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Red Cell Disorders
Disorders of red cells can result in either anemia or polycythemia (an increase in the
number of red cells).
Definition: Anemia is a reduction of the total circulating red cell mass below normal
limits. Anemia reduces the oxygen-carrying capacity of the blood, leading to tissue
hypoxia.
In practice, the measurement of red cell mass is not easy, and anemia is usually
diagnosed based on a reduction in the:
●Hematocrit (the ratio of packed red cells to total blood volume) and the
●Hemoglobin concentration of the blood to levels that are below the normal range. These
values correlate with the red cell mass except when there are changes in plasma volume
caused by fluid retention or dehydration.
Effects of anemia: The decrease in tissue oxygen tension that is associated with anemia
triggers increased erythropoietin production (the exception is that of anemia related to
chronic renal failure, in which erythropoietin-producing cells in the kidney are lost).
Increased erythropoietin production leads to compensatory hyperplasia of erythroid
precursors in the bone marrow and, in severe anemias, the appearance of extramedullary
hematopoiesis within the secondary hematopoietic organs (the spleen, liver, and lymph
nodes).
The hallmark of increased marrow output is reticulocytosis, the appearance of increased
numbers of newly formed red cells (reticulocytes) in the peripheral blood. In contrast,
disorders of decreased red cell production (aregenerative anemias) are characterized by
reticulocytopenia.
Classification of anemia:
Morphologic classification: is based on the morphology of red cells; this is often
correlates with the cause of their deficiency. Specific red cell features that provide
etiologic clues include:
●Cell size (normocytic, microcytic, or macrocytic).
●Degree of hemoglobinization, which is reflected in the color of the cells (normochromic
or hypochromic).
●Shape of the cells.
These features are judged subjectively by visual inspection of peripheral smears (blood
film) and are also expressed quantitatively through the following indices:
Mean cell volume (MCV): the average volume per red cell, expressed in femtoliters
(cubic microns)
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Mean cell hemoglobin (MCH): the average content (mass) of hemoglobin per red cell,
expressed in picograms
Mean cell hemoglobin concentration (MCHC): the average concentration of
hemoglobin in a given volume of packed red cells, expressed in grams per deciliter.
Iron Deficiency Anemia:
It is the most common form of nutritional deficiency.
Iron deficiency anemia can result from a variety of causes:
1. Low intake and poor availability from predominantly vegetarian diets are an important
cause of iron deficiency.
2. Malabsorption can occur with sprue and celiac disease or after gastrectomy.
3. Increased demands not met by normal dietary intake occur around the world during
pregnancy and infancy.
4. Chronic blood loss is one of the most important causes of iron deficiency anemia. This
loss may occur from the gastrointestinal tract (e.g., peptic ulcers, colonic cancer,
hemorrhoids, hookworm disease) or the female genital tract (e.g., menorrhagia,
metrorrhagia, cancers).
Regardless of the cause, iron deficiency develops insidiously. At first iron stores are
depleted, leading to a decline in serum ferritin and the absence of stainable iron in the
bone marrow. This is followed by a decrease in serum iron and a rise in the serum iron-
binding capacity. Ultimately the capacity to synthesize hemoglobin is diminished, leading
to anemia and even reduced immunocompetence.
Pathologic features (Lab findings):
●The red cells are microcytic and hypochromic, reflecting the reductions in MCV and
MCHC.
●For unclear reasons, iron deficiency is often accompanied by an increase in the platelet
count.
●Although erythropoietin levels are increased, the marrow response is blunted by the iron
deficiency, and thus the marrow cellularity is usually only slightly increased.
Diagnostic criteria include:
●Anemia, hypochromic and microcytic red cell indices.
●Low serum ferritin and serum iron levels.
●Low transferrin saturation.
●Increased total iron-binding capacity.
It is important to remember that in reasonably well-nourished persons, microcytic
hypochromic anemia is not a disease but rather a symptom of some underlying disorder.
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Anemia of Chronic Disease:
This is the most common form of anemia in hospitalized patients. It superficially
resembles the anemia of iron deficiency, but it stems from inflammation-induced
sequestration of iron within the cells of the mononuclear phagocyte (reticuloendothelial)
system. It occurs in a variety of chronic inflammatory disorders, including the following:
●Chronic microbial infections, such as osteomyelitis, bacterial endocarditis, and lung
abscess
●Chronic immune disorders, such as rheumatoid arthritis and regional enteritis
●Neoplasms, such as Hodgkin lymphoma and carcinomas of the lung and breast
The serum iron levels are usually low, and the red cells can be normocytic and
normochromic, or, as in anemia of iron deficiency, hypochromic and microcytic.
However, the anemia of chronic disease is associated with increased storage iron in the
bone marrow, a high serum ferritin concentration, and a reduced total iron-binding
capacity, all of which readily rule out iron deficiency. This combination of findings is
attributable to high concentrations of circulating hepcidin, which inhibits ferroportin and
thereby block the transfer of iron from the mononuclear phagocyte storage pool to the
erythroid precursors.
Megaloblastic Anemias:
In megaloblastic anemia the red cells are abnormally large (MCV >95 FL).
There are two principal causes of megaloblastic anemia:
●Folate deficiency.
●Vitamin B
12
deficiency.
Both vitamins are required for DNA synthesis, and, hence, the effects of their deficiency
on hematopoiesis are quite similar.
Pathogenesis: The morphologic hallmark of megaloblastic anemias is an enlargement of
erythroid precursors (megaloblasts), which gives rise to abnormally large red cells
(macrocytes). The other myeloid lineages are also affected. Most notably, granulocyte
precursors are enlarged (giant metamyelocytes) and yield highly characteristic
hypersegmented neutrophils. Underlying the cellular gigantism is an impairment of DNA
synthesis, which results in a delay in nuclear maturation and cell division. Because the
synthesis of RNA and cytoplasmic elements proceeds at a normal rate and thus outpaces
that of the nucleus, the hematopoietic precursors show nuclear-cytoplasmic asynchrony.
