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International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

REVIEW ARTICLE INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Laboratory diagnosis of anemia: are the old and new red cell
parameters useful in classification and treatment, how?
M. BUTTARELLO

Clinical Pathology Laboratory, S U M M A RY


Hospital of Adria, Adria (RO),
Italy Introduction: Anemia is a global problem affecting the population in
both developing and developed countries, and there is a debate on
correspondence:
Mauro Buttarello, Clinical
which hemoglobin level limit should be used to define anemia in
Pathology Laboratory, Hospital general population and particularly in the elderly. We present
of Adria, ULSS 19 Piazza degli herein a laboratory approach to diagnosing the possible causes of
Etruschi, 45011 Adria (RO), anemia based on traditional and new erythroid parameters. In this
Italy. Tel.: 00393407719537;
E-mail: mbuttarello@yahoo.it article, we provide practical diagnostic algorithms that address to
differential diagnosis of anemia. Based on both morphological and
kinetic classifications, three patterns were considered: microcytic,
doi:10.1111/ijlh.12500 normocytic, and macrocytic.
Methods: Main interest is on the clinical usefulness of old and new
accepted for publication 4 April parameters such as mean cell volume (MCV), red blood cell distri-
2016
bution width (RDW), hypochromic and microcytic erythrocytes,
Keywords
immature reticulocyte fraction (IRF), and some reticulocyte indices
Anemia, red cell indices, reticu- such as reticulocyte hemoglobin content and mean reticulocyte
locytes, immature reticulocyte volume. The pathophysiologic basis is reviewed in terms of bone
fraction, reticulocyte hemoglo- marrow erythropoiesis, evaluated by reticulocyte count (increased
bin content, mean reticulocyte
volume or normal/decreased) and IRF. The utility of reticulocyte indices in
the diagnosis of iron-deficient erythropoiesis (absolute or func-
tional) and in monitoring of response to treatment in nutritional
anemia (iron and cobalamin) was also investigated.
Results: For each parameter, the availability, the possible clinical
applications, and the limitations were evaluated. A discussion on
intraindividual biological variation and its implication on the use-
fulness of conventional reference intervals and in longitudinal
monitoring of the patients was also reported.
Conclusion: Red cell parameters and reticulocyte indices play an
essential role in differential diagnosis of anemia and in its treat-
ment. More efforts are needed in harmonizing parameters whose
results are still too different when produced by different analyzers.

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132 123
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124 M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA

