CE UPDATE —ANEMIAS I

Powers Peterson, MD
Margaret F. Cornacchia, MS, MT(ASCP) DLM

Anemia: Pathophysiology,
Clinical Features, and
Laboratory Evaluation
Consider two adult patients, a woman with a hemo- ABSTRACT Anemia is a common and frequently overlooked
globin level of 9.6 g/dL (96 g/L) and a man with a clinical problem. This article discusses the physiology of RBC
level of 13.0 g/dL (130 g/L). The woman is clearly production and destruction and the pathophysiology, clinical
anemic. Is the man also anemic? He may well be.
features, and treatment of anemias. The classification system used
Why? We tend to think of anemia as synonymous
with low hemoglobin level. We may forget that ane- presents anemia in the context of more than hemoglobin level
mia is defined pathophysiologically as impaired alone. In some cases the combination of clinical signs and specific
ability to meet the body’s demands for oxygen. In the laboratory test results may warrant a diagnosis of anemia even
case of the man with a level of 13.0, the hemoglobin when the hemoglobin level is in the reference range.
level alone may not tell the whole story. This is the first article in a three-part continuing education series on anemia. On
completion of this article, the reader will be able to discuss the pathogenesis of
Physiology of RBC Production and Destruction anemia in terms of the structure and functions of the hematopoietic organs, interpret
The bone marrow, the largest and most widely dis- the results of a CBC and a peripheral blood smear in conjunction with other laboratory
persed organ of the body, is the production center for test results, recommend appropriate additional laboratory studies, and discuss
RBCs. The pluripotential stem cell is the precursor of relevant matters, such as diagnostic probabilities and cost-effective testing strategies.
the marrow cell lines. Whether through self-renewal
of the pluripotential stem cell or by differentiation to a
specific lineage, hematopoiesis is coordinated by
cytokines, proteins released by one cell to transmit cytokines. They “control” the proliferation and
information to another cell.1 Human growth factors differentiation of progenitor cells and are produced

Section rWjfi Scientific Communications

(HGF) are one group of by both hematopoietic and nonhematopoietic cell
types. Erythropoietin is a hormonal HGF
Glossary
Culling—Process by which the spleen identifies
and removes damaged, worn out, or potentially Progenitor—Cell from which another cell is formed.
dangerous cells from the blood. Reticulocyte count, corrected—Corrects a
Cytokine—Any of a class of immunoregulatory reticulocyte count for degree of anemia:
substances secreted by cells of the immune system.
Hematopoiesis—Formation of blood or blood
cells in the living body. Hematocrit x Reticulocyte
Hypoxia—Deficiency of oxygen reaching the Corrected (g/dL) count (%)
reticulocyte =
tissues of the body. count Mean normal hematocrit
Interendothelial slits—Minute spaces in the blood (based on gender and age
vessels of the spleen for passage of RBCs. range)
Macrophage—Phagocytic cell of the Reticulocyte production index—Rate of
reticuloendothelial system that can be fixed or effective erythropoiesis in anemia:
freely motile; is derived from a monocyte; also
called histiocyte. Reticulocyte Corrected reticulocyte count
production =
Pitting—Process by which the spleen removes index
Maturation time in peripheral
remnants of nuclear DNA from young RBCs. blood (based on hematocrit)