Erythrocyte, Granulocyte and platelet precursors are all affected. As a result, most
patients with megaloblastic anemia develop pancytopenia (anemia, thrombocytopenia,
and granulocytopenia).
Pathologic features (Lab findings):
●The anemia is macrocytic (MCV >95 fL).
●The macrocytes are typically oval in shape.
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The reticulocyte count is low.
●The total white cell and platelet counts may be moderately reduced, especially in
severely anaemic patients.
●A proportion of the neutrophils show hypersegmented nuclei (with six or more lobes).
●The bone marrow is markedly hypercellular, as a result of increased numbers of
megaloblasts.
●These cells are larger than normoblasts and have a delicate, finely reticulated nuclear
chromatin (suggestive of nuclear immaturity) and an abundant, strikingly basophilic
cytoplasm.
●The granulocytic precursors also demonstrate nuclear-cytoplasmic asynchrony, yielding
giant metamyelocytes.
●Megakaryocytes, too, may be abnormally large.
Causes of vitamin B12 deficiency:
●Nutritional: Especially vegans.
●Malabsorption: Gastric causes = Pernicious anemia. Congenital lack or abnormality of
intrinsic factor. Total or partial gastrectomy.
Intestinal causes: Intestinal stagnant loop syndrome-jejunal diverticulosis, blind-loop,
stricture, etc. Chronic tropical sprue. Ileal resection and Crohn's disease.
(Pernicious anemia: This is caused by autoimmune attack on the gastric mucosa leading
to atrophy of the stomach).
Pernicious anemia: This disease results from an autoimmune reaction against parietal
cells and intrinsic factor itself, which produces gastric mucosal atrophy (autoimmune
chronic gastritis).
Several associations favor an autoimmune basis:
●Autoantibodies are present in the serum and gastric juice of most patients with
pernicious anemia.
Three types of antibodies have been found:
■Parietal canalicular antibodies, which bind to the mucosal parietal cells.
■Blocking antibodies, which block the binding of vitamin B
12
to intrinsic factor.
■Binding antibodies that react with intrinsic factor-B
12
complex and prevent it from
binding to the ileal receptor.
●An occurrence of pernicious anemia with other autoimmune diseases such as
Hashimoto thyroiditis, Addison disease, and type I diabetes mellitus is well documented.
●The frequency of serum antibodies to intrinsic factor is increased in patients with other
autoimmune diseases.
Patients with pernicious anemia have an increased risk of gastric carcinoma.
The diagnostic features of pernicious anemia include:
●Low serum vitamin B
12
levels.
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●Normal or elevated serum folate levels.
●Serum antibodies to intrinsic factor.
● Megaloblastic anemia.
●Leukopenia with hypersegmented granulocytes.
●A dramatic reticulocytic response (within 2-3 days) to parenteral administration of
vitamin B
12
.
Causes of folate deficiency:
●Nutritional: Especially old age, institutions, poverty, famine, special diets, goat's milk
anemia, etc.
●Malabsorption: Tropical sprue, gluten-induced enteropathy (adult or child). Possible
contributory factor to folate deficiency in some patients with partial gastrectomy,
extensive jejunal resection or Crohn's disease.
●Excess utilization:
■Physiological: Pregnancy and lactation, prematurity.
■Pathological: Hematological diseases: hemolytic anemias, myelofibrosis.
■Malignant disease: carcinoma, lymphoma, myeloma.
■Inflammatory diseases: Crohn's disease, tuberculosis, rheumatoid arthritis, psoriasis.
●Excess urinary folate loss: Active liver disease, congestive heart failure.
●Drugs: Anticonvulsants, sulfasalazine.
●Mixed: Liver disease, alcoholism, intensive care.
The diagnostic features of pernicious anemia include
●Low serum vitamin B
12
levels.
●Normal or elevated serum folate levels.
●Serum antibodies to intrinsic factor.
●Moderate to severe megaloblastic anemia.
●Leukopenia with hypersegmented granulocytes.
●A dramatic reticulocytic response (within 2-3 days) to parenteral administration of
vitamin B
12
.
Aplastic Anemia:
Aplastic anemia is “a disorder in which multipotent bone marrow stem cells are
suppressed, leading to marrow failure and pancytopenia.” ,
Etiology:
Aplastic anemia is divided etiologically in to:
■Primary (idiopathic) (50% of cases)
■Secondary to damaging agent to the BM:
●Known toxic agent to the BM:
*Predictable damage, which is dose related, and usually reversible. Included in this
category are antineoplastic drugs, benzene, and chloramphenicol.
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*Unpredictable ("idiosyncratic" or hypersensitivity) damage to small doses of known
myelotoxic drugs (e.g., chloramphenicol) or to drugs such as sulfonamides, which are not
myelotoxic in other persons.
●After certain viral infections, most often community-acquired viral hepatitis.
Marrow aplasia develops several months after recovery from the hepatitis and follows a
relentless course.
Pathogenesis: Autoreactive T cells may play an important role in marrow failure. This is
supported by the observation that in 70% to 80% of cases aplastic anemia responds to
immunosuppressive therapy aimed at T cells. Perhaps viral antigens, drug-derived
haptens, and/or genetic damage create neoantigens within stem cells that serve as targets
for the T cells.
A small fraction of patients with "acquired" aplastic anemia have inherited defects in
DNA telomerase, which is needed for the maintenance and stability of chromosomes. In
these settings, the outcome is direct damage to and senescence of hematopoietic stem
cells.
Pathologic features (Lab findings):
●The bone marrow is markedly hypocellular, with greater than 90% of the intertrabecular
spaces occupied by fat.
●The limited cellularity often consists of only lymphocytes and plasma cells. These
changes are better appreciated in bone marrow biopsy specimens than in marrow
aspirates, which often yield a "dry tap."