underestimate Hct in hypochromic red cells. In this last


INTRODUCTION
condition, the use of Hct rather than the more accurate
Anemia is when blood hemoglobin (Hb) concentra- measured Hb can overestimate the diagnosis of anemia
tion is below the lower limit of the reference interval in subjects with iron deficiency (7). Also, the optical-
stated for age, sex, race, and altitude. The commonly based instruments with isovolumetric sphering provide a
accepted lower limits for adult population are the falsely elevated Hct when they analyze sickle red cells
WHO criteria suggested by an expert committee that cannot be sphered. A typical artifactual dissociation
nearly 50 years ago: 130 g/L in men and 120 g/L in with all the automated analyzers between Hb result
women, without the distinction between age and race (usually correct) and Hct (underestimated) is the pres-
(1). The definition of anemia has attracted interest in ence of red blood cell (RBC) agglutinates. Because the
recent years because epidemiologic studies suggest upper volumetric threshold to consider cells as RBC is
that anemia may be associated with poor prognosis in between 200 and 300 fL according to the analyzer, the
many different diseases, particularly among aged peo- large RBC clumps are not counted as RBC. This causes a
ple. A recent large population survey based on WHO spuriously low RBC count and low Hct. In contrast, Hb
criteria (NHANES-III) (2) showed that nearly ten per- is measured after RBC lysis and is unaffected by agglu-
cent of men and woman older than 65 years were tinins. As a consequence, MCHC is abnormally high,
anemic. These percentages rose to 26% in males and usually greater than 360 g/L. Moreover, also Hb can be
20% in females older than 85. It is not clear whether erroneously overestimated, although more rarely, in
the difference in lower limits, justified in androgen- subjects with severe hypertriglyceridemia or receiving
dependent age, should be continued after 65 years of an intravenous administration of fat emulsions, or with
age. Many of these subjects were apparently healthy, high WBC counts, due to the excessive turbidity.
and in most cases, clinical investigations did not
uncover a specific cause of anemia. These results sug-
O L D E RY T H R O C Y T E I N D I C E S
gest that somewhat lower limits than ‘normal’ might
be used in the elderly. Nevertheless, the too easy Maxwell Wintrobe 80 years ago proposed the anemia
acceptance of mild anemia as physiologic in the classification based on the mean cell volume (MCV)
elderly runs the risk of ignoring an underlying dis- obtained by Hct/RBC ratio from the measurement of
ease. There is a debate on which hemoglobin lower spun Hct and manual hemocytometric RBC count. The
limit should be used to define anemia in general pop- MCHC was calculated as Hb/Hct ratio, where Hb was
ulation and particularly in the elderly (3). Two differ- also based on manual measure (8). Of these two
ent, relatively recent, large databases (NHANES-III indices, which allowed to classify the anemia as micro-
and Scripps-Kaiser) (4, 5) in which the hemoglobin cytic, normocytic, and macrocytic based on MCV value
determination was carried out with standardized auto- and hypochromic, normochromic or hyperchromic
mated methods obtained a good agreement and new based on MCHC, only MCV has survived as key
lower limits are proposed (6). It would seem that parameter for the classification of anemia with auto-
these limits (5% of normal distribution) are 137 g/L mated hematology analyzers (Figures 1–3). With the
in white men (20–59 years) and 132 g/L for men after data collected on a cell-by-cell basis, modern instru-
the age of 60; the corresponding value for women is ments generate a histogram of erythrocytes size distri-
122 g/L independently of age. In Afro-Americans, bution. From this histogram, an index of
these limits are lower: 129 g/L in younger men and heterogeneity referred to as red cell distribution width
127 g/L in men older than 60, while the correspond- (RDW) can be determined. This is almost always
ing value for women is 115 g/L at all ages. expressed as percentage coefficient of variation and,
For many practical approaches, a decrease in hemat- less frequently, as standard deviation. The possibility
ocrit (Hct) is considered equivalent to a decreased of a quantitative, nonsubjective measurement of an
hemoglobin concentration, but this simplification is not anisocytosis index has rewakened interest. Bessman
always correct. All hematology impedance-based analyz- et al. (9) in the early 1980 proposed a classification of
ers falsely overestimate Hct in erythrocytes with a high anemia based on both MCV and RDW: homogeneous
mean hemoglobin concentration (MCHC) and (with normal RDW) and heterogeneous (with

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
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M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA 125

MICROCYTIC ANEMIA NORMOCYTIC ANEMIA


(MCV < 80 fL) (MCV 80 - 100 fL)

Reculocyte count Reculocyte count


(20 - 100 X 109/L) (20 - 100 X 109/L)

Normal or decreased Increased Normal or decreased Increased

Reculocyte hemoglobin % Hypochromic RBC Reculocyte hemoglobin - Anemia of renal Markers suggesve of Markers not suggesve of
content / reculocyte volume content / reculocyte volume insufficiency hemolysis: Haptoglobin, hemolysis
(< 28 pg / < 100 fL) % Microcyc RBC (< 28 pg / < 100 fL) LDH, bilirubin
- Anemia of chronic diseases
- Acute infecons
- Primary bone marrow
disorders
- Aplasc anemia Peripheral blood smear Blood loss
Decreased Increased Decreased

- βthalassemia trait - Early response to iron


- Iron deficiency treatment Other findings (not Fragmented erythrocytes
- Chronic diseases - βthalassemia diagnosc): consider
- Membrane defect: PNH
- Enzymopathy (G6PDH, PK)
- Hemoglobinopathy Microangiopathy Spherocytosis
% Micro / % Hypo rao

