HEMATOPATHOLOGY
Original Article
A Field Evaluation of the Coulter STKS®
BRIAN A. WARNER, F.I.M.L.S., AND DAVID M. REARDON, F.I.M.L.S.
The performance of the Coulter STKS® (Coulter, Hialeah, FL)
was evaluated in a busy computerized teaching hospital laboratory. The STKS was compared with a Coulter S Plus IV and
manually performed 400 white blood-cell differentials. The
measured blood-count parameters (i.e., white blood cells |WBCs|,
red blood cells |RBCs), hemoglobin |Hb|, mean corpuscular volume |MCV), and platelets |PLTs]), compared very well between
the two aperture impedance-based systems; precision, linearity,
and lack of carryover were excellent. The STKS WBC differential
(DIFF), derived from a combination of aperture impedance, aperture conductance, and laser light scatter, also was precise; linear
and carryover were insignificant. The DIFFs (n = 424) compared
well to the manual WBC differentials, with r values of 0.97,
0.97, 0.73, and 0.86 for neutrophils, lymphocytes, monocytes,
and eosinophils, respectively. The DIFF and Suspect Flagging
system produced 6.2% false negatives and 2.6% false positives
when compared with the manual technique. These were further
investigated and discussed. STKS DIFFs were stable for 18 to
24 hours in normal samples anticoagulated with K2EDTA and
stored at 20 °C prior to analysis. Storage in the same anticoagulant at 4 °C and immediate aspiration preserved the DIFF
analysis for considerably longer than 24 hours. These performance characteristics make the STKS a significant advancement
in automated hematology. (Key words: Automated hematology;
Blood count; WBC differential) Am J Clin Pathol 1991;95:207217
During the last ten years considerable improvements have
been made to hematology analyzers. Platelets are enumerated, in the presence of red blood cells (RBCs), from
one aspiration of whole blood, and at the same time cell
sizing parameters are produced, including white blood
cell (WBC) differential screening information. The principles of aperture impedance or light scatter have been
used in this process.
The automation of the full WBC differential count1 has
proven more difficult. Light scatter, coupled with automated cytochemistries2 or pattern recognition systems,3
have been two contrasting technologies employed to provide this information. More recently conductivity has been
developed to complement aperture impedance, and coupled with either differential cell lysis4 or laser light scatter,5
to produce data comparable to the traditional WBC differential. From a managerial point of view, it has proven
preferable to have these combination technologies harnessed together in one analyzer capable of simultaneously
producing full blood count, sizing information, and full
WBC differentials from one aspiration of whole blood.
The complexity and cost of such analyzers necessitates
detailed evaluation procedures at various levels using recommended protocols.6 These protocols suggest comparing
the new technologies with reference methods using normal
blood samples and a diverse range of pathologic samples.
Unfortunately, recent technical innovations have occurred
at a rate that has made full evaluation procedures for each
new analyzer impractical. In contrast, a field evaluation
compares the new technology with the one(s) for which
it is a candidate to supersede.
This paper presents a field evaluation of the Coulter
STKS® (Coulter, Hialeah, FL), which performs the combination of aperture impedance with conductivity and
laser light scatter, to produce full blood counts, including
full WBC differentials from one automated analysis.
. From the Department of Hematology. Addenbrooke's Hospital, Cambridge, England.
Received December 28, 1989; received revised manuscript and accepted for publication May 4, 1990.
Address reprint requests to Mr. Warner: Department of Hematology,
Level 3, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ,
England.
207
INSTRUMENT
The Coulter STKS is a combination of the STKR 7 and
the VCS5 analyzers, incorporating automatic closed-vial
sample handling and a 5-population WBC differential
(DIFF). RBC, WBC, and platelets (PLTS) are enumerated
HEMATOPATHOLOGY
Original Article
208
and sized on the STKS using the Coulter aperture impedance method. 8 The DIFF is obtained by using low-frequency direct current and high-frequency electromagnetic
probes combined with laser light, measuring the cell volume, conductivity, and light scatter, respectively. The differential is performed in the flow cell of the triple transducer located at the front of the instrument.
The triple transducer analyzes as many as 8,192 cells
on each sample, but will stop if this number is not reached
within 20 seconds; this compares to the 90-second count
period on the VCS. Throughout the evaluation the triple
transducer is controlled with Coulter Latron particle control material. Whether in the automated (primary) or
manual (secondary) mode, aspirated blood is divided into
three portions, two for the RBC, WBC, hemoglobin (Hb),
and PLT count, and the third for the DIFF. Therefore,
the WBC is produced from a separate dilution to the DIFF.