●Thrombocytopenia and granulocytopenia may result in hemorrhages and bacterial
infections, respectively.
It is important to distinguish aplastic anemia from anemias caused by:
■Marrow infiltration (myelophthisic anemia).
■Aleukemic leukemia.
■Granulomatous diseases affecting the BM.
Because pancytopenia is common to these conditions, their clinical manifestations may
be indistinguishable, but they are easily distinguished by examination of the bone
marrow.
Hemoglobinopathies and Thalassemia:
The hemoglobinopathies are “a group of hereditary disorders that are defined by the
presence of structurally abnormal hemoglobins”. The prototypical (and most prevalent)
hemoglobinopathy is caused by a mutation in the β-globin chain gene that creates sickle
hemoglobin (HbS). The disease associated with HbS is sickle cell anemia. HbS, like 90%
of other abnormal hemoglobins, results from a single amino acid substitution in the
globin chain. On average, the normal adult red cell contains 96% HbA (α
2
β
2
), 3% HbA
2
7
(α
2
δ
2
), and 1% fetal Hb (HbF, α
2
γ
2
). Substitution of valine for glutamic acid of the β-
chain produces HbS. In homozygotes all HbA is replaced by HbS, whereas in
heterozygotes only about half is replaced.
In parts of Africa where malaria is endemic the gene frequency approaches 30%, as a
result of a small but significant protective effect of HbS against Plasmodium falciparum
malaria. Worldwide, sickle cell anemia is the most common form of familial hemolytic
anemia.
Sickle Cell Anemia:
Pathogenesis:
●Upon deoxygenation, HbS molecules undergo polymerization (gelation or
crystallization). These polymers distort the red cell, which assumes an elongated
crescentic, or sickle, shape.
●Sickling of red cells is initially reversible upon reoxygenation; however, membrane
damage occurs with each episode of sickling, and eventually the cells accumulate
calcium, lose potassium and water, and become irreversibly sickled.
Consequences of sickling :
Two major consequences of RBCs sickling.
●Repeated episodes of deoxygenation cause membrane damage and dehydration of red
cells, which become rigid and irreversibly sickled. These dysfunctional red cells are
recognized and removed by mononuclear phagocyte cells, producing a chronic
extravascular hemolytic anemia.
●The sickling of red cells produces widespread microvascular obstructions, which result
in ischemic tissue damage and pain crises.
Pathologic features (Lab findings):
Homozygous disease:
●The hemoglobin is usually 6-9 g/dL.
●Sickle cells and target cells occur in the blood.
●Features of splenic atrophy (e.g. Howell Jolly bodies) may also be present.
●Screening tests for sickling are positive when the blood is deoxygenated.
●Hemoglobin electrophoresis: In Hb SS: No Hb A is detected.
The amount of Hb F is variable and is usually 5-15%. Larger amounts are normally
associated with a milder disorder.
Sickle cell trait:
●This is a benign condition with no anemia and normal appearance of red cells on a
blood film.
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●Hematuria is the most common symptom and is thought to be caused by minor infarcts
of the renal papillae.
●Hb S varies from 25 to 45% of the total hemoglobin.
Diagnosis:
●In full-blown sickle cell disease, at least some irreversibly sickled red cells can be seen
on an ordinary peripheral blood smear.
●In sickle cell trait, sickling can be induced in vitro by exposing cells to marked hypoxia.
●The ultimate diagnosis depends on the electrophoretic demonstration of HbS.
●Prenatal diagnosis of sickle cell anemia can be performed by analyzing the DNA in fetal
cells obtained by amniocentesis or biopsy of chorionic villi
Thalassemias:
The thalassemias are “a heterogeneous group of inherited disorders caused by mutations
that decrease the rate of synthesis of α- or β-globin chains”. As a consequence there is a
deficiency of hemoglobin, with additional secondary red cell abnormalities caused by the
relative excess of the other unaffected globin chain.
Molecular Pathogenesis: A diverse collection of molecular defects underlies the
thalassemias, which are inherited as autosomal codominant conditions. The adult
hemoglobin, or HbA, is a tetramer composed of two α chains and two β chains. The
mutations that cause thalassemia are particularly common among Mediterranean,
African, and Asian populations.
β-Thalassemia: The β-globin mutations associated with β-thalassemia fall into two
categories:
1. β
0
, in which no β-globin chains are produced; and
2. β
+
, in which there is reduced (but detectable) β-globin synthesis.
The majority of mutations consist of single-base changes.
Individuals inheriting one abnormal allele have thalassemia minor or thalassemia trait,
which is asymptomatic or mildly symptomatic.
Most individuals inheriting any two β
0
and β
+
alleles have β- thalassemia major.
Two conditions contribute to the pathogenesis of the
anemia in β-thalassemia:
1. The reduced synthesis of β-globin leads to inadequate HbA formation, so that the
MCHC is low, and the cells appear hypochromic and microcytic.
2. Red cell hemolysis is even more important is, which results from the unbalanced rates
of β-globin and α-globin chain synthesis. Unpaired α chains form insoluble aggregates
that precipitate within the red cells and cause membrane damage that is severe enough to
provoke extravascular hemolysis. Erythroblasts in the bone marrow are also susceptible
to damage through the same mechanism, which in severe β-thalassemia results in the
destruction of the majority of erythroid progenitors before their maturation into red cells.
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This intramedullary destruction of erythroid precursors (ineffective erythropoiesis) is also
associated with an inappropriate increase in the absorption of dietary iron, which often
leads to iron overload.
Laboratory diagnosis: (β-Thalassemia major):
●There is a severe hypochromic, microcytic anemia.
●Raised reticulocyte percentage.
●Normoblasts, target cells and basophilic stippling in the blood film.
●Hemoglobin electrophoresis reveals absence or almost complete absence of Hb A.
●Almost all the circulating hemoglobin being Hb F.