Direct Coombs test posive Direct Coombs test negave


Decreased Increased - Autoimmune hemolyc - Hereditary spherocytosis
anemia
- Cold agglunin disease
Iron deficiency β-thalassemia trait

Biochemical markers: HbA2 /hemoglobin analysis


ferrin, %TSAT, …
Figure 2. Diagnostic algorithm for normocytic anemia

- Absolute iron deficiency


- Funconal iron deficiency The mean hemoglobin content (MCH), which is
- Chronic diseases
strongly correlated with MCV, is calculated as Hb/RBC
ratio. These last measurements with automated ana-
Figure 1. Diagnostic algorithm for microcytic anemia. lyzers are more accurate and precise than MCV,
which is derived by a direct measurement of a single
cell size using different analytical methods (impedance
increased RDW) erythrocyte population. The former with or without hydrodynamic focusing, or light scat-
includes hypoproliferative anemia, marrow aplasia, tering). Moreover, MCV, different from MCH, is
and thalassemia heterozygosity; the latter includes affected by preanalytical variables such as storage
nutritional anemias (iron, cobalamin, and folic acid temperature and time. A dissociation between MCV
deficiency) and sideroblastic anemia. Although this and MCH was recently described (13): high MCV and
approach was largely accepted and RDW was added to low MCH. This was the case of macrocytic hypochro-
routine analysis in many laboratories, numerous mic anemia, indicating the coexistence of both
exceptions began to be observed. There is a wide distri- macrocytosis due to cobalamin/folate deficiency and
bution of RDW values within a given disease, whose hypochromia due to hemoglobin E disease. Inappro-
usefulness in differential diagnosis has decreased, but priately low MCH with a high MCV can be found also
its utility as a general marker of abnormality has been in anemia due to B12 (or folate) and iron deficiency
maintained. A further complication derives from the (or thalassemia).
difference in reference intervals obtained with analyz-
ers from different manufacturers (10–12). This is
N E W R E D C E L L PA R A M E T E R S
explained by the different algorithms used to cut the
tails of distribution, which is needed to eliminate Some hematology analyzers can quantitate the per-
extreme values often due to artifacts. centage of hypochromic, microcytic, and more rarely

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
1751553x, 2016, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.12500, Wiley Online Library on [23/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
126 M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA

MACROCYTIC ANEMIA hyperchromic cells is useful in the diagnosis of sphe-


(MCV > 100 fL) rocytosis either by hereditary or by immune hemoly-
sis (21, 22). The main limit in the use of these
Reculocyte count
(20 - 100 X 109/L) parameters is due to the fact that they are affected by
Normal or decreased Increased temperature and storage time. In fact, erythrocytes in
the samples stored at room temperature tend to pro-
Immature reculocyte fracon Immature reculocyte fracon gressively swell, with a consequent reduction in cellu-
lar hemoglobin concentration and an increase in
Normal or decreased Increased Increased
hypochromic and decrease in hyperchromic RBC%.

- Myelodysplasc - Hemorrhage
E VA L UAT I O N O F B L O O D S M E A R
syndromes - Hemolysis
Peripheral blood smear - Acute infecons - Response to B12 / folate
treatment All the red blood cell indices, although useful, are the
representation of the mean and overall dispersion of
- Round macrocytes - Oval macrocytes
- No hypersegmentaon of - Hypersegmentaon of the erythroid cellular population and provide little
neutrophils neutrophils
information about specific red blood cell shapes or the
presence of minor populations of abnormal cells.
Nonmegaloblasc Megaloblasc macrocytosis
macrocytosis Examination of blood smear for some specific shapes
as reported in Figures 2 and 3 can provide a valuable
- Hypothyroidism information to aid in the diagnosis of the underlying
- B12 or folate deficiency
- Myelodysplasc syndromes
- Liver diseases
- Therapy (hydroxyurea, disease.
zidovudine, methotrexate,
- Alcohol chronic abuse
..)

