HEMATOPATHOLOGY
Original Article
Evaluation of Automated Reticulocyte
Counts and Their Reliability in the Presence
of Howell-Jolly Bodies
KAREN G. LOFSNESS, MS, MONICA L. KOHNKE, BS, AND NANCY A. GEIER, BS
An automated reticulocyte procedure using a flow cytometer and the
fluorescent dye thiazole orange was evaluated for clinical use. The
mean reticulocyte count on 118 hematologically healthy adults was
1.56% (standard deviation |SD] 0.54), with virtually no difference in
percentage between women and men. The mean absolute values were
68.4 X 1O'/L (SD 24.6) and 75.7 X 10»/L (SD 27.2), respectively.
When compared with the standard microscopic technique, the automated method showed excellent correlation (r = 0.98) and greatly improved precision (coefficient of variation |CV] 4.1%) over the manual
method (CV 22.8%). Preanalytic storage of blood samples at 4 °C for
up to 48 hours did not significantly affect results, nor did varying the
incubation time of diluted samples from */i to 2 hours. In a group of
patients with appreciable numbers of Howell-Jolly bodies, automated
reticulocyte counts were spuriously elevated. The difference between
the manual and automated counts on these patients approximated the
percentage of Howell-Jolly bodies observed on their Wright-Giemsa
stained blood smears. (Key words: Automated counting; Flow cytometry; Howell-Jolly bodies; Reticulocytes) Am J Clin Pathol 1994;101:
85-90.
Reticulocytes are immature red blood cells (RBC) that have
recently left the bone marrow and still contain intracellular
RNA. The effectiveness of erythropoiesis can be measured by
staining blood with a supravital dye and determining the number of RBCs that contain this residual RNA. Reticulocyte
counts provide clinically useful information, not only in the
diagnosis and classification of anemias, but also in monitoring
therapeutic response.
The most widely used method for counting reticulocytes is a
manual microscopic procedure.1 Although it is inexpensive
and relatively simple to perform, this method is labor intensive
and imprecise. Individual differences in the preparation and
staining of the sample, distributional variation on the smear,
the limited number of cells actually counted, and interobserver
subjectivity in identifying reticulocytes all contribute to the
poor reproducibility of the manual procedure.
Recently, automated reticulocyte methods, using flow cytometry and fluorescent dyes that bind RNA, have shown improved precision and cost effectiveness.2"12 However, most of
the currently used fluorescent dyes bind polynucleotides nonspecifically, and thus stain DNA as well as RNA.'3 Nucleated
cells, because of their high DNA content, have fluorescent activity that is many times greater than that of reticulocytes and can
usually be excluded or "gated out" from the RBC population.
Small lymphocytes, such as those seen in chronic lymphocytic
leukemia, occasionally cause interference. Although the rela-
tive concentration of nucleated RBCs and erythrocytes containing malarial parasites or Howell-Jolly bodies is usually very
low, these inclusions also fluoresce and have the potential to
spuriously elevate an automated reticulocyte count.
The objectives of this study were to evaluate the Coulter
Epics Profile II flow cytometer (Coulter Electronics, Hialeah,
FL) for reticulocyte counting in a routine hematology laboratory, to use this instrument to establish reference values, and to
study the effect of significant numbers of Howell-Jolly bodies
on automated reticulocyte counts.
MATERIALS AND METHODS
Automated Reticulocyte Counting
Automated reticulocyte counts were performed on a Coulter
Epics Profile II flow cytometer, using a modification of the
method of Lee and coworkers.13 A 1:10,000 working solution
of thiazole orange was prepared fresh daily by diluting 5 juL
stock solution (1 mg/mL thiazole orange in methanol) in 50
mL Isoton II (Coulter Electronics, Hialeah, FL); the solution
was then stored in a brown bottle.
For each blood specimen, two dilutions (labeled "blank" and
"thiazole orange") were prepared in 10 X 75 mm capped polypropylene tubes. Each blank tube contained 5 nL blood diluted
in 2.0 mL Isoton II, and each thiazole orange tube contained 5
nL blood diluted in 2.0 mL working thiazole orange solution.
Both tubes were mixed well and incubated in the dark at room
temperature for more than 30, but less than 120, minutes before analysis. The tubes were remixed two to three times during
this incubation period, and again immediately before counting.
The following instrument settings were used for reticulocyte
counting. The laser was set at 15 m W. The green fluorescence
From the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis. Minnesota.
Manuscript received October 8, 1992; revision accepted February 1,
1993.
