Laboratory Approach to Anemia

Abstract

Anemia is a major cause of morbidity and mortality worldwide and can be defined as
a decreased quantity of circulating red blood cells (RBCs). The epidemiological studies
suggested that one-third of the world’s population is affected with anemia. Anemia is not
a disease, but it is instead the sign of an underlying basic pathological process. However,
the sign may function as a compass in the search for the cause. Therefore, the prediagnosis
revealed by thorough investigation of this sign should be supported by laboratory
parameters according to the underlying pathological process. We expect that this review
will provide guidance to clinicians with findings and laboratory tests that can be followed
from the initial stage in the anemia search.

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Chapter 12
Laboratory Approach to Anemia
Ebru Dündar Yenilmez and Abdullah Tuli
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.70359
Provisional chapter
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
DOI: 10.5772/intechopen.70359
Laboratory Approach to Anemia
Ebru Dündar Yenilmez and Abdullah Tuli
Additional information is available at the end of the chapter
Abstract
Anemia is a major cause of morbidity and mortality worldwide and can be dened as
a decreased quantity of circulating red blood cells (RBCs). The epidemiological studies
suggested that one-third of the world’s population is aected with anemia. Anemia is not
a disease, but it is instead the sign of an underlying basic pathological process. However,
the sign may function as a compass in the search for the cause. Therefore, the prediag-
nosis revealed by thorough investigation of this sign should be supported by laboratory
parameters according to the underlying pathological process. We expect that this review
will provide guidance to clinicians with ndings and laboratory tests that can be followed
from the initial stage in the anemia search.
Keywords: anemia, complete blood count, red blood cell indices, reticulocyte
1. Introduction
Anemia, the meaning of which in Greek is “without blood,” is a relatively common sign and
symptom of various medical conditions. Anemia is dened as a signicant decrease in the
count of total erythrocyte [red blood cell (RBC)] mass, although this denition is rarely used
in clinical seings. According to the World Health Organization, anemia is a condition in
which the number of red blood cells (RBCs, and consequently their oxygen-carrying capacity)
is insucient to meet the body’s physiologic needs [1, 2]. The individual variation such as a
person’s age, gender, residential elevation above sea level (altitude), and dierent stages of
pregnancy changes the specic physiologic requirements of the body. Anemia is not a dis-
ease, but is instead the sign of an underlying basic pathological process. Nonetheless, the sign
may function as a compass in the search for the cause, as well as function as a road marker
© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

in the investigation of underlying pathological process [3]. Hence, the diagnosis according to
the symptoms obtained by history and physical examination of patients with anemia should
be supported by laboratory parameters related to the underlying pathological cause. The rst
step in the diagnosis of anemia is detection with predictive, accurate tests so that important
clues to underlying disease are not missed and patients are not subjected to unnecessary tests
for and treatment of nonexistent anemia. Instead, clinicians rely on several other measures to
identify the degree and the cause of anemia in a given patient.
The purpose of this chapter is to discuss the clinical approaches with which a practicing physician
is able to evaluate a patient with underlying anemia.
2. Classication of anemia
Based on determination of the red blood cell mass, anemia can be classied as either relative or
absolute. Relative anemia is characterized by a normal total red blood cell mass in an increased
plasma volume, resulting in a dilution anemia, a disturbance in plasma volume regulation.
However, dilution anemia is of clinical and dierential diagnostic importance for the hema-
tologist [4]. Classication of the absolute anemias with decreased red blood cell mass is dif-
cult because the classication has to consider kinetic, morphologic, and pathophysiologic
interacting criteria. Anemia of acute hemorrhage is not a diagnostic problem and is usually a
genitourinary or gastrointestinal event, not a hematologic consideration.
Initially, anemias should be classied into two groups as diminished production and
increased destruction of RBCs. The number of reticulocytes is a remarkable parameter in the
materialization of this classication. Then, diagnostic analysis is able to be based upon both
morphologic and pathophysiological hallmarks.
Anemias can morphologically be classied into three subgroups as macrocytic, normocytic,
and microcytic hypochromic anemias. This classication is based on mean corpuscular vol-
ume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of complete blood
count (CBC) and aids the physician to the diagnosis and monitoring of anemias that can be
easily cured, such as deciency of vitamin B12, folic acid, and iron.
Pathophysiologic classication is best suited for relating disease processes to potential treat-
ment (Figure 1). In addition, anemia resulting from vitamin- or iron-deciency states occurs
in a signicant proportion of patients with normal red blood cell indices.
Each step indicated in Figure 1 can be disrupted and cause anemia. Identifying the aected
step is important for therapeutic intervention and specic treatment. The limitation of
pathophysiologic classication is that pathogenesis involves several steps in most anemias.
