples (Tables 4-6). Atfirst,such
data seem contrary to previously published reports,912 but
if a need for conventional differentials is based on both
instrument-generatedflagsand check and review limits,14
we found the S + 4 missed only 5% and the H-1 only
4.4% of the abnormal adult samples. Both instruments
did better than our technologists, who had a 9.3% error
rate on adult samples. Such results clearly suggest that
the combination of instrumentflagsand check and review
limits, used by the operator or an online computer system,
can yield results at least as accurate as conventional differentials when used for patients similar to those in our
adult ER.
A similar statement cannot be made for the performance of either instrument in the NICU. Both the S + 4
and the H-1 generated some type of WBCflagfor almost
all (97%) samples from the NICU. Increasedflaggingwith
NICU samples appeared to result largely from two factors.
Many patients in the NICU were very sick, but, perhaps
more important, both the S + 4 and the H-1 have their
WBCflaggingcriteria based on adult population norms.
Neonates (especially premature infants) generally have
larger and less mature appearing lymphocytes than adults.1
The S + 4 generated R-2flagsfor these larger cells (110
R-2 or R-M combinations/176 counts), and the H-1 generated 150 false positive blastflags(Table 6). The increased
proportion of large unstained cells (H-1) that we observed
(6.5% NICU mean, normal adult range, 0-4%) may reflect
a similar problem with age-related norms.
For both the ER and the NICU patients, the reliability
and frequency of individual WBCflagsprovides some
additional insight into instrument capabilities (Tables 5
and 6). In both locations, physicians are particularly interested in accurate assessment of the proportion of immature neutrophils. Among ER samples, the H-1 was
slightly better atflaggingleft shifts than the S + 4 ("left
shift" vs. "R3"flags,49 vs. 37% true positive results).
Among NICU samples, the H-1 performed well at flagging
A.J.C.P.-May 1989
younger granulocytes (86% true positive results), whereas
the S + 4 was slightly better atflagginghigh band counts
(85% true positive results). Overall, the H-1 missed about
as many abnormal samples as did the S + 4 (33 vs. 27
false negative results of 176 NICU samples).
Regardless of patient population, recognition of the
presence of nucleated RBCs is important not only because
large numbers of them can affect the WBC count, but
because smaller numbers can have ominous significance
in certain patient populations. Among ER samples, the
S + 4 outperformed the H-1 in this regard (73% true positive vs. 14% true positive results), but the two instruments
were quite similar when analyzing samples from the NICU
(77% vs. 86% true positive results).
From a laboratory standpoint, the Coulter S + 4 and
H-1 have various advantages and disadvantages, depending on the patient population and laboratory needs, and
both are useful in laboratory practice. WBCflagsserve as
a useful adjunct, guiding the technologist to possible leukocyte abnormalities and the need for a conventional differential. If the initial instrument count were released to
the physician as an interim count, before a conventional
differential were performed, the H-1 report would be more
advantageous. Knowledge of the eosinophil count from
the H-1 could be quite valuable in some circumstances,
and the H-1 could guide the physician to other specific
WBC abnormalities (left shift, atypical lymphocytes, nucleated RBCs, blasts). The S + 4flags,on the other hand,
are so general that the R-2, R-M, and . . .flagswould
alert the physician to anything from increased eosinophils
to blasts. From the point of view of a physician in an
acute care situation, the important question may not be
whether or not a patient's blood film is abnormal, but
whether or not a "left shift" is present. In such a circumstance, if one interpreted flags literally (contrary to manufacturers' instructions), the H-1 would be a more useful
instrument for rapid test results.
Our study reflects instrument operations in actual clinical practice. None of our samples received special treatment, and none of our analyses were repeated. We believe
such a test environment is a more useful evaluation of an
instrument's capabilities than its use in either a basically
normal population or an extremely abnormal population
(leukemia patients).7 Ourfindingsindicate that by combining WBCflagswith check and review limits, a differential generated by either instrument in an adult ER can
be a safe and effective screening technique. It is unlikely
that conventional differential counts can be completely
replaced by electronic or cytochemical blood examinations, but the data presented suggested that they could be
safely reduced by 64-75% in the adult ER. Our current
turnaround time from the ER (including transit time) is
about 148 minutes for conventional differentials, depending upon the time of day. If automated differentials
Vol.91 -No. 5
EVALUATION OF DIFFERENTIAL WHITE BLOOD CELL COUNT
were used, the reporting interval could be shortened to
about 80 minutes for most samples. For those requiring
manual differentials, accuracy could be increased if technicians spent more time at the microscopes with each
slide, probably with little or no increase in overall turnaround time.
Acknowledgments. The authors thank Dr. E. Simson and Dr. D. Saw
for providing them with the opportunity to evaluate the Technicon
H-l and for help with correlation and regression analysis. They also
thank the staff of the Hematology Laboratory for their valuable assistance
and the staff of the Adult Emergency Room and Neonatal Intensive Care
Unit for their cooperation.
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