Clin. Lab. Haem.

2001, 23,271-283

REVIEW
A systematic approach to the assessment
of erythropoiesis

H.M. WATERS*.
L.H. SEALf

Summary

Keywords

*Utiiversity Department of Haematology, Manchester Royal Infirmary and 1-Department of

Biological Sciences, Manchester Metropolitan University, Manchester, UK

The pathogenesis of anaemia may be simple or complex and the differential diagnosis
can be difiicult. An appreciation of the erythropoietic processes is required, together
with regular review of investigations, to ensure that appropriate protocols are adopted.
The application of tests, which define different facets of erythropoiesis, should be
appropriate to the clinical circumstances. In some situations, such as the anaemia of
chronic disorders, pregnancy and chronic renal failure, a detailed analysis of
erythropoiesis is often required.
Guidelines for investigating anaemia due to megaloblastosis or haemoglobinopathy are
well established, whereas disturbances of iron metabolism are often difficult to classify.
These require a clear distinction between storage and functional iron to differentiate
whether the defect is due to readily treatable simple iron deficiency or more complex
mechanisms, which do not respond to iron supplementation. Determination of red cell
haemoglobin content, reticulocyte analysis and the assay of serum transferrin receptors are new generation parameters developed to address this.
Practice pressures and new treatment options have contributed to investigations
becoming more complex, especially those of the secondary anaemias, as new tests have
become more readily available and often automated. This has resulted in reduced
turnaround times and clinical demand has driven request patterns. Initiatives to
develop evidence-based anaemia management protocols are welcomed but, wherever
possible, should he developed through collahoration hetween the haematology
department and the user unit, and hased on available guidelines.
Erythropoiesis, review, anaemia, chronic, parameters, secondary

Anaemia can be a feature ofthe disease processes in nearly

180 disorders. Whilst, in some of these, the pathogenesis
may be simple, in others it can be complex, which can
mai^e differential diagnosis more difficult. An appreciation
of the cell biology, physiology and pathophysiology of
erythropoiesis is essential to ensure that appropriate
investigation protocols are adopted in individual cases.
This approach cuts across traditional ciinicai and

laboratory classifications. As improvements in the understanding of erythropoiesis continue, it is also important to

re-evaiuate approaches and protocols periodically to
incorporate new ideas and evaluate any new or improved
analytical methods for the investigation of anaemia.
Normal erythropoiesis is the result of a steady-state
baiance between red cell mass (RCM) and erythropoietin
(EPO), with each influencing the other (Figure 1). In
uncomplicated sudden blood loss, RCM falls, EPO rises and
RCM is restored by a phase of hyperproliferative erythropoiesis. Three main variations from this EPO/RCM relationship have been identified in anaemias.

Accepted for publication 7 August 2001

Correspondence: H. M. Waters. University Department of
Haematology. Manchester Royal Infirmary, Oxford Road, Manchester
M13 9WL. UK. E-mail: hwaters@labmed.cmht.nwest.nhs.uk

1. Hypoproliferative erythropoiesis in which either the

bone marrow is unabie to respond to an enhanced EPO
signal, as seen in iron deficiency - sideropenic, or where

Introduction

2001 Blackwell Science Limited

A systematic approach to the assessment of erythropoiesis

Red Cell Mass

Bone
Marrow

Kidney

Erythropoietin

relationship between EPO and RCM but, because of

failure of O2 delivery to the kidney, RCM increases as in
the secondary polycythaemias. Alternatively there may
be a hyperproliferative response because of loss of the
physiological link between RCM and EPO, as seen in
primary polycythaemia when there is an erythropoietic clone with a reduced or absent dependency on
EPO,

Figure 1, Components of erythiropoiesis.

Traditional parameters

the EPO control mechanism or response to it is

diminished, as seen in chronic renal failure (CRE) and
the anaemia of chronic disease (ACD) - non-sideropenic.
The latter two are complex conditions and are expanded
upon later.
2. Ineffective erythropoiesis in which an increased
response is found but the output of cells is inadequate,
as seen in megaloblastic anaemia, aplastic anaemia,
thalassaemia, abnormal haemoglobins and iron deficiency.
3. Hyperproliferative erythropoiesis in which an increased
response occurs but the demand for cell replacement
exceeds the compensatory capacity of the bone marrow, as seen in haemolytic anaemias.

Investigations: past

Some types of anaemia show more than one type of

pathogenesis, for example, thalassaemia can have both
hyperproliferative and ineffective erythropoiesis, whereas
iron deficiency can have hypoproliferative and ineffective
elements. Analyses are available which define different
facets of erythropoiesis. Some are basic descriptors, for
example, the red cell count or haemoglobin concentration,
whereas others are functional response markers, for
example, reticulocytes or soluble transferrin receptors.
When investigating anaemia, these analyses must be used
in an appropriate sequence or algorithm. Initially it is
sufficient to establish whether anaemia is due to an inability
to make red cells or an inability to make haemoglobin. In
some instances this approach will be all that is required.
However, in others, a full profile of erythropoiesis is
essential and may require quite different thinking, including using different reference ranges for some investigations. The approach will also difi'er depending on whether
the investigation is trying to establish a diagnosis or looking
for a transition point during the course of a disorder and its
treatment, for example, checking iron availability when
patients with CRF are treated with EPO. Table 1 summarizes the application of parameters described below.
In erythrocytosis, although the outcome is different,
the approach is similar. There may be an appropriate