R E T I C U L O C Y T E A N D I M M AT U R E
Figure 3. Diagnostic algorithm for macrocytic anemia. R E T I C U L O C Y T E F R AC T I O N
In addition to red cell indices and morphological crite-
hyperchromic erythrocytes (Table 1). In iron-deficient ria, anemia may be classified by kinetic approach, that
erythropoiesis, a greater fraction of RBC is hypochro- is, the degree of bone marrow response evaluated by
mic rather than microcytic, and the microcytic/hypo- the reticulocyte count. The reticulocyte count is clini-
chromic ratio shows the best diagnostic efficiency in cally important both for the pathophysiological classi-
the differential diagnosis with beta-thalassemia trait fication of anemia (due to an inadequate production
(14, 15). Many other discriminant algorithms have of erythrocytes by the bone marrow, in which case
been recently proposed including conventional and there is a decreased number of reticulocytes, or to an
new RBC parameters (15, 16). The results were in excessive loss or the destruction of erythrocytes, in
general better than the traditional discriminant func- which there is an increase in reticulocyte count) and
tions, but the performance of any index seems to for the early identification of the normalization of ery-
depend on the geographical origin of the population thropoiesis by the marrow after therapeutic interven-
in which it is applied (17). Recent studies have shown tion (iron, cobalamin, folic acid, ESAs, etc.), after
that hypochromic erythrocytes are useful in identify- spontaneous or pharmacologically induced aplasia of
ing iron-restricted erythropoiesis in anemic patients the marrow, or following bone marrow transplanta-
treated with erythropoiesis-stimulating agents (ESAs), tion. However, the imprecision of the manual micro-
particularly anemia of the chronic kidney disease or scopic method (coefficient of variation (CV) between
in hemodialyzed patients. The response to ESAs is 68.6% at low concentration and 16% at high level)
strictly dependent on iron availability and is limited (23) makes it almost useless mainly in severe reticulo-
by iron deficiency that can be absolute or functional cytopenia. It does not allow for the observation of
(i.e., limitation of bone marrow erythropoietic activity small but significant variations during the early recov-
by the inability to mobilize the sufficient iron from ery of erythropoietic bone marrow activity, nor does
body storage sites) (18–20). The presence of it clearly define the difference between normal and

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
Table 1. Old and new RBC parameters and their clinical applications

Proposed clinical
Parameter Availability applications Limitations References

Old RBC parameters

• Mean cell volume All the analyzers Anemia classification Affected by preanalytical 8, 45
(MCV) (fL) based on morphological variables (storage
approach temperature, time)
• Mean cell hemoglobin All the analyzers Useful when hemoglobin Highly correlated with MCV 13, 46
content (MCH) (pg) synthesis is impaired as
in iron deficiency
anemia
• Mean cell hemoglobin All the analyzers Increased in spherocytosis With some impedance 7, 47
concentration (MCHC) (g/ because of a reduced analyzers, the value is
L) surface/volume ratio clamped around the mean
• Red cell distribution All the analyzers Generic marker of Of little usefulness in the 9–12
width (RDW) (%) abnormality when differential diagnosis of

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
increased anemia. Reference intervals
are method dependent
New RBC parameters

• Percentage of Hypo% (Siemens Advia Assessment of iron Affected by preanalytical 18, 19, 48, 49
hypochromic red cells 2120); %Hypo-He availability (absolute or variables (storage
(Sysmex XE/XN); % functional) for temperature, time).
HPO (Abbott erythropoiesis. Related Reference intervals and
Sapphire); LHD% to iron status in the last diagnostic thresholds are
(Beckman-Coulter 3 months. method dependent. Limited
LH/DxH 800). value in the presence of b-
thalassemia.
• Percentage of Hyper% (Siemens Diagnosis of hereditary/ Reference intervals and 21, 22
hyperchromic red cells Advia 2120); % immune spherocytosis diagnostic thresholds are
Hyper-He (Sysmex method dependent.
XE/XN); %HPR
(Abbott Sapphire).