Address correspondence to Ms. Lofsness: Box 198 UMHC, University of Minnesota, Minneapolis, MN 55455.
85
86
HEMATOPATHOLOGY
Original Article
of 50,000 RBC (measured with 525 nm band pass and 550 nm
short pass dichroic filters) was collected on a 256 channel 4
generation log analyzer. Forward scatter and log side scatter
gates were used to encompass the RBC in a bitmap. The high
voltage and gain were set so that 99.9% of thefluorescenceof an
unstained sample fell between channels 0 and 50. The sample
flow rate was set at 50 ^L/min (3000-4000 cells/sec). The instrument start-up procedure recommended by the manufacturer was performed daily, and any necessary adjustments were
made before the analysis of the blood samples.
For each determination, the blank sample was analyzed first,
and results were checked to ensure that the background count
was approximately 50 events, the reported percentage was
0.1%, and the histogram did not appear abnormal. The range
bar cursor was adjusted if necessary, and the thiazole orange
sample was analyzed using the same settings as the blank (Figure 1). To correct for the background count, 0.1% was subtracted from the reticulocyte percentage obtained on the thiazole orange sample.
LFL1
1
M R MAX CODNT PBRCBHT
0.102 102.3
0.1
51
HEAR
1.519
SD
0.378
I gPCV
HEAR
SO \ BPCJ
ÜTÍ7T
Manual Reticulocyte Counting
Equal amounts of blood and new methylene blue were combined and incubated at room temperature for 5-15 minutes.
The dilutions were remixed, and three wedge smears were prepared from each sample. The number of reticulocytes per 500
erythrocytes was determined microscopically by each of two
technologists on separate slides (1000 cells total). If the number
of reticulocytes did not agree withinfivecells, a third technologist counted 500 cells on the third slide, and results were averaged. A reticulocyte was defined as an RBC containing at least
two granules of reticulum.
Specimen Collection and Method Evaluation
Venous blood samples were collected in Na2EDTA (1.5 mg/
mL), and reticulocyte counts were determined as described.
Complete blood counts (CBC) were performed by either a
Coulter S-Plus IV or STKR (Coulter Electronics, Hialeah, FL),
and blood smears were prepared and stained by a WrightGiemsa method. Results were analyzed statistically using Statview II software (Abacus Concepts, Berkeley, CA).
Healthy Subjects
Reference values were determined by performing automated
reticulocyte counts on blood samples from 118 healthy nonhospitalized subjects, aged 16 to 80 years. All 67 women and 51
men were judged to be hematologically healthy on the basis of
their automated CBC. Absolute reticulocyte counts were calculated by multiplying the relative count (in percent) by the RBC
count determined on the same blood sample.
Patient Samples
For correlation studies, 215 random blood samples from hospitalized patients, on whom reticulocyte counts had been ordered, were analyzed by both the manual and automated methods. Reproducibility was evaluated by performing three
replicate analyses on 10 patient samples by both the manual
and automated methods. To determine the effect of preanalytic specimen storage time on automated reticulocyte results, 15
patient blood samples were first analyzed within 4 hours of
LFL1
B1
MIR
MAI
COURT PEBCEHT
O.ÍOTTÍÍX 1527
—rr
TUT
FIG. 1. Histograms of blank (A) and thiazole orange (B) dilutions. Cell
number is shown on the vertical axis (count), andfluorescentintensity,
plotted on a four-generation log scale, is shown on the horizontal axis
(LFL1). The range bar cursor [1] is set so that the event count on the
blank is around 50, and the percentage of cells showingfluorescenceis
0.1%. The thiazole orange sample is analyzed using the same settings,
and 0.1 % is subtracted from the result. This patient's reticulocyte count
was 4.9%.
collection, then stored at 4 °C and reanalyzed at 24 and 48
hours. The stability of the fluorescence in the blood-working
thiazole orange dilution was measured by comparing the results on 10 patient samples after 'A, 1, and 2 hours incubation
time.