Clinical skills and observation originally formed the basis

of a patient profile. These, together with a limited, manual,
blood count, assessment of the reticulocyte response and
examination of blood film and bone marrow morphology
all played major roles. Diagnosis was presumptive and
often unreliable in differentiating anaemias with different
pathophysiologies.
Investigations: present
The differential diagnosis now includes a much extended
blood count profile which includes a readily available,
accurate measurement of the mean cell volume (MCV).
Assay of circulating levels of ferritin, vitamin B12 and
folate are carried out by most laboratories. Differential
studies into the patient's vitamin B12 absorptive capacity
are undertaken routinely in most large hospitals. Tests
for serum autoantibodies are offered by fewer centres, but
are also readily available. Some functional tests tend to
be available only from a few specialized laboratories.
Blood film
Automated erythrocyte analysis has reached the stage
where examination of the peripheral blood film is used
mainly to verify indices. This is particularly important in
the case of mixed cell populations and for red cell changes
which do not affect blood count parameters.
Bone marrow examination
This provides a general evaluation of erythropoiesis and
has been largely superseded by non-invasive methods.
Sufficient information can usually be derived from peripheral erythrocyte analysis to indicate erythropoietic
drive. The estimation of stainable iron in bone marrow
biopsies is a time-honoured method for assessing iron
stores, although the procedure is traumatic for the patient

2001 Blackwell Science Ltd., Clin. Lab. Haem., 23. 271-283

H. M. Waters & L H. Seal

Table 1, Summary of parameters useful in evaluating erythropoiesis

Test

Sample

Application

Blood fllm

Peripheral blood

Important for mixed cell populations and for red cell changes which do not affect
blood count parameters.

Bone marrow

Marrow aspirate

General evaluation of erythropoiesis. Gold standard for iron stores.

MCV

Peripheral blood

General indication of the size of circulating red cells. Non-speciflc. Cut-off values
need to be reconsidered in some situations.

Hypochromia

Peripheral blood

Reflned MCH measurement indicating haemoglobin content of individual

erythrocytes only available with some analysers. Provides a direct indication
of iron delivery to the bone marrow.

Reticulocytes

Peripheral blood

Manual methods of limited value. Automated analysis provides a range of

parameters including reticulocyte haemoglobin content, a sensitive indicator
of functional iron deflciency.

Ferritin

Serum

Reliable estimate of iron stores but can be disproportionately increased due

to acute phase response. Used in haematinic screen.

sTfK

Serum

Generally considered a sensitive indicator of iron deflcient erythropoiesis

unaffected by acute phase response. Results more meaningful when expressed
as a ratio with serum ferritin.

Serum iron/TIBC

Serum

Only reliable application is in screening for idiopathic haemochromatosis.

ZPP

Peripheral blood

Rapid analysis indicative of both absolute and functional iron deflciency.

Not always reliable in monitoring iron supplementation.

B]2 assay

Serum

Differential diagnosis of macrocytosis. Reference ranges derived from white

populations may not be generally applicable. Used in haematinic screen.

Folate assay

Serum/red cell

DiiTerential diagnosis of macrocytosis. Serum and red cell assays each have
limitations therefore both should be assayed. Used in haematinic screen.

IFAB

Serum

Diagnostic test for pernicious anaemia but only 70% sensitivity.

Bi2 absorption studies

/n vivo

Differential diagnosis of B12 malabsorption. Does not exclude malabsorption

of food-bound vitamin.

du suppression test

Marrow aspirate

May be useful to conflrm mild B]2 or folate deficiency.

Methylmaionic acid

Serum

Increased in B12 deflciency.

Homocysteine

Serum

Increased in B12 and folate deflciency.

and the method only semiquantitative. Observing iron in

developing cells provides direct information to differentiate
between reduced stores in iron deficiency and impaired
functional release from reticuloendothelial cells in the
chronic anaemias (Baynes, 1996; Worwood, 1997). For
this reason most investigations, which attempt to evaluate
methods for assessing iron stores and functional iron, have
used bone marrow iron observation as the reference
method.

Mean cell volume and mean cell haemoglobin

A low MCV indicates a general decrease in the size of
circulating red cells and this effect may be seen in more
than one condition. In contrast, although discovery of a
2001 Blackwell Science Ltd.. Clin. lab. Haem., 23, 271-283

high MCV can facilitate the early diagnosis of megaloblastic anaemia, since this will often precede clinical
anaemia by months or even years, up to 4% of all
patients may have abnormally high MCVs in the
absence of any other abnormality (Lindenbaum,
1983), Natural and biological variations, sometimes
compounded by coexisting pathological conditions often
result in great variation in the MCV rendering it an
unreliable, if not misleading, measurement on occasion.
Thus, a normal, lowered or raised MCV cannot always
be equated to normo-, micro- or macrocytosis, respectively, often making examination of the blood film
essential. Cavill (1997) describes the MCV as an 'elastic
parameter' and suggests that, since the haemoglobin
content of red cells is fixed throughout their lifespan, the