Therefore, the provided chapter is a guideline for the practical understanding of the processes
underlying the production and destruction of RBCs. Despite all these morphological clas-
sication is more useful in terms of convenience and clinical usage. Hence, morphological
classication serves to support the diagnosis and indirectly treatment in connection with the
laboratory and clinic. The major limitation of such a classication is that it tells nothing about
the etiology or reason for the anemia [5].
Current Topics in Anemia236

3. Laboratory evaluation
A comprehensive laboratory evaluation is required for denitive diagnosis and treatment
for any anemia, although the anamnesis (history of patient) and physical examination of the
patient may indicate the presence of anemia and propose its cause. As appropriate to this aim,
the various tests for the diagnosis of anemia are done with routine hematological tests such
as CBC and reticulocyte counts as well as studies of iron status that serve as a leaping point
to the diagnosis (Figure 2). When the diagnosis of specic anemic conditions is conrmed, a
large number of other specic tests are used [6]. Laboratory tests used in the diagnosis of ane-
mia are roughly summarized in Figure 2. The laboratory investigation of anemias involves
the quantitative and semiquantitative measurements of RBCs and supplementary testing of
blood and body uids. The laboratory results obtained from these parameters are important
arguments in the diagnosis, treatment, and monitoring of the anemias.
3.1. Complete blood count
Prior to the development of modern hematology blood analyzers, blood counts included
hemoglobin (Hb) concentration, white blood cell (WBC) count, and manual platelet count. The
other parameters like mean corpuscular volume (MCV) had to be mathematically calculated
by using the measured parameters such as Hb, RBC count, and hematocrit (Hct). Modern ana-
lyzers provide CBC indices by using various physical and chemical methods such as electronic
impedance, laser light scaering, light absorption, and staining properties [7].
How will CBC parameters such as Hb concentration, Hct, RBC count, MCV, MCHC, WBC
count, platelet count, and other parameters related to formed elements of blood measured
by modern blood analyzers help the diagnosis or management of the patient? CBC identies
Figure 1. Classication of anemia according to pathophysiologic characteristics (gure has been modied from Ref. [4]).
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237

several dierent parameters and can provide a great deal of information. Hematologic and
biochemical variations of red blood cells determine whether the patient is anemic or not. If
anemia is present, MCV is likely to provide clues about the cause of anemia. While an infection
can lead to increased WBC, lymphocytosis can be seen in viral infections (but not always so).
Abnormal size or number of platelets may be either due to the direct eect of any underlying
blood disease or may simply be the reection of the presence of some other underlying pathol-
ogies. Because of all these, CBC parameters obtained as a result of clinical evaluation should
be reassessed more carefully and curiously [7]. Therefore, the fundamental parameters of CBC
such as Hb concentration, RBC, Hct, MCV, mean corpuscular hemoglobin (MCH), MCHC,
and red blood cell distribution width (RDW) which plays an important role in the diagnosis,
treatment, and monitoring of the anemic patient will be explained below.
3.2. Hemoglobin concentration
Determination of Hb is a part of CBC. Hemoglobin is intensely colored, and this property
has been used in methods for estimating its concentration in the blood. Erythrocytes contain
a mixture of hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, and minor
amounts of other forms of hemoglobin [4].
Monitoring the response to treatment of anemia and to evaluate polycythemia, Hb concentration
is used to screen for diseases associated with anemia and to determine the severity of anemia [6].
Finding an increased Hb concentration requires a systematic clinical approach for dierential
diagnosis and further investigation. The conditions such as polycythemia vera, congestive heart
failure, chronic obstructive pulmonary disease, etc., can cause Hb levels to rise.
Figure 2. Laboratory tests used in anemia diagnosis (gure has been modied from Ref. [4]).
Current Topics in Anemia238

Decreased Hb levels are found in anemia. Hb must be evaluated along with the RBC and Hct.
In iron deciency, hemoglobinopathies, pernicious anemia, liver disease, hypothyroidism,
hemorrhage (chronic or acute), hemolytic anemia (caused by transfusions, reactions to chemi-
cal or drugs, infectious and physical agents), and various systemic diseases (e.g., Hodgkin’s
disease, leukemia, etc.), decrease in Hb levels can be observed.
Variations in Hb levels occur after hemorrhages, transfusions, and burns (Hb and Hct are both
high during and immediately after hemorrhage). Hb and Hct supply valuable information in
an emergency situation [8].
Excessive uid intake, pregnancy, and drugs, etc., which cause increase in plasma volume and
decrease the Hb values, are interfering factors. Drugs such as methyldopa and extreme physi-
cal exercise can give rise to increased Hb levels. In addition, people living in high altitudes
have increased Hb concentration, Hct, and RBC count [8].