The instrument can be operated with or without the DIFF
function.
STKS reagents include those used for current Coulter
instruments, namely Isoton® HI diluent, Coulter Clenz®
and Lyse S® III diff. The Scatterpak (which also is used
on the VCS) contains Erythrolyse (specially formulated
for the STKS) and Stabilyse. Isoton III, as well as being
used as a diluent for cell counting, is used as a sheath
fluid to hydrodynamically focus WBCs during the DIFF
analysis. WBCs are differentiated in their near native state.
Lyse S HI diff is used for the lysing of red cells for the
WBC count and the estimation of hemoglobin.
Instrument data are handled by the Data Management
System (DMS), which consists of a microcomputer with
a 40-megabyte fixed and 1.2-megabyte floppy disk. The
microcomputer is multitasking and has the facility for
bidirectional communication with a host computer. The
software (edition 1 A) enables the storage of all information, including scattergrams on as many as 1,000 samples.
Early editions of the software documented the number of
cells counted and the time taken to count the cells passing
through the triple transducer during each analysis. This
information has been removed from the final evaluation
of edition 1 A. Since the evaluation, the software has been
developed to an edition ID. This differs from 1A only in
the addition of control files, data base interrogation, and
help screens.
The STKS flagging system is different from that used
on other Coulter instruments, particularly in interpretive
reporting (IR) and region alarms (R or X flags). These are
replaced by more specific flags, which are either software
generated (suspect flags) or user definable (definitive flags).
Stored in the microcomputer and available for hard-copy
printout (monochrome or color) from the DIFF process
are the conventional XY scattergrams, histograms, and
digital information. Similarly, the aperture impedance
information for RBC and PLT parameters, including volumetric histograms, is stored available to print. A composite of two scattergrams indicating the position of normal cell types derived from aperture impedance volume
and light scatter (DF1) and aperture impedance volume
and conductivity (DF2) is shown in Figure 1.
MATERIALS AND METHODS
Carryover
Carryover was established by using the method of
Broughton and colleagues.9 A sample with high levels of
analyte was aspirated three times (a b a2, a3), this was then
immediately followed by three aspirations of a sample
with low analyte levels (b|, b 2 , b 3 ). Carryover percentage
was then calculated from the following:
(b, - b3)
X 100%
(a3 - b3)
Carryover also was evaluated by a program in the DMS.
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Conductivity
A.J.C.P. • February 1991
FlG. 1. Normal scattergrams illustrating
(A) aperture impedance volume (y-axis)
against light scatter (x-axis) and (B) aperture
impedance volume (fL) (y-axis) against aperture conductivity (x-axis). The positions
of the normal cell populations are indicated
(a) lymphocytes, (b) monocytes, (c) neutrophils, and (d) eosinophils. Signals occurring
in position (e) are classified as cellular debris.
Basophils are identified as a discrete population by using both light scatter and conductivity.
209
WARNER AND REARDON
Coulter
This method differed only in that diluent (Isoton III) was
used to replace the low analyte.
Precision
Intrabatch precision was performed by replicate analysis
(n = 30) of the same sample. This exercise was repeated
on a number of occasions, using either fresh blood or
control material, covering a wide range of cell counts.
Interbatch comparability was determined by analyzing
batches of samples (n = 30) over varying time periods.
Three batches were analyzed within 30 minutes of venepuncture, one batch was reprocessed at 2 hours, the second
at 6 hours, and the third at 12 hours. The exercise also
was performed when the instrument had been shut down
and restarted between batch analysis.
The precision of the primary and the secondary mode
was compared by analyzing batches of the same samples
(n = 20) in both operating modes.
Linearity
Linearity was established over as wide a range of cell
counts as possible. Samples were concentrated by centrifugation. Then ten accurate, evenly spaced dilutions
(10-100%) were performed on each sample. Triplicate
analyses were made at each dilution point. Dilutions were
made in either autologous plasma or AB plasma. The
following ranges were used: WBC, 1.0-100 X 109/L; RBC,
0.8-8.66 X 10 I2 /L; Hb, 28-280 g/L; PLT, 10-1,000
X 109/L. Linearity curves were assessed statistically by
using the method of England and colleagues.10
Storage in Di-Potassium Ethylene diamine
Tetraacetic Acid
Blood was collected from 25 laboratory staff and anticoagulated with di-potassium ethylenediamine tetraacetic acid (EDTA) at a concentration of 1.5 mg/mL blood.
