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d
Little hole
Side Scattering Detector
ing
Light source
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+
External
electrode
Fig. 15.1 Diagram of the electrical impedance cell counter (section).
The cell suspension is drawn (arrows directed upward) from the outer
container (bath apertures) to the inner container (tube apertures)
through a capillary bore (apertures). The current ow that is maintained
between the inner (+) and outer (−) electrodes passes only through the
small hole; it is constant until a cell also passes through it. Since the
current is weak and the cell has dielectric properties this results in a
momentary reduction in charge ow, the magnitude of which is proportional to the volume of the cell. (Copyright EDISES 2021. Reproduced
with permission)
–
Bath openings
Internal
electrode
Suspension
of red bloo
cells
Tube
openings
elements at the same time, which increases as the number of
cells in the sample increases (e.g., a sample with two million
erythrocytes has a lower coincidence than one with four million erythrocytes). A special statistical algorithm corrects the
count for this predictable phenomenon, which would otherwise result in a default assessment of the number of cells.
Forward Scatter
Detector
Fig. 15.2 Light scattering is observed when a beam of light impinges
on a cell without being absorbed. Part of the photons are deected with
a low angular degree (forward scattering) in relation to the size of the
cell, part is deected with a high angular degree (side scattering) in
relation to the contents inside the cell. Appropriate detectors collect the
signal obtained from the cell ow. (Copyright EDISES 2021.
Reproduced with permission)
Flow Cytometry
Flow cytometry is based on the detection of the absorption
and diffraction of light at certain angles and the differential
evaluation of the signals received. Current instruments operate with laser lights at certain wavelengths, which can guarantee the constancy of the light source. Each stream of cells
suspended in solutions in which reagents chosen to identify
distinct characteristics are premixed is passed through a thin
optical glass cell so that the cells are aligned in a single row
during the transit. The beam of light passing through each
cell is partly absorbed (photons passing through the cell) and
partly deected (scattering photons impinging at an angle on
the cell or being deected by structures within the cell, such
as the nucleus and granules) (Fig.15.2). The detection of the
magnitude of absorption and the signal from different angular degrees of deection are useful elements to diversify individual classes of cells by number, size, and peculiar
characteristics (Fig.15.3). Flow cytometry is widely used in
modern hematological instruments in association with electrical impedance so as to guarantee high counting accuracy
and allow correct detection of all the cellular elements of the
Fig. 15.3 Flow cytometer scheme. Each cell passing through the laser
light beam generates different signals that are collected by appropriate
detectors. The combination of the signals, through a special algorithm,
allows each cell to be assigned to its own distinctive class. (Copyright
EDISES 2021. Reproduced with permission)
blood, including both immature elements and elements
found in hematological disorders (Fig.15.4).
Fluorescence Cytometry
An extension of the principle of ow cytometry is present in
uorescence cytometry, in which the ow of cells is suspended in solutions containing uorochromes that bind specically to nuclear structures or contain nucleic acids,
166
1000
FSC-H
SSC-H
ab
ard
Fluorescence
Sample
Waste
M
=×/10
MCH Hb RBC=×/10
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Granulocytes
800
38.7
600
400
200
Lymphocytes
0
Fig. 15.4 Scattering graph. Each cell class is reported in the graph to
the area identied by the intersection of signals from FSC-H (forward
scattering) and SSC-H (side scattering). The graduation of colors within
each area is in relation to the cell density
PEROXIDASIS
28.7
400
200
0
Monocytes
4.87
600
1000
800
PEROXIDASIS
Lamp Beam
(UV)
side scatter
Diode
laser beam
Ar laser beam
Fig. 15.6 Graph of dual laser beam ow cytometric cell with multicolor uorescent signal emission. (Copyright EDISES 2021.
Reproduced with permission)
Forw
scatter
(Hydrodinamic
focusing)
Erythrocyte Indices andDistribution
Measurements
The erythrocyte indices are derived from the measurements
described above. They are mainly used as rst-line diagnostic indicators for different types of anemia.
Fig. 15.5 Myeloperoxidase activity graph. In abscissa, the peroxidase
activity increases toward the right, and the number of cellular elements
increases toward the top. Shown on the right is a normal specimen in
which cellular activity in neutrophil granulocytes is evident in the
upper-right portion; lower-right is eosinophils; and middle-upper is
monocytes. The middle-left band has lymphocytes, which lack myeloperoxidase activity. The bottom left are platelets lacking activity. On the
left is a sample with myeloperoxidase deciency
allowing further elements of characterization of certain
classes of cells (e.g., reticulocytes, platelets).
The manufacturers personalize their instruments by varying the combinations of the counting principles or by modifying the performance with the intention of introducing
improvements to the diagnostic possibilities. Mention should
be made of the use of reagents that detect the peroxidase
activity typical of cells of myeloid origin that allows, for
example, the distinction between polymorphonucleates and
monocytes (Fig.15.5). Others have made the measurement
of hemoglobin more sensitive and reduced analytical interference, and still others have employed ow cytometry systems with multicolor uorescence and multiangle scattering
detection to increase the specicity of detection of different
leukocyte classes (Fig.15.6).
