Fe b ru ary 2013 Vol. 34 No.1

44 Pages·2013·4.57 MB·English

f
e
b
r
u
a
r
y
2
0
1
3
V
O
L
.
3
4
N
o
.
1
P
a
g
e
s
1
-
4
0
a
u
S
T
r
a
L
I
a
N
J
O
u
r
N
a
L
O
f
M
e
D
I
C
a
L
S
C
I
e
N
C
e
3 1 ORIGINAL ARTICLE ORIGINAL ARTICLE HAEMATOLOGY UPDATE
1 .
o
0 Diagnosis of iron deficiency and Low temperature, heat-induced epitope Transformation from a transient
N
2 anaemia of chronic disease retrieval on free-floating/whole-mount myeloproliferative disorder of Down
y archival human gynaecological pathology syndrome to acute megakaryoblastic
leukaemia
r paraffin sections
a
4
u
3
r

b .
l
e o
F V
Compact Max
STA
ntry
u
o
ur c
o
n y
d i
ali
or v
e
bl
essi
acc
ot
n
e
wis
er
h
ot
n
o
ati
m
or
nf
or i
ails
et
d
uct
d
o
pr
n
ai
nt
o
d c
ul
ReliabMiliatyx InnovaMtioanx PerformaMncaex EfficieMncayx gnostica Stago - All rights reserved - Non-contractual photos - 01/2013mation on products which is targeted to a wide range of audiences and co
n L2R - ©2012 Diament contains infor
Innovation born from Expertise ConceptioThis docu
STA Compact Max® is the new generation of analyser
developed by the Coagulation company to meet labs’
expectations.
Diagnostica Stago Pty. Ltd.
Ask for the new design of expertise!
651 Doncaster Road
Doncaster, Victoria, 3108
Australia
Phone (AUS): 1800 4 STAGO
Phone (NZ): 0508 4 STAGO
Fax: [phone]
[email protected]
www.stago.com.au
A u s t r a l i a n J o u r n a l o f
M e d i c a l S c i e n c e
A D M I N I S T R A T I O N C O N T E N T S
February 2013 Vol. 34 No.1
AIMS National Office
Review Article
Chief Executive: Ms Meredith Liddy, BSc GradDipMedTech
GradCertMan
Diagnosis of iron deficiency and anaemia of chronic disease
Administration: Ms Melanie Cleal
Email: [email protected] Julie R Fielding 2
Website: www.aims.org.au
Telephone: [phone]
Facsimile: 61 7 3876 2999
Address: PO Box 1911 Milton Qld 4064 Australia Original Article
Editorial Board Low temperature, heat-induced epitope retrieval on free-
Editors floating/whole-mount archival human gynaecological
Mr John Stirling, BSc(Hons) MLett AFRCPA MAIMS
Honorary Lecturer pathology paraffin sections
Flinders University, South Australia 14
Frank Manconi, Eleanor Kable, Robert Markham & Ian
Assoc Prof Tony Woods, BA BSc(Hons) PhD MAIMS FFSc(RCPA)
School of Pharmacy and Medical Sciences Fraser
University of South Australia
Board Members Original Article
Dr Ross Brown, PhD MSc MBA FAIMS FFSc(RCPA)
Principal Hospital Scientist
Royal Prince Alfred Hospital Evaluation of an agar cell block method to improve cell yield
in non-gynaecological cytology specimens 20
Ms Robyn Wells, BAppSc MAIMS
General Haematology Paul W Shield, Dan Duricic &Truc T Truong
Pathology Queensland
Princess Alexandra Hospital
Dr Christopher McIver, PhD MPH GMQ(AGSM) MAIMS Regular Features
Principal Hospital Scientist, Conjoint Associate Professor UNSW
St George Hospital Haematology Update 24
Assoc Prof Rob Siebers
PGCertPH, FNZIC, FNZIMLS, CBiol FSB Journal-based CPD No. 35 30
Research Associate Professor
School of Medicine and Health Sciences, University of Otago, Book Reviews 32
Wellington, New Zealand
Editor, NZ Journal of Medical Laboratory Science Books for Review 34
Prof Adrian Esterman, PhD AStat DLSHTM
Foundation Chair of Biostatistics Convention & Congress Calendar 36
School of Nursing and Midwifery
University of South Australia Instructions to authors 38
AJMS Statistical Adviser
The Australian Journal of Medical Science is the official publication of
the Australian Institute of Medical Scientists.
AIMS
Circulation 2000 per issue. The Journal is circulated to members in
pathology laboratories, universities and research institutes throughout AIMS NSM Scholarships 2013 26
Australia and overseas.
AIMS NSM 2012 Scholarship Winner Report 27
Annual subscription rates are available from the AIMS National
Office.
AIMS QAP 28
Article reprints may be organised on request from AIMS National
Office. AIMS/RCPA QAP Scholarship 2013 29
Advertising rates are available from AIMS National Office.
