Reproducibility in the Hematology Laboratory:
T h e Microhematocrit Determination
PAUL B. GOLDENFARB, M.D.,
FRANK P. BOWYER, M.D.,
AND EFFIE BROSIOUS,
ELMER HALL, P H . D . ,
MT(ASCP)
Hematology Unit, National Communicable Disease Center, Health Services and Mental
Health Administration, Public Health Service, U. S. Department of Health,
Education, and Welfare, Atlanta, Georgia 30333
ABSTRACT
Goldenfarb, Paul B., Bowyer, Frank P., Hall, Elmer, and Brosious, Effie: Reproducibility in the hematology laboratory: The microhematocrit determination. Amer. J. Clin. Path. 56: 35-39, 1971. T o estimate the overall microhematocrit test error and to determine the relative contributions of reading
error, capillary tubes, centrifugation times, and clays to the variation in test
results, a prepared erythrocyte suspension was tested repeatedly on six alternate days. Results indicate that the variation caused by reading the hematocrit to the nearest one-half percentage point exceeds that attributable to
differences among capillary tubes and centrifugation times.
for measuring packed blood cell volume is widely used
in clinical laboratories. Data collected by
the Hematocrit Proficiency Testing Program of the National Communicable Disease Center indicate that more than 75%
of Public Health and licensed commercial
laboratories used this technic. The test itself is simple to perform and requires only
a high speed centrifuge, capillary pipettes,
sealing clay, and a packed cell volume
reader. A single determination can be performed rapidly, and many specimens can
be processed simultaneously.
Among laboratories participating in the
Proficiency Testing Program, microhematocrit centrifugation times usually range from
3 to 5 min. Although the manufacturer of
one popular centrifuge ° recommends a
spin time of 3 min., the importance of 1 or
2 min. deviation from this recommendation
is unknown. T h e microhematocrit method
has been the subject of several precision
studies,2 but few investigations have been
made into the relative contributions of centrifugation time and other test components
to overall variation.
This paper presents the results of a study
undertaken to estimate the microhematocrit
test error under controlled laboratory conditions. The study was designed specifically
to provide quantitative information about
the relative contribution of the following
factors to analytical variability: readings,
capillary tubes, centrifugation times, and
days (among-run variation). Results indicate that when the hematocrit is read to the
nearest 0.5%, the resulting variation in test
values exceeds the variation due to capillary tubes and centrifugation times.
T H E MICROHEMATOCRIT METHOD
Received June 26, 1970; accepted for publication
July 30, 1970.
Dr. Goldenfarb's present address is: Duke University Medical Center, Department of Medicine,
Durham, North Carolina 27706.
Use of trade names is for identification only and
does not constitute endorsement by the Public
Health Service or by the U. S. Department of
Health, Education, and Welfare.
• Adams micro-hematocrit centrifuge, Clay-Adams,
Inc., New York, New York.
35
36
GOLDENFARB ET AL.
A.J.C.P.—Vol.
56
DAY (1-6)
POOL A
C2
C3
C5
POOL B
Cs
C2
AAAA
T|
T2 T|
T 2 T|
T 2 T|
C3
C5
C8
AAAA
T2
T|
T 2 T|
T 2 T|
T 2 T|
ftftiiiiiiiiiiim
T2
R| R2 R| R2 R| ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 ^2 ^1 °2
FIG. 1. Experimental design of the microhematocrit study. C = centiifugation times;
T = tubes; R = readings.
Methods and Materials
from the time of filling and sealing until
The experimental design shown in Fig- centrifugation. In the first centrifugation,
ure 1 was used to provide a data structure two pairs of tubes (one from each pool)
suitable for analysis of variance computa- were spun simultaneously for 8 min. at
tions. In keeping with this model, micro- 15,500 X g. When the spin cycle was comhematocrit determinations were performed pleted, each tube was read twice in rapid
every other clay for 6 days by one technolo- succession.! The hematocrit was expressed
gist. At approximately 8:30 AM on each as the per cent of total blood volume ocstudy day, the technologist received two cupied by packed erythrocytes. Readings
sterile vials of stable erythrocyte suspen- were made to the nearest 0.5%. The same
sion.4 These vials had been filled from two procedure was followed with tubes centriseparate erythrocyte pools having different fuged for 5, 3, and 2 min. After each readhematocrits. The vials were randomly la- ing, tubes were segregated by pool and
beled "A" and " B " on each study clay. From were placed upright in 12 by 75 mm. glass
each vial, the technologist filled and sealed tubes. At the completion of this part of the
eight capillary tubes,t dividing the group study, eight hematocrit readings had been
into four pairs. In reverse order of filling, made in duplicate from each pool. In takindividual pairs from each pool were desig- ing these readings, the technologist of necnated for centrifugation at 8, 5, 3, or 2 min. essity knew the identity of duplicate speciAll tubes were kept upright in sealing clay % mens (hereafter referred to as "known duplicates"). T o "blind" the readings, tubes
f Capilets, Scientific Products, Evanston, Illinois.