Mean Corpuscular Volume
The mean corpuscular volume (MCV) is the value obtained
from the ratio of the Hct to the number of red blood cells:
CV HctRBC
Values are normally expressed in femtoliters
(1fL= 1×10
−15
L), according to the International System
(IS), or in microliters (μL), according to traditional units.
The reference range is between 80 and 100fL, with variations depending on age and status (e.g., pregnancy). Anemic
conditions are dened according to the measured MCV value
as microcytic if MCV <80fL, normocytic if MCV is between
80 and 100fL, and macrocytic if MCV >100fL (Table15.3).
Mean Corpuscular Hemoglobin
The mean corpuscular hemoglobin (MCH) is the value
obtained from the ratio of hemoglobin to the number of red
blood cells and expresses the hemoglobin content as
mass-weight.
Values are normally expressed in picograms
(1pg=1×10–12g) according to IS.The reference range is
27–31pg, with variations depending on age and status (e.g.,
pregnancy). Anemic conditions are dened according to the
measured MCH value as hypochromic if MCH <27pg, normochromic if MCH is between 27 pg and 31pg, and hyperchromic if MCH >31pg.
RBC
Frequency (%)
Average cell volume
CV
MCHCHbHt=×/ 100
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Table 15.3
Microcytic (MCV<80fL) Normocytic (MCV 80–100fL) Macrocytic (MCV>100fL)
Generally microcytic
Sideropenia (iron deciency)
Thalassemias
Hereditary sideroblastosis
Occasionally microcytes
Chronic conditions
Hemoglobinopathies
Classication of anemia according to mean corpuscular volume (MCV) and morphology
Generally normocytic
Hypoproliferative anemia
Anemia secondary to malignancy
Refractory myelodysplastic anemia
Hemolytic anemia
Hemoglobinopathies
Post-hemorrhagic anemia
Chronic conditions
Acquired sideroblastosis
Occasionally normocytic
Early stage iron deciency
Generally macrocytic
Folic acid deciency
Vitamin B12 deciency
Liver diseases
Hemolytic anemia
Post-hemorrhagic anemia
Occasionally macrocytic
Hypoproliferative anemia
Refractory anemia
Mean Corpuscular Hemoglobin Concentration
Mean corpuscular hemoglobin concentration (MCHC) is the
100
value obtained from the ratio of hemoglobin to hematocrit
and expresses the concentration of hemoglobin compared to
Histogram width
the total erythrocyte volume.
20
RDW-SD
The values are normally expressed in g/L according to the
IS or in percentage according to the traditional system. The
50
100 150
reference range is 320–360g/L (IS) or 32–36g/dL or % (traditional system). Its use as a diagnostic index is limited to
conditions affecting the erythrocyte membrane, such as an
increase in its outer surface due to the adhesion of cold
Fig. 15.7 Graph representing the distribution of the erythrocyte population with RDW-CV and RDW-SD. (Copyright EDISES 2021.
Reproduced with permission)
agglutinins to the membrane or a decrease in membrane
thickness, which can be found in some clinical conditions,
such as hereditary spherocytosis (HS) and elliptocytosis. In
cyte volumes within physiological ranges should be
considered:
addition, increased MCHC is found in pathological conditions that cause the concentration of erythrocyte content to
increase due to dehydration, such as some hemoglobinopathies (mainly HbS and HbC).
• Variability of production by the bone marrow, which is
kept within narrow limits and controlled at the site by
eliminating, through the medullary reticuloendothelial
system, cells that cannot be released into circulation.
Red Cell Distribution
Red cell distribution width (RDW) is a measure of the range
of variation in erythrocyte volumes in terms of a coefcient
of variation (CV) in percent. RDW can be reported statistically as a coefcient of variation (CV) and/or standard deviation (SD).
• The progressive aging of the circulating erythrocyte pop-
ulation is accompanied by a reduction in cell volume due
to dehydration. Young red blood cells, since the reticulo-
cyte state, are signicantly more voluminous than red
blood cells that have reached the end of their useful life,
and, passing from one extreme to the other, a progressive
and regular reduction in volume is observed. In many
• RDW-CV, which represents the coefcient of variation in
percent with respect to MCV, is expressed by the formula:
RDW=SD
/MCV×100.
(MCV)
• RDW-SD, which reports in femtoliters the true value of
the standard deviation obtained from the distribution of
erythrocyte volumes measured at the height of 20% on the
curve itself (Fig.15.7).
conditions of anemia, often high values of RDW are
observed, so this index is used in association with MCV
for a rst differentiation among anemias that present similar characteristics; the most typical case is the differential
diagnosis between two common microcytic anemias, the
iron deciency anemia and the β-thalassemic trait anemia, in which, besides a common reduced MCV, a high
RDW value is more indicative of the rst condition than
The reference range is between 12% and 15% (RDW-CV)
and between 40 and 55 fL (RDW-SD), with variations
depending on age and status (e.g., pregnancy). In evaluating
this index, two factors that inuence the variation of erythro-
the second one. Also, macrocytic anemia due to the deciency of folate and vitamin B12 is characterized by an
increase in RDW and an elevated MCV.However, it must
be kept in mind that this is an index with relatively modest
MCV
= RDW-
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G. C. Guidi
specicity and that, therefore, more specic tests must be
associated with it. In recent years, interest in RDW has
increased because an association between RDW and cardiovascular disease has been described. Specically,
increased levels of RDW are a predictor of adverse outcomes in patients with cardiovascular diseases.