Forty-one blood samples from 19 patients, on which HowellJolly bodies had been observed microscopically, were used to
study the potential interference of these inclusions on automated reticulocyte counts. The automated and manual reticulocyte percentages on these patients were obtained by the
methods described above. The percentage of Howell-Jolly bodies was determined independently on a Wright-Giemsa stained
AJ.C.P.-January 1994
87
LOFSNESS, KOHNKE, AND GEIER
Automated Reticulocyte Counts
TABLE 1. AUTOMATED RETICULOCYTE
REFERENCE VALUES
±2SD
Mean
(XIO'/L)
±2SD
0.48-2.64
0.46-2.66
0.47-2.63
71.5
68.4
75.7
19.7-123.7
19.2-117.6
21.3-130.1
Mean
s
118 adults
(ages 16-80 yr)
67 women
51 men
1.56
1.56
1.55
¿5
60
80
100
120
Relic absolute «
blood smear of the same sample by having two technologists
count the number of RBCs containing Howell-Jolly bodies per
500 cells (1000 cells total) in the manner of a manual reticulocyte count.
FIG. 3. Frequency distribution of absolute reticulocyte values (X 109/L)
by flow cytometer on 118 healthy adults (67 women, 51 men).
Reproducibility and Stability
RESULTS
Reference Values and Correlation Studies
Reticulocyte results for 118 hematologically healthy adults
are shown in Table 1. The mean reticulocyte percentage was
1.56%, with a standard deviation (SD) of 0.54%. The average
absolute reticulocyte count was 71.5 X 109/L (SD 25.9). For the
67 women, the means were 1.56% (SD 0.55) and 68.4 X 109/L
(SD 24.6), with observed ranges of 0.4% to 3.4% and 17 to 136
X 109/L, respectively. The 51 men had means of 1.55% (SD
0.54) and 75.7 X 109/L (SD 27.2), and observed ranges of 0.6%
to 2.9% and 30 to 144 X 109/L. Figure 2 shows the frequency
distribution of reticulocyte percentages. Because this distribution was not Gaussian, the 95% reference interval was also
determined by percentile rank. When the lowest 2.5% and
highest 2.5% of the values were excluded, the range was 0.6% to
2.7%. Figure 3 illustrates the frequency distribution of absolute
reticulocyte values for these 118 subjects.
The correlation between manual and automated reticulocyte
results on 215 patient samples with manual percentages ranging from 0.1% to 28.2% is shown as Figure 4. By linear regression analysis, the coefficient of correlation (r) was 0.98. The
coefficient of determination (i2) was 0.95, and y = 0.92x
+ 0.17.
The 10 patient samples used to evaluate reproducibility had
automated counts ranging from 1.2% to 9.3%. For the automated method, the average coefficient of variation (CV) was
4.1%, with a range of 0.9% to 7.7%. By the manual method, the
same samples showed a mean CV of 22.8% (range 11.1% to
36.1%).
The 15 random bloods used to study the effect of preanalytic
specimen storage time had initial automated reticulocyte values ranging from 1.0% to 10.3%, with a mean of 3.3%. After 24
hours of storage at 4 °C, the results ranged from 0.8% to 10.3%,
with a mean of 3.4%. Only one 24-hour sample varied as much
as 0.5% from the original value. After 48 hours at 4 °C, the
mean was still 3.4% and the range was 0.9% to 10.3%. One
48-hour sample was 0.7% higher than the initial result; the rest
were all within ± 0.5%. These differences were not statistically
significant by paired /-tests.
The 10 samples that were analyzed after lh, 1, and 2 hours
incubation in the working thiazole orange solution had initial
automated reticulocyte percentages of 1.1% to 6.8%, with a
mean of 2.4%. After 1 hour of incubation, they ranged from
1.0% to 6.5% (mean 2.4%), and after 2 hours the range was
1.0% to 6.4% (mean 2.4%). One 2-hour dilution was 0.4% lower
than the initial value; the rest were all within ± 0.2%. These
differences were not statistically significant by paired i-tests.
y • Six * .17,
r > 48,
r-aqu«i*d m M
(n • 21S)
45.
40.
30.
25.
20
10
5
0
|,w,..,VK.i...v...,
.
1.5
2
2.5
3.5
Relic %
FIG. 2. Frequency distribution of reticulocyte percentages byflowcytometer on 118 healthy adults.
FIG. 4. Flow cytometer versus manual method, reticulocyte percentages. Scattergram and regression coefficients.
Vol. 101 • N o . I
HEMATOPATHOLOO T
Original Article
88
TABLE 2. EFFECT OF HOWELL-JOLLY BODIES
Patient No.