Each sample was divided into aliquots. A new aliquot was
aspirated at each time point. Storage temperature was
strictly controlled at 20 °C or 4 °C. In all, 17 time points
were used, extending to 48 hours. Sampling was regular
within the first hour, then hourly up to 6 hours, after
which a wider time span was used. Time zero was within
1 minute of the blood coming into contact with EDTA.
Samples stored at 4 °C were initially analyzed cold,
within 1 minute of being taken from the refrigerator, then
reanalyzed after a 15-minute warm-up period at 20 °C.
Blood Count Comparability
The STKS blood count data were compared with two
Coulter S Plus IVs, both being calibrated with the same
batch of S-Cal, and controlled daily with high, normal,
Vol.
and low 4C Plus, and an internal laboratory standard.
The STKS was entered into a weekly regional external
quality assessment scheme (EQA) based at Addenbrooke's
Hospital. A total of 46 instruments were involved in the
scheme.
Differential Correlation
Samples used for the correlation study were randomly
taken from specimens received in the laboratory during
a two-week period and represented a good cross section
of normal and abnormal hematological samples analyzed
at Addenbrooke's Hospital. A wedge film was made from
each sample, which was stained with Wright's stain using
a Hema-tek® (Miles, Slough, England) automatic staining
machine. A 200 WBC manual differential was performed
independently by two experienced technologists, both using the same wedge film. The mean of the differential
results obtained was expressed as a percentage. Any film
showing evidence of changes caused by EDTA storage
was excluded from the study.
A STKS analysis also was performed on each sample.
Again any sample showing significant evidence of an
EDTA storage effect was excluded. Significant dispersion
of the cell clusters and accumulation in the debris box,
as illustrated in Figure 2, were used as exclusion criteria.
Differential
Comparability
Comparability between the manual WBC differential
and the STKS DIFF was established retrospectively on
the random samples used for the correlation study. The
criteria used to judge comparability were as follows:
The STKS was deemed positive when one or more of
the following were present:
1. a distributional abnormality according to the laboratory's reference ranges {e.g., neutrophilia, lymphocytosis);
2. a suspect WBC flag,
imma grans/bands,
blasts,
variant lymphs, or
nucleated red cell flag;
3. an abnormal scattergram, where populations crossed
thresholds but were not flagged.
The blood film was deemed positive when one or more
of the following were present:
1. a distributional abnormality (as previously described);
2. abnormal cells,
any blast cells,
myelocytes,
promyelocytes,
• No. 2
210
HEMATOPATHOLOGY
Original Article
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0.63
0.31
0.17
0.29
0.41
0.24
0.45
Other Aspects
During the evaluation other operating aspects of the
instrument, which are important to routine hematological
practice, were observed and documented. These included
average throughput of samples, blood aspiration volumes,
mixing efficiency in the primary operating mode, DIFF
performance with leukopenic blood samples, analytical
interference from substances or pathological states, reliability of the evaluation analyzer, and ease of use.
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85.0
WBC(X10 9 /L)
6.64
RBC(X10' 2 /L)
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246
PLT(X10'/L)
800.0
Neutrophils (XI0 9 /L)
82.0
Lymphocytes (X109/L) 42.9
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Ranges Tested (Boughton and
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High
Low
colleagues)
DMS
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24 hours RT
FlG. 2. Qualitative effect of K?EDTA storage on STK.S differential
scattergram in one normal individual. Volume (fL) (y-axis) and light
scatter (x-axis) displayed at 1, 12, 18, and 24 hours.
RESULTS
Carryover
Carryover (Table 2), using either calculation, was found
to be acceptable, and significantly less than 1 % on all occasions.
nucleated red blood cells,
variant lymphocytes, or
>0.05 (>5%) metamyelocytes or band cells.
When comparing distributional abnormalities around
the upper and lower limits, differences between STKS and
the manual count were recorded only when the two disagreed by >0.05 (>5%) for neutrophils or lymphocytes
and >0.03 (>3%) for any other cell type.
Table 1 shows the patient data base (n = 424) and the
abnormalities that were found on the manual film examination.