A systematic approach to the assessment of erythropoiesis

mean cell haemoglobin (MCH) provides a more reliable,

and standardizable, index of cell size.
Reticulocytes
Reticulocyte analysis is undergoing fundamental change.
Subjective visual investigations have restricted its usefulness in the past to situations where marked changes in
bone marrow response may be occurring such as haemorrhage, haemolysis or the response to treatment. High
levels of imprecision, with a coefficient of variation (CV)
typically in excess of 25%, have severely limited the
applicability. In an attempt to reduce method imprecision,
the International Council for Standardization in Haematology (ICSH) has proposed a manual reference method
based on the determination of the reticulocyte to red cell
ratio (International Council for Standardization in Haematology, 1998). It is suggested that this method, which
standardizes specimen collection, staining and counting
procedures, should be used to determine the accuracy of
automated counting systems and to provide a means for
instrument calibration. The advent of analysis by flow
cytometry has encouraged a re-evaluation of the role of
reticuloctye analysis. This is discussed further, in 'New
Parameters', below.
Ferritin assay
Body iron in excess of requirements comprises ferritin and
haemosiderin. Most is in the form of ferritin, which is
water-soluble. Iron may be mobilized from both forms, but
is more accessible from ferritin. Found chiefly in the
cytoplasm of cells of the reticuloendothelial system, it was
once thought that ferritin did not appear in plasma or
extracellular fluid under normal conditions but development of a sensitive immunoradiometric technique by
Addison et al. (1972) enabled the demonstration that
ferritin is a constituent of all normal human serum. In
contrast to tissue ferritin, ferritin present in serum is low
in iron content and glycosylated. However, the concentration in serum may be directly related to body iron stores
and the assay has become the most widely applied
evaluation of iron status. The original radioassay techniques have been largely superseded by automated
enzyme-labelled techniques.
In most normal adults, concentrations range from 15 to
300 f.ig/\, but these vary with age and sex (Worwood,
1982, 1997: Baynes, 1996). Serum ferritin concentrations are relatively high at birth. As fetal red cells are
removed from the circulation during the first few weeks of
life, the haemoglobin concentration falls to 10-11 g/dl. At

this time the rate of erythropoiesis is relatively low and

iron released from the destruction of fetal cells is stored in
the tissues. This process is accompanied by an increase in
serum ferritin concentration. Synthesis of adult haemoglobin commences during the following 2 months, causing a rapid fall in both iron stores and the serum ferritin
concentration. Both remain relatively low throughout
childhood and adolescence.
Serum ferritin levels are higher in men than in women
of childbearing age, refiecting difierences in storage iron.
Levels in postmenopausal women are closer to those
found in men. The concentration falls during pregnancy.
The initial reduction is thought to reflect increased
maternal erythropoiesis, whereas in the latter stages
transfer of iron to the fetus is the main cause. From
35 weeks, serum ferritin concentrations are lower in
women who have not received iron therapy than in
those who have.
Addison et al. (1972) found that patients with iron
deficiency anaemia have serum ferritin levels approximately one-tenth of normal subjects, while patients with
iron overload (haemochromatosis, haemosiderosis) have
serum ferritin levels much higher than normal. Serum
ferritin levels may serve as a tool to monitor the effects of
iron therapy. However, raised concentrations must be
interpreted with caution. Ferritin behaves as an acute
phase protein and in some circumstances the serum level
may not illustrate the true state of iron stores since
immunoassay techniques refiect ferritin protein rather
than iron. In both adults and children, chronic infiammation results in a disproportionate increase in ferritin levels in
relation to iron reserves. Elevated ferritin levels, not directly
related to iron stores, are also observed in acute and chronic
liver disease, chronic renal failure and in some types of
neoplastic disease (Baynes, 1996; Worwood, 1997).
Serum iron and total iron binding capacity
Measurement of the serum iron and total iron binding
capacity (TIBC) can provide information relating to
transport iron. Recommendations on methodology have
been made by the ICSH (International Committee for
Standardization in Haematology, 1978, 1990), Iron
deficiency results in low serum iron concentrations,
whereas high levels are found in iron overload conditions.
Unfortunately this is not always the case. In the presence
of inflammation or infection the serum iron is low
irrespective of the level of storage iron present. High
concentrations may also be encountered in liver disease,
hypoplastic anaemias and in conditions where erythropoiesis is inelTective (Worwood, 1997).
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H. M. Waters & L, H. Seal

Isolated sampling from the transport iron compartment

can provide unreliable, if not misleading, information.
Circulating iron, bound to transferrin, comprises only a
very small part (0.01%) of the total body iron and has a
very high turnover rate of 10-20 times per day in normal
subjects (Cavill etal., 1977). These factors contribute to
the variability of measurement encountered and severely
limit the diagnostic usefulness of an individual serum iron
measurement. The TIBC is indicative of the circulating
transferrin concentration and is a more stable measurement than the serum iron (Cavill, 1982). Measurement of
both the serum iron and TIBC provides more information
than individual measurements (Worwood, 1997), Transferrin normally circulates about one-third saturated.
Calculation of the percentage transferrin saturation from
the serum iron and TIBC (serum iron/TIBC x 100) reflects
the transport compartment but, for reasons oudined
above, is also too variable as a single measurement
(Baynes, 1996).
In iron deficiency, a low serum iron is associated with a
raised TIBC, resulting in a low percentage transferrin
saturation. A saturation level of 16% or less refiects
inadequate transport iron to sustain erythropoiesis. Pregnancy and anaemia secondary to chronic disease are also
associated with low transferrin saturation despite the
presence of adequate iron stores. Iron overload results in
high transferrin saturation levels due to the combination
of an elevated serum iron and a normal or depressed TIBC.
The only viable clinical role for serum iron and TIBC is in
screening for idiopathic haemochromatosis. The diagnosis
of this condition, one of the most common inherited
conditions in populations of European origin, should
include the demonstration of a raised transferrin saturation
(Males > 60%, Females > 50%) on at least two blood
samples (Worwood, 1997), In the early stages ofthe disease
this abnormality will be encountered before excessive iron
accumulation and the resulting raised ferritin.
Zinc protoporphyrin
Zinc protoporphyrin (ZPP) is synthesized by developing
erythrocytes when there is insulBcient iron available to
ferrochetalase for haem production. It can be quantified
by spectroscopy and instruments are available for rapid
analysis. Values are standardized by expressing the result
as a ratio to haem. In healthy blood donors, a good
inverse correlation between serum ferritin and ZPP has
been found. Studies in a range of disorders suggest that
ZPP is indicative of both absolute and functional iron
deficiency and may substitute for percentage hypochromia where the instrumentation required for this analysis
2001 Blackwell Science Ltd.. CUn. hah. Haem.. 23, 271-283