(continued)
M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA
127

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128

Table 1. (Continued)

Proposed clinical
Parameter Availability applications Limitations References

• Percentage of microcytic Micro% (Siemens Useful in combination Reference intervals and 14–17
red cells Advia 2120); % with other RBC diagnostic thresholds are
micro-R (Sysmex XE/ parameters (mainly method dependent.
XN); %MIC (Abbott hypochromic
Sapphire). erythrocytes) to obtain
discriminant indices for
the differential diagnosis
of microcytic anemia.
Reticulocyte parameters

• Immature reticulocyte All the analyzers Classification of anemias Reference intervals and 25–35
M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA

fraction (IRF) (fraction) and monitoring of diagnostic cutoff are


treatment. Verify aplastic method dependent.
anemia.
• Reticulocyte mean CHr (Siemens Advia Diagnosis of iron-deficient Limited value in the 35–38
hemoglobin content (pg) 2120); Ret-He erythropoiesis. Early presence of a- or b-
(Sysmex XE/XN); monitoring the response thalassemia.
MCHr (Abbott to iron therapies.
Sapphire); RHE
(Mindray BC 6800);
RHCc (ABX-Horiba
Pentra Nexus DX)
• Mean reticulocyte MCVr (Siemens Advia Diagnosis of iron-deficient Affected by preanalytical 34, 37, 40
volume (fL) 2120); MCVR (Abbott erythropoiesis. Early variables (storage
Sapphire); MVR monitoring of treatment temperature, time).
(Mindray BC 6800); with B12/folate/iron in Reference intervals strictly
MRV (ABX-Horiba nutritional anemia. method dependent.
Pentra Nexus DX);
MRV (Beckman-
Coulter LH/DxH 800).

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
1751553x, 2016, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.12500, Wiley Online Library on [23/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1751553x, 2016, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.12500, Wiley Online Library on [23/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA 129