3.3. Red blood cell count
The quantication of the percentage of microcytic and hypochromic RBCs has proved its
clinical usefulness in the dierential diagnosis of microcytic anemia [9]. RBC count has
been recognized as the most ecient single classical measurement in the dierential diag-
nosis of microcytic anemia [10]. Iron-decient erythropoiesis is characterized by the pro-
duction of RBC with a decrease in Hb content, so a high percentage of hypochromic cells
are present.
In β-thalassemia cases, increased RBC count is a characteristic as a result of chronic increase in
erythropoiesis. Therefore, MCV and MCH are lower in beta thalassemia than in iron deciency
anemia [11].
3.4. Hematocrit
The word hematocrit, also called packed cell volume (PCV), means “to separate blood,” which
underscores the mechanism of the test, because the plasma and blood cells are separated by
centrifugation [6].
Decreased Hct values are an indicator of anemia, in which there is a reduction in the Hct. An
Hct ≤30% means that the patient is severely anemic. Decreased values also occur in leukemias,
lymphomas, Hodgkin’s disease, adrenal insuciency, chronic diseases, acute and chronic
blood loss, and hemolytic reactions (transfusions, chemical, drug reactions, etc.).
Increased Hct values are observed in erythrocytosis, polycythemia vera, and shock (when
hemoconcentration rise) [4].
Interfering factors such as pregnancy, age, sex, and dehydration have dierent eects in Hct.
People living in high altitudes have increased Hct values and RBC count. Hct decreases in
the physiologic hydremia of pregnancy. Hct varies with age and gender. Hct levels are lower
in men and women older than 60 years of age. Severe dehydration from any cause falsely
increases the Hct value [8, 12].
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4. Red blood cell indices
The size and hemoglobin content of erythrocytes (red blood cell indices), based on popu-
lation averages, have traditionally been used to assist in the dierential diagnosis of ane-
mia [13]. Some red blood cell parameters (for instance, RBC count, Hb concentration, MCV,
RDW) are directly measured, while the others (e.g., Hct, MCV, MCHC) are derived from
these primary measurements [14]. These measurements are provided by any of the common
automated instruments. Instruments vary somewhat in their technologies. The most com-
monly used method is either a combination of a highly focused light source, an electric eld,
and a laser-based ow cytometry or a radiofrequency wave to discriminate between cells.
Automated instruments are not only fast but extremely accurate. The coecient of variation
(measurement error) of an automated counter is usually less than 2%, and each of the major
measurements, including the hemoglobin level, red blood cell count, and mean corpuscular
volume, can be standardized independently with commercial red blood cell and hemoglobin
standards [4, 6, 12].
4.1. Mean corpuscular volume (MCV)
MCV has been used to guide the diagnosis of anemia in patients, for example, testing patients
with microcytic anemia for iron deciency or thalassemia and those with macrocytic anemia
for deciency of folate or vitamin B12 [4, 15].
The reference value of MCV ± 2 SD is 90 ± 9 fL and generally coincides with the peak of the
Gaussian distribution of RBC size. Although MCV is both accurate and highly reproducible,
errors may be introduced by RBC agglutination, distortions in cell shape, the presence of very
high numbers of WBCs, and sudden osmotic swelling [8]. MCV results are the basis of the
classication system used to evaluate an anemia (Table 1, Figure 3).
Increased reticulocytes and marked leukocytosis can also increase MCV [8]. The mixed popula-
tion of microcytes and macrocytes results in normal MCV values and is an interfering factor in
evaluating MCV.
4.2. Mean corpuscular hemoglobin (MCH)
MCH, the amount of hemoglobin per red blood cell, increases or decreases in parallel with
MCV and generally provides similar diagnostic information. Because this parameter is
aected by both hypochromia and microcytosis, it is least sensitive as MCV in detecting iron
deciency states [16].
The reference value of MCH is 32 ± 2 pg. This is an excellent measure of the amount of hemo-
globin in individual red blood cell. Patients with iron deciency or thalassemia who are unable
to synthesize normal amounts of hemoglobin show signicant reductions in the MCH [8, 17].
An increase of MCH is associated with macrocytic anemia; a decrease of MCH is associated
with microcytic anemia.
Current Topics in Anemia240

Microcytic anemias (MCV 50–79 fL)
• Disorders of iron metabolism Iron deciency anemia, anemia of chronic disease, congenital
hypochromic-microcytic anemia with iron overload
• Disorders of porphyrin and heme synthesis Acquired sideroblastic anemias, idiopathic refractory sideroblastic
anemia
• Disorders of globin synthesis Thalassemias, hemoglobinopathies, characterized by unstable
hemoglobins
Normocytic normochromic anemia
(MCV 80–98 fL)
• Anemia with appropriate bone marrow
response
Acute posthemorrhagic anemia, hemolytic anemia
• Anemia with impaired marrow response Aplastic anemia, pure red blood cell aplasia, myelobrosis
Macrocytic anemias (MCV 99–150 fL)
• Cobalamin (B12) deciency Lack of animal products, intrinsic factor deciency, pernicious
anemia, hyperthyroidism, pregnancy, enzyme deciencies
• Folate deciency Lack of vegetables, celiac disease, hypothyroidism, folic acid
antagonists, hemodialysis
• Unresponsive to cobalamin or folate Metabolic inhibitors (i.e., 6-mercaptopurine), inborn errors (Lesch-
Nyhan syndrome)
Table 1. Classication and possible diagnosis of anemia according to MCV in clinical use [8].