% Seal-Ease, Clay-Adams, Inc., New York, New
§ Adams micro-hematocrit tube reader, ClayAdams, Inc., New York, New York.
York.
July 1971
37
MICROHEMATOCR.IT T E S T PRECISION
were randomized and reread. They were
then rerandomized and read a second time.
This procedure provided eight duplicate
pairs (hereafter referred to as "blind duplicates") which were free of reading bias and
suitable for comparison with the known
duplicates.
To reduce the possibility of reporting
bias, readings from each clay's tests were
recorded in sections on three separate answer sheets. As each individual sheet was
completed, the technologist submitted it
immediately to one of the study supervisors.
After an entire day's run, answer sheets
were grouped and filed. At the conclusion
of the study, answers were decoded, and
the data were examined by analysis of variance technics.
Results
Table 1 summarizes the analysis of variance calculations for each of the four sets
of data (Pool A and Pool B each with
known duplicate readings and blind duplicate readings). The sources of variation and
degrees of freedom indicated in the two
left-hand columns follow from the structure
of the experiment (Fig. 1). The four columns of "Mean Squares" form the basis
for testing the statistical significance of the
various sources of variation and for estimating the individual variance components
in each data set.
The variances of replicate readings on
the same tube, as estimated by the "Mean
Square" for readings, are similar for all
four sets of data. Pooling these estimates
yields a variance of 0.085. Hence, the standard deviation of replicate readings of the
same tube is 0.29, which indicates that duplicate readings should seldom differ by
more than one percentage point. Visual
inspection of the results in all four sets of
data supports this conclusion. Variation
due to capillary tubes was statistically significant for only one set of data (Pool A,
blind duplicates). T h e standard deviation
of readings on replicate tubes for this set
of data is 0.40, which indicates that tube
differences also should seldom exceed one
percentage point. For the other sets of data,
tube differences were no larger than reading differences on the same tube.
Interpretation of the three remaining
sources of variation (centrifugation times,
days, and an interaction of times and days)
can be aided by examining Tables 2 and
3. Table 2 summarizes the average hematocrit readings for each set of data and centrifugation time. These averages differed sig-
Table 1. Summary of Analysis of Variance Computations for Each Set of Data
Mean Squares
Pool B1
Pool A
Source of Variation
Degrees
of
Freedom
Known
Duplicates
Blind
Duplicates
Known
Duplicates
Blind
Duplicates
Centrifugation times
Days
Times X days interaction
Tubes
Readings
3
5
15
24
48
0.83*
4.23*
0.15*
0.08J
0.09
0.53*
6.15f
0.09J
0.21*
0.11
0.75*
15.60*
0.2U
0.10*
0.07
0.74f
14.64*
0.24f
0.06t
0.07
TOTAL
* Significant at 0.05 level,
t Significant at 0.01 level.
| Not significant.
95
38
GOLDENFARB ET AL.
A.J.C.P.—Vol. 56
Tabic 2. Average* Hematocrit Determinations for Four Centrifugation Times by Pool and Set of Data
Centrifugation Times
Pool
Data Set
8 Min.
5 Min.
3 Min.
2 Mil
Known duplicates
Blind duplicates
25.72
25.85
25.80
25.87
26.00
25.92
26.12
26.17
Known duplicates
Blind duplicates
38.52
38.60
38.54
38.56
38.74
38.60
38.90
38.94
* Each average is based on 24 readings: two readings on each of two tubes on each of six days.
nificantly in three of the four sets of data.
Consistently, average hematocrits were lowered by increasing centrifugation times;
however, in no instance did the difference
in averages between the shortest and longest
times exceed 0.4 percentage points.
