Comparison of three-fold converted hematocrit and micro-hematocrit in pregnant women
Comparison of three-fold converted hematocrit and micro-hematocrit in pregnant women
Girum Tesfaye KiyaID 0 1
Fitsum Mesfin Zewudie 1
0 School of Medical Laboratory Sciences, Institute of Health, Jimma University , Jimma , Ethiopia , 2 Clinical Chemistry Laboratory Unit, Jimma Medical Center, Jimma University , Jimma , Ethiopia
1 Editor: David J. Garry, Stony Brook University Health Sciences Center School of Medicine , UNITED STATES
Anemia is one of the common complications of pregnancy. Hemoglobin concentration, Hematocrit, and Red cell count are laboratory tests used to diagnose anemia. In are source poor setting, there is a practice of using three-fold converted Hematocrit. This study is designed to assess the association and acceptability of three-fold converted Hematocrit as compared to the standard Micro-hematocrit method, in pregnant women.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interest exist.
The cross-sectional study conducted from May 18 to June 12, 2018 involved 200 pregnant
women who visited the Laboratory for a Hematocrit test. Three milliliter of venous blood was
collected with EDTA tube to determine Hematocrit by the Micro-hematocrit method and
Hemoglobin concentration measured by a HemoCue Hemoglobin B analyzer. A scatter plot,
correlation coefficient, Bland and Altman plot, and Area under curve were employed to
assess the agreement and acceptability of the calculated Hematocrit as compared to the
The correlation coefficient, Intraclass correlation coefficient and concordance correlation
coefficient were 0.91, 0.94, and 0.89, respectively. The Bland and Altman plot showed a
mean difference of 0.94 with the limit of agreement ranges from 0.6 to 1.3. The area under
the receiver operating characteristics with cut-off point of Hematocrit <33% was 0.86. The
sensitivity and specificity of the calculated method was 95.5% and 71.4%, respectively.
Generally there is excellent association between the two methods. The two methods were
identical within inherent imprecision of both methods. Hence, the Hematocrit value,
threefold calculated from the Hemoglobin was found to be acceptable to diagnose anemia in
Iron deficiency anemia is one of the most common causes of anemia in pregnancy[
is defined as a Hemoglobin concentration <110g/l for pregnant women according to World
Health Organization (WHO)[
].There are different laboratory tests that are employed to
diagnose anemia such as Hematocrit, Hemoglobin, and Red cell count [
Hematocrit is a test that measures the percentage of blood that is comprised of red blood
]. This is often referred to as packed cell volume (PCV) or erythrocyte volume fraction.
A hematocrit value less than the reference interval is indicative of anemia and greater than the
reference interval is indicative of polycythemia [
]. Micro-hematocrit method is a standard
method for hematocrit determination according to Clinical and Laboratory Standards
Institute (CLSI) [
]. A blood sample is filled three fourth in a capillary tube and centrifuged at
specified revolution per minute (RPM) to find a separated compartments, of which, the ratio of the
packed red cell at the bottom is measured. There is also conductivity method and automated
Hematology analyzer to determine hematocrit value [
Hemoglobin is a protein in red blood cells which carries oxygen molecules to the tissues
]. Hemoglobin measurement methods have shown progress starting from adding distilled
water to measure the volume of blood as a hemoglobin level until its color matches with
], to development of spectrophotometry and the hemoglobin cyanide
(cynamethemoglobin) method, and then to automated hematology analyzer and point-of-care tests
Hemocue HB 301 is a point of care test instrument which allows accurate determination of
hemoglobin at the bedside and found to provide comparable result to other analyzers [
There is a disposable micro cuvette in it which has reagents necessary for both release of
hemoglobin and conversion of it to a stable colored product for color intensity measurement [
In anemia diagnosis, hemoglobin gives direct measurement of oxygen carrying capacity
while the hematocrit provides an indirect one [
]. Standard conversion has been used
between these two tests (Hemoglobin = Hematocrit X3) as a cut-off point to estimate anemia
].However, the conversion of hematocrit by Micro-hematocrit to hemoglobin has shown
bias as compared to the direct measurement of hemoglobin, particularly in malaria endemic
]. This discrepancy might be due to the mean corpuscular volume (MCV) which
is related to quantity of trapped plasma during centrifugation .The other way round
conversion(Hematocrit = Hemoglobin x3) has not yet been evaluated for its comparability with
the standard measurement which necessitates this study.