Figure 3. Flowchart to follow in the diagnosis of anemia according to MCV [4].
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Hyperlipidemia is one of the interfering factors of MCH because it falsely increases MCH
values. WBC counts >50,000/mm3 also falsely provide increased level for MCV as well as for
Hb. In addition, high heparin concentrations also falsely elevate MCH value [8].
4.3. Mean corpuscular hemoglobin concentration (MCHC)
MCHC is not used frequently for diagnostic purpose, but is primarily useful for quality con-
trol purposes, such as detecting sample turbidity. Because MCHCs are average quantities in
the blood with mixed-cell populations, it is dicult for these red blood cell indices to detect
abnormalities in the blood [4].
The reference value of MCHC is 33 ± 3 g/dL. The principal purpose of MCHC is to detect
patients with hereditary spherocytosis who has very small, dense spherocytes in the circula-
tion. These spherocytes represent cells that have lost considerable intracellular uid because
of a membrane defect. In situations such as sideroblastic anemia, recently transfused patients,
patients with severe pernicious anemia with red blood cell fragmentation, and in conditions
where both folate and iron deciency are present, both large and small red blood cells are
observed, which compromise the value of MCV. When present in signicant numbers, they will
cause MCHC to increase to levels in excess of 36 g/dL [4, 6, 15].
Decreased MCHC indicates that packed RBCs (a unit volume) contain less Hb than normal.
MCHC is decreased in hypochromic anemia (MCHC < 30 g/dL) observed in iron deciency,
microcytic anemias, chronic blood loss anemia, and some thalassemias.
Increased MCHC levels (RBCs cannot accommodate more than 37 g/dL Hb) occur in sphero-
cytosis, in newborns and infants.
Because of falsely elevating MCHC, lipemia, cold agglutinins or rouleaux, and high heparin
concentrations may be among the interfering factors. MCHC cannot be greater than 37 g/dL
because the RBC cannot accommodate more than 37 g/dL Hb [8].
4.4. Red blood cell distribution width (RDW)
RDW is an estimate of the variance in the volume within the population of red blood cells [4].
RDW, provided by automated counters, is an index of the distribution of RBC volumes. RDW is
derived from pulse height analysis and can be expressed as an SD (fL) or as a coecient of vari-
ation (%) of the red cell volume. Automated counters use two methods to calculate RDW [6].
The rst is referred to as RDW-CV. RDW-CV is the ratio of the width of the red blood cell
distribution curve at 1 SD divided by MCV (normal RDW-CV = 13 ± 1%) (Figure 4). Since it
is a ratio, changes in either the width of the curve or MCV will inuence the result. In micro-
cytosis, any changes in the RDW-CV simply reduce the denominator of the ratio. Conversely,
in macrocytosis the change in the width of the curve will minimize the change in RDW-CV. A
second method of measuring the RDW is RDW-SD and is independent of MCV. RDW-SD is
measured by calculating the width at the 20% height level of the red blood cell size distribu-
tion histogram (normal RDW-SD = 42 ± 5 fL) [6, 8, 15].
Current Topics in Anemia242

Both measurements of RDW are essentially mathematical statement of anisocytosis. Increases
in the RDW suggest the presence of a mixed population of cells. Double populations, whether
microcytic cells mixed with normal cells or macrocytic cells mixed with normal cells, will
widen the curve and increase the RDW. The RDW-SD is more sensitive to the appearance of
minor populations of macrocytes or microcytes since it is measured lower on the red blood
cell volume-distribution curve (Figure 4) [4, 8].
The RDW can be used to distinguish thalassemia (normal RDW) from iron deciency anemia
(high RDW). Also, it can be used to distinguish chronic disease anemia (normal RDW) from
early iron deciency anemia (elevated RDW). RDW increases in iron deciency anemia, vita-
min B12 or folate deciency (pernicious anemia), abnormal Hb (S, S-C, or H), S-β thalassemia,
immune hemolytic anemia, marked reticulocytosis, and posthemorrhagic anemia.
The RDW may be an alternate marker for systemic inammation and/or oxidative stress;
however, the predictive value of RDW is independent of other inammatory markers. This
suggests that this biomarker also follows other nonempirical processes [8, 17]. The determina-
tion of the physiological and biological mechanisms that associate RDW to adverse clinical
results is important in using these prognostic biomarkers to therapeutic decisions [18].