Stem cell—Unspecialized cell that gives rise to

differentiated cells.®

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Fig 1. A, Peripheral blood smear from a patient with ongoing slight hemolysis. The
reticulocytes (arrows) have prominent basophilia and are slightly larger than RBCs
(Wright stain, original magnification x 250); B, Peripheral blood smear from a patient
recovering from an episode of brisk hemolysis. Note that the reticulum, from which
reticulocytes derive their name, is well demonstrated (Brilliant cresyl blue, original
magnification x 250).
Fig 2. Section of
spleen from a patient
with idiopathic
thrombocytopenic
purpura shows the
vascular pattern of the
red pulp. Blood cells
traverse the cords (c)
where old or damaged
RBCs are sequestered
by macrophages, while
young and healthy
RBCs are released into
the sinuses (s), to
return to circulation.
Note that cords are
widened and stained associated with erythropoiesis. The drug Epogen
material is present in (epoetin alfa [Amgen, Thousand Oaks, Calif]), an
macrophages (periodic erythrocyte-specific HGF, was produced by means
acid-Schiff, x 400).
of genetic engineering. Two other HGFs important
in erythropoiesis are the c-kit ligand, also known as
stem cell factor, and insulin-like growth factor I.
The pluripotential stem cell gives rise to the
committed hemopoietic cells that migrate into the
marrow cavity. Maturation of RBCs occurs outside
the marrow vascular compartment, and RBCs enter
the circulation through the thin- walled sinusoids. In
humans, erythropoiesis is found in the liver after 6
weeks of gestation, in the spleen by 12 weeks, and in
the marrow by 20 weeks. Intramedullary blood cell
formation progressively increases during the second
half of intrauterine life. At birth and for the first
years of life the marrow is completely cellular. In the
adult, however, hematopoietic marrow is almost
exclusively confined to the axial skeleton and the
proximal ends of the femur and humerus.
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Knowledge of the kinetics of RBC production is
necessary to understand anemias. Under steady-state
conditions, the marrow can produce RBCs in 6 to 8
days. Under maximal stress and with adequate
supplies of building blocks (iron, vitamin B12, folic
acid), the marrow can increase production as much
as five to six times normal. The total RBCs in the
circulation of the average 170-pound man weigh
more than 2.5 kg (5 lb).
Total erythropoiesis is the total number of RBCs
produced. It does not indicate whether adequate
numbers of RBCs are being produced, nor whether
adequate numbers of RBCs are available to the
peripheral (nonmarrow) organs. Effective
erythropoiesis is the number of viable and functional
RBCs available to meet physiologic needs. Effective
erythropoiesis reflects the balance between the
number of cells produced and their life span. One
measure is the reticulocyte count; another is the
corrected reticulocyte count, a number that reflects
RBC production in comparison with steady-state
conditions (ie, hematocrit 45%).2,3 The corrected
reticulocyte count is used in calculating the
reticulocyte production index (RPI), a more accurate
measure of effective erythropoiesis. An elevated RPI
usually indicates a hemolytic process; for example,
RPI 3.5 means that RBC production is three and a
half times normal.
A reticulocyte is a young RBC that has extruded
its nucleus but still contains a large amount of
cytoplasmic RNA (Fig 1). Reticulocytes are visible
on a peripheral blood film after staining with a
supravital dye such as methylene blue. The number
of reticulocytes in the peripheral circulation depends
on their rate of release from the marrow, their degree
of maturity at the time of release, and the rate of
disappearance of the RNA. Reticulocytes are
commonly enumerated on automated analyzers (eg,
Cell-Dyn 4000, Abbott Laboratories, Abbott Park,
111; Advia-120, Bayer Diagnostics, Tarrytown, NY;
and Coulter GEN-S, Beckman Coulter, Fullerton,
Calif) with flow cytometric methods in a manner
analogous to RBC counting.
The spleen is the primary organ of RBC
destruction. The red pulp of the spleen consists of a
branching system of cords and venous sinuses (Fig
2). The primary function of this meshwork is
phagocytosis, specifically the destruction of old or
damaged RBCs. The spleen separates the RBCs
from the plasma and temporarily retains them in
 Table 1. Pathophysiologic Classification of Anemias

the red pulp. Young and viable RBCs pass through Blood loss
the spleen rapidly, and defective and older RBCs are
Acute
culled out and destroyed. Passage from cord to sinus
is the ultimate test of size, shape, compliance, and Chronic
stretch.4 For example, aging RBCs are targeted for Impaired production (hypoproliferative)
destruction through exposure of galactose moieties in
Anemia of chronic disease
the membrane. Rigid cells that cannot squeeze
through, such as sickled cells, are caught in the Aplastic anemia
interendothelial slits of the vascular meshwork. In Congenital or acquired
pliant RBCs with rigid inclusions, such as malarial Iron deficiency
parasites, the inclusion may be pitted out. Megaloblastic (nuclear-cytoplasmic dyssynchrony)
Vitamin B12 or folic acid
Clinical Setting
Many anemias are initially asymptomatic. The Myelodysplasia
diagnosis may be unexpected because the anemia Myelophthisic (infiltrative)
developed over so long a time that the patient Increased destruction (hemolytic)
adjusted to the symptoms without realizing it. The
clinical manifestations of anemia vary with age, Extrinsic to RBCs
rapidity of onset, and concurrent clinical conditions. Autoimmune or isoimmune
The tissue hypoxia that results from decreased Infections
oxygen-carrying capacity is responsible for the
clinical signs and symptoms, which include fatigue, Physical or chemical agents
shortness of breath, altered menses, and pallor of Intrinsic to RBCs
skin and mucous membranes; potentially more
Enzyme deficiencies (metabolic)
serious manifestations include tachycardias,
congestive heart failure, and icterus. Hemoglobinopathies
Whether detected unexpectedly or because of Amino acid substitutions Chain
patient complaints, the diagnosis of anemia requires
synthesis defects (thalassemias)
that the patient’s history be meticulously taken and a
physical examination be performed. Broadly directed Combinations Membrane defects
questions may allow a physician to make an early
Classification of Anemias No classification
diagnosis and facilitate ordering the most appropriate
system of anemias is perfect.
studies. Areas covered should include family history,
We prefer a pathophysiologic system, but a system based on
dietary and social habits (eg, alcohol consumption,