TABLE 1. PATIENT D A T A BASE
Abnormality
No. of Patients
Demonstrating
Abnormality
% of Total
Patients
(n = 424)
No abnormality
Neutrophilia
Lymphocytosis
Monocytosis
Eosinophilia
Basophilia
Lymphopenia
Neutropenia
Myelocytes
>0.05 (>5%) Metamyelocytes/bands
Nucleated red blood cells
Blast cells
Variant lymphocytes
210
128
11
17
7
1
84
8
36
65
20
6
8
49.5
30.0
2.6
4.0
1.7
0.2
19.8
1.9
8.5
15.3
4.7
1.4
1.9
Precision
All of the precision and batch comparability studies
(Tables 3 and 4) were acceptable and within the manufacturer's claims for the STKS. Coefficients of variation
(CV) are given for all of the measured parameters, the
five from the standard blood count and thefivepopulation
DIFF, for replicate sampling (Table 3). The CVs of the
five measured blood count parameters are, not surprisingly, similar to those published from aperture impedance
devices. The CVs for the DIFF are superior to those obtainable routinely when performing manual WBC differTABLE 3. INTRABATCH PRECISION
WBC(X10 9 /L)
RBC(X10 ,2 /L)
Hb (g/L)
MCV (fL)
PLT(X10 9 /L)
Neutrophils (XI0 9 /L)
Lymphocytes (XI0 9 /L)
Monocytes (X109/L)
Eosinophils (X109/L)
Basophils (X109/L)
A.J.C.P. • February 1991
Range
Average CV%
4.0-19.0
2.41-5.20
64-173
75.6-94.8
68-435
53.3-54.7
35.4-37.5
5.0-6.0
2.5-3.7
0.0-1.0
1.42
0.48
0.46
0.24
3.28
0.7
1.9
5.9
11.0
53.4
WARNER AND REARDON
Coulter
211
STKS®
TABLE 4. INTERBATCH COMPARABILITY
Batch 2
Batch 1
WBC(X10 9 /D
RBC(X10 ,2 /L)
Hb (g/L)
MCV (fL)
PLT(X10 9 /L)
Neutrophils (XI0 9 /L)
Lymphocytes (X109/L)
Monocytes (XI0 9 /L)
Eosinophils (X109/L)
Basophils (XI O'/L)
Batch 3
<0.5h
2h
<0.5 h
6h
<0.5 h
12 h
7.7
4.07
128
92.5
294
65.5
26.6
5.0
2.1
1.0
1.1
4.07
128
92.6
295
65.3
26.9
4.9
2.2
0.8
8.3
4.47
135
94.1
318
60.1
28.9
7.5
3.3
0.1
8.5
4.53
136
94.0
325
58.7
31.7
6.2
3.1
0.2
12.1
4.54
133
91.3
197
68.9
20.7
6.2
3.6
0.5
12.1
4.46
131
91.1
190
65.0
25.4
6.5
2.8
0.2
Means of three batches analyzed within 30 minutes of blood collection and reanalyzed after 2-hour (batch 1), 6-hour (batch 2) and 12-hour (batch 3) intervals.
ential counts and comparable to the diverse group of
technologies now used and previously used for this procedure.
No significant difference between the precision of the
primary and the secondary operating modes was observed.
Linearity
The linearity studies, throughout a very wide concentration range for all of the measured parameters, showed
an apparently excellent response, slope and intercept. The
calculated RBC indices responded as expected in a linearity study using the data from the RBC and Hb dilution
experiments. However, using the formulae advocated by
the ICSH,10 the Hb and RBC apparently were nonlinear
at the lower end of the dilution range. This may be attributable to the extent of the experimental range employed. While this was statistically significant, it was not
of practical importance. Similar findings have been reported previously when assessing aperture impedance"
and light scatter technology.12
Storage in EDTA
The qualitative effect of blood storage in EDTA on the
differential scattergram of one individual is shown in Figure 2. A loss of neutrophils into the debris box was evident
at 12 hours and became more significant at 24 hours. This
resulted in a relative increase in lymphocytes. Eosinophils
and monocytes also showed a reduction over time, with
changes at 24 hours of storage apparently significant. The
storage effect was quantified by studying 25 normal individuals at regular time points for 48 hours of storage at
20 °C. Each data point represents the mean of the 25
analyses. In Figures 3 and 4, mean percentage changes in
neutrophil, lymphocyte, and monocyte counts were plotted against storage time. In Figure 5 the eosinophil and
basophil counts were plotted against storage time. Changes
of 10-20% of the original value occurred in the STKS
DIFF at 18-24 hours storage at 20 °C in EDTA. In ad-
dition, suspect flags appeared in 4 of 25 of the normal
samples studied at the 24-hour time point. Acceptable
DIFFs were obtained at time zero, eliminating the need
for a stabilization period in EDTA, presumably because
of the measurement of cells in their "native" state.