Variation among clays was statistically
significant in all four sets of data. As indicated in Table 3, successive daily means decreased from the first day through the sixth
day (excepting the sixth day for Pool A
results). Because of the consistency of the
trend from clay to day, this source of variation cannot be considered completely random; that is, an additional factor is probably confounded with days such as deterioration of the erythrocyte suspension. T h e
largest differences between day means for
each set of data were 1.3, 1.5, 2.5, and 2.3
percentage points, respectively. Note that
greater changes occurred in Pool B (higher
hematocrits) than in Pool A. T h e significant interaction components denoted in
Table 1 for only two of the sets of data
indicate that the effect of centrifugation
times was not consistent from clay to day
(or, vice versa, that day-to-day effects were
not consistent among centrifugation times).
However, the interaction component was
relatively small and can be ignored.
Discussion
Differences among centrifuges, capillary
tubes, and hematocrit readers have all been
mentioned as possible sources of variation
in hematocrit testing. 1 - 3 In the present
study, reading the packed cell volume to
the nearest one-half percentage point caused
a variation in test results which encompassed the effects of different capillary tubes
and centrifugation times. Nevertheless, the
range of daily readings from the highest to
the lowest values from each pool rarely exceeded one percentage point. I n clinical
terms, this variation is relatively small. It
Table 3. Average* Hematocrit Determinations on Six Days by Pool and Set of Data
Day
Pool
Data Set
1
2
3
4
5
6
A
Known duplicates
Blind duplicates
26.64
26.68
26.34
26.50
26.00
26.28
25.72
25.66
25.36
25.18
25.41
25.41
B
Known duplicates
Blind duplicates
39.91
39.88
39.50
39.59
38.94
38.88
38.61
38.46
37.72
37.66
37.38
37.59
* Each average is based on 16 readings: two readings on each of two tubes for each of four centrifugation times.
July 1971
39
MICROHEMATOCR1T TEST PRECISION
compares favorably with the results of other
hematocrit studies,2 and to a great extent
reflects only the lack of sensitivity of the
reading method. The minor effect of differences among capillary tubes supports the
continued use of these commercially available items. The effect of varying the centrifugation time from 3 to 5 min. is also
relatively unimportant and indicates that
maximal cell packing probably occurs after
3 min. This finding supports the manufacturer's recommendation (3 min.), and suggests that laboratories which now use 4- to
8-min. centrifugation times can economize
on time without objectionable changes in
accuracy.
The finding of a statistically significant
variation among days is difficult to interpret. The trend toward decreasing hematocrit values during the study period (Table
3) probably reflects pool deterioration, a
complication which was not anticipated and
which was not noted in previous batches
of erythrocyte suspension.4 T o estimate the
random clay component of the observed
day-to-day variation, we could remove the
deterioration trend from (lie data, but the
reliability of results as calculated from so
small a data sample would not be great.
In summary, the daily variation among
replicate hematocrit readings on the same
tube or on different tubes from the same
erythrocyte pool should rarely exceed one
percentage point. Centrifugation time has
little, if any, practical effect on the results.
Day-to-day differences, although relatively
large, may possibly be attributed to deterioration of the suspension. From the data
at hand, we cannot reliably estimate the
random day-to-day component of test variation.
References
1. DeBoroviczeny ChG: On the standardization of
packed cell volume determination. Standardization in hematology. Biol Haemat 24:83-100,
1966
2. McGovern JJ, Jones AR, Steinberg AG: T h e
hematocrit of capillary blood. New Eng J Med
253:308-312, 1955
3. Strumia MM, Sample AB, Hart ED: An improved
micro hematocrit method. Araer J Clin Path
24:1016-1024, 1954
4. Zucker S, Brosious E: Preparation of quality control specimens for erythrocyte counting, hematocrit and hemoglobin determinations. Amer J
Clin Path 53:474-480, 1970
Erratum
I wish to r e p o r t an error in my article "A Practical A p p r o a c h to Identification of Yeast-like Organisms" (Amer. J . Clin. Path., 55: 580-590, 1971). O n p a g e
581, line 33 should r e a d "6.7 G m . " r a t h e r t h a n "11.7 Gin." for the a m o u n t of
yeast nitrogen base dissolved in 100 ml. of distilled water.
C. T E R R E N C E D O L A N ,
M.D.
Mayo
Clinic
Rochester, Minnesota
55901