β-Thalassemia trait (minor): This is a common, usually symptomless, abnormality
characterized by:
●A hypochromic, microcytic blood picture (MCV and MCH very low) and mild anemia
(hemoglobin 10-12.g/ dL).
●A raised Hb A2 (>3.5%) confirms the diagnosis.
α-Thalassemia syndromes: These are usually caused by gene deletions.
As there are normally four copies of the α-globin gene:
The clinical severity can be classified according to the number of genes that are missing
or inactive.
Loss of all four genes completely suppresses α-chain synthesis. Because the α chain is
essential in fetal as well as in adult hemoglobin: This is incompatible with life and leads
to death in utero (hydrops fetalis).
Three α gene deletions leads to a moderately severe (hemoglobin 7-11 g/dL) microcytic,
hypochromic anemia with splenomegaly. This is known as Hb H disease because
hemoglobin H (β4) can be detected in red cells of these patients by:
●Electrophoresis or
●In reticulocyte preparations.
In fetal life: Hb Barts (γ4) occurs.
The α-thalassemia traits are caused by loss of one or two genes and are usually not
associated with anemia, although the mean corpuscular volume (MCV) and mean
corpuscular hemoglobin (MCH) are low. Hemoglobin electrophoresis is normal.
Hemolytic Anemias:
Normal red cells have a life span of about 120 days. Anemias that are associated with
accelerated destruction of red cells are termed hemolytic anemias. Destruction can be
caused by:
1. Inherent (intracorpuscular) red cell defects, which are usually inherited, or
2. External (extracorpuscular) factors, which are usually acquired.
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There are certain general features of hemolytic anemias. All are characterized by:
●An increased rate of red cell destruction.
●A compensatory increase in erythropoiesis that results in reticulocytosis. In severe
hemolytic anemias, extramedullary hematopoiesis often develops in the spleen, liver, and
lymph nodes.
Extravascular and Intravascular hemolysis:
There are two main mechanisms whereby red cells are destroyed in hemolytic anaemia.
●Extravascular hemolysis: There is excessive removal of red cells by cells of the
reticuloendothelial system.
●Intravascular hemolysis: The red cells are broken down directly in the circulation.
Whichever mechanism dominates will depend on the pathology involved.
Non-Immune Hemolytic Anemia:
Glucose-6-Phosphate Dehydrogenase Deficiency (G6PDD):
The red cell is vulnerable to injury by endogenous and exogenous oxidants, which are
normally inactivated by reduced glutathione (GSH). Abnormalities affecting the enzymes
that are required for GSH production reduce the ability of red cells to protect themselves
from oxidative injury and lead to hemolytic anemias. The prototype (and most prevalent)
of these anemias is that associated with a deficiency of glucose-6-phosphate
dehydrogenase (G6PD). The G6PD gene is on the X chromosome.
G6PD deficiency produces no symptoms until the patient is exposed to an environmental
factor (most commonly infectious agents or drugs) that results in increased oxidant stress.
The drugs incriminated include antimalarials (e.g., primaquine), sulfonamides,
nitrofurantoin, phenacetin, aspirin (in large doses), and vitamin K derivatives. More
commonly, episodes of hemolysis are triggered by infections, which induced phagocytes
to produce free radicals as part of the normal host response. These offending agents
produce oxidants such as hydrogen peroxide that are sopped up by GSH, which is
converted to oxidized glutathione in the process. Because regeneration of GSH is
impaired in G6PD-deficient cells, hydrogen peroxide is free to "attack" other red cell
components, including globin chains, which have sulfhydryl groups that are susceptible
to oxidation. Oxidized Hb denatures and precipitates, forming intracellular inclusions
called Heinz bodies, which can damage the cell membrane sufficiently to cause
intravascular hemolysis. Other cells that are less severely damaged nevertheless suffer
from a loss of deformability, and their cell membranes are further damaged when splenic
phagocytes attempt to "pluck out" the Heinz bodies, creating so-called bite cells. All of
these changes predispose the red cells to becoming trapped in the splenic sinusoids and
destroyed by the phagocytes (extravascular hemolysis). Drug-induced hemolysis is acute
and of variable clinical severity. Typically, patients develop evidence of hemolysis after a
lag period of 2 or 3 days. Because the G6PD gene is on the X chromosome, all the red
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cells of affected males are affected. Most carrier females are asymptomatic. In a variant
known G6PD Mediterranean, found mainly in the Middle East, the enzyme deficiency
and the hemolysis that occur upon exposure to oxidants are more severe.
Hereditary Spherocytosis (HS):
Is characterized by an inherited (intrinsic) defect in the red cell membrane that renders
the cells spheroidal, less deformable, and vulnerable to splenic sequestration and
destruction. It is transmitted most commonly as an autosomal dominant trait;
approximately 25% of patients have a more severe autosomal recessive form of the
disease.
Pathogenesis:
● HS is usually caused by defects in the proteins involved in the vertical interactions
between the membrane skeleton and the lipid bilayer of the red cell.
Various mutations involving spectrin and ankyrin that weaken the interactions between
these proteins cause red cells to lose membrane fragments.
The loss of membrane may be caused by the release of parts of the lipidbilayer that are
not supported by the skeleton.
●The spleen plays a major role in the destruction of spherocytes. The marrow produces
red cells of normal biconcave shape but these lose membrane and become increasingly
spherical (loss of surface area relative to volume) as they circulate through the spleen and
the rest of the RE system. Ultimately, the spherocytes are unable to pass through the
splenic microcirculation where they die prematurely.
Pathological features:
●On smears, the red cells lack the central zone of pallor because of their spheroidal
shape.
●Spherocytosis, though distinctive, is not diagnostic; it is seen in other conditions, such
as immune hemolytic anemias, in which there is a loss of cell membrane relative to cell
volume.
●Because of their spheroidal shape, HS red cells show increased osmotic fragility when
placed in hypotonic salt solutions, a characteristic that is helpful for diagnosis.
