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Received: 24 August 2018    Revised: 10 February 2019    Accepted: 19 February 2019

DOI: 10.1111/ijlh.13009

ORIGINAL ARTICLE

Percentage of hypochromic red cells as a potential screening

test to evaluate iron status in blood donors

Noraini Amir1 | Sabariah Md Noor1 | Indhira Subbiah2 | Malina Osman3 |

Zainina Seman1

1
Hematology Unit, Department of
Pathology, Faculty of Medicine and Health Abstract
Sciences, Universiti Putra Malaysia, Serdang, Introduction: Haemoglobin (Hb) levels are used to assess eligibility for blood dona‐
Malaysia
2 tion but are not correlated with iron status. The percentage of hypochromic red cells
Department of Pathology, Hospital
Sultanah Aminah, Johor Bahru, Malaysia (%Hypo‐He) has been suggested as a useful screening parameter for iron deficiency.
3
Department of Medical Microbiology and The aim of this study was to determine the cut‐off level and accuracy of %Hypo‐He
Parasitology, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, Serdang,
screening among blood donors.
Malaysia Materials and Methods: A total of 170 blood donors were recruited into the study.

Correspondence
Blood donors were classified into three groups: normal, latent iron deficiency and
Zainina Seman, Department of Pathology, iron deficiency anaemia based on their Hb, serum ferritin and transferrin saturation
Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, Serdang, Malaysia.
(TSAT) levels. The diagnostic performance of %Hypo‐He was evaluated with a valida‐
Email: zainina@upm.edu.my tion group comprising 160 blood donors.

Funding information
Putra Grant ‐ Inisiatif Putra Siswazah (IPS),
Universiti Putra Malaysia, Grant/Award
Number: 9495500
Results: Receiver operating characteristic (ROC) curve analysis showed that %Hypo‐He
is an excellent parameter for detecting iron deficiency, with an area under the curve
(AUC) of 0.906, a confidence interval (CI) of 0.854‐0.957 at a cut‐off of 0.6%, and 74.51%
sensitivity and 88.24% specificity. A moderate negative correlation between %Hypo‐He
and TSAT (ρ = −0.576 [P < 0.001]) and a strong negative correlation between %Hypo‐
He and serum ferritin (ρ = −0.703 [P < 0.001]) were found. A cut‐off value of 0.6% was
applied to the validation group and showed 82.9% sensitivity and 96% specificity.
Conclusion: %Hypo‐He with a cut‐off value of 0.6% is a potential parameter with
high sensitivity and specificity for evaluating iron status among blood donors. This
parameter is suitable for screening because its measurement has a faster turnaround
time than biochemical markers.

KEYWORDS
blood donors, percentage of hypochromic red cells, screening parameter, serum ferritin,
transferrin saturation

1 |  I NTRO D U C TI O N regular blood donation.1 A donation of 450 mL of blood contributes

Iron deficiency anaemia (IDA) is among the most common factors
contributing to the global burden of anaemia. Blood donation is
recognized as the most common iatrogenic cause of iron deficiency
among healthy adults, which arises as a direct consequence of
to 236 mg of iron loss. Without iron supplementation, the recovery
times are more than 56 days. 2
Iron plays a central role in erythropoiesis and is an important el‐
ement in cellular metabolism. Iron store depletion (ID) occurs when
there is a negative iron balance resulting in mobilization of iron