AIMS NSM 2013 Call for Abstracts 37
Abstraction of the Australian Journal of Medical Science is through the
following serial catalogue listings: Australasian Medical Index, Chemical
Abstracts, and EMBASE/Excerpta Medica. Advertising Index
The Australian Journal of Medical Science is listed on the Register
Diagnostica Stago IFC
of Refereed Journals by the Australian Commonwealth Department
of Education Employment and Workplace Relations.
LabTech Training 19
ISSN 1038-1643
Printed by Westminster Eagle Eye Printing, PO Box 161, Paddington
Qld 4064.
Design Cover and layout design by Kim Brown, 23 Denman St Exeter SA
5019 [email protected]. Cover photograph courtesy of Prof Ian
Gibbins, Flinders Medical Centre, Adelaide.
Instructions to authors are available on the AIMS website www.aims.org.au
Copyright: All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic, mechanical, including photocopying, recording, or by
an information storage and retrieval system, without permission in writing from the AIMS. Copyright by the Australian Institute of Medical Scientists, 2013.
Disclaimer: The opinions expressed in this Journal including those of the technological and advertisement sections are not necessarily those of the Editorial Board. 1
Australian Journal of Medical Science February 2013 Vol. 34 No. 1
REVIEW ARTICLE
Diagnosis of iron defi ciency and anaemia of chronic disease
Julie R Fielding
Pathology Queensland Cairns Laboratory, Cairns Base Hospital, Queensland
Abstract
Aberration in iron homeostasis can be clinically signifi cant and extreme eff ects manifest as either iron defi ciency anaemia
or iron toxicity. Many patients suff ering from iron defi ciency (ID) or anaemia of chronic disease (ACD) will present with
complex medical conditions including infl ammation, infection or malignancy. Iron status in disease may be classifi ed as
early ID, iron defi ciency anaemia (IDA), functional iron defi ciency (FID), ACD or iron defi ciency anaemia combined with
ACD (IDA/ACD). It is important to diff erentiate between these clinical entities as the treatment regime will vary. A failure
to diagnose anaemia or inappropriate treatment may lead to poorer clinical outcomes particularly in the newborn. Th e bone
marrow iron stain was the initial standard for diagnosing iron defi ciency but is impractical as a screening test. Th is test was
superseded by the serum ferritin which is a very good indicator of iron status when infl ammation is absent. Alternative or
supplementary tests proposed to improve the diagnosis of IDA and ACD include serum transferrin receptor, reticulocyte
haemoglobin content/equivalent, C-reactive protein and the Ferritin Index. Cut-off points for these tests may vary with
diff erent clinical presentations and a single reference range for such tests reduces their diagnostic eff ectiveness. Th e recently
identifi ed iron regulatory proteins like hepcidin are still research subjects but off er the potential for novel therapeutic agents
in addition to better diagnosis of iron status. Th ose who are most at risk of IDA and/or ACD often reside in less developed
or remote locations and would benefi t from a more clinically eff ective diagnostic test such as a point-of-care (POC) test.
Keywords: Anaemia chronic disease, functional iron defi ciency, iron defi ciency, iron defi ciency anaemia
Introduction diagnostic technology and are often in composite clinical
settings. In such situations a test for assessing iron status
Iron homeostasis is critical to an individual’s well-
needs to be diagnostically robust and unambiguous. For
being because iron is central to oxygen delivery, energy
maximum clinical eff ectiveness, these tests should have
production and cellular metabolic processes. Iron is a key
universal availability, a rapid test result turn-around-time
component in DNA synthesis. Extremes of iron levels
(TAT) and low cost.
manifest clinically as hypoxia (ID) versus iron toxicity
(iron excess). Detrimental consequences include ID causing
Ideally a diagnostic test for iron status should be able
anaemia or iron excess causing free radical formation and
to diagnose the presence of ID with or without anaemia
potential organ dysfunction.
and also to diff erentiate early ID from IDA, ACD, FID and
IDA/ACD; fi ve diff erent clinical presentations. Th is would
Anaemia aff ects at least two billion people worldwide
provide clinical staff with a tool to enact early intervention,
(WHO 2004). ID and/or chronic infl ammation are the
choose appropriate treatment options and signifi cantly
most common causes with almost a billion people having
improve world-wide health morbidity and mortality.
IDA (Breyman et al 2002, Sheftel et al 2011). Populations
Australians still die of IDA (ABS 2006) and quality of life
in less developed nations or in tropical locations in
is signifi cantly reduced in those with ACD.
developed nations like northern Australia have a higher
incidence of IDA and ACD and it is particularly prevalent
Th is aim of this paper is to review the tests currently
in the indigenous paediatric population. In Australia it
available for diagnosing IDA and ACD and their cited
has been estimated that the incidence of anaemia is 8%
ability to diff erentiate between ID, IDA, ACD, FID and
in preschool children, 12% in pregnant women and 15%
IDA/ACD.
in non-pregnant women. Indigenous communities have
been shown to have a much higher prevalence of anaemia
Current understanding of iron
i.e. 55% in women and 18% in men (Pasricha et al 2010).