●The excessive red cell destruction and resultant anemia lead to a compensatory
hyperplasia of marrow red cell progenitors and an increase in red cell production, which
is marked by peripheral blood reticulocytosis.
●The other general features of hemolytic anemias described earlier are also present,
pigmented gall stone, which occurs in upto 50% of HS patients.
Traumatic Hemolytic Anemia:
These arise through physical damage to red cells either on:
●Abnormal surfaces: (e.g. artificial heart valves or arterial grafts), arteriovenous
malformations or
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●A microangiopathic hemolytic anemia.
This is caused by red cells passing through abnormal small vessels.
The latter may be caused by:
●Deposition of fibrin strands often associated with disseminated intravascular
coagulation (DIC) or
●Platelet adherence as in thrombotic thrombocytopenic purpura (TIP) or
●Vasculitis (e.g. polyarteritis nodosa).
All of the above produce vascular lesions that predispose the circulating red cells to
mechanical injury.
The morphologic alterations in the injured red cells (schistocytes) are striking and quite
characteristic; "burr cells," "helmet cells," and "triangle cells" may be seen.
Immune Hemolytic Anemias:
Autoimmune Hemolytic Anemias:
Autoimmtme hemolytic anemias (AIHAs) are caused by antibody production by the body
against its own red cells.
They are characterized by a positive direct antiglobulin test (DAT) also known as the
Coombs' test . Divided into: ●Warm. ●Cold types.
According to whether the antibody reacts more strongly with red cells at 37°C or 4°C. A.
Classification of immune hemolytic anemias:
A. Warm type:
●Autoimmune:
▪Idiopathic.
▪Secondary: SLE, other 'autoimmune' diseases. CLL, lymphomas. Drugs (e.g.
methyldopa).
●Alloimmune:
▪Induced by red cell antigens: Hemolytic transfusion reactions. Hemolytic disease of the
newborn.
▪Drug induced: Drug-red cell membrane complex. Immune complex.
B. Cold type:
●Idiopathic.
●Secondary:
▪Infections: Mycoplasma pneumonia. Infectious mononucleosis.
▪Lymphoma. ▪Paroxysmal cold hemoglobinuria (rare, sometimes associated with
infections, e.g. syphilis).
Laboratory findings (Warm type):
The hematological and biochemical findings are typical of an extravascular hemolytic
anemia with spherocytosis prominent in the peripheral blood.
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The DAT is positive as a result of Ig G, Ig G and complement or Ig A on the cells.
Laboratory findings (Cold type):
Are similar to those of warm AIHA EXCEPT that:
●Spherocytosis is less marked.
●Red cells agglutinate in the cold.
Laboratory Diagnosis of Anemias:
The diagnosis is established by
●Decrease in the Hb and the hematocrit (PCV) to levels that are below normal.
●The red cell hemoglobin content and size of the RBCs are discriminatory in that the
results can place the anemia into one of three major subgroups:
*Normocytic Normochromic. *Microcytic Hypochromic. *Macrocytic.
●The presence of red cells with a particular morphology, such as spherocytes, sickled
cells, and fragmented cells, provide additional etiologic clues.
●Specialized tests are particularly important in establishing the diagnosis of certain
classes of anemia; these include:
■Gel electrophoresis: used to detect abnormal hemoglobins, such as HbS.
■Coombs test: used to diagnose immunohemolytic anemias.
■Reticulocyte counts: used to distinguish between anemias caused by red cell destruction
(hemolysis) and depressed production (marrow failure).
■Iron indices (serum iron, serum iron-binding capacity, transferrin saturation, and serum
ferritin concentrations): used to distinguish between hypochromic microcytic anemias
caused by iron deficiency, anemia of chronic disease, and thalassemia minor.
■Serum and red cell folate and vitamin B
12
concentrations: used to identify the cause of
megaloblastic anemia.
■Plasma unconjugated bilirubin and haptogloblin concentrations: used to support the
diagnosis of hemolytic anemia.
In isolated anemia, tests performed on the peripheral blood are usually sufficient to
establish a cause. In contrast, when anemia occurs in combination with thrombocytopenia
and/or granulocytopenia, it is much more likely to be associated with marrow aplasia or
infiltration; in these instances, BM aspiration & biopsy are often important for diagnosis.
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Polycythemia (Erythrocytosis):
Polycythemia (Erythrocytosis): This term signifies an increase in the blood concentration
of red cells, which usually correlates with an increase in the hemoglobin concentration.
Polycythemia are of two types:
●Relative polycythemia that is associated with hemoconcentration caused by
dehydration, such as with water deprivation, prolonged vomiting, diarrhea, or the
excessive use of diuretics.
●Absolute polycythemia, when there is an increase in the total red cell mass. Absolute
polycythemia is either:
■Primary when the increase in red cell mass results from an autonomous proliferation of
the myeloid stem cells
■Secondary when the red cell progenitors are proliferating in response to an increase in
erythropoietin.
Primary polycythemia (polycythemia vera [PCV]) is a clonal, neoplastic proliferation of
myeloid progenitors.
The increases in erythropoietin that are seen in secondary polycythemias have a variety
of causes:
●Appropriate: lung disease, high-altitude living, cyanotic heart disease
●Inappropriate: erythropoietin-secreting tumors (e.g., renal cell carcinoma, hepatoma,
cerebellar hemangioblastoma).
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HEMATOPATHOLOGY
WHITE CELL DISORDERS
Disorders of white cells include deficiencies (leukopenias) and proliferations (leukocytosis),
which may be reactive or neoplastic.
NON-NEOPLASTIC DISORDERS OF WHITE CELLS
Leukopenia is a decrease in the number of white cells in the peripheral blood, most commonly
the result of a decrease in neutrophils (the most prevalent circulating white cells).
Neutropenia
Neutropenia signifies a reduction in absolute neutrophil number below normal in peripheral
blood; when severe (reduced less than 500 cells/µl), where affected persons are extremely
susceptible to bacterial and fungal infections, which can be severe enough to cause death.