Additionally, high RDW values have been described in
several clinical conditions, including thromboembolic
diseases, various cancers, diabetes, pulmonary diseases,
kidney diseases, liver diseases, and other chronic disorders. The pathophysiological mechanisms underlying the
RDW increase in these clinical conditions are not entirely
understood, but erythrocyte eriptosis or apoptosis have
been hypothesized. Eryptosis is a suicidal mechanism of
the cell triggered by the entry of calcium ions, oxidative
stress, alterations in the regulation of cytokines, and other
mechanisms that occur in many chronic inammatory
diseases (e.g., heart and kidney failure), metabolic diseases (e.g., diabetes), and others such as neoplasms.
Platelet Indices andPlatelet Count
Modern hematological instruments calculate several indices
related to platelets, which provide useful information regarding the morphology and turnover of these cellular elements.
Mean Platelet Volume
The mean platelet volume (MPV) population, unlike erythrocytes, does not have a Gaussian distribution but is rather
shifted to the left so that some graphs represent the curve
after log transformation. The reference interval is between 7
and 12fL.Newly produced platelets from the marrow are
larger in size than older platelets, and greater MPV is
observed when platelet production increases. MPV may be
useful as an index to assess the extent of platelet production
when accelerated destruction is suspected. In the latter case,
increased MPV can be found in several inammatory gastrointestinal diseases, thrombotic thrombocytopenic purpura
(TTP), Bernard–Soulier syndrome, myeloproliferative diseases, preeclampsia, and some cancers. In many of these
clinical conditions, an increase in MPV is accompanied by a
signicant decrease in the number of circulating platelets
due, as mentioned, to early destruction in the circulation. On
the contrary, a decrease in MPV can be observed in some
genetic thrombocytopenias (Wiskott–Aldrich syndrome) and
in bone marrow aplasia.
can be expressed both as a coefcient of variation(PDW-CV)
or standard deviation (PDW-SD):
• PDW-CV=SD
/MPV× 100
(MPV)
• PDW-SD, which reports in femtoliters the true value of
the standard deviation obtained from the platelet volume
distribution measured at the height of 20% on the curve
itself.
The mode of expression depends on the type of instru-
mentation. The reference range for PDW-CV reported in the
literature is 10.0–17.9%, with a mean value of 13.3%,
depending on the instrumentation used. PDW generally
increases in relation to an increase in the number of young
circulating platelets, as seen in idiopathic thrombocytopenic
purpura (or immune thrombocytopenia or immune thrombocytopenic purpura [ITP]) or macrothrombocytopenia.
Although PDW is signicantly increased in ITP compared to
controls, its diagnostic power is limited when analyzed on its
own. PDW should be considered a useful supporting parameter in association with MPV and immature platelet fraction,
a parameter known as IPF. For example, PDW should be
evaluated together with other platelet parameters for differentiating idiopathic thrombocytopenic purpura from
Wiskott–Aldrich syndrome in patients with macrothrombocytopenia. In ITP, young platelets are produced with a larger
size, and, thus, MPV is often elevated along with a high
PDW.On the other hand, patients with Wiskott–Aldrich syndrome have uniformly large platelets, thus a high MPV but a
normal PDW.
Immature Platelet Fraction
Immature platelet fraction (IPF) is a parameter recently
introduced in automated analyzers. It provides the fraction
of cross-linked platelets on the total number of platelets,
that is, the youngest platelets, which, similar to reticulocytes, have a cytoplasmic reticular structure due to polyribosomal RNA.The reference range is between 1% and 8%
of the total platelets, and the value increases when the production of platelets by the marrow increases. It is therefore
indicative of the efciency of the bone marrow and, in particular, the proportion of megakaryocytes produced. The
parameter is useful in the diagnosis and treatment of thrombocytopenia, such as idiopathic thrombocytopenic purpura
and thrombotic thrombocytopenic purpura, as well as in the
differentiation between forms with destruction in the circulation and those due to suppression of bone marrow
production.
Platelet Distribution Width
The platelet distribution width (PDW) represents a measure
of the range of variation of platelet volumes in terms of the
percentage coefcient of variation. Similarly to RDW, PDW
Plateletcrit
The plateletcrit (PCT), similar to the hematocrit for erythrocytes, represents the portion of the volume occupied by
platelets in the blood as a whole. The value of the PCT is
PC
=×
000
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expressed as a percentage and is calculated according to the
following formula:
Tplateletcount MPV
/,10
The reference range for PCT is 0.22–0.24%. In normal
subjects, the total mass of platelets is a constant ratio between
the number of platelets and MPV, which therefore shows an
inverse correlation between them. This ratio is altered in
some conditions, including physical efforts, consumption of
substances of abuse, such as alcohol and smoking.