In are source poor setting where there is no automation and even in some Hospitals with
automated Hematology analyzer, there is a practice of reporting hematocrit derived from
hemoglobin as a three-fold conversion. Though the Micro-hematocrit is a standard method to
measure hematocrit, it is time consuming and demands careful preparation and reading of the
ratio. It?s use in under-resourced laboratories may also be limited as it demands a specialized
centrifuge and a reliable supply of capillary tube [
]. In this case, Hemocue is a method mostly
used to calculate hematocrit as it is easily portable and rapid test. Therefore, this study is
aimed at determining the association and acceptability of a hematocrit value calculated from
HemoCue HB 301as compared to the standard Micro-hematocrit method.
Materials and methods
Facility based cross-sectional study was conducted in Jimma University Medical center
(JUMC), Jimma, Southwest Ethiopia. The study was conducted from May 18 to June 12, 2018.
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A total of 200 pregnant women were involved in this study. All pregnant women who visited
JUMC laboratory for Hematocrit test during the study period were included consecutively.
Data collection and analysis
Socio-demographic and obstetric data were collected using structured questionnaire (S1 File).
Three milliliter of blood sample was collected from each participant by a tube containing
EDTA anticoagulant. A drop of blood from well mixed sample was used to fill the
microcuvette of the HemoCue HB analyzer to determine Hemoglobin. The micro-cuvette was then
inserted to the instrument which is factory pre-calibrated using Hemiglobincynanide (HiCN)
standard and hence absorbance of the test solution is automatically converted to hemoglobin
concentration and displayed in less than a minute[
].The hemoglobin concentration from
Hemocue was converted to estimate Hematocrit, multiplying it by 3.Hematocrit was also
measured by using manual Micro-hematocrit method. Three-fourth (75%) of capillary tube was
filled, sealed on one side by a sealant and centrifuged at 12,000 RPM for 5 minutes to find
layers of blood components. It is then placed on a Micro-hematocrit reader and the proportion of
the bottom red pack was measured[
].Blood samples were measured by both methods within
2 hours of collection. A hematocrit value of less than 33% is used as a cut-off value to diagnose
anemia in pregnant women.
All laboratory activities were performed by strictly following manufacturers? instruction and
specific standard operating procedures in the laboratory. All reagents and quality control
materials used were checked for their expiry date and their functionality. All instruments and
materials were calibrated before use. A daily optic check was performed using the red control
cuvette when using the HemoCueHB301 analyzer. Both a high and a low liquid control were
run each week to ensure functionality of the analyzer. Hematocrit results of both methods
were recorded on standard report formats according to study participants? unique
identification number and attached to the respective questionnaire.
Descriptive statistics was used to determine frequency and percentage. The data were plotted
from the calculated Hematocrit on the Y axis and Micro-hematocrit on the x axis to make
visual assessment of the relation between the two methods. A correlation coefficient was
analyzed to assess the association between the two methods. Interclass correlation coefficient and
concordance correlation coefficient were also computed as an improved version to indicate
the strength of association. Bland and Altman method was used to assess the agreement
between the two methods which recommends the use of plots with bias and precision statistics
and the plot consists of average of the two methods on X axis and the difference of the two
methods on Yaxis [
]. The area under Receiver operating characteristics (ROC) was also
determined with Hematocrit of less than 33% as a cut-off point, along with sensitivity and
Ethics approval and consent to participate
Ethical clearance was obtained from the ethical review board of Faculty of health sciences,
Jimma University. Permission was obtained from Medical director of JUMC and head of
Laboratory of the Medical center. The purpose and importance of the study were explained and
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oral informed consent was taken from each participant. Confidentiality was maintained at all
levels of the study.
A total of 200 pregnant women participated in this study. The majority (160(80%)) were
within the age range of 20?34, of which 19 were anemic while 106(53%) were from rural area,
of which 8 were anemic. Among the participants, 41(20.5%) attended either college or
University whereas 31(15.5%) were illiterate. The estimated annual family income was 32.5% for the
range of 10,000 to 15,000ETB. From the total pregnant women, 53% were on third trimester
followed by 35% on 2nd trimester and 12% in 1sttrimester(Table 1).
The overall magnitude of anemia among study participants based on Hemoglobin
measurement (Hb<11) by making use of Hemocue HB 301 was 20(10%) whilst by threefold converted
Hematocrit (calculated), it was found to be 21(10.5%). This is also comparable when
Hematocrit (Hct<33%) by Micro-hematocrit method is employed, which is 19(9.5%). Generally, 172
(86%) pregnant women were within the normal range in both methods whilst there were 9
(4.5%) women in Micro-hematocrit method and 7(3.5%) women in three fold converted
method, with value above the upper limit.