Etiology and Pathogenesis
The mechanisms that cause neutropenia can be broadly divided into two categories:
1. Inadequate or ineffective granulopoiesis, which is a manifestation of:
A. Generalized marrow failure as in:
- Aplastic anemia
- A variety of leukemias
B. Isolated neutropenia; there is involvement of neutrophilic precursors only as is seen with
- Congenital
- Idiopathic benign (racial or familial)
- Cyclical neutropenia syndrome (with 3-4 weeks periodicity)
2. Accelerated removal or destruction of neutrophils:
- Acquired: drug-induced (immune-mediated or direct toxicity)
- Overwhelming infections
- Splenomegaly that leads to sequestration and accelerated removal of neutrophils
Lymphopenias are associated with
1. Congenital immunodeficiency diseases
2. Acquired in association with:
advanced HIV infection
treatment with corticosteroids and other immunosuppressive therapy
Hodgkin disease
Widespread irradiation
Reactive Leukocytosis
An increase in the number of white cells is common in a variety of reactive inflammatory states
caused by microbial and non-microbial stimuli.
Neutrophilic Leukocytosis (Neutrophilia)
a. Acute bacterial infections (especially pyogenic)
b. Sterile inflammation caused by tissue necrosis (myocardial infarction, burns)
c. Metabolic disorders (uremia, eclampsia, acidosis, gout)
d. Neoplasms of all types
e. Acute hemorrhage or hemolysis
2
f. Treatment with myeloid growth factors (G-CSF, GM-CSF)
Eosinophilic Leukocytosis (Eosinophilia)
a. Allergic disorders such as asthma, hay fever, allergic skin diseases (e.g., pemphigus,
dermatitis herpetiformis)
b. Parasitic infestations
c. Drug sensitivity
d. Collagen vascular disorders (polyarteritis nodosa, vasculitis)
e. Certain malignancies (e.g., Hodgkin disease and some non-Hodgkin lymphomas)
f. Hypereosinophilic syndrome
g. Myeloproliferative neoplasms, Chronic eosinophilic leukemia
h. Treatment with GM-CSF
Basophilic Leukocytosis (Basophilia): this is rare, often indicative of myeloproliferative
neoplasms (e.g., chronic myeloid leukemia). Reactive increase is seen in myxoedema,
smallpox or chickenpox infections and in ulcerative colitis.
Monocytosis
a. Chronic bacterial infections (e.g., tuberculosis, brucellosis, endocarditis, typhoid)
b. Collagen vascular diseases (systemic lupus erythematosus, rheumatoid arthritis)
c. Hodgkin disease, AML and other malignancies
d. Myelodysplastic syndrome (especially chronic myelomonocytic leukemia)
e. Inflammatory bowel diseases (e.g., ulcerative colitis)
Lymphocytosis
a. Acute infections: infectious mononucleosis, rubella, pertussis, mumps, hepatitis A,
cytomegalovirus, HIV, herpes, Epstein-Barr virus
b. Chronic infections: tuberculosis, toxoplasmosis, brucellosis, syphilis
c. Chronic lymphoid leukemias
d. Acute lymphoblastic leukemia
e. Non-Hodgkin lymphoma (some)
f. Thyrotoxicosis
NEOPLASTIC PROLIFERATIONS OF WHITE CELLS
A. Lymphoid neoplasms, which include non-Hodgkin lymphomas, Hodgkin lymphomas, acute
and chronic lymphoid leukemias, and plasma cell dyscrasias and related disorders.
B. Myeloid neoplasms arise from stem cells that normally give rise to the formed elements of the
blood: granulocytes, red cells, and platelets. The myeloid neoplasms fall into three fairly distinct
subcategories:
1. Acute myeloid leukemias, in which immature progenitor cells accumulate in the bone
marrow (BM).
2. Chronic myeloproliferative neoplasms, in which inappropriately increased production of
formed blood elements leads to elevated blood cell counts.
3. Myelodysplastic syndromes, which are characteristically associated with ineffective
hematopoiesis and cytopenias.
ACUTE LEUKEMIAS (AL)
There are two major types of AL; acute lymphoblastic (ALL) and acute myeloblastic (AML).
3
Acute leukemia is usually an aggressive clonal malignant transformation involving the
hematopoietic stem cells or early progenitors and characterized by uncontrolled proliferation of
blasts in the BM with spillage into the peripheral blood and variable infiltration of other organs.
Etiology of AL
Several factors have been linked to the occurrence of AL including:
I. Environmental Agents
A. Ionizing Radiation
Exposure to atomic bomb explosions is associated with increased incidence of AL;
younger age and those who are closer to the hypocenter are at particularly high risk.
The predominant type is AML though ALL is reported in younger individuals. Infants
whose mothers were exposed to X-rays during pregnancy are at higher risk. Exposure to
diagnostic X-rays or radioisotopes at diagnostic levels (low dose) does not increase the
risk.
B. Chemicals
Exposure to the following agents have been noted to be associated with a higher
incidence
Benzene
a. Benzene and other petroleum derivatives
b. Shoe makers and plastic glues
c. Handling buses and trucks
Alkylating agents: (cytotoxic drugs used in the treatment of certain malignancies)
II. Host susceptibility to AL is determined by
A. Genetic factors
Fraternal twins and siblings of affected children are at a 2- 4 fold greater risk of
leukemia during the first decade of life than are unrelated children.
If one identical twin is affected, the other twin has a 20% chance of developing ALL.
Those with Down's syndrome have 10-30 fold ↑ risk.
B. Acquired factors; AL show increased incidence in association with the following:
Myelodysplastic syndrome
After chemotherapy + radiotherapy
Chronic myeloproliferative neoplasms
Aplastic anemia
III. Oncogenic viruses: there is no good evidence except for HTLV-1, which may cause
adult T-cell leukemia/lymphoma.