A different pattern was observed in samples stored at
4 °C (Figs. 6 and 7). Samples (n = 6), when analyzed
cold, appeared to remain more stable than the same samples allowed to warm to room temperature before analysis.
When the same samples were warmed for 15 minutes at
20 °C, some lymphocytes were counted as neutrophils.
Clear discrimination between cell types was maintained
for considerably longer if the analysis was made immediately from storage at 4 °C. There were no significant
changes in any of the count parameters as a result of storage at 4 °C.
Blood Count Comparability
The blood count data derived from the STKS compared
excellently with the Coulter S Plus IV data. This was expected because the analytical principles of both analyzers
are precisely the same. Therefore, the statistical analysis
of the data is not shown. Moreover, the S Plus series of
instruments has been extensively evaluated and is commonly employed in routine practice.
During the three-month evaluation period, the STKS
performance within the Addenbrooke's quality assurance
scheme was good. Each of the weekly results fell well
within 2 SDs of the population mean. The cumulative
performance over a nine-week period is shown in Figure
8. Similar observations were made with the limited number of National External Quality Assurance Scheme13
samples received during the evaluation period.
Differential Correlation
The correlation coefficients and linear regression analysis for the differential parameters are shown in Table 5.
Vol. 95 • No. 2
212
HEMATOPATHOLOGY
Original Article
110
NEUTROPHIL/LYMPHOCYTE STORAGE
AT ROOM TEMPERATURE (n=25)
90
70
50
111
30
§
10
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—•—
*»••••
-50
-70
-90
I
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5 10 15 30 45
(min)
Neutrophils
Lymphocytes
I
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I
" I
1
2
4
5
3
I
1
I
T " I
I —
6 12 18 24 30 48
(h)
Time
110
MONOCYTE STORAGE AT
ROOM TEMPERATURE (n=25)
90
70
UJ 50
O 30
7
< 10
O -10
analyses. This is reflected in the scatterplots and statistical
analysis of the data comparing observers 1 and 2. Furthermore, a stronger relationship between the two manual
techniques is evident from this data than from data previously published in similar evaluation studies. 1415 Consequently a degree of confidence was expressed when the
automated DIFF procedure was compared to the manual
data. Therefore, the NCCLS recommended method H20T 16 for manual WBC differential counts was not used in
this study.
Acceptable correlation coefficients of 0.97, 0.97, 0.73,
and 0.86 for neutrophils, lymphocytes, monocytes, and
eosinophils, respectively, were obtained in this study.
These compare favorably to other technologies in similar
studies. Comparison of the two techniques for basophils
is statistically invalid. Unfortunately, a significant number
MiHii
40
-30
-70
-90
NEUTROPHIL STORAGE AT 4°C
(n=6)
30
-50
20"
-i
1
0
5
1
1
1
1
10 15 30 45
(min)
1
1
1
1
1
2
3
4
1
1
1
1
1
1
r—
5
6 12 18 24 30 48
(h)
10
<D
Time
a
.c
O
S5
EOSINOPHIL/BASOPHIL STORAGE
AT ROOM TEMPERATURE (n=25)
0
-10
-20
—•—
—«•—
-30
-40
%
1h
2h
6h
2-
Cold Analysis
warm Analysis
12h 16h 24h 30h 36h 48h 60h 72h
Time
40-
0
—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—
5 10 15 30 45 1 2 3 4 5 6 12 18 24 30 48
(min)
(h)
T i m e
FlGS. 3 {upper), 4 (center), and 5 (lower). Quantitative effect of K2EDTA
storage on STKS differential parameters. Storage time on a nonlinear
scale up to 48 hours on the x-axis and, on the y-axis, mean percentage
change for neutrophils and lymphocytes (Fig. 3), monocytes (Fig. 4),
and mean count for eosinophil and basophils (Fig. 5). Each data point
represents the mean of 25 normal individuals (error bars ± I SD).