metabolism
Th ese high risk populations usually have limited access to
Traditionally the focus has been on those proteins
involved in the storage (ferritin) and transport (transferrin)
of iron. Additional proteins have recently been identifi ed
Address correspondence to:
Julie R Fielding which appear to play an important regulatory role in
jrfi [email protected] cellular and mitochondrial iron haemostasis. Assays for
these new iron regulatory proteins such as the liver hormone
hepcidin are still mostly at the research level. Translation to
2
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
routine pathology assays is eagerly anticipated as the 'holy like the serum ferritin the use of a bone marrow iron stain
grail' in the diff erential diagnosis of IDA/ACD. Other to assess iron status has declined.
molecules that have been identifi ed are ferroportin, iron
2. Biochemical markers of iron status
regulatory proteins 1and 2 (IRP1 and IRP2), divalent metal
and infl ammation
transporter-1 (DMT1), bone morphogenic proteins 2, 4, 5,
6, 7, 9 (BMPs), growth diff erentiating factor 15 (GDF15) Th e most commonly used biomarkers for iron status
and matriptase-2 (TMPRSS6) (Huang et al 2011). all have advantages and disadvantages in regard to their
ability to accurately and quickly diff erentiate between ID,
Ferroportin has been identifi ed as the main cellular IDA, ACD, FID and ID/ACD.
iron export protein in the enterocytes of the gastro-intestinal
Serum ferritin
tract and placenta and also in the macrophages of the
Serum ferritin is an early marker of ID with levels
reticulo-endothelial system (RES).
falling below normal limits well before morphological
changes appear in the mature red cell. Values of <15ug/L
Hepcidin is now recognized as the major systemic
are diagnostic of iron defi ciency (Lewis et al 2006). In
(negative) regulator of iron through its ability to degrade
childhood lower ranges of serum ferritin correspond to the
ferroportin and subsequently to inhibit extracellular iron
higher demands for iron required for growth.
transit (Th eurl et al 2009). An increase in hepcidin levels
leads to reduced dietary iron absorption in intestinal
enterocytes and also a blockade of iron export from the IDA can develop within three months in premature
RES. Hepcidin has been shown to respond rapidly to infants in contrast to, four years for an adult male to
changes in haematological statuswith anaemia, hypoxia deplete body iron stores and develop IDA due to dietary
and erythropoiesis all suppressing hepcidin expression insuffi ciency or malabsorption.
(Th omas et al 2011). Synthesis of hepcidin is stimulated
by iron overload, an increase in BMPs or the presence Th e major limitation of the diagnostic ability of serum
of infl ammatory cytokines such as interleukin-6 (lI-6), ferritin to detect ID is the sensitivity of serum ferritin to
interleukin-1 (IL-1), tumour necrosis factor (TNFβ) or infl ammation. Patients with ACD who have serum ferritin
interferon gamma (IFγ). values within the reference range may still be iron defi cient.
Patients with chronic renal disease (CRD) on regular
Laboratory testing
haemodialysis and undergoing treatment with recombinant
Due to the lack of clear cut diagnostic criteria for erythropoietin have been shown to respond to iron therapy
determining iron status it is not uncommon for medical even with serum ferritin levels above 200ug/L. Serum
personnel to forgo laboratory testing and prescribe a ferritin levels are also known to be increased independent of
treatment of oral iron when presented with clinical signs iron status in various clinical conditions and cannot be used
and symptoms that suggest anaemia. For those patients as a reliable indicator of iron storage. Such clinical situations
who do not have ID/IDA such a regime has the potential include hyperthyroidsim, liver disease, some malignancies,
to produce iron toxicity and a further deterioration in the alcohol consumption and oral contraceptive pill therapy
patients’ health. Because of the high incidence of anaemia (Brugnara 2003, Cook 2005). If serum ferritin is to be
and ID there has been a drive to improve existing testing used to diagnose ID in the presence of infl ammation then
and to identify new biomarkers for iron. diagnostic cut-off points need to be carefully evaluated to
refl ect the incidence of infl ammation and reference ranges
1. Bone marrow iron stain need to be tailored to the local patient demographic.
Initially the standard for determining the level of Serum iron
storage iron present in an individual was a bone marrow
Serum iron lacks specificity as values are low in
aspirate stained by Perl’s method to demonstrate the
IDA, ACD and FID. Th e concentration of serum iron is
presence of hemosiderin and ferritin (Dacie et al 2006).
variable and changes with recent iron ingestion and diurnal
variation; iron levels are highest in the morning and lowest
In IDA neither macrophage nor erythroblast iron is
at midnight. Individuals may show a physiological variation
present (Hoff brand et al 2005), whereas in ACD or FID
of up to 50%.
iron is usually present but not able to be utilised.