Other Indexes
• Percentage of large platelets (>12fL) over total platelets
(platelet larger cell ratio, [P-LCR]), with normal values
between 15% and 35%. Since the largest platelets are the
youngest and most active, the index would provide an
indirect estimate of platelet activity.
• The mean platelet component (MPC) indirectly provides
an evaluation of the state of platelet activation through the
scatter of incident light, whereby a lower deection cor-
responds to lower granule content and, therefore, is indic-
ative of the previous activation.
• Platelet component distribution width (PCDW) and mean
platelet mass (MPM) are linked to the P-LCR and MPC.
• Immature platelet fraction (IPF) is a percentage index
obtained by detecting the uorescent signal emitted by
platelets due to the presence of RNA within them. This
is obtained in the same ow channel, which contains a
special dye to detect the RNA, which is indicative of
the percentage of maturation of reticulocytes. IPF
increases with increased platelet production and
decreases in thrombocytopenia due to reduced
thrombocytopoiesis.
Reticulocytes andrelated Indices
Reticulocyte count is performed by microscopy or by electronic instruments. In both cases, the distinctive marker is
polyribosomal RNA, which is well represented in reticulocytes. Preparation for microscopic counting is performed by
supravital staining of a part of incoagulable blood suspended
in an equal part of an isotonic solution of brilliant cresyl
blue. Other supravital stainings are the new methylene blue
and Azur B.Also, dyes for uorescence microscopy, such as
acridine orange and auramine, are commonly used. The
observation is done by immersion (1000×) on a freshly
swiped slide (Fig.15.8). The reticulocytes observed are compared to 1000 erythrocytes, and their number is expressed as
a percentage; a CV of 10% is acceptable, but this value must
consider the variability among operators. Therefore, in practice, even higher values are reported to the point of invalidat-
ing their clinical-diagnostic signicance. Although the
advent of automatic instruments has displaced microscopic
counting, it is still used in less-developed healthcare
facilities.
Automated instruments allow not only obtaining a more
accurate and precise reticulocyte count than microscopybased methods but also measuring reticulocyte characteristics that cannot be captured by microscopic observation. In
the automatic procedure, the blood is incubated with an isotonic solution of a dye, which crosses the cytoplasmic membrane and binds to RNA; the detection of reticulocytes is
carried out by colorimetry or uorescence through the use of
supravital dyes, such as the new methylene blue, or uorescent dyes, such as thiazole orange, osazine 750, and others.
The reticulocyte count allows us to evaluate the production rate and, indirectly, the replacement rate of the
erythrocyte population. It can be reported as an absolute
count or as a percentage of reticulocytes relative to erythrocytes. In this way, it is possible to distinguish between
anemias of different origins, such as those resulting from
hemolysis or other destructive processes, and those due
to deficient production or deficiency of essential factors
for erythropoiesis. The reticulocyte count allows determining not only if their production is qualitatively
increased during anemia but also if the entity of increased
production is adequately sustained and leads to the recovery of the state of normality. On the other hand, a decrease
in the reticulocyte count can be a consequence of conditions such as deficiency states, bone marrow insufficiency, and cancer (Table15.4).
The reference range for the adult percentile count is 0.5–
1.5%. The absolute reticulocyte count is obtained by calculating its value over the total number of red blood cells; for
example, in a subject with 4,800,000/fL erythrocytes and
1.5% reticulocytes: 4,800,000/(100×1.5)×72,000/fL.
Hematological instruments automatically provide this
calculation. If there is a state of anemia, the percentage of
reticulocytes does not accurately reect the true responsiveness of the bone marrow to the anemic state. It is necessary
Fig. 15.8 Reticulocytes after supravital staining
170
Hematocr
EMENT”
correction factor
Normoblasts and
Reticulocytes in
=×
/%
=×
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G. C. Guidi
Table 15.4
count
Increase in count from
increased production
Post-hemorrhage (e.g.,
trauma, gastrointestinal
hemorrhages, obstetricgynecological
hemorrhages)
Post-hemolysis (e.g.,
hemolytic anemia, neonatal
hemolytic disease)
Response to ad hoc
treatments (e.g.,
administration of iron,
vitamin B12, folic acid)
Recovery of bone marrow
function after
chemotherapy/irradiation
Recovery of bone marrow
function after
transplantation
Conditions accompanying changes in the reticulocyte
Decreased count from decreased
production
Iron deciency, vitamin B12
deciency, folic acid deciency
Decreased production of erythropoietin
(e.g., in chronic kidney failure)
Aplastic anemia and/or bone marrow
failure syndromes
Chemotherapy and/or post-irradiation
Bone marrow replacement from benign
conditions (e.g., metabolic and storage
diseases, infections, sarcoidosis) and/
or malignant (e.g., leukemias,
lymphomas, metastatic tumors)
to correct the percentage value according to the hematocrit
value. While it appears evident that the absolute value does
not need any correction as it expresses the actual number of
reticulocytes, the percentage value needs correction for two
main reasons: one intrinsic to the data itself and the other due
to the period of maturation of the reticulocytes.