LYMPHOCYTE STORAGE AT 4°C
(n=6)
3020'
o> oco
O -io-
*^«
--•*""*-*•-»
Cold Analysis
Warm Analysis
Both the correlation between the STKS and the manual
technique, and the correlation between the two technologists (observers 1 and 2) are shown. The regression analysis scatterplots for the same data, excluding basophils,
are shown in composite Figures 9 and 10. Regression
analysis was performed on all samples regardless of
whether or not flags were present. The technologists performing the manual film analyses were highly experienced,
significantly limiting any observer errors in the manual
0
1h
2h
6h
12h
18h
24h
30h
36h
48h 60h 72h
Time
FIGS. 6 (upper) and 7 (lower). Quantitative effect of K2EDTA storage
at 4 °C on STKS differential parameters. Immediate aspiration (cold
analysis) and aspiration after allowing to stand at room temperature for
15 minutes (warm analysis). Storage time on a nonlinear scale up to 72
hours on the x-axis and on the y-axis mean percentage change in neutrophils (Fig. 6) and lymphocytes (Fig. 7). Each data point represents
the mean of 6 normal individuals.
A.J.C.P. • February 1991
213
WARNER AND REARDON
Coulter STKS®
Hospital ADDENBROOKES
Standard 241
Machine
Date
COULTER STKS
27/07/89
•2SD
•2S0
RCC
PLT
-2S0
-250
•2»
•2$0
MCC
ncu
-2S0
-2S0
•2«
•2$o
HB
HCT
-2$»
-2«
241
241
l
FIG. 8. Coulter STKS performance in Addenbrooke's EQA scheme for blood count analyzers. Cumulative graphical plot ±2 SD for count parameters.
Displayed is week No. 241 of the scheme and the previous nine weeks. (Units WBC, X109/L; RBC, X10'2/L; Hb, g/L; PLT, X109/L;MCV fL.)
of basophilias were unobtainable during the evaluation
period.
Another 32 results were excluded from the data, 17
because of evidence of storage on both the scatterplot and
blood film, 12 because of storage changes in the film alone,
and 3 because no differential analysis was obtained from
the STKS.
Differential Comparability
In the comparability study (n = 424), 199 (46.9%) of
the cases were true negatives and 188 (44.3%) were true
positives, when determined by using the comparative criteria previously mentioned.
The causes of 11 (2.6%) false-positive cases are shown
in Table 6. The immature neutrophils and band cell flags
(imma grans/band) and nucleated red blood cell flags
(NRBC) were found to be the least specific of the softwaregenerated suspect flags.
The 26 (6.2%) false-negative cases (Table 6) were subdivided into three groups of increasing importance. In
Group A, 16 cases were observed in which low levels of
abnormal cells were detected by the manual 400-cell differential. These were usually the result of one observer
noting the presence of one abnormal cell. Abnormalities
present in Group A are below the detection level of the
instrument and probably are below the detection level of
TABLE 5. WBC DIFFERENTIAL CORRELATION BETWEEN STKS/MANUAL AND OBSERVER 1/2 (n = 424)
Correlation Between STKS and
Manual Differential
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Correlation Between Observer 1 and Observer 2
r
Slope
Intercept
r
Slope
Intercept
0.967
0.966
0.734
0.860
0.280
0.958
0.945
0.645
0.817
0.331
2.193
2.345
1.408
0.625
0.603
0.937
0.938
0.561
0.717
0.390
0.907
0.907
0.596
0.781
0.631
6.580
1.975
2.455
0.433
0.121
Correlation studies were performed on all samples regardless of suspect flags.
Vol. 95 • No. 2
214
HEMATOPATHOLOGY
Original Article
LYMPHOCYTES
NEUTROPHILS
(b)
to
20
40
FIG. 9. Regression analysis plots for STKS
results against manual 400-WBC differential
for neutrophils (a) and lymphocytes (b).
Regression analysis plots for 200-WBC differential observer 1 against observer 2 for
neutrophils (c) and lymphocytes (d).