low reticulocyte levels. Automated analyzers represent with positive covariance corresponding to the healthy
a revolution for this cell type using dyes to bind retic- subjects and to accelerated erythropoiesis; (iii) for
ulocyte RNA and flow cytometers to perform rapid marked reticulocytosis, the covariance is negative,
and objective counts. The possibility to analyze tens of suggesting a gradual deceleration of erythropoiesis
thousands of cells per sample has reduced imprecision (28). Therefore, IRF and reticulocyte count may vary
(CV between 25% at low concentration and 3.0% at in a concordant or independent way according to the
high counts) (24) (M. Buttarello, personal observa- erythropoietic conditions and can be hypothesized the
tions). Furthermore, using an absolute reticulocyte IRF as an index of acceleration and the absolute retic-
count (expressed as the number of cells per unit of ulocyte count as a quantitative measure of the effec-
volume: x 109/L) rather than proportion no longer tiveness of erythropoiesis (28, 29). This parameter is
requires correction for reduced hemoglobin concentra- therefore useful in distinguishing (i) anemia charac-
tions. A little complication derives from the differ- terized by an increase in erythropoiesis, like acquired
ences in the reference intervals that are strictly hemolytic anemias or the loss of blood, which pro-
method dependent (lower limit of the 95% interval duces an increase both in total reticulocytes and in
between 19 and 30 9 109/L and upper between 85 IRF; (ii) anemias due to the reduced marrow produc-
and 130 9 109/L) (24). What has created an addi- tion (i.e., chronic renal disease) in which both values
tional interest about automated reticulocyte analysis is are found to be decreased, and (iii) anemia of acute
the availability of a new parameter called immature infections or myelodysplastic syndromes in which
reticulocyte fraction (IRF) based on reticulocytes RNA there is a dissociation between total reticulocyte count
content (25). Reticulocytes originate from orthochro- (reduced or normal) and the IRF which can be
matic erythroblasts following ejection of the nucleus, increased (30–33). Other uses include monitoring the
and they gradually mature, partly in the marrow therapy efficacy in nutritional anemia (e.g., cobal-
(3 days on average) and partly in the peripheral blood amin, folates, and iron) because the increase in IRF
(1 day). Reticulocytes gradually lose their RNA and precedes the increase in total reticulocyte count by
ultimately become RNA-free red cells, while some several days. In subjects with iron-deficient anemia
RNA-rich, more immature reticulocytes are found in treated with iv iron, it increased at day 1 and contin-
relatively narrow proportion in the peripheral blood ued to increase until reaching the maximum value at
of the healthy subjects. There are, however, various day 5 (34). This value was correlated with the ery-
expressions according to the analyzer used, and thus, thropoietin concentration at the beginning of therapy
the reference intervals are different (low level (M. Buttarello, data not published). The combination
between 0.012 and 0.20 and high level between 0.14 of reticulocyte count >80 9 109/L with a reticulocyte
and 0.40) (26, 27). Independently by the way on as it count/IRF ratio >7.7 is considered useful for the
is produced, the IRF is an early and sensitive index of screening of trait and mild hereditary spherocytosis
erythropoiesis; in fact, immature reticulocytes appear (35).
in a larger proportion when red cell production
increases. The IRF has a weak but significantly posi-
RETICULOCYTE INDICES
tive correlation with the absolute reticulocyte count,
indicating that it is an additional useful parameter to The latest generation of hematology analyzers pro-
evaluate the erythropoietic activity. The greatest vides some reticulocyte indices analogous to the
clinical usefulness, especially in the classification of equivalent RBC indices (Table 1). Among these,
anemia based on marrow response, is found using the most promising from a clinical point of view are
two-dimensional matrices of IRF vs. the absolute retic- the hemoglobin content of reticulocyte and the mean
ulocyte count (25, 26). With covariance analysis (AN- reticulocyte volume. The hemoglobin content, which
COVA), it is possible to identify some well-differentiated directly reflects the synthesis of hemoglobin in mar-
behaviors in certain areas of the matrices: (i) In retic- row precursors, is a measure of adequacy of iron
ulocytopenia, there is no covariance, both in marrow availability (34, 36–38). This parameter is important
aplasia and in early erythropoietic response; (ii) in because its reduction indicates iron-deficient erythro-
normal or mild reticulocytosis, there are two subsets poiesis, even in conditions in which traditional

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
1751553x, 2016, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.12500, Wiley Online Library on [23/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
130 M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA

150 30
6 I N T R A I N D I V I D UA L B I O L O G I C A L VA R I AT I O N
125

RETICULOCYTES (%)
25
One source of variability of clinical laboratory results
4
(besides preanalytic and analytic variations) is the
Hb (g/L)

CHr (pg)

100
intraindividual variation around the homeostatic set-
20
2 ting point (41). Several studies have investigated this
75
biological variation and the results are similar even if
carried out at different time points and in different
50 15 0
-1 21 42 63 84 105 126 147 168 189 210 231 252 geographical areas (42–44). The reported values varied
DAYS CH r
START (OS) IRON THERAPY
END (IV) IRON
THERAPY Hb between 1.9 and 2.8% for Hb and Hct; between 0.6
START (IV) IRON RET %
THERAPY
and 1.3% for MCV; and between 5.8 and 9.5% for
Ferr µg/L : 5.7 Ferr µg/L : 3.4 Ferr µg/L : 74
Iron µmol/L : 3.58 Iron µmol/L : 3.22 Iron µmol/L : 12.0
Ferr µg/L :
Iron µmol/L :
43
8.24
reticulocytes. From these values, an index of individu-
TRF g/L: 3.71 TRF g/L: 3.50 TRF g/L: 3.41 TRF g/L: 2.73
ality as ratio of intraindividual to interindividual
coefficient of variation can be calculated. For the
Figure 4. Response in hemoglobin concentration (Hb), above-mentioned parameters, it is between 0.19 and
reticulocyte hemoglobin content (CHr), and 0.42 for Hb, between 0.17 and 0.27 for MCV, and
reticulocyte% (Siemens Advia 120 analyzer) to between 0.18 and 0.30 for reticulocytes, according to
intravenous (iv) iron administration in a patient
the different studies. A low index of individuality
unresponsive to oral iron therapy. Ferr: serum
ferritin concentration; Iron: serum iron (<0.5) indicates that conventional reference intervals
concentration; TRF: serum transferrin concentration. may be of little usefulness, especially when deciding if
the change observed in a subject is clinically