Reticulocyte Production Index
The rst correction is performed by the reticulocyte production index (RPI) or corrected reticulocyte count using the
following calculation:
RPIReticulocyteCount patient
hematocrit Hematocrit
.45
A hematocrit of 45% is taken as a reference. The RPI is
normally between 0.5% and 2.5%. Alternatively, there are
formulas based on hemoglobin, such as the following:
RPIReticulocyteCount patient
hematocrit normalhemoglobin
/
A normal hemoglobin value is 150g/L.
The second condition is a little more complex and depends
on the period of maturation of reticulocytes from the loss of
the nucleus (erythroblast stage) to maturation into circulating erythrocytes. Usually, this maturation takes place over a
period of 4.0–4.5days, of which 3.0–3.5days are spent in
the marrow and one in the circulation. However, under conditions of erythropoietic stress, such as hypoxia due to anemia, premature release of reticulocytes occurs from the
marrow, and the days of maturation in the circulation increase
up to 2–3. This leads to increased reticulocyte counts that do
not accurately reect normal production and release kinetics.
lymphocytes in the
bone marrow (days)
it (%)
45
35
25
15
Fig. 15.9 The correction factor is the divisor to be applied to the percent count to modify the effect due to the lengthening of circulating
maturation in case of anemia. (Copyright EDISES 2021. Reproduced
with permission)
peripheral
blood (days)
3.5 1.0
3.0 1.5
2.5 2.0
1.5 2.5
“DISPLAC
Graphs such as the one shown in Fig.15.9 have been proposed to correct this effect.
Immature Reticulocyte Fraction
In the evaluation of anemia, the state of maturation of reticulocytes can introduce elements of complexity when a correct
meaning must be attributed to their count. However, it provides further diagnostic opportunities that have been progressively perfected with automation. The microscopic
observation gives the opportunity to detect different entities
of staining in relation to the progressive reduction of RNA
until its disappearance when the reticulocyte becomes an
erythrocyte. To dene this phenomenon, as evidenced very
well by automatic instruments, an index called immature
reticulocyte fraction (IRF) has been proposed and adopted
internationally. IRF considers the reticulocytes subdivided
into populations of different maturation degrees. Generally,
instruments divide reticulocytes into three populations of
different immaturities: low, medium, and high. The IRF
index groups the fractions of medium and high immaturity.
The reference range is 0.11–0.38% (percentage of medium
and high immature reticulocytes in the total erythrocyte
count), with possible variations related to the instrumentation used. The parameter represents an early and sensitive
index of bone marrow erythropoietic activity, with the following main diagnostic features:
• After marrow transplantation, IRF may demonstrate successful engraftment earlier than other laboratory parameters, including absolute neutrophil granulocyte count. An
increase in IRF of more than 20% over the post-transplant
value is indicative of successful erythroid engraftment.
• IRF is a sensitive and early index of the recovery of bone
marrow activity after chemotherapy treatment.
• IRF, measured in patients with anemia associated with
chronic renal failure, neoplasms, infections, and chronic
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171
Table 15.5
reticulocyte fraction (IRF) and reticulocyte count
Clinical condition IRF
Medullary aplasia
Early erythropoietic response following
anemia or after rooted bone marrow
transplantation
Response to erythropoietin or early
post-hemorrhage recovery or postradiation treatment
Hemolytic anemia or bleeding
Clinical conditions using the combination of immature
Absolute
reticulocytes
count
↓ ↓
↓ / ≈↓
↑ ↑
↑ ↑
conditions, is an early and reliable index of response to
erythropoietin therapy.
• In surgical procedures with preservation of the patient’s
blood, it may be useful to monitor the response to
erythropoietin.
• It can be used to monitor the effectiveness of treatments
for decient anemias, such as those caused by iron, vitamin B12, and folic acid deciency.
Evaluation of IRF along with reticulocyte counts may
provide additional insight, as shown in Table15.5.
Reticulocyte Hemoglobin Content
Hemoglobin content in reticulocytes (reticulocyte hemoglobin content [CHr]) provides a measure of the efciency of
hemoglobin synthesis that the reticulocyte has implemented
in the previous few (3–4) days. The reference range is
approximately 24–32pg. Values <24pg are indicative of iron
deciency. Since hemoglobin synthesis depends on iron
availability, CHr provides a measure of functional iron and,
therefore, is an index for early assessment of iron deciency.
Accordingly, it could be useful in the following conditions:
• Pediatric age.
• Pregnancy.
• Complex clinical pictures, such as chronic inammation
and chronic kidney disease, as the ferritin level in these
conditions, is elevated as an acute-phase reactant, despite
the reduced iron stores.
• Nonmacrocytic anemias, which may be a better predictor
of iron stores than traditional indices.
Reticulocyte Hemoglobin Equivalent
The hemoglobin equivalent in reticulocytes (Reticulocyte
Hemoglobin equivalent [Ret-He]) is a parameter with a very
similar meaning to the previous one. The reference range is
approximately 28–35pg. Values <28pg are indicative of iron
deciency.