Manual (%)
(d)
20
40
60
80
20
100
Observer 1 (%)
40
Observer 1 (%)
MONOCYTES
EOSINOPHILS
(b)
to
5
10
5
15
10
Manual (%)
Manual (%)
15 -
(d)
10 •
5-
0 •
5
10
Observer 1 (%)
15
5
10
Observer 1 (%)
A.J.C.P. • February 1991
FIG. 10. Regression analysis plots for
STKS results against manual 400-WBC differential for moncytes (a) and eosinophils
(b). Regression analysis plots for manual
200-WBC differential observer 1 against
observer 2 for monocytes (c) and eosinophils (d).
WARNER AND REARDON
215
Coulter STKS®
Aspiration
TABLE 6. CAUSES OF FALSE POSITIVES (11/424)
In the primary mode the STKS aspirated between 270
and 290 uL, but because of the depth of penetration of
the aspiration needle, 1.3 mL of blood was required in
the sample tube. Between 150 and 170 fiL was aspirated
in the secondary mode. Aspiration volumes remained
the same whether the differential function was turned on
or off.
Cause
No.
Imma grans/band flag present
NRBC flag present
Abnormal scattergram
CAUSES OF FALSE NEGATIVES (26/424)
No.
12
4
1
5
2
1
1
Cause
<0.01 (<1%) Myelocytes
<0.01 (<1%)NRBC
> 0.023 (2.3%) Myelocytes
> 0.10 (>10%) Band cells
14% difference (neutrophils)
Variant lymphs
8% Blasts
Volumes
Group
A
Mixing Efficiency
B
The efficiency of the rocker bed mixing was established
by first analyzing a batch of 12 samples, then reanalyzing
the samples after they had been centrifuged at 1,500 g for
15 minutes. Results obtained were within the range observed in the interbatch comparability studies.
the manual 100-WBC differential. In Group B, the manual
differential identified moderately significant abnormalities
that were not identified by the STKS and are above the
detection rate of the instrument.
In Group C, two important cases were noted in which
the STKS DIFF analysis failed to detect morphologically
significant abnormalities. Both cases were investigated
further. The variant lymphocytes recorded by the manual
observers referred to approximately 5% "reactive" lymphocytes in a patient with treated lymphoma. In the second case, the manually recorded blast cells were from a
patient with AML in relapse. When the complete analysis
from the STKS was scrutinized, a pancytopenia definitive
flag was revealed. (Despite demonstrating distributional
abnormalities, the second case was recorded as positive
because of the importance of the lack of blast flag.) Cumulative patient data also were available. Therefore, it
was thought that when the complete case profile was taken
into account, a blood film would have been examined in
routine practice despite the lack of a blast flag with the
STKS analysis.
Further retrospective analysis of all data during a threemonth period showed the "variant lymphocyte" flag to
be activated in 25 of 27 known cases of lymphoproliferative disorders. Similarly, 15 of 16 cases with blast cell
were flagged with the suspect flag blast cells.
Throughput
Throughput was found to be between 90 and 102 samples per hour, with a mean of 98. This was established
using representative batches of samples but included no
control material. When the differential function was
turned off, the throughput increased to 111 samples per
hour.
Leukopenic
Samples
The Coulter VCS shows an increase in incomplete
computation for WBC differentials on white cell counts
of less than 1.0 X 109/L-5 Because of the reduced analysis
time of the STKS, an extended study was performed on
leukopenic samples to ascertain if the failure rate increased. A total of 46 samples with WBCs between 0.1 2.5 X 109/L were analyzed. Of these, 24 were found to
have incomplete differential flags; only one had a WBC
of greater than 1.0 X 109/L.
Interfering
Substances
Previously documented instances of interfering substances or pathologic states still affect the aperture impedance blood count aspect of the STKS. When taking the
differential analytical system in isolation, only two instances throughout the evaluation were found to produce
an abnormal scattergram. The first was shown to be a
plasma effect by correction of the abnormal scattergram
after washing and resuspending the patient's cells in AB
plasma. The effect also was reproduced by suspending
normal cells in the patient's plasma. The patient showing
this effect had hyperbilirubinemia as the most striking
biochemical abnormality. However, patients with higher
bilirubin levels failed to reproduce this phenomenon. The
second case was a patient with Hb SC. This case gave
evidence that red cell lysis was incomplete in the reagent
system for the WBC differential. Unfortunately, an insufficient number of cases with hemoglobinopathies has
been analyzed to show this finding to be consistent. However, hemoglobinopathies have been reported as causes
of anomalous results in other automated WBC differential
systems, 41718 and this was not unexpected with the STKS.