4.5. Stained peripheral blood smear
Peripheral blood smears can provide important additional information about RBC morphology
in anemia and are easily prepared manually using glass slides. The hematology laboratory usu-
ally examines a peripheral blood smear if the patient’s indices are abnormal (unless there has
been no major change from previous CBCs). If an underlying blood disorder is suspected, a lm
should be requested. Automated instruments ensure accurate RBC counts and indices and WBC
counts and dierentials in both healthy and diseased individuals [8, 19].
Figure 4. Red blood cell distribution width. Automated counters provide measurements of the width of the red blood
cell distribution curve. RDW-CV is calculated from the width of the histogram at 1 SD from the mean divided by
MCV [6].
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The peripheral blood smear complements the automated countermeasurements of MCV
and MCH. Visible changes in cell diameter, shape, and hemoglobin content can be used
to distinguish both microcytic and macrocytic cells from normocytic/normochromic RBCs
(Table 2) [6].
In clinical cases, the variation such as staining, color, shape, and inclusion bodies in the
blood smear of RBCs is not only an indication of RBC abnormalities but also a diagnosis
of diseases.
4.6. Reticulocyte count
Reticulocyte count is an essential component of CBC and has a substantial role in initially clas-
sifying any anemia. Reticulocytes are newly formed red blood cells with residual strands of
nuclear material called “reticulin” that remain following extrusion of the nucleus from bone mar-
row normoblasts [20]. The reticulocyte is a young red blood cell containing residual ribosomal
RNA that can be stained with a supravital dye such as acridine orange or new methylene blue [4].
The reticulocyte count can be used in dierentiation of the patients with a functionally nor-
mal marrow response to anemia/hypoxia and those with a failed marrow response. Whenever
the reticulocyte production index (RPI) increases to levels greater than three times normal in
response to an anemia (hematocrit <30%), it can be assumed that the patient has normal renal
function with an appropriate erythropoietin response and a normal erythroid marrow with an
adequate supply of key nutrients (iron, folic acid, and vitamin B12) [6, 15].
The paerns of some
abnormal RBCs
Comment
Macrocyte Larger than normal (>8.5 μm diameter)
Microcyte Smaller than normal (<7 μm diameter)
Hypochromic Less hemoglobin in the cell. Enlarged area of central pallor
Spherocyte Loss of central pallor, stains more densely, often microcytic. Hereditary spherocytosis
and certain acquired hemolytic anemias
Target cell Hypochromic with central “target” of hemoglobin. Liver disease, thalassemia, Hb D,
and postsplenectomy
Leptocyte Hypochromic cell with a normal diameter and decreased MCV. Thalassemia
Elliptocyte Oval to cigar shaped. Hereditary elliptocytosis, certain anemias (particularly vitamin
B12 and folate deciency)
Stomatocyte Slit-like area of central pallor in erythrocyte. Liver disease, acute alcoholism,
malignancies, hereditary stomatocytosis, and artifact
Acanthocyte Five to ten spicules of various lengths and at irregular intervals on surface of RBCs
Echinocyte Evenly distributed spicules on surface of RBCs, usually 10–30. Uremia, peptic ulcer,
gastric carcinoma, pyruvate kinase deciency, and preparative artifact
Sickle cell Elongated cell with pointed ends. Hb S and certain types of Hb C
Table 2. Various forms and interpretations of RBCs observed in the peripheral blood smear examination [31].
Current Topics in Anemia244

Reticulocytosis, increased RBC production, occurs when the bone marrow is replaced, is lost,
or has prematurely destroyed cells. Identifying reticulocytosis is important for the recogni-
tion of other clinic conditions such as hidden chronic hemorrhage or unrecognized hemoly-
sis (e.g., thalassemia, sickle cell anemia). Reticulocyte levels increase in hemolytic anemia,
immune hemolytic anemia, primary RBC membrane problems, hemoglobinopathy, RBC
enzyme decits, and malaria.
Increased reticulocyte count after hemorrhage (3–4 days) or after treatment of anemias can
be used as an index for an eective treatment. In iron deciency anemia, reticulocytes may
increase to more than 20% after sucient doses of iron. A proportional increase in reticulocytes
can also be seen when pernicious anemia is treated by transfusion or vitamin B12 therapy.
If there is not enough erythrocyte production in the bone marrow, the reticulocyte count
decreases in untreated iron deciency anemia and aplastic anemia, untreated pernicious anemia,
anemia of chronic disease, radiation therapy, endocrine problems, tumor in the marrow (bone
marrow failure), myelodysplastic syndromes, and alcoholism.
Interfering factors: Reticulocytes are normally increased in infants and during pregnancy.
Recently transfused patients have a lower count because of the dilution eect. The presence
of Howell-Jolly bodies falsely elevates reticulocyte count when automated methods are used.