is unavailable. However, ZPP does not predict a response

to iron supplementation in some renal patients. There are
some instances when fluorescent substances in samples
interfere with the assay, giving falsely elevated results.
Washing the erythrocytes before analysis can reduce this
but some of the test's facility is lost (Braun, 1999),
Vitamin B^ and folate assays
Vitamin B12 and folate act as coenzymes in two important
metabolic functions vital to normal cell growth and DNA
synthesis: (1) the synthesis of methionine, and (2) the
conversion of methylmalonyl CoA to succinyl CoA (Weir &
Scott, 1983; Chanarin et al, 1992; Hoflbrand & Jackson,
1993). In the first of these, vitamin Bj2 deficiency interferes
with the regulation of folate metabolism, resulting in
impaired DNA synthesis and consequently megaloblastic
anaemia. Because both vitamins are linked by the reaction
pathway for methionine synthesis, a deficiency in either
will disrupt this and produce the same symptoms. However, the causal mechanism remains a topic of some
discussion (Chanarin et al, 1992; Hoffbrand & Jackson,
1993). Much ofthe research on this pathway resulted from
the discovery that nitrous oxide inhibited thymidylate
synthesis through inactivation of vitamin B12 (Amess
et al, 1975, 1978). In the second reaction, vitamin B12
deficiency results in impaired fatty acid metabolism which
may in turn lead to abnormal myeiin lipid formation,
contributing to neurological lesions (Weir & Scott, 1983).
In general, the serum or plasma vitamin B]2 concentration is related to the degree of tissue depletion, lowest levels
occurring in the most severe cases (Chanarin, 1990; British
Committee for Standards in Haematology General Haematology Task Force, 1994a). Normal levels vary depending
on the method used and range between 120 and 1100 ng/l.
Results falling within plus or minus 20% ofthe lower end of
the reference interval should be regarded as Indeterminate',
and the sample repeated (British Committee for Standards in
Haematology General Haematology Task Force 1994a),
Carmel (1999) suggests that ethnic and racial factors
should be considered when investigating vitamin B12
disorders and metabolism, and questions the adequacy of
reference ranges derived largely from white populations.
Elevated vitamin B12 levels have been associated with the
use of oral contraceptives and multivitamins, and in
myeloproliferative diseases such as chronic granulocytic
leukaemia and myelomonocytic leukaemia. An elevated
vitamin Bj 2 level in itself has not been known to cause
clinical problems (Chanarin, 1979, 1990),
Folate levels in both serum and red cells are used to
assess folate status. Normal serum concentrations range

A systematic approach to the assessment of erythropoiesis

between 3.0 and 20.0 fig/l In contrast, red cells have

much higher folate levels, i.e. between 150 and 600 fig/l.
A low serum folate is indicative of deficiency in the
absence of recent folate intake. Red cell folate is the best
indicator of long-term folate stores (Chanarin, 1979,
1990). A low red cell folate value is suggestive of a
prolonged folate deficiency. However, a consequence of the
common metabolic pathway is that B12 deficiency disrupts
the uptake of folate into red cells. This can lead to a low
red cell folate value even with adequate folate intake. For
this reason, it is often necessary to measure both vitamins
in a clinical workup. A case can be made for either the
serum or red cell folate to be assayed but, because of the
limitations of each, it would seem reasonable to assay both
or to assay the red cell concentration whenever the serum
folate is low. The true incidence of folate deficiency is not
well known and most figures are derived from the
frequency of anaemia in pregnancy. Globally, malnutrition is the most common cause of both folate and vitamin
Bj2 deficiency (Dawson & Waters, 1994),
Both vitamins can be measured in serum by competitive
protein binding assay. Most vitamin B12 methods use
intrinsic factor (IF) as the specific binding protein,
although radioimmunoassays have been described
(O'Sullivan et al, 1992). ^-Lactoglobulin from cow's milk
is the binding agent commonly used in folate assays.
During the past 10 years, fully automated non-radioisotopic systems have largely replaced radioisotopic manual
methods. In these systems, monoclonal antibodies are
often used to set up the conditions whereby the specific
binder is attached to a solid phase.
Intrinsic factor antibodies and assay
The demonstration of circulating intrinsic factor antibodies (IFAB) is virtually diagnostic of pernicious anaemia
(Lindenbaum, 1983), They are rarely found in other
autoimmune diseases such as disorders of the thyroid,
diabetes mellitus and myasthenia gravis. Two types of
IFAB are known to exist. Type I blocks the IF binding site
for B12, preventing uptake of the vitamin, whereas type II
reacts with an alternative antigen site on the IF molecule
and may prevent attachment ofthe IF-B12 complex to the
ileal binding sites (Roitt et al, 1964; Chanarin, 1979).
Traditionally, techniques of testing for IFABs have been
functional methods for the detection of type I antibody
(Chanarin, 1979). Immunoassay methods have also been
described which are capable of detecting both type I and
type II IFAB (Conn, 1986; Waters et al, 1989), or type II
alone (Sourial, 1988). Subsequent studies using this
methodology have indicated that type II IFAB occur.