Serum transferrin and total iron binding
Th e bone marrow iron stain is not infallible: one study capacity (TIBC)
reported incorrect results in 30% of cases reviewed (Cook
Transferrin is a glycoprotein which binds iron in the
2005). A bone marrow iron stain is unsuitable as a screening
circulation and then transports it to transferrin receptors
test because of the invasive nature of the test, the expense
located on the surface of cells needing iron. It is synthesized
and often the lack of clinical expertise required to perform
in the liver in response to cellular demand and levels will
this procedure. Since the advent of biochemical blood tests
be elevated in ID and reduced in iron overload. Th e life
3
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
span of transferrin is eight days and the concentration will of standardization between methods and this is a major
change with diurnal variation (Uhr 1998). analytical limitation of the sTfR in detecting ID.
Zinc protoporphyrin (ZPP)
Th e TIBC is an estimate of the capacity of transferrin
Zinc protoporphyrin is a product of abnormal heme
to bind iron and is derived from the transferrin level. A rise
synthesis. When iron supply for incorporation into the heme
in TIBC is characteristic of IDA whereas a reduced TIBC
moiety of haemoglobin is restricted, there is an accumulation
indicates a response to infl ammation or infection. TIBC
of the immediate precursor protoporophyrin IX. In such
and transferrin are both indicators of transport iron; their
instances zinc is readily substituted in the absence of iron
levels will be increased in ID reduced in infl ammation or
to form ZPP (Hoff brand et al 2005).
remain normal when IDA and ACD co-exist.
Transferrin saturation % (TSAT)
Elevated levels of ZPP may detect the presence and
Each transferrin molecule can bind two iron atoms and severity of IDA but ZPP is not suitable for early detection of
usually carries between 13–47% of its maximum carrying ID as the associated rise in protoporphyrin may take weeks
capacity. Th is percentage is reduced in IDA but increased in to become evident. An increase in ZPP is not specifi c to
people taking iron and also in those with haemochromatosis. ID as increased levels are also seen in lead poisoning and
Transferrin becomes overloaded if the bone marrow is unable ACD. False elevation in ZPP values may be seen in patients
to accommodate the iron delivered to it (McNeil et al 2006). on haemodialysis or with elevated serum bilirubin levels
TSAT is calculated from the TIBC: TSAT = [conc. Serum (Cook et al 2005).
Iron / TIBC] x 100 %.
Th e upper limit of the reference range for ZPP has
Th e reference range for TSAT is 16–50% (Dacie et al been quoted by various authors as 40 (Hastra et al 1996),
2006). A TSAT <16% indicates insuffi cient iron to support 60 (Lynch et al 1998) or 80 umol/mol heme (Lasocki et
normal erythropoiesis whereas a TSAT >50% indicates al 2011). IDA is said to present at levels of >80 umol/mol
parenchymal iron loading e.g. haemochromatosis. heme and severe IDA is associated with ZPP levels of >200
umol/mol heme, however, ACD is also associated with
C – reactive protein (CRP)
elevated ZPP levels (>100umol/mol of heme) (Hastka et
Th e CRP level in serum is considered to be one of al 1993).
the best markers of infl ammation and cited cut-off values
3. Haematological markers of iron
indicating inflammation vary from >5 to >10 mg/L.
status
Increased levels are seen in FID, ACD and IDA/ACD. In
uncomplicated IDA CRP levels are normal. Automated haematology analysers provide accurate,
rapid and reproducible cell measurements. Th e haemoglobin,
Serum transferrin receptors (sTfR)
red cell count and associated indices may be used to screen
Serum transferrin receptors are derived from membrane for the presence of ID and IDA. Many of these parameters
transferrin receptors found on the surface of cells which lack sensitivity and/or specifi city as they are based on
utilize iron. Receptor concentration is stimulated by cellular erythrocytes which have a life span of up to 120 days.
iron need and/or cell growth. An up-regulation in the
Reticulocytes mature in two to four days and are
number of cell membrane transferrin receptors allows the
a subset of erythrocytes. . Th e haemoglobin content of
cell to compete more eff ectively for iron. Th e level of serum
reticulocytes will fall within days of the onset of ID and
sTfR corresponds to the concentration of membrane bound
provide an indication of the most recent iron uptake in
receptors and is indicative of the body’s requirement for iron,
the bone marrow.
which is mainly for erythropoiesis (Baillie et al 2003).
Haemoglobin (Hgb)
In ID levels of sTfR do not rise until all available Haemoglobin carries the largest component of
storage iron is depleted and the serum ferritin level is below iron in the body. There are 400 million molecules of
the reference range. As the sTfR is unaff ected by the presence haemoglobin in a single erythrocyte and thus any alteration
of infl ammation, it has potential to be a useful marker for in haemoglobin synthesis, erythrocyte production or life-
ID or FID when the serum ferritin is no longer suitable. span will infl uence the body’s iron levels. A decreased level
However in a diverse hospital population sTfR levels may of haemoglobin generally indicates the severity of ID after
be up-regulated for reasons other than iron depletion. anaemia has developed. But haemoglobin synthesis is also
An increase in sTfR concentration may also occur as a aff ected by a number of other clinical disorders and lacks
response to pathological conditions like haemolytic anaemia, specifi city for the diagnosis of IDA. In the early stages of
hypoxia, malignancy, or physiological states like pregnancy. ID before erythropoiesis is aff ected, the haemoglobin level
Conversely, reduced sTfR levels occur when erythropoiesis may still be normal.
is hypoplastic as in aplastic anaemia and chronic renal
failure (Th orstensen et al 1993). Diagnostic cut-off values
and reference ranges vary considerably because of the lack
4
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
Mean cell haemoglobin (MCH) not identify ID in a complicated and/or remote clinical
setting.