Some other laboratory tests are useful to dene the physiologic defects responsible for ane-
mia. Indirect serum bilirubin and lactic dehydrogenase (LDH) levels increase in patients with
increased hemolysis and in ineective erythropoiesis. Indirect bilirubin levels correlate with
RBC turnover rate. Serum LDH is exceedingly responsive to increased rates of RBC destruction
(because of the excess levels of LDH 1 in RBCs) [8, 21].
Reticulocyte hemoglobin content (CHr or Ret-He) measurement demonstrates Hb synthe-
sis in marrow precursors. Ret-He also reects the early stages of iron deciency. Ret-He is
dened as an auxiliary parameter in the dierential diagnosis of anemias.
5. Additional new red blood cell and reticulocyte indices
Current high-end automated cell counters measure unique properties of mature red blood
cells and reticulocytes on a cell-by-cell basis, not just as population averages. This results a
plethora of new indices that are in many cases specic to an instrument manufacturer, pre-
senting diagnostic opportunities but also a confusing nomenclature and a potential lack of
comparability. Some examples of parameters that have been studied include hypochromic
erythrocytes (HypoHe%), percentage microcytic red blood cells (MicroR%), reticulocyte
hemoglobin equivalent (Ret-He), reticulocyte hemoglobin content (CHr), red blood cell size
factor (RSf), low hemoglobin density (LHD%), and fragmented red blood cells (FRCs) [22–24].
Ret-He demonstrates the real-time information on the synthesis of young RBCs in the bone
marrow. Other available parameters are the percentage of RBCs with Hb content equivalent
≤17 pg (HypoHe%) and the percentage of RBCs with a volume of <60 fL (MicroR%), which
reects the subpopulation of mature RBCs exhibiting evidence of insucient iron content [6, 8].
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Estimates of reticulocyte-specic hemoglobin content (which are comparable) by light-scaer
measurements of reticulocytes are closely related to adequacy of iron availability to erythroid
precursors during the preceding 24–48 hours and have been described as diagnostically useful
in detecting functional iron deciency [8, 22].
The CHr may be a beer predictor of depleted marrow iron stores than traditional serum iron
parameters in nonmacrocytic patients and is a more sensitive predictor of iron deciency than
hemoglobin for screening infants and adolescents for iron deciency [25, 26].
Schistocytes or FRC is also used as new red blood cell indices. Nevertheless only a few
studies have been published on this parameter, but concerns have been expressed for false
positivity in the presence of hypochromic samples. Schistocytes are elevated in thrombotic
microangiopathies [1].
5.1. Marrow examination
Bone marrow examination has a special place in the cause of anemia since it is the organ of
blood production [20]. The marrow examination is of greatest value in patients who fail to
show an appropriate increase in the reticulocyte production index in response to anemia. A
sample of the marrow can easily be obtained by needle aspirate or biopsy to evaluate overall
cellularity, the ratio of erythroid to granulocytic precursors (E/G ratio), and cellular morphol-
ogy. In these patients defects in erythroid precursor proliferation or maturation play a major
role. Examination of any marrow aspirate should include a careful assessment for evidence of
a red blood cell maturation abnormality, especially changes in cell size, nuclear morphology,
and hemoglobin production. A number of anemias are characterized by distinct abnormalities
in the maturation sequence and the morphology at each stage of maturation [6]. The assess-
ment of the bone marrow is the gold standard in iron deciency. The presence of the mineral
in reticuloendothelial cells is the key to the diagnosis [20].
5.2. Tests of iron
Iron supply tests (serum iron level, transferrin iron-binding capacity, and serum ferritin level)
play an important role in the initial dierential diagnosis of an anemia. They are essential
components to the marrow iron stain whenever a marrow aspirate is performed [8, 22, 25].
1. Serum iron levels. This is serum iron (SI) measurement which reects an amount of iron
bound to transferrin. The reference range of SI level is 50–150 μg/dL for an individual. The
proliferative capacity of the erythroid marrow and its ability to synthesize hemoglobin are
assessed by serum iron level [6].
2. Total iron-binding capacity (TIBC). The amount of iron which is bound to transferrin is
called TIBC. Actually, it is equivalent to measuring the level of transferrin. The reference
value of TIBC is 300–360 μg/dL. TIBC increases in excess of 360 μg/dL in patients with
severe iron deciency.
3. Serum ferritin level. Ferritin is a spherical protein and is used clinically to evaluate total body
storage iron (body iron stores). A normal adult male has a serum ferritin level of between 50
and 150 μg/L, reecting iron stores of 600–1000 mg. Serum ferritin levels decrease when the
Current Topics in Anemia246

iron stores are depleted. Levels below 10–15 μg/L indicate iron deciency due to exhaustion
of iron store [6].