Int J Lab Hematol. 2019;1–6. © 2019 John Wiley & Sons Ltd |  1
wileyonlinelibrary.com/journal/ijlh  
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2       AMIR et al.
stores. At this stage, serum iron and Hb levels are normal despite low
serum ferritin levels. Further iron depletion will lead to iron‐deficient
erythropoiesis (IDE), in which serum ferritin and serum transferrin
saturation (TSAT) will be reduced such that the Hb level remains nor‐
mal. IDE is referred to as iron deficiency without anaemia or latent
iron deficiency. Persistent noncorrected iron depletion leads to true
IDA, which manifests as a low Hb level with increased hypochromic
microcytic red cell levels and a reduction in serum iron, serum ferri‐
tin and serum TSAT levels.
In Malaysia, a minimum haemoglobin (Hb) requirement of 12.5 g/
dL is used as a prerequisite for blood donation to prevent anaemia.
However, the Hb level does not exclude the state of latent iron de‐
ficiency. The Hb level discriminates only blood donors with IDA,
which is the end stage of iron deficiency. Many studies have shown
a higher prevalence of iron deficiency in regular donors than in new
donors.3,4 Without prevention and appropriate counselling, this
group of blood donors will ultimately develop IDA.
The diagnosis of absolute iron deficiency is based on a low
level of serum iron, low level of TSAT and a low level of ferritin.
Unfortunately, the techniques for measuring all these biochemi‐
cal parameters have a long turnaround time and are costly. These
parameters are not suitable for use as screening parameters in the
setting of blood donation. The inability to review the results while
blood donors are still at the site further delays the counselling.
Percentage of hypochromic red cells (%Hypo‐He) is a new pa‐
rameter available in the course of determining the full blood count
with a Sysmex (Sysmex, Kobe, Japan) automated haematology analy‐
ser. Imbalances between iron requirements and the actual iron sup‐
ply cause a reduction in red cell haemoglobin content, which causes
the formation of hypochromic red cells and reticulocytes. This pa‐
rameter reflects iron status, specifically, Hb content <17 pg in the
last 2‐3 months, and has been suggested to be a reliable indicator
of iron deficiency,6 and %Hypo‐He is a suitable screening parame‐
ter because the measurement is rapid, has no incremental costs and
does not require extra blood. Previous work has shown that %Hypo‐
He is not influenced by blood donation, infection or inflammation,
unlike biochemical tests.7,8
Our aim is to identify the potential utility of %Hypo‐He in the
detection of iron deficiency among blood donors and to establish
a cut‐off value with good sensitivity and specificity as a marker for
iron deficiency. Thus, this parameter can be used as a screening pa‐
rameter for iron deficiency among blood donors.
2 |  M ATE R I A L A N D M E TH O DS
2.1 | Study population
This cross‐sectional, prospective study involved unselected new
and regular blood donors at Hospital Sultanah Aminah, Johor Bahru,
Malaysia. Regular blood donors were defined as blood donors who
had made at least two previous donations within 24 months, while
new blood donors had never donated blood before. Informed con‐
sent was obtained from those who agreed to participate in this
study. Universal blood donor deferral criteria were used to exclude
noneligible blood donors.
2.2 | Blood sampling
All blood donors were screened for anaemia at a cut‐off level of
12.5 g/dL using finger prick capillary blood by a HemoCue system
(Hb 301 System, Sweden). HemoCue was validated in a previous
study and showed a sensitivity of 61.9% and specificity of 100%.9
Six millilitres of blood was taken, and 2 mL was placed into each of
the following: an ethylenediaminetetraacetic acid (EDTA) tube for
full blood count and 2 lithium heparin tubes for serum iron, serum
ferritin, total iron binding capacity (TIBC) and unsaturated iron bind‐
ing capacity (UIBC).
2.3 | Analytical methods
A full blood count, including the percentage of hypochromic red
cells and reticulocyte count, was performed by a Sysmex XN 9000
(Sysmex, Kobe, Japan) automated haematological analyser. Serum
iron, TIBC and UIBC, including TSAT, were measured by a Cobas
8000 analyser (Roche Diagnostics, Germany). Serum ferritin was
determined by a Cobas 6000 (Roche Diagnostics) automated ana‐
lyser. All samples were analysed within 2 hours of collection because
%Hypo‐He is influenced by sample stability.8,10
TIBC is a parameter derived from the sum of serum iron and
UIBC. TSAT was calculated as the percentage of serum iron per
TIBC.
2.4 | Patient classification
Blood donors were divided into three groups. Three main param‐
eters (serum ferritin [reference range: 30‐400 µg/L for males and
13‐150 µg/L for females], TSAT [reference range: 15%‐45%] and Hb
level [reference range: 13 g/dL for males and 12 g/dL for females])
were used to classify the groups into groups. In the normal group, all
the parameters were within a reference range. The latent iron defi‐
ciency group was defined by low serum ferritin (<30 µg/L in males,
<13 µg/L in females) and low TSAT (<15%) with normal Hb levels.
The third group was the iron deficiency anaemia group, in which
all three parameters were low serum ferritin (<30 µg/L in males,
<13 µg/L in females), low TSAT (<15%) and low Hb levels (<13 g/dL
for males and <12 g/dL for females).
2.5 | Statistical analysis
Statistical analyses of the data were performed using the IBM
Statistical Package for the Social Sciences (SPSS) version 25. The
diagnostic performance of %Hypo‐He in discriminating between
iron‐deficient and normal blood donors was assessed with receiver
operating characteristic (ROC) curve analysis. The area under
the curve (AUC), sensitivity and specificity were determined. A
P‐value < 0.05 was considered statistically significant. Spearman's
AMIR et al. |
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correlation was applied to find the correlation of %Hypo‐He with
TSAT and serum ferritin.
The diagnostic performance of %Hypo‐He was evaluated with
a validation group comprising a mixture of 21 new and 139 regular
blood donors. True positive, true negative, false‐positive and false‐
negative rates were determined. Sensitivity and specificity in the
validation group were obtained.
When a 0.6% cut‐off value for %Hypo‐He was applied to the
validation group comprised of 160 subjects with a mixture of new
(n = 21) and regular blood donors (n = 139), 93.1% were correctly
classified into the iron‐deficient and normal groups with 82.9% sen‐
sitivity and 96.0% specificity. The findings are summarized in Table 4.
4 | D I S CU S S I O N