Th e MCH is a product of the mean erythrocyte cell
volume (MCV) and the mean cell haemoglobin concentration Red cell distribution width (RDW)
(MCHC) and is an average of the haemoglobin content per
Th e red cell distribution width (RDW) indicates the
red cell. An MCH <26pg is a more sensitive indicator of ID
variation in size of the erythrocyte. It is measured as the
than an MCV <80fL (Bain 2006) unless the patient has large
standard deviation (SD) and coeffi cient of variation (CV)
erythrocytes which are hypochromic; then the MCH value
of the erythrocyte histogram from which the erythrocyte
may fall within the reference range (Jandl 1996).
count and MCV are derived. The reference range for
Mean cell haemoglobin concentration RDW CV is approximately 11–15% but will vary slightly
(MCHC) depending on the technology used. An increase in the
RDW is a very sensitive indicator for iron defi ciency as
The MCHC is the average concentration of
erythrocyte anisocytosis precedes anaemia and the RDW is
haemoglobin in circulating erythrocytes. Values of <320g/l
usually the fi rst FBC parameter to become abnormal in ID.
have been used to screen for ID but reference ranges are
However, the RDW lacks specifi city as raised RDW values
specifi c to the analysis technique. Also normal MCHC
are also associated with an increased reticulocyte count,
values may be seen in patients who have hypochromic
other haematinic defi ciencies and numerous haematological
erythrocytes but a proportionally low red cell mass (Jandl
disorders.
1996). In vitro infl uences that aff ect the erythrocyte volume
due to hypo-osmolar states may produce incorrect MCV, Reticulocyte and hypochromic/microcytic
MCH and MCHC measurements (Bain 2006). red cell indices
Th e most immediate consequence of reduced iron
In a study of Malawian children with severe anaemia, supply is an inhibition in the haemoglobin formation in
it was proposed that “the MCHC may be an acceptable reticulocytes. Th e reticulocyte haemoglobin which measures
alternative screening test (for IDA) in resource-poor settings” the haemoglobin content of newly produced erythrocytes
(Phiri et al 2009). has been proposed as an indicator of the most recent iron
availability in the bone marrow.
Erythrocyte mean cell volume (MCV)
Haemoglobin contributes to over 90% of the
Some of the newer reticulocyte and red cell indices
erythrocyte and reticulocyte content. The cell size is
are unique to the model and/or individual brands of
considered to be an indirect estimate of the amount
haematology instruments and reference ranges for these
of haemoglobin present and therefore an indication of
values are usually analyser specifi c.
iron supplied for erythropoiesis (Urrechaga et al 2011).
Microcytosis develops when the haemoglobin content in
Th e reticulocyte haemoglobin content (CHr), mean
mature erythroblasts falls below 20pg/cell and an extra cell
cell volume of reticulocyte (MCVr), percentage hypochromic
division occurs (Jandl 1996).
red cells (%HYPO) and percentage microcytic red cells
(%Micro) are produced by the Advia 120 & 2120i ® analysers
In the manufacture of haemoglobin, globin synthesis is
(Siemens Healthcare, Dublin Ireland).
coordinated with the synthesis of porphyrin, the precursor to
haem. Microcytosis and hypochromia are both caused by a
Th e reticulocyte haemoglobin equivalent (Ret-He),
decreased concentration of haem. Haem regulates eIF2alph
hypochromic red cell equivalent (Hypo-He) and percentage
kinase (HRI), a key step in the synthesis of alpha and beta
microcytic red cells (%MicroR) are produced by the Sysmex
globin chains in erythrocytes. When intracellular haem is
XE5000® (Sysmex Corporation, Kobe, Japan).
decreased the translation initiation factor eIF2α is inhibited
and globin synthesis is reduced (Brugnara 2003).
Th e red cell size factor (RSf) and low haemoglobin
density percentage (LHD%) are produced by the LH 750®
Th e MCV lacks specifi city for the diagnosis of ID as
(Beckman Coulter Inc. California USA).
low MCV values are also seen in ACD, thalassaemia, and
sideroblastic anaemia. Conversely renal dialysis patients Reticulocyte haemoglobin content (CHr)
may show a higher MCV compared to their base line values
Th e CHr is the mean value of haemoglobin mass of
pre renal disease. Combined haematinic defi ciencies of ID
each reticulocyte in pictograms. It is determined by two
and megaloblastosis may give a normal MCV albeit with
independent fl ow cytometric measurements:CHr(pg) =
a raised RDW.
MCVr x CHCMr.