alone and in combination with type I, in a much higher

proportion of pernicious anaemia samples than was
previously recognized (Conn, 1986; Waters et al, 1993),
Assay of gastric IF is a direct measurement of the defect in
pernicious anaemia but this requires an invasive technique and is rarely used.
Absorption studies
The urinary excretion method of Schilling (1953) remains
the most commonly used and best standardized test for
absorption of free vitamin B12, The recommended procedure (International Committee for Standardization in
Haematology, 1981) is based on the Schilling test and
employs a 1.0-jUg aqueous oral dose of cyanocobalamin
radiolabeiled with cobalt-57. In this form, the test is carried
out as a two-part procedure, without IF (part I) and with IF
(part II) added to the oral dose. Dual-labelled methods,
based on the Schilling test, where the two stages are
combined by simultaneously administering two radiolabeiled versions of cyanocobalamin, one bound to IF, the
other unbound, were developed in the 1960s (Katz, DiMase
& Donaldson, 1963; Bell, Bridges & Nelson, 1965).
Although the original adaptations held potential advantages over the two-stage approach, particularly in terms of
patient attendance and sample collection, doubts have been
expressed over a later commercial modification (Payne &
Finney, 1972; McDonald, Barr & Barton, 1975; Fairbanks,
Wahner&Phyliky, 1983;Zuckier&Chervu, 1984; Atrah&
Davidson, 1989). This test has recently been withdrawn
from the market.
Demonstration of normal absorption of free vitamin B12
does not exclude malabsorption of the food-bound vitamin. In patients with a subnormal serum B]2. a normal
Schilling test and satisfactory dietary intake, a food-bound
absorption test should be considered (British Committee
for Standards in Haematology General Haematology Task
Force, 1994a). Vitamin B12 deficiency due to food-bound
malabsorption does not reach the same degree of severity
as that which may occur in pernicious anaemia because
the enterohepatic circulation of the vitamin remains intact
whilst IF is available. The incidence of the condition is
unknown but Carmel (1995, 1996) believes it to be at
least as common as pernicious anaemia and a major cause
of vitamin B12 deficiency.
Deoxyuridine suppression test
The deoxyuridine suppression test can be used to confirm
mild vitamin B12 or folate deficiency, although in some
deficient patients the test can be normal. Bone marrow is
2001 Blackwell Science Ltd., Ctin. Lab. Haem., 23, 271-283

H. M. Waters & L. H. Seal

the recommended sample as opposed to peripheral blood

lymphocytes. The test is labour-intensive and expensive,
and is only available in a few specialized centres (British
Committee for Standards in Haematology General Haematology Task Force, 1994a),

type of disorder the pathogenesis is typically more

complex. The main situations where this arises are in
the ACD in rheumatoid patients, pregnancy and chronic
renal failure patients being treated with recombinant
human erythropoietin (r-HuEPO),

Methylmaionic acid and homocysteine

Anaemia of chronic disorders

Both methylmaionic acid (MMA) and homocysteine (Hey)

are increased in the serum of patients with vitamin B12
deficiency, whereas only Hey is increased in folate
deficiency. Measurement of MMA in a random urine
sample can be used as a screening test, although the 24-h
excretion level is more sensitive. Serum is the sample of
choice. These metabolite assays can be automated using
capillary gas chromatography-mass spectrometry, high
performance liquid chromatography-mass spectrometry or
immunoassay, but the equipment required is expensive
and currently only available in a few specialized centres.
However, new generation, integrated, systems are likely to
become more available to a larger number of laboratories.
Thin-layer chromatography is recommended as an inexpensive but sensitive screen for urinary MMA (British
Committee for Standards in Haematology General Haematology Task Force, 1994a).

Means (1999) has reviewed the pathophysiology of this

syndrome. Often confused with and sometimes wrongly
referred to as 'iron deficiency anaemias', ACD has a much
more complex pathogenesis than simple iron-deficient
erythropoiesis, ACD is associated with infiammatory
states, including chronic infection, rheumatoid arthritis
and neoplasia. It is one of the most common disorders
found in medicine and accounts for a significant proportion of anaemia investigations. There is evidence that the
common factor causing anaemia is the change in immune
and inflammatory mediators common to all of these
disorders. Increased concentrations of tumour necrosis
factor, interleukin-1 and interferons have been implicated
and the mechanisms by which they disrupt erythropoiesis
explored. The effect on erythropoiesis is threefold. A
shortened red cell survival producing a slight increase in
red cell production, impaired erythropoiesis due to reduced
erythropoietin production and reduced progenator sensitivity, and impaired release of reticuloendothelial iron. The
resulting hypoproliferative erythropoiesis has in the past
been characterized by a low serum iron and a normal or
raised serum ferritin. This confiicting information has
made it difficult to draw conclusions about true iron status
in relation to the anaemia. Recombinant erythropoietin
has already been used to correct the anaemia in some
cases of ACD. Better measures of erythropoiesis will have
to be used to ensure adequate iron is available to support
the erythropoietic drive and for monitoring treatment
response. A fundamentally different approach is therefore
required to differentiate whether the defective red cell
output is due to simple iron deficiency, which is readily
treatable, or more complex mechanisms which do not
respond to iron supplementation.