Section Wi Scientific Communications

numerical data including RBC indices is equally clinically
strict vegetarianism), medications (prescribed or
useful in classification and differential diagnosis. A
otherwise), occupation and hobbies, travel, menstrual
pathophysiologic approach divides anemias into
history, transfusion history, and concurrent and
three categories: anemias due to blood loss,
significant past medical disorders (eg, thyroid or
impaired production (hypoproliferative), or
renal disease, rheumatoid arthritis, hepatitis).
increased destruction (hemolytic) (Table 1).
Particular findings on physical examination may
Of Wintrobe’s original RBC indices, only mean
suggest a more likely cause, such as
corpuscular volume (MCV) is still clinically useful,
hepatosplenomegaly, lymphadenopathy, ascites,
although this opinion is not universally held.5,6
specific neurologic defects, evidence of mucosal or
Many anemia classification schemes now include Knowledge
gastrointestinal bleeding, and icterus.
RBC distribution width (RDW). Automated Look for the CE
hematology analyzers generate these numbers as Update exam on
Anemias (906) in the
part of the CBC. September issue of
Laboratory Medicine.
Participants will earn
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465
Fig 3. Peripheral blood
smear from a patient
with megaloblastic
anemia due to vitamin
B12 deficiency shows a
spectrum of
morphologic changes.
Note the marked
variation in size and
shape of RBCs.
Several target cells
also are present
(Wright stain, original
magnification x 250).
Fig 4. Fragmented and misshapen RBCs show the spectrum of morphologic changes
found in hemolytic anemia. A, Peripheral blood smear from a patient with
disseminated intravascular coagulopathy. RBC remnants are the result of a
microangiopathic (or intravascular) process. Fibrin spiderweb-type strands in the
capillaries create an obstacle that is difficult for RBCs to negotiate intact (Wright stain,
original magnification x 250). B, Peripheral blood smear from a patient with glucose-6-
phosphate dehydrogenase deficiency. Morphologic changes result from extravascular
hemolysis. Splenic cord macrophages have pitted portions of RBC membranes with
resultant bite cells (Wright stain, original magnification x 250).
Laboratory Evaluation To return to the question
posed at the beginning of this article, how can a man
with a hemoglobin level of 13 g/dL (130 g/L) have
anemia? Whatever the patient’s hemoglobin level,
the MCV determines which category or subcategory
of causes is relevant. If the MCV is elevated above
102 to 104 fL, the morphologic appearance of the
RBCs is described as megaloblastic. The explanation
is that DNA synthesis is impaired, but RNA
synthesis is not. DNA is synthesized in the nucleus
of the cell and requires vitamin B12 and
tetrahydrofolate (folic acid). If either or both of these
nutrients is absent, nuclear maturation is overly
prolonged. But cytoplasmic RNA synthesis is
unimpaired, resulting in nuclear-cytoplasmic
dyssynchrony (Fig 3). [Ralph Green, MD, addresses
megaloblastic anemias in part III of this series.]
If the MCV is less than 70 to 72 fL, the RBCs are
described as microcytic. Indeed, such cells are
smaller, and many of the microcytic conditions and
disorders also show target cells. Unlike
megaloblastic anemias, microcytic processes are
seen in the three major pathophysiologic categories.
Chronic blood loss (menstruation, inflammatory
bowel diseases, alcoholic gastritis) commonly results
in iron deficiency.7 One milliliter of whole blood
contains 1 mg of heme iron.8 The body’s total supply
of iron is 3.5 to 4.0 g. Iron deficiency due to dietary
constraints may also cause microcytic anemia. Many
of the hemolytic processes intrinsic to the RBCs
commonly are associated with microcytic anemia.
Membrane defects such as hereditary spherocytosis,
enzyme abnormalities such as glucose-6- phosphate
dehydrogenase deficiency, and many
hemoglobinopathies are characterized by low MCV
levels. This is particularly true of the thalassemias,
disorders of globin-chain production. Hemolysis due
to causes external to the RBCs (eg, Clostridium
welchii sepsis, cobra snakebite poisoning, hemolytic
disease of the newborn) often results in
fragmentation and disruption of the RBCs (Fig 4).
[Nancy Rosenthal, MD, discusses microcytic
anemias in part II of this series.]
In outpatient populations the RDW is also useful
in the initial evaluation of the CBC. The RDW can
be interpreted as reflecting the degree of
anisocytosis; thus an elevated RDW suggests a het-
erogeneous RBC population. The RDW is extremely
useful in detecting early iron deficiency or
depletion.7,9-11 (Note that its utility is not limited only
to evaluation of iron deficiency.) Because the RBCs
conserve hemoglobin at the expense of cell volume,
the increasingly smaller RBCs produced increase the
RDW, but the MCV is in the reference range, and the
hemoglobin level may not be numerically indicative
of anemia.
Having classified the anemia into its appropriate
category, one thinks next of further studies. Any
subsequent test must take into account the patient’s
age, gender, physical condition, medications,
ethnicity, and often additional factors. Two other
easily obtained studies are the reticulocyte count and
the total and unconjugated serum bilirubin levels. If
total bilirubin is elevated and if the unconjugated
fraction is more than 50%, hemolysis is the likely
cause. Because of the preva-
 Table 2. Anemia of Chronic Disease