Th e MCV is subject to in vitro instability due to values
Th e MCVr is the reticulocyte MCV and the CHCMr
increasing with specimen age and ambient temperature
is the cellular haemoglobin content mean of reticulocytes.
and therefore the MCV will not detect early ID and may
5
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
An increase in hypochromic reticulocytes produces a low Th e Ret-He has also been used in haemodialysis
CHr value whereas replenishment of iron status stimulates an patients to detect a response to iron therapy within two
elevation in CHr levels. weeks of treatment (Miwa et al 2010).
Th e CHr has been shown to detect early ID in adults For patients in remote locations the Ret-He may be
and healthy adolescents. In one study the CHr reference range useful. A study by Brugnara and colleagues demonstrated
in healthy adults was cited as 29-32pg with a cut-off value of a stability of at least 48 h with a change in values of <1SD
<29pg used to detect ID/IDA. Slightly lower cut-off values (0.35pg) in samples stored at room temperature (Brugnara
were used in infants (27.2pg) and toddlers (28.4pg) (Stoff man et al 2006).
et al 2005). Such cut-off values may vary depending on co-
Red cell size factor (RSf)
existing clinical conditions. Th alassaemia patients have been
shown to demonstrate a CHr value which was signifi cantly Th e Red cell size factor (RSf) uses the volume of both
lower than those seen in ID. Patients with megaloblastic/ mature (MCV) and immature red cells (MRV):
macrocytic erythropoiesis who have macrocytic reticulocytes
(e.g. some chemotherapy patients) will have an increased CHr
(Brugnara 2003).
Changes in volume refl ect changes in the cellular
haemoglobin content. Correlation of the RSf with the
CHr values have also been used to reduce dosages in CHr was observed to be 0.85 (Urrechaga 2009). In another
iron therapy for haemodialysis patients who are refractory to study the
recombinant erythropoietin treatment. A combination of CHr
<29pg and percentage hypochromic red cell (%HYPO) >6%
RSf was found to correlate with the Ret-He (r2 =
was found to have the best discriminative ability for detecting
0.8154). Th is was in both normal subjects and in patients
a response to intra-venous iron therapy (Tessitorce 2001).
with ID, beta thalassemia minor, or those undergoing
Reticulocyte haemoglobin equivalent (Ret- haemodialysis with iron therapy and erythropoiesis
He) stimulating agents (ESA). Th e mean RSf for iron defi cient
patients was found to be 82.1 fL, compared with 76.9 fL for
Th e Ret-He is analogous to the CHr and is measured in
beta thalassaemia trait patients. Both of these patient groups
the same units but as the measurement is indirect it has been
had signifi cantly diff erent values compared to the reference
termed an ‘equivalent’. It is calculated from the RET-Y which
range mean of 100.9 fL. However there was some overlap
is the mean value of forward light scatter by reticulocytes and
in RSf values between healthy patients and those with IDA
is a relative measure of the reticulocyte size (Bain 2006). Th e
and also between chronic kidney disease (CKD) patients and
RET-Y is combined with the optical refractive index of the
those with ID. Th e RSf had a higher SD compared to the
reticulocyte in a regression plot to obtain an estimate of the
Ret-He in all four patient groups (Urrechaga et al 2011).
haemoglobin concentration of the reticulocyte:
Percentage hypochromic red cells (%HYPO),
Ret-He (pg) = 5.5569e0.001RET-Y hypochromic red cell equivalent (Hypo-He)
and low haemoglobin density percentage
(LHD%)
Investigations into the clinical usefulness of the Ret-He
based on biochemical iron markers and the CHr showed it When iron supply for erythropoiesis diminishes
to be similar to the CHr in terms of reference ranges and hypochromic erythrocytes are produced (Hoff brand et
ability to diagnose ID. Th e reference range for Ret-He (28.6- al 2005). Hypochromia manifests before the advent of
36.3pg) was similar to that of the CHr (27.8-34.5pg). Receiver microcytosis (Urrechaga et al 2009).
operator characteristics curve (ROC) analysis for the diagnosis
of ID gave a Ret-He cut-off of 27.2pg with a sensitivity of
The %HYPO is exclusive to the Advia 120 and
93.4% and specifi city of 83.2% (AUC = 0.913) P<0.0001
2120i®. Th e Sysmex XE5000® uses a similar but indirect
(Brugnara et al 2006). Th e Ret-He was approved for clinical
measurement called the Hypo-He, and the Beckman
application in the USA by the FDA in 2005 (Jayaranee et al
Coulter LH 750® generates the LHD%.
2010).
Th e %HYPO measures the percentage of red cells
A recent study showed that when diagnosing ID in
with a cellular haemoglobin content mean (CHCM) <280
haemodialysis patients the CHr and the Ret-He gave similar
g/l (David et al 2006). For a diagnosis of ID a %HYPO
diagnostic performances but with higher values than that seen
value of >6% was found to be comparable to a CHr cut-
in non-haemodialysis patients. ROC analysis gave diagnostic
off <29pg (Wish 2006). Th e %HYPO is also considered
cut-off values of 31.2pg for the CHr and 30.9pg for the Ret-
to be a sensitive marker of FID if measured within six to
He. Th is was higher than the target of 29pg proposed by the
eight hours of collection. Limitations of the %HYPO relate
American Kidney Foundation (KDOQI) Guidelines 2006
to inaccuracies in the measurement of the CHCM and
(Buttarello et al 2010).