3 | R E S U LT S Repeated blood donations will result in IDA if no precaution is taken.

IDA is among the most common causes of donor deferral.11,12 A
A total of 170 new and regular blood donors were recruited for the deferred blood donor may not return to donate blood, which make
study. Three main laboratory parameters used to classify iron sta‐ the blood donor pool smaller. Approximately 50 blood donors in
tus among blood donors, as summarized in Table 1. Thirteen blood
donors who were eligible to donate blood during prescreening
TA B L E 2   Iron status among blood donors
(HemoCue) were found to be anaemic when the Hb level was re‐
tested using an automated haematology analyser (Sysmex XN 9000). Iron status n (%)
With the three main parameters in Table 1 (Hb level, TSAT, serum
Iron
ferritin), blood donors were classified as normal (120; 70.6%), latent Latent iron deficiency
iron deficiency (37; 21.8%) and iron deficiency anaemia (13; 7.6%).   deficiency anaemia Normal
The findings are summarized by gender, type of donor, and propor‐ Gender
tions of normal and iron‐deficient donors in Table 2. All 50 blood
Male (n = 153) 32 (20.9) 12 (7.8) 109 (71.2)
donors who exhibited iron deficiency were regular blood donors.
Female (n = 17) 5 (29.4) 1 (5.9) 11 (64.7)
Figure 1 illustrates the receiver operating characteristics (ROC)
Type of donor
curve analysis for %Hypo‐He in discriminating iron‐deficient blood
New donor 0 (0.0) 0 (0.0) 19 (100.0)
donors and normal blood donors.
(n = 19)
Table 3 shows the optimal cut‐off value for the detection of
Regular donor 37 (24.5) 13 (8.6) 101 (66.9)
iron deficiency using %Hypo‐He with the corresponding sensitivity, (n = 151)
specificity and AUC.
Spearman's correlation was used to assess the relationship of
%Hypo‐He with TSAT and serum ferritin, which are the gold standard
for determining iron deficiency. A statistically significant negative cor‐
relation was observed in the studied setting, as displayed in Figure 2.

TA B L E 1   Demographic factors and laboratory parameters of

iron‐deficient blood donors (n = 170)

Variable Number, n = 170 (%)

Sex
Male 153 (90.0%)
Female 17 (10.0%)
Type of donor
New 19 (11.2%)
Regular 151 (88.8%)
Serum ferritin
Male (<30 µg/L) 44 (25.9%)
Female (<3 µg/L) 6 (3.5%)
TSAT (<15%) 50 (29.4%)
Haemoglobin
Male (<13 g/dL) 12 (7.1) F I G U R E 1   Receiver operating characteristics (ROC)
curve analysis for %Hypo‐He [Colour figure can be viewed at
Female (<12 g/dL) 1 (0.6)
wileyonlinelibrary.com]
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4       AMIR et al.

TA B L E 3   ROC curve analysis for %Hypo‐He in the detection of latent iron deficiency based on an optimal cut‐off value

Parameter Sensitivity (%) Specificity (%) AUC 95% CI Cut‐off P value

Hypo‐He (%) 74.51 88.24 0.906 0.854‐0.957 0.6 <0.001

AUC, area under curve; CI, confidence interval.