Dierential Leukocyte Count (Leukocyte
Formula)
The differential leukocyte count provides the relative percentage of each class of white blood cells. In addition to
these values, hematological instruments generally report the
absolute values of the various classes. The differential count
also allows the detection of cells normally not present in the
circulation, such as immature elements, or abnormal populations, such as, for example, leukemic cells. The differential
count is obtained mainly by two methods: microscopic and
automated. Leukocytes are classied into granulocytes, lymphocytes, and monocytes.
Granulocytes owe their name to the presence of distinct
cytoplasmic granulations that dene three classes: neutrophils (also called polymorphonuclear), eosinophils, and
basophils. Neutrophil granulocytes are mature cells that
originate from an initial morphologically identiable precursor in the marrow, the myeloblast. As maturation progresses,
the myeloblast becomes rst a promyelocyte and then a
myelocyte. In these proliferative stages, the number of cells
increases geometrically. The metamyelocyte, the next cell,
no longer has mitosis and then transforms into a granulocyte
with a banded nucleus (band granulocyte). At this stage, the
cell is released into the circulation, where it completes its
maturation or enters medullary compartments to mature,
from which it is later released into the circulation. About half
of the intravascular polymorphonucleates circulate, maintaining a dynamic equilibrium with the other half, which is
“marginalized” on the vascular endothelium. Therefore, only
the circulating neutrophils are considered in the differential
count. The half-life of mature circulating neutrophils is about
7 h. They irreversibly cross the vascular endothelium,
migrate into the tissues to perform antibacterial surveillance,
and die after 1 or 2days. Eosinophilic and basophilic granulocytes have a similar development. After their release from
the bone marrow, eosinophils rapidly leave the intravascular
compartment and migrate into tissues, especially the gastrointestinal tract, lung, and skin, and never return to circulation. The function of these cells is probably to defend against
multicellular parasites and to control the mechanisms associated with allergies and asthma, as well as inammatory phenomena. Basophil granulocytes constitute about 1–2% of
circulating leukocytes. Their physiological role is not yet
completely claried. They are present in inammatory reactions during the immune response, as well as in allergic manifestations. Their granules contain heparin and histamine.
Lymphocytes and monocyte-macrophages are known as
mononuclear leukocytes. Both play an important role in cellular and humoral immunity. These cells can get in and out of
172
Granulocytes and Monocytes Lymphocytes
Head
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circulation, maintaining their function, and can spend a long
time in tissues or lymph nodes.
Lymphocytes are the key immune cells for cellular and
humoral immunity. In the blood, they represent 20–45% of
leukocytes. Morphologically indistinguishable, they belong
to two distinct systems called T (thymus) and B (bursa or
bone marrow), having different functions. B lymphocytes
are responsible for the synthesis of antibodies. When a
lymphocyte of the B system (humoral system) is properly
stimulated, it rst proliferates and then turns into a plasma
cell, the effector part of the immune response. Each B lymphocyte can synthesize only one type of antibody. The T
lymphocyte system constitutes the cellular immune system
and regulates the entire immune system. Different subsets
of T cells can be identied by monoclonal antibodies specic against different membrane antigens. For example, T
helper lymphocytes promote the function of B lymphocytes, while T suppressor lymphocytes inhibit them. Some
T cells are responsible for cell-mediated cytotoxicity; natural killer (NK) lymphocytes are responsible for the nonspecic lysis of certain cells, for example, cancer cells. In
peripheral blood, about 15–25% of lymphocytes are B
cells, and about 40–75% are T cells.
Monocyte-macrophages originate in the bone marrow,
from where they are rapidly released into the circulation,
where they account for ∼5% of leukocytes. Once they
migrate into tissues, they become macrophages.
Monocyte- macrophages play phagocytic functions on
bacteria and particulate material, participate in inflammatory reactions, and are important to the immune system as they process antigenic material and communicate
related information to T lymphocytes through a process
of cell–cell interaction. Monocytes can also secrete interleukins, which boost B and T lymphocytes. Additionally,
they participate in fibrinolysis by secreting plasminogen
activators.
Microscope Dierential Count
It is performed under the microscope after staining a smear
with the May-Grünewald-Giemsa technique (more used in
Europe) or with Wright’s technique (in the USA and AngloSaxon countries). The 100× immersion objective is used
with a 10× eyepiece, and then the nal magnication is
1000×. Running the slide in the tail of the smear, according
to a Greek prole that includes in part the cells on edge and
in part those more centrally located, a total of 100 leucocytes
are counted, classifying them in percentages according to the
class to which they belong (Fig.15.10). Greater accuracy is
obtained by counting 200 cells.
G. C. Guidi
ThinRight Thick
Tail
Fig. 15.10 Correct procedure for differential leukocyte count.