Data were plotted from Micro-hematocrit method (Observed) on the X-axis and three fold
converted (calculated) on the Y-axis to make an overall visual assessment and the data were
linear and distributed around the line; most of them within the reference range(Fig 1).
The correlation coefficient to measure the association between Micro-hematocrit method
and three fold converted (calculated) method was = 0.91. The intraclass correlation coefficient
was 0.94 while the concordance correlation coefficient was 0.89.
aHemoglobin level of less than 110g/L
Fig 1. A scatter plot of the observed and calculated methods.
The mean difference of measurements of the two methods based on Bland and Altman plot
was 0.94 while the 95% limit of agreement ranges from 0.6 to 1.3(Fig 2).
The area under the ROC curve with a cut-off point of hematocrit less than 33% has shown
that AUC = 0.86 with 95% confidence interval of 0.79 to 0.93 and p-value of 0.003(Fig 3). The
sensitivity and specificity of the calculated method was also 95.5% and 71.4% respectively,
taking Micro-hematocrit as a gold standard method of hematocrit determination.
A plot has shown that there is a linear association between the three-fold converted
Hematocrit and Micro-hematocrit to measure Hematocrit in pregnant women. Furthermore, the
correlation coefficient as well as the intraclass correlation coefficient and the concordance
correlation coefficient showed that there is strong association between the two methods. It is
important to note that while most of previous studies have shown association between
observed hemoglobin and three fold converted hemoglobin, from hematocrit, this study is the
other way round(hemoglobin to hematocrit). Previous studies has shown that the standard
three fold conversion from hematocrit to hemoglobin underestimates the prevalence of low
hemoglobin (<11g/dl) in children under five years of age in malaria endemic area [
]. It also
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Fig 2. The Bland and Altman plot of limit of agreement.
underestimates the prevalence of anemia and low level hemoglobin in adults and children in
malaria endemic area [
]. This might be due to trapped plasma during centrifugation which
accounts for 3% in normal MCV, slightly higher value in macrocytic cells and a value as high
as 5?6% in microcytic cells (14).
Another study in pregnant women has shown that hemoglobin from Drabkin?s method
was significantly greater than three-fold converted hemoglobin which tends to be more
prominent in anemic pregnant women (r = 0.5). It also suggested that there is no simple conversion
factor between the two measurements [
]. Long interval between sample collection and
analysis may be a reason for this discrepancy.
In the present study, Bland and Altman plot showed the acceptability (Mean
difference = 0.94) of the calculated hematocrit as compared to the observed hematocrit seeing that
the mean difference is not significantly different from zero. This indicates the two methods are
Identical within inherent imprecision of both methods. A study in India showed that there was
a correlation between calculated hemoglobin and observed hemoglobin (r = 0.94) but
acceptability has failed based on the limit of agreement of the Bland and Altman method [
might be due to the fact that the study only involved women on 3rd trimester and the
repeatability also gets worse as the severity of anemia increases.
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Fig 3. The area under the ROC curve.
The ROC curve is plotted to quantify how the new method can discriminate between
anemic and non-anemic individuals. The area under the ROC curve which is 0.86 showed that the
three-fold converted (calculated) method has a high predictive capacity to discriminate
between anemic and non-anemic Individuals.
Hematocrit calculated from Hemocue HB 301 Hemoglobin has acceptable agreement with
Hematocrit determined by Micro-hematocrit method in pregnant women. Therefore, this
study recommends that calculated Hematocrit as a three-fold conversion from Hemocue HB
301 Hemoglobin can be used to diagnose Anemia in pregnant women.
S1 File. Data collection tool for the study.
I would like thank all the study participants for taking part in the study. I sincerely thank staffs
of Jimma university Medical center Laboratory unit and school of Medical laboratory sciences
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for their support during the study. My heartfelt gratitude also goes to Dr. Colin Mudd who
reviewed the paper for its grammar, spelling and texts.
Conceptualization: Girum Tesfaye Kiya.
Data curation: Girum Tesfaye Kiya, Fitsum Mesfin Zewudie.
Formal analysis: Girum Tesfaye Kiya, Fitsum Mesfin Zewudie.
Investigation: Fitsum Mesfin Zewudie.
Methodology: Girum Tesfaye Kiya.
Supervision: Girum Tesfaye Kiya.
Writing ? original draft: Girum Tesfaye Kiya, Fitsum Mesfin Zewudie.
Writing ? review & editing: Girum Tesfaye Kiya, Fitsum Mesfin Zewudie.
8 / 9
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