Guidelines
A clinical history and blood count with appropriate
follow-up tests usually leads to a rapid laboratory
diagnosis and protocols for investigating anaemia due to
megaloblastosis (British Committee for Standards in
Haematology General Haematology Task Force, 1994a)
or haemoglobinopathy (British Committee for Standards
in Haematology General Haematology Task Force, 1994b,
1994c, 1998) are well established. The pathogenesis of
disturbances of iron metabolism is more difBcult to
classify, and requires a clear distinction between storage
and functional iron. Most of the newer generation tests
have been developed to address this. Therefore the
remainder of this article will concentrate on hypoproliferative variants around functional iron deficiency and
anaemia of chronic disorders.
Problem areas
The mechanism underlying some anaemias is relatively
simple and the diagnosis straightforward, e.g. simple iron
deficiency. In some situations a more detailed analysis of
erythropoiesis may be necessary to ensure that an
adequate response to treatment may be achieved. In this
2001 Blackwell Science Ltd.. CUn. Lab. Haem.. 23. 271-283

Pregnancy
A number of physiological changes occur in pregnancy,
including expansion of plasma volume, an EPO-driven
increase in erythropoiesis to provide an expansion in RCM
and increasing demands of the fetus for haematinics.
These adaptations complicate the assessment of haematinic deficiency; for example, a reduction in haemoglobin
concentration cannot be relied upon as a first-line

A systematic approach to the assessment of erythropoiesis

screening test. Iron stores are challenged by normal

pregnancy. Research, by tracking EPO concentration, has
shown that some benefit is likely to be gained by iron
supplementation (Milman et al, 1997), Better criteria are
therefore needed to provide a reliable indication of iron
stores and functional iron in this situation. Using a study
population which included anaemia, chronic inflammation and some thalassaemias, van den Broek et al. (1998)
explored measures of iron status referenced against bone
marrow-stainable iron. They confirmed that the MCV is
unreliable as a screen due to the erythropoietic drive
causing shortened transit times. Using a typical cut-off
value of 1.2 ^mol/1, these authors showed ZPP to have
very poor specificity and that this could lead to significant
over-diagnosis of iron deficiency. When referenced against
bone marrow iron they suggest that serum ferritin can be
used as a reliable indicator of stores provided that the
cut-off value is raised to 30 /ig/l. They also suggest
specificity can be further improved (to 89%) by using
multiple parameters, e.g. by combining serum ferritin with
serum transferrin receptor (sTfR) and C-reactive protein
measurements.
Similar questions have been raised about the significance of estimates of B12 and folate status in pregnancy
(House et al, 2000). Reliability of single estimates is
questionable. It may be necessary to follow serum
vitamin assays with biochemical indices (serum Hey
and MMA) before advising supplementation. In another
study, Pardo et al. (2000) confirmed that serum B12
assay alone in non-anaemic patients may be unreliable.
Hey and MMA values indicated there was no difference
between a group with reduced B12 and one with normal
levels. More extensive studies are needed to define
variability in all of these parameters in pregnancy before
a protocol can be recommended.
Renal failure
Decreased production of erythropoietin is the main cause
of anaemia in chronic renal failure. Introduction of
r-HuEPO provided a major advance in the treatment of
this form of anaemia. To achieve the optimum response
from r-HuEPO therapy, the supply of iron to sites of
erythropoiesis must be adequate to maintain the resulting increase in red cell mass. Failure to provide sufficient
iron is considered to be the single most important reason
for r-HuEPO hyporesponsiveness, as iron supply limits the
stimulation rate of erythropoiesis (Horl et al, 1996;
Cavill etal, 1997). A small proportion of patients may
respond poorly to r-HuEPO for reasons other than lack of
iron. These may be infection, infiammatory disease.

malignancy, blood loss or hyperparathyroidism. Aluminium overload can produce a functional deficiency that
does not respond to iron therapy (Cavill et al, 1997;
Tarng et al, 1999). Best practice guidelines on the
strategies for managing anaemia in renal failure are
available (Cameron, 1999).
The iron status of patients on r-HuEPO therapy may
have to be assessed by a variety of methods at different
stages of the treatment. Haemoglobin and ferritin concentrations may be used to assess iron stores. Transport
iron is reflected by transferrin saturation but is variable.
An assessment of the erythron iron supply can be obtained
from the measurement of hypochromic cells (Horl et al,
1996; Cavill et al, 1997). The serum ferritin level should
be measured prior to r-HuEPO treatment and, where
necessary, iron supplementation should be provided until
the ferritin concentration is raised to more than 200 ^ig/\
(Cavill etal, 1997).
New parameters
Hypochromic cell analysis
Functional iron deficiency arises when the rate of
erythropoiesis exceeds the supply of iron to the erythron,
irrespective ofthe level of storage iron present (Cavill et al,
1997). With the appropriate analyser it is possible to
determine the haemoglobin content of individual erythrocytes. This measurement refiects the iron content of
circulating red cells and provides a direct indication of iron
delivery to the bone marrow (Macdougall et al. 1992).
The emergence of a subpopulation of hypochromic
erythrocytes is an indicator of lack of functional iron
availabifity to developing cells. Normally less than 2.5% of
erythrocytes have a haemoglobin concentration of less
than 28 g/dL. In studies on renal patients this cut-off
value gave a sensitivity in detecting iron deficiency of 91%
but a specificity of only 54%. The specificity can be
increased by raising the cut-off level but then sensitivity
falls (Cullen et al, 1999). Baumann Kurer et al (1995)
used a cut-off of 11% in a study on iron deficiency in
chronic infiammatory disease, giving a specificity of 90%
but sensitivity was less than 77%. More studies are
required on this parameter but at present availability is a
limiting factor.