(Copyright EDISES 2021. Reproduced with permission)
Body
Automated Dierential Count
Multiparametric ow cytometry is currently the most popular (and preferred) alternative to microscopic differential
counting since it allows analyzing thousands of cells in a
standardized way in a short time, with the possibility of
detecting not only the populations usually recognized and
classied in a blood smear, that is, neutrophils, eosinophils,
basophils, lymphocytes, monocytes, immature granulocytes,
and blasts, but also other subsets of cells, including various
types and subpopulations of blasts and lymphocytes. In addition, recent studies have demonstrated the feasibility of ow
cytometry using combinations of monoclonal antibodies that
allow the detection and quantication of up to 16 cell subsets. The instruments most commonly used in laboratories
are based on two technologies, either alternative or combined, depending on the complexity of the instrumentation
and the manufacturer, namely electrical impedance and
cytochemistry.
The differential leukocyte count is primarily necessary
for the following reasons:
• To search for quantitative anomalies in a population of
morphologically normal leukocytes, such as in infectious
or allergic diseases.
• To therapeutic monitoring of cytotoxic or myelotoxic
drugs. Of course, this requires a high level of precision
and accuracy.
• To detect morphological abnormalities of white blood
cells (e.g., when abnormal populations of white blood
cells are circulating or when the presence of immature or
atypical cells is suspected). This requires a high level of
clinical sensitivity, that is, the ability to identify all
patients with abnormal circulating leukocytes.
Automated differential counting provides a high level of
accuracy and precision in quantifying and identifying normal white blood cells. However, the method is not always
sufciently sensitive in identifying abnormal or immature
uation
Fetus and
Tr imester
Iron requirement (mg/day)
15 Hematological Diagnostics
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173
cells, and it cannot identify and classify all types of white
blood cells accurately. To overcome this problem, many
automated analyzers ag the presence of possible abnormal
white blood cell populations, thereby indicating the need for
a peripheral smear examination by personnel capable of
identifying abnormal cells. Monocytes and basophils are the
most difcult populations to count, and as a result, counting
them has a low level of precision and accuracy. In addition,
automated analyzers tend to underestimate basophil counts
in true basophilic conditions. It should also be noted that
both automated and manual methods are unable to detect a
small number of abnormal cells. The false-negative rate
reported in the detection of abnormal cells varies from 1% to
20%, depending on the instrument and the required detection
limit. The identication of lymphoma cells and reactive lymphocytes is the most difcult condition for both automated
instruments and microscopic examination. The value of
reporting the presence of banded core granulocytes is questionable. However, the measurement of immature cells in
myeloid lines, particularly bands, has been considered clinically useful in the diagnosis of infections, particularly neonatal sepsis. However, it should be considered that neutrophil
granulocytes with banded nuclei cannot be enumerated by
automated analyzers, as they are reported together with segmented neutrophils as absolute neutrophil counts.
Identication of neutrophils with banded nuclei by microscopic examination is neither precise nor consistent due to
the high variability in morphological classication since
quantication of banded neutrophils reects interexaminer
variability.
The Primary Forms ofAnemia
Microcytic Anemias
As reported in Table15.3, there are three forms of microcytic
anemia: sideropenic, thalassemias, and hereditary sideroblastosis. The rst two have a higher incidence, even if sideropenic anemia (from iron deciency) is the most prevalent
worldwide. Thalassemia is more frequent in regions (centralnorthern Africa, southern Asia, and the Mediterranean area)
in which the contemporary presence of favorable factors, in
particular the malarial disease, has increased its diffusion.
Sideropenic andAssimilated Anemia
These are disorders in which the production of red blood
cells is decreased due to low iron stores in the body and/or
reduced iron utilization of the stores. The former is the most
common nutritional disorder worldwide and accounts for
about half of all cases of anemia. The development of anemia
can result from insufcient iron intake, decreased iron
absorption, increased demand for iron by the body, and
placenta
7
6
5
4
Menstruation Menstr
3
2
1
Not
pregnant
Fig. 15.11 Iron demands in different physiological states in women.
(Copyright EDISES 2021. Reproduced with permission)
Body iron loss
First Second Third Postpartum
Red
blood
cells
Lactation
increased iron losses. Correct identication of the underlying
etiology and administration of appropriate therapy are the
keys to the evaluation and management of this condition. As
mentioned at the beginning, sideropenic anemia should be
differentiated rst from the anemia of chronic conditions,
which generally occurs in subjects affected by chronic
inammatory diseases and presents many clinical and laboratory features similar to sideropenic anemia, even if it differs in evolution and treatment.
The regulation of the body’s iron balance is based on
the meticulous control of intestinal absorption, as there is
no excretory regulation. Daily losses, which in a normal
male subject are between 1mg and 2mg and in the female
are greater but variable depending on the physiological
state (as shown in Fig.15.11, they range from values simi-
lar to the male in childhood and menopause to values
30–100% greater in childbearing age, to values more than
3.5 times greater in pregnancy), are then rebalanced by
absorption. In turn, absorption depends on the iron content of the diet. An average European diet contains 6mg
of iron per 1000kcal; this corresponds to total values of
10–30 mg/day (Table15.6). It should be borne in mind
that, under conditions of normal balance regulation, the
iron absorbed corresponds to 5–10% of the total content,
and that under decit conditions, this percentage may
increase by 3–5 times. The localization of absorption
begins in the stomach, proceeds to a maximum in the central part of the proximal jejunum, and then decreases caudally. Absorption is inuenced by pH and redox potential.