MCV. A false increase in %HYPO will occur when the
erythrocyte swells with specimen age and higher ambient
6
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
storage temperatures (Brugnara 2003). Th e %HYPO has bone marrow iron stain, patients with stainable iron had
been recommended for use in conjunction with biochemical mean values which were signifi cantly diff erent to those with
iron markers in the American Kidney Foundation (KDOQI) depleted iron stores i.e. 25.0 +/- 19.1 compared to 54.9
Guidelines for monitoring erythropoietin therapy in +/- 27.9. Unfortunately the ranges were so wide that there
haemodialysis patients. was considerable overlap between the two groups (Ervasti
et al 2004).
Th e Hypo-He calculates the percentage of red cells
4. Iron regulatory proteins (IRPs)
with an Hgb of <17pg (Urrechaga et al 2010), and utilizes
both the absolute amount of haemoglobin present in the
Hepcidin
mature red cell as well as the size of the cell (Wish 2006).
Initial methods to measure urine and plasma hepcidin
A reference range for the Hypo-He has been established as
have been cumbersome and lacked standardization. Some
0–0.6% and a cut-off value of >1.8% was proposed for the
commercial assays have become available but further
diagnosis of ID (AUC = 0.963, a sensitivity of 98.3% and
harmonization is required before a hepcidin assay becomes
a specifi city of 91.1%) (Urrechaga et al 2009).
routine (Galesloot et al 2011).
In a study by Grillo et al 2006, the red cells of renal
Hepcidin has been found to be more responsive to
dialysis patients appeared to be recognized as hypochromic
erythropoietic demands for iron than to the stimulus of
by the %HYPO but normochromic by the Hypo-He.
infl ammation. Patients with IDA are able to absorb dietary
Th e Hypo-He was deemed to be unreliable in detecting
iron and mobilize iron from macrophages even in the
hypochromia in renal dialysis patients. Another study
presence of infl ammation. Patients with ACD/IDA have
investigating the diagnosis of ID in renal haemodialysis
lower levels of circulating hepcidin than those with ACD
patients and also for predicting response to iron therapy
alone. Th e most signifi cant increase in hepcidin values are
obtained cut-off values of >5.8% for %HYPO (AUC = 0.72)
seen at ferritin concentrations >200ug/l, when iron overload
and >2.7% for Hypo-He (AUC = 0.68). Hypo-He values
begins (Th eurl et al 2009).
were found to decrease with specimen age and produce false
negatives. It was concluded that the CHr and Ret-He were
the best predictors of response to iron therapy followed by Using regression analysis it was proposed that hepcidin
the %HYPO (Buttarello et al 2010). levels below 0.23nmol/L confi rmed ID and values above
4 nmol/L excluded ID (Th omas et al 2011). A recent
study in critically ill patients with anaemia proposed a
Th e low haemoglobin density percentage (LHD%) is
plasma hepcidin cut off value of 129.5ug/l for diagnosing
derived from the MCHC using the formula:
ID. Plasma hepcidin was undetectable (< 0.05ug/L) in
patients with pure IDA compared to patients with ACD
where the median plasma hepcidin was 566ug/L (range
and it correlated with the %HYPO (r2 = was 0.869).
297–1040). Critically ill patients who had ID either alone
Using a LHD cut-off value of >4% for the diagnosis of or in combination with infl ammation gave a range of 0.05–
IDA, ROC curve analysis gave an AUC = 0.976, a sensitivity 548.3ug/L with a median of 80.5 (Lasocki et al 2010).
of 95.2% and specifi city of 93.3%. But the distribution
Other IRPs
for LDH% was found to be non-Gaussian. Th e LHD%
of normal subjects and beta thalassaemia minor patients Duodenal iron absorption is also inhibited by down-
demonstrated a narrow standard deviation, whereas patients regulation of DMT-1), a transporter of iron, lead, zinc
with IDA, CKD and ACD had large standard deviations and copper across the apical boarder of enterocytes in the
(Urrechaga et al 2010). proximal duodenum.
Percentage Microcytic Red Cells
(%MicroR) Newly discovered erythropoiesis driven regulators of
iron homeostasis are GDF15 and TMPRSS6. Th e former
Th e %MicroR is a research parameter available on the
is a hormone that inhibits hepcidin expression and appears
Sysmex XE5000® and has the potential to detect aberrations
to be induced by infl ammation. Levels of GDF15 are
in iron metabolism and measure response to iron medication.
higher in both ACD and ACD/IDA compared to those
Clinical and analytical limitations encountered with the
with IDA alone (Cullis 2011). TMPRSS6 is sensitive to
MCV also apply to the %MicroR. A study performed by
iron and blocks hepatic hepcidin synthesis by clearing
Urrechaga et al 2009 established a reference range of 0.2 –
membrane-bound Hemojuvelin (Th eurl et al 2009; Lasocki
1.9 % (median 1.1, SD 0.44).
et al 2011).