ρ = –0.576*

ρ = –0.703**

F I G U R E 2   Relationship of %Hypo‐He with ferritin and TSAT. Moderate negative correlation between %Hypo‐He and TSAT (ρ = −0.576*
[P < 0.001]). Strong negative correlation between %Hypo‐He and serum ferritin (ρ = 0.703** [P < 0.001]) [Colour figure can be viewed at
wileyonlinelibrary.com]

TA B L E 4   Sensitivity and specificity of %Hypo‐He in the protein and the level increases in inflammatory states. TSAT will
validation group of 21 new blood donors and 139 regular blood
have a lagging in declining during the early phase of iron deficiency.6
donors
In addition, diurnal variations in TSAT and serum ferritin make these
  Iron deficient Normal parameters unsuitable for screening tests.8,14
%Hypo‐He ≥ 0.6 29 5 The Hb level measured using capillary blood samples obtained

%Hypo‐He < 0.6 6 120
this study exhibited iron deficiency, including 13 blood donors who
exhibited IDA despite being eligible for blood donation. This find‐
ing showed that using the Hb level to screen blood donors did not
provide a true reflection of the iron status. The haemoglobin level is
suitable only for identifying blood donors with IDA for deferral but
is unable to detect the early stage of iron deficiency in donors. Iron
metabolism is a dynamic process; therefore, no definite single pa‐
rameter can be used to define iron status. Serum ferritin and serum
transferrin receptor‐ferritin index are superior indicators of an in‐
dividual's iron status, but these tests are costly and relatively time‐
consuming and thus not suitable for a blood centre's routine donor
testing repertoire.13 Furthermore, ferritin is an acute phase reactant
by finger prick is being used to qualify blood donors to avoid do‐
nation by people with underlying anaemia. In Malaysia, regardless
of sex and age, blood donors are deferred from donating blood
if their capillary haemoglobin measurement is less than 12.5 g/
dL. However, the Hb level measured using capillary blood tends
to be higher than the venous blood Hb level.12,15 We also found
that approximately 13 blood donors who passed the predonation
screening for a capillary Hb level of 12.5 g/dL using the HemoCue
method had an Hb level below the required level when retested
by venous haemoglobin measurement using an automated haema‐
tological analyser (Sysmex XN 9000). We confirmed the previous
finding, in general, that the Hb value obtained was higher than the
reference method.12 The use of capillary Hb levels using HemoCue
alone as a screening test would result in the recruitment of iron‐
deficient and anaemic donors. This result showed that higher Hb
AMIR et al.
levels should be implemented for predonation screening to de‐
crease the risk of donors developing IDA.
This study confirmed the findings of previous studies that the
prevalence of iron deficiency increases with the frequency of blood
donations.3,4 All iron‐deficient blood donors were regular blood
donors; 37 (24.5%) had latent iron deficiency, while 13 (8.6%) had
IDA. Further removal of blood from these blood donors would pro‐
mote a late stage of iron deficiency and ultimately donor deferral.
Consequently, our national guideline recommended that blood do‐
nors who donate blood every eight weeks or the maximum of four
whole blood donations over 12 months should receive an iron profile
assessment at least once a year.16
The AUC for the %Hypo‐He (0.906, 95% CI 0.854‐0.957) indicates
that this parameter is a good parameter discriminator of iron defi‐
ciency. Our specificity for %Hypo‐He (88.24%) was comparable to that
in previous studies (specificities of 88.46% and 88.1%), and our sensi‐
tivity and AUC were better even though the population of interest was
8,17
different. Our AUC was almost comparable to that of Buttarello
et al18 (AUC: 0.96, 0.93), although their findings favoured reticulocyte
haemoglobin content. Compared to the values in another study by this
author, our AUC, specificity and sensitivity were much better, possibly
19
due to the different populations in the studies. Another study of a
menometrorrhagia population showed an AUC of 0.901 for %Hypo‐
He with 87.2% specificity and 85% sensitivity; however, this compar‐
ison was made with haematological parameters such as Hb, mean cell
volume (MCV) and red cell distribution width (RDW).20
At the cut‐off of 0.6%, %Hypo‐He seems to have a good spec‐
ificity of 88.24% and a sensitivity of 74.51%. We suggest using a
cut‐off of 0.6% to screen for iron deficiency among healthy regular
blood donors. Therefore, blood donors with a %Hypo‐He greater