biochemical markers such as ferritin and transferrin


120
saturation are inadequate (e.g., in inflammations or 40 150 5

anemia from a chronic disease), and besides, it is use- 110


MCVr

CHr
ful for monitoring early response to intravenous iron 140
RET % 4
35
therapy because it increases significantly after only 100
Hb g/L

130
Hb (gL)

48–72 h (Figure 4) (34, 36). Exceptions are heterozy- 3

RETICULOCYTES (%)
MCVr (fL)

gotes for beta-thalassemia whose reticulocyte hemo- 90


CHr (pg)

30 120

globin content is found to be always reduced 2

independently of iron stores (39). Low values of this 110 80

25
index are indicative even in functional iron deficiency 1
100 70
which appears in patients treated with erythropoietin
(38). Few studies are available on the clinical useful- 20 90 0
60

ness of mean reticulocyte volume. In subjects with -5 0


DAYS
5 10 15

depleted iron stores, this index increases rapidly fol- START OF IRON (IV) THERAPY
START OF COBALAMIN (IV) THERAPY
lowing iron therapy and decreases equally as rapidly
with the development of iron-deficient erythropoiesis.
The reticulocyte volume decreases dramatically and Figure 5. Changes in mean reticulocyte volume
(MCVr), reticulocyte hemoglobin content (CHr),
reticulocytes are smaller than the circulating RBCs, in
reticulocyte % and hemoglobin concentration (Hb)
nutritional macrocytosis after therapy with vitamin (Siemens Advia 120 analyzer) to intravenous
B12 and/or folic acid (Figure 5) (37). The main limit cobalamin administration, followed by intravenous
of the use of these indices is the difficulty to compare iron due to the functional iron deficiency
numeric results obtained from the analyzers of differ- development. MCVr and CHr decreased significantly
ent manufacturers (the lower limit of the 95% inter- after iv cobalamin therapy. The excessive decrease in
CHr suggests an iron deficiency, promptly corrected
val between 91 and 100 fL and the upper limit
by iv iron administration.
between 111 and 120 fL) (40).

© 2016 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2016, 38 (Suppl. 1), 123–132
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M. BUTTARELLO | LABORATORY DIAGNOSIS OF ANEMIA 131

remarkable. Useful in serial monitoring of physiologic


CONCLUSIONS
or pathologic conditions is the critical difference
(called also reference change value: RCV), which Red cell parameters and reticulocyte indices play an
defines the percentage change that should be essential role in the differential diagnosis of anemia
exceeded (given the analytic and intraindividual bio- and in its treatment. More efforts are needed in har-
logical variations) so that there is a significant differ- monizing parameters whose results are still too differ-
ence between two consecutive measurements. The ent when produced by different analyzers. Moreover,
significant percentage changes (for probabilities of it should be remembered that despite the essential
95%) for the named red cell parameters are (43, 44) role of automation, microscopic control of pathologic
as follows: between 6.22 and 6.82% for Hb and Hct, samples remains indispensable.
between 2.35 and 3.12% for MCV, and between 36.7
and 41.7% for reticulocytes. Differences depend on
COMPETING INTERESTS
biological variation (42–44) and on the analytical
variability of analyzers, and these differences are The author has no competing interest.
smaller for instruments with smaller imprecision.

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