Percentage hypochromic reticulocyte
(%HYPOr) Hypoxia-inducible transcription factor (HIF) is an
oxygen sensitive nuclear transcription factor which has been
Th e %HYPOr is considered to be an early marker
shown to stimulate expression of duodenal cytochrome b
of diminished iron supply due to the rapid maturation of
reticulocytes. In a study comparing %HYPOr against a
7
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1
(DcytB) and DMT-1 resulting in an increase in intestinal Th e use of this index was found to ameliorate the
iron uptake (Shah et al 2009). eff ects of infection on serum ferritin and also haemolysis
on sTfR when investigating severe anaemia in Malawian
5. Algorithms for diagnosis ID/IDA/ACD
children (Phiri et al 2009).
Rather than use a single test to determine a patient’s
Ferritin index vs. CHr (Thomas Plot 1)
iron status, a fl ow chart or algorithm combining a number
of tests may be used to interpret a patient’s biochemical and In 2002 Th omas and Th omas proposed a graph of
haematological profi le (Fig.1 and Table 1). CHr against the Ferritin Index (sTfR/log Ferritin). A CHr
cut-off of <28pg and a Ferritin Index cut-off of >0.8 (if

the CRP >5), or a Ferritin Index cut-off of >1.5 (when
Hgb < 130 g/l in (cid:387) no infl ammation was present) was used to produce four
< 120 g/l in (cid:386)
quadrants:
(adult reference ranges)
1. Normal: iron replete (Ferritin Index <0.8/1.5)
TSAT < 16%
with normal erythropoiesis (CHr >28pg)
2. ID: reduced iron supply (Ferritin Index >0.8/1.5)
Exclude other causes of
but normal erythropoiesis (CHr >28pg)
anaemia
3. IDA: depletion of iron (Ferritin Index >0.8/1.5)
with ID erythropoiesis (CHr <28pg)
4. FID: iron replete (Ferritin Index <0.8/1.5) with
reduced haemoglobinisation of red cells (CHr
<28pg).
Hepcidin vs CHr (Thomas Plot 2)
Ferritin reduced Ferritin normal Ferritin normal / Th e ‘Th omas plot’ was later updated by substituting
<15–30ug/l in adults >15-30 <100ug/l increased the serum hepcidin concentration for the Ferritin index.
<10–12ug/l in >100ug/l
children and Th e hepcidin cut-off used for ID was <4.0nmol/L (Th omas
and et al 2010).
and sTfR/log ferritin >2
sTfR/log ferritin <1
MCH <27pg or 6. Point-of-care (POC)
or
= IIDA hepcidin normal hepcidin raised Th ere are a number of POC devices that can detect
= AACD/IDA anaemia by measuring haemoglobin but none that can
= AACD
indicate the cause of anaemia or diff erentiate between IDA
and ACD.
Figure 1. Possible algorithm for the diff erential diagnosis of IDA,
ACD and ACD/IDA (Adapted from Cullis et al 2011 modifi ed from HemoCue® Haemoglobin
Weiss & Goodnough 2005)
HemoCue (Angelholm, Sweden) have a number
of models including the Hb201+ (measurement range
Table 1. Anaemia screening
20-250g/L). Ten microliters (uL) of capillary, venous or
Iron defi ciency Anaemia of
IDA arterial blood is required. Th e haemoglobin is measured
anaemia chronic
+ACD at two wavelengths in order to compensate for turbidity.
(IDA) disease (ACD)
The Hb201+ system is factory calibrated against the
Serum ferritin Low 30-100 >100
hemiglobincyanide method and needs no further calibration.
CRP Normal High High Turnaround time is one minute and precision is <4%.
CHr / Ret-He Low Low Normal
Pronto-7® Haemoglobin
sTfR High High Normal
Th e Pronto-7 (Masimo, Irvine, California, USA) is a
Hepcidin Low/
Low Raised palm-sized handheld device which measures the haemoglobin
(urine or plasma) Normal
(SpHb), arterial oxygen saturation (SpO2), pulse rate and
perfusion index, by pulse oximetry. Th e SpHb measurement
Ferritin index (sTfR/log ferritin)
range is 0–250g/L with a SD of <1.1g/L. SpHb comparison
Th e use of the transferrin-ferritin index was fi rst with reference laboratory haemoglobin readings were within
proposed by Punnonen et al in 1997 to improve the 2g/L for more than 95% of measurements. Th e Pronto-7 is
detection of ID particularly in settings with a high incidence non-invasive, provides results within one minute and does
of infection: sTfR Index = sTfR / [log serum ferritin]. not require any consumables.
Measurement of haemoglobin mass via
A ratio of <1 suggests ACD whereas a ratio of >2 carbon monoxide rebreathing
suggest IDA+/-ACD.
Direct measurement of haemoglobin mass (Hbmass)
via carbon monoxide (CO) rebreathing has been used
8
Australian Jour nal of Medical Science February 2013 Vol. 34 No. 1