5.3. Other measurements
For the diagnosis of specic hematopoietic disorders, there are some other laboratory tests.
Table 3 demonstrates some of the special assays for such disorders [1, 4].
The owchart that follows is intended as a rst approach for the diagnosis of anemia and is
a supplement to this chapter to demonstrate how the steps might be placed in a logical order
(Figure 3).
5.4. Evaluation and investigation of the patient with anemia according to laboratory
parameters
A CBC and dierential and reticulocyte counts together with stained peripheral blood smear
examination should be the starting point of investigations. These conrm the clinical suspi-
cion of anemia and direct further investigation [5].
The points to be followed in Figure 5 (A–F) may help to begin anemia investigation:
A. Check RBC status of the patient.
The RBC performs some functions such as transportation of O2 and CO2. An increase in RBC
is referred as polycythemia. Patient may have α- or β-thalassemia. Conrm abnormal hemo-
globins with electrophoresis, Hb A2 value in β-thalassemia will be >3.5%, check if there are
target cells, etc. A decrease in RBC accounts for less hemoglobin. If RBC count is low, patient
iron status should be checked (iron, TIBC, Sat%, ferritin, etc.).
B. If red blood cell morphology demonstrates schistocytes.
1. Red blood cell fragmentation can be investigated. Fragmented red blood cells (FRCs)
and hemolysis occur when RBCs get stressed through partial vascular occlusions or over
Hypoproliferative anemias Maturation disorders Hemolytic anemias
*Cytometric assay of CD59/CD55
levels (paroxymal nocturnal
hemoglobinuria)
*Chromosomal analysis (leukemias)
*Marrow aspirate/biopsy special
stains
• Trichrom stain, silver stain for
reticulin (myelobrosis)
*Serum vitamin B12 level (vitamin B12
deciency)
*Serum RBC folate level (folic acid
deciency)
*Hb electrophoresis (abnormal
hemoglobins)
*Hb A2 level-HPLC (β-thal)
*Hb F level-HPLC (β-thal) RBC
protoporphyrin level (iron deciency)
Brillant aresyl blue stain
*Hb electrophoresis and HPLC
(hemoglobinopathies)
*Coombs test (autoimmune
hemolytic anemia)
*Cold aglutinin titer
(autoimmune hemolytic anemia)
*Haptoglobin level (hemolysis)
*G6PD screen (G6PD deciency)
Table 3. Specic hematopoietic disorders and the associated laboratory tests [6].
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abnormal vascular surfaces. “Split” RBCs, or schistocytes, are considered on peripheral
blood smears under these conditions; signicant quantities of lactate dehydrogenase are
released into the blood from injured RBCs [4].
2. The ethnicity of the patient is important for this situation.
Check the patient for sickle cell disease (SCD). If the patient is normal for SCD, investigate
iron deciency anemia.
C. Screen for uncorrected reticulocyte count.
Reticulocyte count and indices: Reticulocytes are stained by supravital staining. Typical normal
range is 0.5–1.5%. The count depends on total RBC count [12]. For both the pathophysiological
classication of anemia and to monitor marrow response after therapeutic interventions, reticu-
locyte count is clinically important [11, 19].
Reticulocyte count was used in the clinical and laboratory practice for a long time due to
three main factors: technical limitations in the detection of cell, the imprecision of manual
microscopic method, and high coecient of variations in counts [28, 29]. The index is the
corrected value in relation to total red blood cell mass and Hb%. Increased count indi-
cates increased red blood cell turnover. Reticulocyte count can be used as a measure of red
blood cell production by correcting red blood cell count for both changes in hematocrit.
The result of correction reects the eect of erythropoietin on reticulocyte release from the
marrow [6].
Obtain single correction reticulocyte count (reticulocyte index) (S):
S = Reticulocyte count ×
(
Patient Hct
/
0.45
)
(1)
Double-corrected reticulocyte count or reticulocyte production index (RPI) is calculated by
dividing the single correction reticulocyte count by the maturation index.
In situations where the reticulocyte count is elevated, other possibilities should be inves-
tigated, for example, serum haptoglobin and hemopexin, which are degraded hemo-
globin-bound complexes, are impaired and can’t be monitored in acute intravascular
hemolysis. Unconjugated bilirubin in serum and urobilinogen in urine should be also mea-
sured. Unconjugated hyperbilirubinemia in the absence of urobilirubinogen in urine is a
marker of hemolysis [12].
If reticulocyte production index (RPI) is ≥3, peripheral blood smear should be examined for
abnormal morphology, and the values of bilirubin, LDH, serum-free Hb, urine Hb, urine
hemosiderin, and haptoglobin should be evaluated.
If Haptoglobin is >40 mg/dL, the patient probably has/had an acute hemorrhage or is respond-
ing to hematinic. Patient should be evaluated for external or internal bleeding.