Vol. 95 • No. 2
216
HEMATOPATHOLOGY
Article
Reliability
Throughout the evaluation period, downtime was
minimal. The only major problem with the instrument
was a fault with the initial flow cell during thefirsttwo
weeks. The replacement flow cell gave no problems.
DISCUSSION
Complete blood counts are possible in automated hematology from a technologically diverse range of analyzers. The need for full WBC differentials with every analysis
has been a matter of considerable clinical and laboratory
debate. However, the significantly improved precision of
the automated techniques, when compared with the
manual technique, has been clearly established.
The accuracy of automated WBC differentials has been
difficult to establish because of the lack of a suitable reference method. Consequently, careful comparability
studies have to be undertaken using the tentative standard
manual method,16 or suitable variations of it, before implementing the automated system. The comparability of
alternative WBC differential technologies with each other
remains to be established and will be the subject of another
report. Minor differences have been reported between alternative automated technologies when enumerating
blood cells. These differences were more significant when
sizing the same cells.19 Therefore, complete reference
ranges require compilation for each technology.20 Thus,
mixing technologies in the same laboratory requires careful consideration.
The STKS performance during thefieldevaluation at
Addenbrooke's Hospital is no exception to the technological advancement process. The evaluation instrument
was precise, carryover was minimal, and its response was
apparently linear throughout a very wide range of concentrations for all the measured parameters. Sample handling and data management are considerable improvements when compared with the Coulter S Plus IV. The
combination of both blood count and full WBC differential analysis within one analyzer is beneficial to the organization of a busy hematology laboratory. The STKS
was reliable, downtime was virtually nonexistent, and an
average throughput of 98 samples per hour was achieved
in the evaluation. The DIFF analysis may be disabled,
increasing throughput to 111 samples per hour, with a
concomitant reduction in reagent costs of approximately
60%. Complete analysis was possible with 150 fiL of blood
in the secondary mode. At least 1.3 mL of blood was
necessary in the primary automatic sampling mode.
In the comparability studies, using a representative cross
section of the work load at Addenbrooke's Hospital, blood
counts were comparable with the S Plus IV, and the STKS
performed consistently well in an external quality assessA.J.C.P. •
ment scheme. The DIFFs compared well to carefully performed manual 400-cell WBC differentials. The DIFFs
were stable in K2EDTA anticoagulated blood samples for
between 18 and 24 hours when stored at 20 °C and for
considerably longer if stored at 4 °C and aspirated immediately. Individual users will have to determine the
storage time that they find acceptable in K2EDTA for the
STKS differential.
False negatives (6.2%) and false positives (2.6%) were
observed when comparing the STKS DIFF and suspect
flags with the manual differential and interpretation. Several false negatives were considered insignificant. The most
significant false-negative case wasflaggedwhen the definitiveflaggingsystem was included in the comparison. It
is doubtful that an automatic differential system can be
relied upon to identify every abnormal cell (specificity).
However, the sensitivity offlaggingsystems is paramount
to good laboratory practice. The STKS definitive flagging
system would be improved further if the user-programmable reference ranges were age and sex linked via the
bidirectional facility of the DMS.
Anomalous results observed with the STKS can be
subdivided into two groups: those attributable to interference with the blood count and sizing aspect of the analyzer are well documented21 and those attributable to
interference with the Erythrolyse reagent system used in
the DIFF analysis. The latter group was comprised of two
cases, one due to a presumed plasma-quenching effect
and the other due to a presumed red cell alteration in a
S/C hemoglobinopathy. Both cases were easily identified
by an abnormal scattergram, suspect flags and flow cell
analytical information (number of cells counted and time
taken for the analysis). Unfortunately, the latter is deleted
from version 1A of the DMS software but does represent
an independent method of quality controlling the flow
cell function of the analyzer.
In conclusion, the STKS was evaluated as an acceptable
hematology analyzer capable of producing full blood
counts, sizing information, and WBC differential counts
in a large teaching hospital environment. Univariate reference ranges remain to be established. The STKS is not
expected to completely replace the manual WBC differential and interpretation.
Acknowledgments. The authors thank the staffof Addenbrooke's Haematology Department, particularly Jess Fletcher, F.I.M.L.S., Olwyn
Tarrant, F.I.M.L.S., David Bloxham, F.I.M.L.S., and Jack Chambers,
F.I.M.L.S. They also thank Coulter Electronics for facilitating the evaluation.
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