Reticulocyte analysis
Flow cytometry, in addition to providing information on
reticulocyte numbers, also indicates the amount of RNA
present on a cell-to-cell basis and hence their maturity.
2001 Blackwell Science Ltd., Clin. Uib. Haem.. 23, 271-283

H. M. Waters & L. H. Sea/

This shows potential as a measure of erythropoiesis. Due
to the much larger number of cells analysed, flow
cytometry has made this investigation viable when the
proportion of reticulocytes is around 1% of the red cell
population, often where the interest is greatest. Some
standardization of methodology is still required and a
standard material for method comparison is needed (Cavill
& Rowan, 1996). From this new analysis, two parameters
are available to investigate anaemia. The reticulocyte
count, which indicates effective erythropoiesis, and the
proportion of reticulocytes with a high RNA content,
which indicates the intensity of reticulocyte stimulation
(d'Onofrio et ah, 1996).
The clinical value of these parameters has been studied
in a wide range of haematological disorders. Whilst they
are useful, there is some question whether, for example,
they give any better indication than white cell changes in
a bone marrow transplant setting. Once reticulocytes
could be isolated in flow cytometry, furtber analysis could
be made on individual cells and population characteristics
sucb as reticulocyte volume, reticulocyte haemoglobin
concentration and reticulocyte haemoglobin content could
be measured. It would appear that reticulocyte haemoglobin content, in particular, shows promise as a sensitive
indicator of functional iron deficiency (Brugnara, 1998).
Cullen et al (1999) have shown that reticulocyte
haemoglobin content may have better diagnostic merit
than hypochromic cell analysis. Tbese authors used a cutoff for reticulocyte haemoglobin content of < 26 pg in their
renal study. This produced a sensitivity and specificity for
detecting iron deficiency of 100% and 73%, respectively.
Brugnara et al. (1999) have also found reticulocyte
haemoglobin content to be a strong predictor of iron
deficiency in children. It may also be useful for treatment
monitoring as early changes are seen in response to
therapy. Where the appropriate instrumentation is available this could be a valuable parameter for assessing
effective iron availability to developing erythrocytes.
Serutn transferrin receptor assay
Recent study of the control of iron metabolism at the
cellular level, particularly the relationship between apoferritin and transferrin receptor (TfR) synthesis, has
significantly improved understanding of iron metabolism
in health and disease. Transferrin receptor molecules are
eventually cleaved from the cell surface and form a free
pool in plasma TfR (measured as sTfR). As erythropoietic
cells have the highest concentration of receptors, they are
the main contributors to sTfR concentration. In negative
iron balance, serum ferritin concentration falls with iron
2001 Blackwell Science Ltd.. Clin. Uib. Haem., 2 3 , 271-283

stores. No significant change occurs in sTfR concentration

until iron stores are exhausted or availability is restricted.
At this point the sTfR concentration rises in response to
tbe lack of iron. Unlike serum ferritin, sTfR levels are not
influenced by infection or infiammation and have therefore been monitored in a wide range of anaemias including
those with more complex pathogenesis, e.g. anaemias of
chronic disease. The assay is generally considered to be a
sensitive indicator of iron-deficient erythropoiesis which is
not affected by the acute phase response. Reference values
and changes in sideropenia and anaemia have been
explored but the parameter has not gained favour as a
standalone measure of iron status. It has been suggested
that combination with ferritin is more informative.
Punnonen, Irjala & Rajmaki (1997) have suggested
calculation ofthe ratio sTfR/log ferritin (sTfR-F Index) as a
way of combining sTfR and ferritin results. They found
this ratio was higher in iron-depleted patients with or
without an accompanying infectious or infiammatory
condition compared with iron replete ACD patients and
provided a useful parameter for the identification of
patients with depleted iron stores. Means et al. (1999)
studied sTfR in patients with a wide range of anaemia
aetiologies. Their study was referenced against bone
marrow aspirate iron and found sTfR assay significantly
more sensitive but less specific than other iron status
assays in identifying the absence of stainable iron. In
comparison, serum ferritin alone was highly specific but
insensitive. Serum ferritin predicted aspirate iron stores
accurately when values were above or below the reference
range. They suggested a decision algorithm combining
serum ferritin and sTfR sequentially. sTfR should be
assayed when serum ferritin falls in the range of 2 5 300 /ig/1. This improved sensitivity and specificity.
Flowers & Cook (1999) used the ratio of sTfR/ferritin
obtained from dried plasma spots to identify iron deficiency. These authors suggest that dried plasma spots may
be used to study the prevalence of anaemia in epidemiological studies. However, they acknowledge that ACD
patients were not included in the study.
In a study using response to iron supplementation as
the main indicator, Suominen et al. (2000) compared
bone marrow iron level, sTfR concentration, serum ferritin
and sTfR-F index as indicators of functional iron deficiency
in ACD patients. The sTfR concentration and sTfR-F index
were botb more effective indicators of patients who would
benefit from supplementation tban either bone marrow
iron or serum ferritin, STfR and sTfR-F index both
returned to normal after supplementation. One patient
with zero bone marrow iron levels bad normal baseline
values and did not respond to supplementation. These

A systematic approach to the assessment of erythropoiesis

authors concluded that sTfR and sTfR-F index are useful

parameters in detecting functional iron deficiency irrespective of storage iron level. At present, tbe assay is not
widely used and it remains to be seen whether the sTfR
assay, alone or in combination with the serum ferritin,
gains wide acceptance.