Of the various forms of iron presented for absorption,
hemic iron (Fe
2+
) derived from hemoglobin, myoglobin,
and hemic enzymes from animal-derived foods is the most
easily absorbed. Proteases and gastric acid juice release
heme from the apoproteins to which it is bound. The iron
in heme is oxidized to hemin (Fe3+), and the molecule
enters the enterocyte intact, from which it will then pass
into the blood. European nonvegetarian diets contain
174
Basolateral
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G. C. Guidi
Table 15.6
Sex/eating habits mg/day
Nonvegetarian woman in premenopause 18
Postmenopausal nonvegetarian man/woman 8
Vegetarian man/woman
Maximum iron intake in the diet 45
Recommended daily intake of iron in food
♂14 and ♀33
approximately 10–15% heme iron. The latter is not
affected by diet, in contrast to inorganic iron (Fe3+). It is
generally complexed with phytates, oxalate, citrate, lactate, sugars, and amino acids and must be reduced to Fe2+
to be absorbed. Other foods and substances present in the
diet can favor its absorption (ascorbates and animal meats
through low molecular weight factors due to proteolysis
that carry inorganic Fe−, keto sugars, amino acids, organic
acids, and human milk) or slow down or even hinder its
absorption (vegetable proteins, soy, egg white, cow’s
milk, phytates, bran and vegetable bers, polyphenols,
phosphates and phosphoproteins, tannin, and other metals
that compete for absorption, such as Zn, Cd, and Ca). The
recommended amount of iron in the diet also depends on
dietary habits; subjects with a predominantly or totally
plant- based diet should include higher amounts of iron in
the diet. Once entering the enterocyte cytoplasm through
the apical membrane, absorbed Fe3+ is reduced to Fe2+ by
a ferro- reductase and internalized via the divalent metal
transporter (Fig. 15.12). In contrast, heme iron (Fe2+),
transported internally by heme carrier protein 1, is
released from the heme by heme oxygenase. Fe2+ in the
cytoplasm can now take two routes: (1) the pathway by
which iron is released into the circulation for use in erythropoiesis, which is mediated by ferroportin, a molecule
located on the basolateral membrane that releases Fe2+,
which is simultaneously oxidized to Fe3+ by the ferroxidase hephaestin and immediately bound to circulating
transferrin; and (2) the intracellular deposition pathway,
whereby iron is incorporated into the cell’s ferritin molecules when the cell receives the signal that no more iron is
needed for erythropoiesis. Since the enterocyte is exfoliated after a few days, the iron inside it is eliminated with
feces. A molecule, hepcidin, interferes with the mechanisms of iron entry into the circulation. It performs its
function by inducing the lysosomal degradation of ferroportin. Hepcidin is expressed in the liver and is regulated
by the BMP6 protein (bone morphogenic protein 6),
together with its co-receptor hemojuveniline, and thus
represents a mechanism for regulating iron absorption
according to the needs and deposits existing mainly in the
liver. Its action also occurs at the level of the reticuloendothelial macrophages.
Since hepcidin behaves as an acute-phase protein, it
increases during inammation upon induction by interleukin- 6 and inhibits iron absorption. In chronic inammation,
this prolonged effect results in anemia with features similar
Non-heme iron
2+
2+
Fe
Fe
DMT-1
Ferritin
Lysosomal
degradation
of ferroportin
3+
2+
Fe
Transferrin
3+
Fe
Apical
membrane
Ferro-
reductase
Enterocyte
Fe
Peroxidase
(Hephaestin)
membrane
Fig. 15.12 Molecules contributing to the iron cycle in the enterocyte.
(Copyright EDISES 2021. Reproduced with permission)
Heme Iron
HCP1
oxygenase
2+
Fe
Ferroportin
Eme
Eme
Heme
Lysosome
+
Hepcidin
to those of iron deciency anemia, from which, however, it
must be differentiated. Indeed, anemia due to chronic inammation results from the inability to allocate sufcient iron to
erythropoiesis as it remains sequestered in the reticuloendothelial system.
Genetic mutations involving hepcidin are also known to
cause juvenile hemochromatosis. This pathology can also
be due to mutations in the hemojuvenilin regulator, which
are the most frequent causes. Other important genes
involved in iron regulation are transferrin receptor 2 (TfR2)
and HFE. Some mutations of the latter, mainly C282Y and
H63D, are responsible for hereditary hemochromatosis in
adults, which occurs around 40years of age. The laboratory
analysis useful for the diagnosis of hemochromatosis is
shown in Table15.7.
The measurement of serum or plasma iron (sideremia)
reects the amount of iron bound to transferrin, which is
continuously variable throughout the day as a function of
diet and activity. Generally, sideremia values tend to be
higher in the morning than in the evening and higher after
meals than during fasting. For these reasons, sideremia is not
reliable for assessing the iron status of the body.
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