HJ (%)
Manual reticulocyte (%)
HJ + manual reticulocyte (%)
Automated reticulocyte (%)
1
2
41
18
04
09
59
5.9
6.0
13
7.6
9.3
1.4
4
08
17 5 l
5.9
7.0
5
6
88 1 2
5 8 49
14.6 6 1
14.3 7.3
7
03
02
OS
0.7
8
9
10
5 3 09 ? 1
3 1 1 "> 1 4
8.4 1 1 3 7
11.0 3.0 6.6
11
12
13
14
15
20
07
43
50
4.9
05
05
13
18
31
1.0
34
44
6.0
21
4.1
3.3
3.6
36
4.2
16
n
13
07
24
06
19
2.5
18
13
18 7
3 1 20.0
2.8 24.3
29
0?
I3
15
1.5
HJ = Howell-Jolly bodies.
Effect of Howell-Jolly Bodies
Forty-one samples in which Howell-Jolly bodies were easily
found on the Wright-Giemsa smear were selected for reticulocyte analysis by both the manual and automated methods.
These samples had manual reticulocyte counts ranging from
0.2% to 19.0%, and automated counts from 0.7% to 24.3%. In
every case, the automated percentage was higher than the manual. The percentage of Howell-Jolly bodies was determined microscopically and ranged from 0.2 to 8.8%. When this percentage was added to the manual reticulocyte count, the result
approximated the automated count in most cases. Table 2
shows data from the initial samples of the 19 patients with
increased Howell-Jolly bodies. The flow cytometer histogram
patterns on these samples with Howell-Jolly bodies did not
appear to show any unusual characteristics (Fig. 5). When the
thiazole orange dilutions from one of the Howell-Jolly samples
was examined by fluorescent microscopy, the Howell-Jolly bodies appeared as bright green discrete intracellular dots, whereas
the reticulocytes were diffusely fluorescent.
DISCUSSION
The clinical utility of the reticulocyte count is compromised
by the fact that, in most laboratories, it is still determined microscopically. Although other routine hematologic counts are
1
NIK
0.715 102.3
LFL1
COOHT PERCENT
2753
5.5
HEAR
SD
3.097
0.236
\ HPCV
FIG. 5. Histogram of thiazole orange dilution on a patient with an
automated reticulocyte count of 5.4%, a manual count of 2.2%, and a
Howell-Jolly body count of 2.5%.
now performed by complex instruments that give highly accurate and reproducible results, the standard method for counting reticulocytes has not changed for many years and remains
imprecise and unreliable.14'15 The recent development of practical techniques usingflowcytometry and fluorescent dyes will
allow many clinical laboratories to automate reticulocyte
counting and bring it to an acceptable level of reliability.
On the basis of an internal study performed in 1966, the
reference range for reticulocytes in our laboratory was considered to be 0.5% to 3.3% for women and 0.5% to 2.1% for men.
As a result of the present study, the range has been adjusted to
0.5% to 2.7% for both sexes. Although we found virtually no
difference between the reticulocyte percentages for women and
men, reports in the literature vary on this point. Earlier studies
by both manual16 and automated4 methods describe significantly higher reticulocyte percentages in women than men.
However, other authors report no appreciable difference by
flow cytometry,2'3'7 and one study of 265 adults actually found
a slightly lower mean percentage in women (0.92%) than in
men (1.07%)."
Because method comparison for reticulocyte counting necessitates using the admittedly imprecise manual technique as
the reference by which to judge a new automated system, it is
somewhat surprising to find a high degree of correlation between the two methods, especially in the higher clinical ranges.
Our correlation and reproducibility data were similar to those
reported by others,4-6'7'10'12 and showed that the automated
method agreed closely with the manual, and was far more precise. The greatly improved precision obtained withflowcytometry reflects not only the much larger number of cells counted
(~ 50,000 versus 1000 with the microscope), but also the elimination of subjectivity on the part of the observer. In a preliminary report from the CAP reticulocyte project, Savage and coworkers15 recommend that the r value for an automated
reticulocyte system should be greater than 0.95, with a y intercept of 0.25 or less when correlated with manual reticulocyte
results. They also state that the system CV should be 15% or
less. The automated system evaluated in this study, with r
equal to 0.98, a y intercept of 0.17, and a CV of 4.1%, easily
meets these criteria.
The stability of this system was not affected by specimen
storage time, either before analysis or following dilution in
thiazole orange. Although some studies of sample storage reported a progressive fall in reticulocytes after blood was stored
more than 24 hours at 4 °C,4-9 we found no significant differences up to 48 hours. Because refrigerated blood may be held as
long as 2 days without appreciable changes in reticulocyte values, routine samples can be batched and run every other day,
with specimens requiring immediate attention being performed by the manual method. Selected samples can also be
stored at 4 °C and reanalyzed with the next batch as a quality
control check of system reproducibility. Because the thiazole
A.J.C.P.-January 1994
LOFSNESS, KOHNKE, AND GEIER
89
Automated Reticulocyte Counts
LFL1
HIN HAX COUNT PERCENT
SD % HPCV
HEAD
27T TTJOT X5IT
132T
1 0.517 102.3
0.699
3.1
1536
2 0.499 1023.