IV. Others: there is a significant correlation between infants with AL and alcohol intake,
smoking, and exposure to benzene and petroleum derivatives of their mothers during
pregnancy.
Pathophysiology of Acute Leukemias
In acute leukemia there is a block in differentiation. This leads to the accumulation of
immature leukemic blasts in the BM, which suppress the function of normal hematopoietic
stem cells by physical displacement and other poorly understood mechanisms.
Eventually BM failure results, which accounts for the major clinical manifestations of AL.
4
The acute leukemias have the following clinical characteristics:
Variable age of onset: ALs can occur at any age, however, childhood AL (age <15 years) is
usually ALL (80%) whereas adult AL (age ≥15 years) is usually AML (80%).
Abrupt stormy onset especially in children
Symptoms and signs related to BM failure. These include;
a. Pallor, weakness, fatigue, lethargy, dyspnea on exertion, angina, and palpitation (due
mainly to anemia)
b. Fever (reflecting mainly infections resulting from neutropenia)
c. Bleeding such as; petechiae, ecchymoses, epistaxis, and gum bleeding (secondary to
thrombocytopenia).
Symptoms related to organ or tissue infiltration:
Generalized lymphadenopathy, splenomegaly, and hepatomegaly, these are more
pronounced in ALL than in AML. Central nervous system manifestations these include
headache, vomiting, and nerve palsies resulting from meningeal spread; these features are
more common in children than in adults and are more common in ALL than AML. Gum
infiltration is more common in AML. Testicular involvement is more common in ALL.
Arthralgia, bone pain and tenderness.
Laboratory diagnosis of Acute Leukemias
The diagnosis of AL is based on the presence of > 20 % blasts in the BM and/or peripheral
blood. However; it can be diagnosed with even < 20 % blasts if specific leukemia-associated
cytogenetic or molecular genetic abnormalities are present.
Because of different responses to therapy, it is of great practical importance to distinguish
ALL from AML. The nuclei of lymphoblasts have somewhat coarse and clumped
chromatin and one or two nucleoli; myeloblasts tend to have finer chromatin with multiple
nucleoli and more cytoplasm, which may contain granules or Auer rod(s).
Blood film (BF):
a. RBCs: anemia is usually normochromic normocytic and is almost always present.
b. WBCs: the total WBC count is variable. There may be;
leukocytosis, where blasts are self-evident, or
leukopenia, blasts may be present or absent, or
it may show normal count.
Neutropenia is also a common finding in the peripheral blood.
c. Platelets: the count is reduced in most cases (i.e. <150,000/μL or <150 × 10
9
/L).
BM aspirate is necessary to confirm the diagnosis (especially when low counts).
BM trephine biopsy is only essential when:
1. BM aspirate is inadequate; commonly due to BM fibrosis.
2. To distinguish whether a poor aspirate is due to hypocellularity or persistent
leukemia.
Investigations
Hematological: BF and BM findings are already mentioned.
Biochemical tests may reveal increased S. uric acid, S. LDH, and hypercalcemia.
Liver & Renal Function Tests are performed as a baseline before treatment begins.
Radiological Examination may reveal,
a. Lytic bone lesions.
5
b. Mediastinal widening caused by enlargement of the thymus and/or mediastinal
lymphadenopathy.
CSF examination may show blast cells infiltration, indicating CNS involvement.
Cytochemistry is useful if the leukemia is not obviously myeloid.
Immunophenotyping is indicated in all patients in whom the leukemia is not obviously
myeloid.
Cytogenetic analysis is essential in all patients, best performed on BM aspirate.
Classification of acute leukemia is based on:
1. Morphology of blasts
2. Cytochemistry through the use of special stains like; SBB, PAS, MPO, Estrases…etc
3. Immunophenotyping (analysis by flow cytometry and immunohistochemistry).
4. Genetic analysis includes;
Cytogenetic analysis
(applied by conventional karyotyping and FISH
techniques)
Molecular genetic analysis (applied by PCR and FISH techniques)
Morphological classification
I. French American British (FAB) classification (1976)
A. Acute Lymphoid Leukemia (ALL) is classified into three subtypes:
ALL- L1: Monomorphic blasts, majority are small, high nucleo-cytoplasmic (N/C) ratio,
and scanty cytoplasm with small or inconspicuous nucleoli.
ALL- L2: Heterogeneous blasts, variable sizes and N/C ratios, more prominent nucleoli
with nuclear membrane irregularities.
ALL- L3: Monomorphic large blasts with prominent nucleoli and strongly basophilic,
vacuolated cytoplasm.
B. Acute Myeloid Leukemia (AML) is classified into eight subtypes:
M0: AML with minimal evidence of myeloid differentiation
M1: AML without maturation
M2: AML with maturation
M3: Acute promyelocytic leukemia
M4: Acute myelomonocytic leukemia
M5: Acute monoblastic M5a/monocytic M5b leukemia
M6: Acute erythroleukemia
M7: Acute megakaryoblastic leukemia
II. WHO classification (2000-2002)
There is a consensus that FAB L1, L2 and L3 of ALL are no longer relevant, since L1 & L2
morphology do not predict immunophenotype, genetic abnormalities, or clinical behavior.
ALL-L3 is generally equivalent to Burkitt lymphoma in leukemic phase and should be
diagnosed as such.
The WHO Classification of AML had reduced the blast threshold for diagnosis from 30% (in
FAB classification) to 20% in the peripheral blood and/or BM. In addition, patients with
certain clonal, recurrent cytogenetic abnormalities should be considered to have AML
regardless the blast percentage.
Cytochemistry of AL
6
ALL: is negative for Myeloperoxidase, Sudan Black B, and Non-specific estrases. Periodic
Acid Schiff is positive in many cases.
AML: is positive for Myeloperoxidase, Sudan Black B, and Non-specific estrases. PAS is
positive in AML-M6.