than 0.6% should be encouraged to adhere to an iron supplemen‐
tation regimen and advised to increase their interdonation interval.
%Hypo‐He has been studied in many populations, such as dialysis
patients and subjects with various types of anaemia and pregnancy,
and the cut‐off value varies according to their population of inter‐
est.8,17,18 The reported diagnostic cut‐off values vary according to
the study population and diagnostic inclusion criteria. A cut‐off
of >2.4%‐2.7% has been proposed for %Hypo‐He for evaluating
iron responses in haemodialysis patients,17,19 whereas a cut‐off of
>0.2% has been suggested for the pregnant individuals.8 Buttarello
et al reported a best cut‐off value of >0.9% to distinguish between
healthy and iron deficiency groups; this value is quite close to the
cut‐off in the current study.18 Urrechaga et al suggested a cut‐off
of 3.5%‐3.6% for detecting iron deficiency in healthy vs various
groups. 21,22 This variation shows the need to establish cut‐off limits
for specific populations.
It has also been shown that %Hypo‐He has a moderate inverse
correlation with TSAT and a strong negative correlation with serum
ferritin. Therefore, an increase in a %Hypo‐He is associated with
a reduction in serum ferritin and TSAT levels. This finding means
that an imbalance between iron supply and iron requirements for
erythropoiesis leads to a reduction in Hb content in mature RBCs
and reticulocytes. Another study also showed an inverse correlation
|
      5
between %Hypo‐He and Hb levels. 22,23 This relation will result in
an increased production of hypochromic red cells. Furthermore,
%Hypo‐He is more likely to provide a stable parameter of iron avail‐
ability than TSAT because TSAT has large circadian variations and
%Hypo‐He is also not affected by inflammation, in contrast to ferri‐
tin and transferrin.7,20,24
In the validation group of blood donors, we found that a %Hypo‐
He of more than 0.6% had 96.0% specificity and 82.9% sensitivity,
even though the ideal screening test would provide 100% specificity.
One limitation of our study is that thalassaemia was not excluded.
Thalassaemia could lead to an increase in the percentage of hypo‐
chromic red cells,18 which might contribute to our finding of five blood
donors with a false‐positive result when the diagnostic cut‐off value
was applied. A local study found that approximately 5% (36 out of 738)
of healthy blood donors were presumed to be thalassaemia carriers
based on red blood indices.25 However, in the current study, approxi‐
mately 93.1% were correctly classified into the iron‐deficient and iron‐
normal groups. If thalassaemia syndrome was excluded, the possibility
of false‐positive results would decrease, and our specificity would be
more than 96%. This analysis confirmed the validity of this parameter
with the suggested cut‐off value. We believe that our results are valid
and will contribute to the growing literature on the subject.
4.1 | Study limitations
A limitation of our study is that we did not assess any inflammatory
markers, such as C‐reactive protein, to exclude the effects of inflam‐
mation in blood donors. The difference in the number of recruited
subjects between males and females and between regular and new
blood donors may bias the results. Another limitation is that we did
not exclude blood donors who were thalassaemia carriers, which
may result in an increased percentage of hypochromic red cells.
5 | CO N C LU S I O N
In conclusion, %Hypo‐He could serve as an inexpensive, reliable
and fast screening parameter to evaluate iron status among healthy
blood donors. Therefore, %Hypo‐He could improve blood donor
management because early detection of iron deficiency before do‐
nors develop IDA is important. Thus, blood donor deferral can be
prevented, and the blood donor pool can be maintained.
AC K N OW L E D G E M E N T
The authors would like to thank Putra Grant (Inisiatif Putra Siswazah),
Universiti Putra Malaysia for the sponsorship of this study.
AU T H O R C O N T R I B U T I O N
Noraini Amir and Indhira Subbiah: conceived and project execution.
Zainina Seman: conceived project, data analysis and wrote manuscript.
Malina Osman: data analysis. Sabariah Md Noor: reviewed manuscript.
 |
6       AMIR et al.
ORCID
Zainina Seman  https://orcid.org/0000-0003-3850-6974
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How to cite this article: Amir N, Md Noor S, Subbiah I,
Osman M, Seman Z. Percentage of hypochromic red cells as a
potential screening test to evaluate iron status in blood
donors. Int J Lab Hematol. 2019;00:1–6. https://doi.
org/10.1111/ijlh.13009