lence of both iron deficiency anemia and anemia of Underlying disorder

chronic disease, the serum ferritin level is also often
requested. Ferritin is an indirect measure of the
body’s total iron stores and can be useful in
distinguishing iron deficiency from anemia of
chronic disease7,12 (Table 2).
Treatment of Anemias Treatment of anemia is
based on its cause. Some anemias can be cured; iron
deficiency due to transient nonrecurring blood loss
may not even require iron replacement therapy.
Other anemias can be treated reasonably well; for
example, hereditary spherocytosis is “cured” with
splenectomy. The macrophages in the splenic
sinusoids recognize the membrane abnormalities and
cull such cells from the circulation. Removal of the
spleen removes the anatomic site of this
extravascular destruction. Some anemias can be
treated only symptomatically. Sickle cell disease is
one such disorder; another is anemia due to marrow
replacement by metastatic carcinoma, unless a stem
cell or bone marrow transplant is feasible and
available. In some conditions, whether temporary or
permanent, erythropoietin therapy may be
appropriate.
Conclusion
Anemias are common clinical problems with various
and varied causes. Understanding the normal
physiologic processes of RBC production and
destruction is crucial to elucidate the pathogenesis of
anemia.®
Chronic inflammatory states Chronic osteomyelitis Chronic
pulmonary infections Subacute bacterial endocarditis
Tuberculosis Metabolic or endocrine
Chronic adrenal insufficiency
Chronic renal failure
Hypothyroidism Rheumatoid
arthritis Systemic lupus
erythematosus
Laboratory test results
Hemoglobin 9-11 g/dL (90-110 g/L) (slight to moderate
anemia)
Normal or slightly decreased mean corpuscular volume with
normal RBC distribution width
Decreased serum iron with normal or low transferrin saturation
Total iron-binding capacity always decreased Elevated serum
ferritin concentration Increased marrow iron stores
Reticulocyte production index <2
Absence of indicators of hemorrhage, hemolysis, or marrow
infiltration (by infection, metastases, fibrosis)
Differential diagnosis
a- or (3-Thalassemia
Iron-deficiency anemia
Lead or other heavy metal poisoning
Refractory anemia with ringed sideroblasts (myelodysplasia)

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