If Haptoglobin is <30 mg/dL, probably the patient has hemolytic anemia. Bilirubin is usually
between 1.0 and 5.0 mg/dL. Mostly indirect bilirubin is present.
Current Topics in Anemia248

Screen for uncorrected reticulocyte count if the patient has any morphological abnormalities
screen for uncorrected reticulocyte count.
Often, the etiology of a patient’s anemia can be determined if the shape or size of RBCs is
altered or if they include inclusion bodies (Table 2). Plasmodium falciparum malaria is sug-
gested by the presence of more than one ring form in an RBC, and the infection produces
pan-hemolysis of RBCs of all ages [30].
D. If normocytic, heterogeneous anemia is present.
The levels of ferritin and RBC folate/vitamin B12 should be examined to conrm/exclude the
possible early diagnosis of iron deciency anemia, sideroblastic or megaloblastic anemia,
mixed deciency, and myeloproliferative disorder. Serum transferrin receptor, homocysteine,
and methylmalonic acid levels can be also considered.
E. Check pyruvate kinase and glucose-6-phosphate dehydrogenase (G6PD) enzyme.
In severe hemolytic anemia, spherocytosis and RBC fragmentation may be seen in the stained
lm. Although drug-induced hemolysis may indicate “bite cells” in the blood of patients with
G6PD deciency, this may not always be associated with G6PD deciency because such cells
are generally not found in patients with acute hemolytic conditions of chronic G6PD vari-
ants or patients with chronic hemolytic G6PD deciency [4]. Repeat the history and physical
examination for splenomegaly.
F. If macrocytic, heterogeneous anemia is present.
The paern of folate or vitamin B12 of the patient should be checked. The homocysteine, meth-
ylmalonic acid, LDH, and indirect bilirubin values can be investigated. It is necessary to eval-
uate the intrinsic factor and parietal cell antibody to conrm or exclude pernicious anemia.
Figure 5. Flowchart as a rst approach to diagnose anemia. In anemic patients, approaches should follow according to
MCV and RDW because of their comprehensibility and simplicity [27].
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249

The proper use and interpretation of laboratory tests are important in the diagnosis and treat-
ment of anemia. Whether the patient is anemic can be determined by using Hb, Hct, or RBC
count and the reference intervals for age and sex or the patient’s previous values [31]. Routine
examination of the blood includes CBC and examination of a stained peripheral blood smear.
The values could be normal in mild anemia with RBC count in normal range [32].
6. Conclusion
There is no single optimal marker or test combination in the dierential diagnosis of ane-
mias [33]. The knowledge and experience of the physician who demands appropriate hema-
tological and biochemical tests related to preliminary diagnosis have the important role in
the diagnosis of anemias. It is recommended to use algorithms as a tool in determination
of anemias in order to reduce the laboratory tests and accurately diagnose the underlying
cause(s) in patients.
For the past decade, remarkable progress has been made in the procedures and algorithms in
the dierential diagnosis of anemias. CBC is the main procedure for investigating anemia. The
percentage of microcytic RBCs is considered in the rst step. In the second step, MCV, RDW,
and RBC count should be examined. It is advocated that innovative algorithms, including
parameters reecting hemoglobinization of RBCs and reticulocytes, are integrated to improve
the dierentiation between anemias. Subsequently, new algorithms, including conventional
as well as innovative hematological parameters, were assessed for subgroups with microcytic
erythropoiesis. Nowadays automated reticulocyte counts provide new parameters to evalu-
ate marrow activity [29]. It is therefore important to establish accurate and reliable criteria for
both identifying the specic causes of anemia and evaluating the impact of intervention strate-
gies. These should be followed by laboratory tests that are mandatory and simple to perform.
7. Key points of this chapter
CBC is the most sensitive measure in the routine use to obtain the information about the pres-
ence and severity of anemia.
For the evaluation of anemia, there are some essential basic laboratory tests such as CBC,
reticulocyte count, blood smear morphology changes, iron balance studies, and bone marrow
morphology reports.
Severity of the hematocrit/hemoglobin changes in MCV, RDW, and blood smear morphology
are the rst parameters to evaluate anemia. These help to dene the anemia as normocytic,
microcytic, or macrocytic.
Reticulocyte index denes the adequacy of the erythropoietin and red blood cell production
response.
Current Topics in Anemia250

Bone marrow examination can also provide information about proliferative response and
whether there is any defect in precursor maturation.
Iron studies should also be included in the investigation of anemia.
In conclusion, identication of the cause of anemia by the clinician with the support of laboratory
data is an important step to diagnose, treat, and monitor the underlying pathological process.
Author details
Ebru Dündar Yenilmez* and Abdullah Tuli
*Address all correspondence to: edundar@cu.edu.tr
Department of Medical Biochemistry, Faculty of Medicine, Çukurova University, Adana,
Turkey
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