6.541
0.349
2.3
3 0.499 64.09
1173
2.720
FIG. 6. Histogram of thiazole orange dilution on patient with chronic
lymphocytic leukemia. The second peak caused by the WBC (200 X
109/L) is evident. The initial range bar cursor setting [1] included some
of the WBC, and gave a reticulocyte value of 2.6%. Range bar cursor
setting [2] was extended to include all of the WBC, whereas setting [3]
excluded the WBC peak and gave afinalresult of 2.2%.
orange incubation timing is somewhatflexible,individual samples do not have to be analyzed in any specific order. Samples
are easily added or repeated, and batches may be even interrupted for a reasonable period of time.
Thiazole orange and the otherfluorescentdyes used in automated reticulocyte counting bind to DNA as well as RNA, thus
nuclear material can potentially cause interference. Most leukocytes are so large and highly fluorescent that they are easily
excluded from the RBC population being analyzed. Occasionally, it is difficult to "gate out" the small lymphocytes seen in
chronic lymphocytic leukemia. However, the second peak they
produce is evident on the histogram, and adjustment of the
bitmap and the upper range bar cursor will usually provide
acceptable results (Fig. 6). Large numbers of nucleated RBC
would probably require similar adjustments.
Erythrocytes containing smaller inclusions of DNA, such as
malarial parasites or Howell-Jolly bodies, are more similar to
reticulocytes in both size and fluorescent activity. When present in appreciable numbers, these RBC inclusions have the
potential to elevate automated reticulocyte counts incorrectly.
A recently published method for quantitating malarial parasites in in vitro culture systems actually uses flow cytometry
with thiazole orange as the binding dye.17 Some studies evaluating automated reticulocyte counting mention the possibility of
spurious results in samples containing Howell-Jolly bodies.78
However, others state that either Howell-Jolly bodies do not
interfere,2'10'" or their concentration is generally so low that
they have no significant effect.61'8
Our investigation of the interference caused by Howell-Jolly
bodies was prompted by a patient sample that had a manual
reticulocyte count of 1.8% and an automated count of 6.0%.
Howell-Jolly bodies were noted on the new methylene blue
preparation, and examination of the patient's Wright-Giemsa
stained blood smear showed several Howell-Jolly bodies in
everyfield.By counting the number of Howell-Jolly bodies per
1000 RBC in the manner of a manual reticulocyte count, 4.1%
Howell-Jolly bodies were found on this blood smear. Because
the total number of microscopically determined reticulocytes
and Howell-Jolly bodies so closely approximated the automated reticulocyte count, it was decided to select samples with
noticeable numbers of Howell-Jolly bodies deliberately to see if
this pattern persisted. Although this method of determining the
percentage of Howell-Jolly bodies is admittedly as imprecise as
the manual reticulocyte method itself, in every case the automated count was demonstrated to be higher than the manual,
and the numeric difference between the two was somewhat
close to the Howell-Jolly percentage.
Howell-Jolly bodies are nuclear remnants that may be seen
in erythrocytes following splenectomy. They may also be seen
in hemolytic anemia, megaloblastic anemia, and hyposplenic
states.19 The question might well be raised as to the prevalence
of samples containing appreciable numbers of Howell-Jolly
bodies in a patient population. The samples in this study were
from 19 patients who were seen in our 550-bed tertiary care
hospital over a span of 4 weeks. Because such samples are not
rare we recommend screening Wright-Giemsa blood smears on
all samples submitted for reticulocyte counts, and performing
manual counts on those that have more than one Howell-Jolly
body per oil immersion (X 1000) field.
Automated reticulocyte counting, which has consistently
proven superior to the manual technique, should eventually
replace the manual technique as the method of choice. In our
experience, agreement between the two methods has been
good, except for the occasional sample containing Howell-Jolly
bodies or otherfluorescinginterferences. Errors associated with
these factors can be minimized by carefully monitoring histograms and screening Wright-Giemsa smears for RBC inclusions. Although automated reticulocyte counting may not be
feasible for smaller laboratories with low test volume, it should
be a practical alternative for many clinical laboratories, particularly those that have already acquired aflowcytometer to perform immunologic marker studies.
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