Practice pressures

Non-invasive analyses, which describe different facets of

the erythropoietic process, should be applied in an
appropriate, sequential, manner to obtain maximum
information. This investigative approach can be modified
depending on whether a diagnosis is to be established or
the patient's condition or treatment are being monitored.
Periodic re-assessment of existing procedures and evaluation of new, or modified, methods is necessary to improve
the investigation and understanding of erythropoiesis.
Since so many disorders have anaemia as a complication,
investigation of these anaemias exerts significant demands
on pathology services. With new treatment options
making it possible to improve the quality of life (Florencio,
1981; Scholz et al, 1997; Silverberg et al, 2001), the
investigation has become more complex, especially that of
the secondary anaemias. However, current trends towards
non-investigative profiling can work against this. As new
tests for the investigation of anaemia were developed, the
traditional approach was to use tbese as follow-up tests
and to treat them as 'specials' with particular attention
from the haematologist guiding the investigation to
conclusion. This practice is changing and, as tests have
become more readily available, clinical demand has driven
requesting patterns. Now results are often reported direct
to the requesting clinician who will then make tbeir own
interpretation. The use (and abuse) of any test is directly
related to availability. Establishment of a method, with
increased confidence in the relevance of the results,
usually leads to more requests, with a corresponding rise
in laboratory workload.
In some respects a non-investigative strategy may be
viewed as a positive factor. From tbe clinicians viewpoint a
screening approacb where most laboratory testing is
carried out at tbe first visit, whether known at the time
to be relevant or not, is efficient. In primary care this results
in fewer journeys to the general practice surgery for the
patient. In the outpatient clinic, or on the ward, fewer
episodes will be required, resulting in fewer appointments
or faster bed turnover, respectively. However, the transition to sucb a service also has drawbacks. In an
uncontrolled service, relevant abnormal results will constitute an even smaller minority ofthe total produced, most

ofthe remainder being unnecessary to the well-being ofthe

patient. Because time spent examining data is inversely
related to its volume there is a danger that tbe relevant
may be obscured by the irrelevant, making it even more
difficult to define the pathophysiology of anaemia (or
any otber disorder) in individual cases. Production of
redundant information also bas cost implications.
Advances in instrumentation have resulted in automation of many routine assays. Available systems fall into
two main categories; 'closed' which restrict the user to tbe
instrument manufacturer's reagents and preclude development or running of in-house assays; and 'open' which
perform liquid handling robotic functions, allowing any
reagents to be used. Non-isotopic assay systems are
available for use with random access automated analysers,
offering the main advantages of increased throughput of
samples and reduced turnaround times of patient results.
The choice of system will depend upon the investigations
carried out together witb the circumstances witbin the
individual department. Adequate independent evaluations
of available systems are required, particularly for tbe large
volume measurements such as vitamin B12. folate and
ferritin. Precision for within-batch assay should be < 5%
CV and for between-batcbes < 10% CV. The best precision
is usually required at the lower end of tbe reference
interval. Accuracy should be controlled by the standardization of calibrators against international standards
[National Institute for Biological Standards and Control
(NIBSC), Potters Bar. UK, http://www.nibsc.ac.uk].
Reagent stability over a reasonable period is important
for the laboratory carrying out small numbers of tests.
Systems that can accept a variety of anticoagulated
specimens as well as serum are beneficial.
Witb fully automated systems most errors are likely to
be limited to the pre- and postanalytical stages. These may
be dilutional, for example in the preparation of red cell
lysate, or transcription errors. Consideration therefore
needs to be given to sample labelling and tracing features.
The use of barcoded labels reduces tbe possibility of
transcription faults in addition to speeding up patient data
input. Adequate interfacing to a host computer should
allow two-way transfer and matching of patient identification and results. Linkage to an electronic transfer
system reduces the chance of error at tbe request receipt
and result delivery stages. Significant laboratory investment, eitber in terms of equipment capital costs, reagent
rental arrangements or electronic data processing and
transfer of results is required to maximize efficiency. A
multidisciplinary or multicentre approach may have to be
adopted to guarantee a sufficiently high throughput of
samples to ensure tbat associated costs are maintained at
2001 Blackwell Science Ltd.. Clin. lah. Eaem., 2 3 , 271-283

H. M. Waters & L. H. Seal

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Baumann Kurer S., Seifert B., Michel B., Ruegg R. & Fehr J.
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Baynes R.D. (1996) Assessment of iron status. Clinical Biochemistry 29 (3), 209-215.
Bell T.K., Bridges J.M. & Nelson M.G. (1965) Simultaneous free
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Braun J. (1999) Erythrocyte zinc protoporphyrin. Kidney International 55, S57-S60.
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Clinical acceptability
and diagnosis of cobalamin and folate deficiencies. Clinical and
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