Immunophenotyping of AL
This is very useful in typing and subtyping of AL. CD79a is a specific marker for B-cells and
CD3 for T-cells. The most specific myeloid marker is anti-myeloperoxidase (MPO).
Karyotyping of AL
ALL
: the most common karyotypic abnormalities in pre-B-cell ALL is hyperploidy (>50
chromosomes/cell), which is associated with t(12: 21) chromosomal translocation involving the
TEL1 and AML1 genes. The presence of these aberrations correlates with a good prognosis.
Poor outcomes are observed with pre-B-cell ALL that have translocations involving the MLL
gene on chromosome 11q23 or the Philadelphia (Ph
+
) chromosome.
AML
: good outcome correlates with t(8:21) & t(15:17). Conversely, poor outcome correlates
with Ph
+
, t(6:9) and hyperploidy.
Course & Prognosis of AL
If untreated, patients will only survive for few months, and they will usually die either of severe
infection or bleeding. ALL, in general, carries a better prognosis than AML. T-ALL patients
have a better prognosis in adult than in children.
Treatment of childhood ALL (2-10 year age) represents one of the great success stories in
oncology and has the best prognosis; most can be cured. Other groups of patients do less well.
CLASSIC CHRONIC MYELOPROLIFERATIVE NEOPLASMS (MPN)
This term covers a group of clonal disorders of the hematopoietic stem cells that lead to effective
proliferation of one or more hematopoietic component in the BM, and in many cases, in the liver
and spleen leading to elevated blood levels of one or more myeloid cell lineages (i.e.
erythrocytosis, leukocytosis, and thrombocytosis). The classic MPNs include:
1. Chronic myeloid leukemia (CML - Ph
+ve
)
2. Polycythemia vera (PV)
3. Essential thrombocythemia (ET)
4. Primary myelofibrosis (MF)
These disorders are closely related to each other and transitional forms and evolution from one
entity into another occurs during the course of the disease.
Karyotype and Molecular Features
The vast majority of CML show the Philadelphia chromosome, in (90-95%) and M-
BCR-ABL p210 in (99% of patients). Ph chromosome is a minute chromosome 22 from
which the long arms are deleted (22q-). It is part of reciprocal translocation between
chromosome 9 & 22 t(9; 22) in which part of 22 is clearly visible on 9 but the part of 9
on 22 is too small to be distinguished cytogenetically. This translocation is detected by
PCR or FISH techniques.
Almost all PV patients, and about 55% of ET and MF cases show an acquired
mutation of cytoplasmic Janus-Associated Kinase 2 (JAK2) that occurs in the BM and
in the peripheral blood granulocytes. JAK2 plays a major role in normal myeloid
development.
7
POLYCYTHEMIA
True polycythemia refers to an absolute increase in total body red cell volume (mass), which
usually manifests itself as a raised hemoglobin (Hb) concentration and/or packed cell volume
(PCV) above the upper limit of normal for the patient's age and sex in specific population.
A raised Hb (or PCV) can also be due to a reduction in plasma volume, without an increase in
total red cell volume; this is known as apparent (or relative) polycythemia
.
Polycythemia is classified according to its pathophysiology:
A. Absolute
1. Primary
o
Polycythemia (rubra) vera
o
Familial (congenital) Polycythemia.
2. Secondary
Caused by compensatory erythropoietin increase in:
High altitudes
Pulmonary disease and alveolar hypoventilation (sleep apnoea)
Cardiovascular disease, especially congenital with cyanosis.
Increased affinity hemoglobin (familial Polycythemia).
Heavy cigarette smoking
Caused by inappropriate erythropoietin increase in:
Renal diseases (e.g. hydronephrosis, vascular impairment, cysts, carcinoma)
Tumors (such as uterine leimyoma, renal cell carcinoma.and hepatocellular carcinoma).
B. Relative (Stress or pseudopolycythemia):
a. Cigarette smoking
b. Dehydration: water deprivation, vomiting.
c. Plasma loss: burns, enteropathy.
POLYCYTHEMIA RUBRA VERA (PV)
PV is characterized by generalized hyperplasia of all marrow elements, but dominated by
expansion of the red blood cell mass. Although the diagnostic finding is the increase in red
cell volume (>125%), Hb >18.5 g/dl for men, >16.5 g/dl for women, in many patients there
is also neutrophilia and thrombocytosis in 50% of cases.
The clinical features are headache, dyspnea, blurred vision, night sweats, pruritus
(characteristically after hot bath) and plethoric appearance. Splenomegaly occurs in 75%
of patients
.
Thrombosis and hemorrhage are the major clinical problems. Typically, the
prognosis is good with a median survival of 10-16 years. Transition from PV to MF and AL
may occur.
ESSENTIAL THROMBOCYTHEMIA (ET)
ET is characterized by a sustained increase in platelet count, because of megakaryocytic
proliferation and overproduction of platelets.
A persisting platelet count > 400 × 10
9
/L (400,000 /µL) is the central diagnostic feature but
other causes of raised platelet count need to be fully excluded before the diagnosis can be
made. Many cases are symptomless and diagnosed on routine blood counts. Thrombosis is
a risk in about 25% of the patients. Hemorrhage as a result of abnormal platelet function.
Erythromelalgia is a characteristic symptom (it is a burning sensation felt in hands or feet
and relieved by aspirin). Up to 40% of patients will have palpable splenomegaly, whereas
in others there may be splenic atrophy because of infarction. Abnormal large platelets and
8
megakaryocyte fragments may be seen on blood film. BM typically shows an excess
proliferation of abnormal large and mature megakaryocyte, and no or little granulocyte or
erythroid proliferation. Often the disease is stationary for 10-20 years or more and has a
lesser risk to transform to MF, AL and PV.
PRIMARY MYELOFIBROSIS (MF)
MF is characterized by proliferation of multiple cell lineages and accompanied by progressive
BM fibrosis, with development of hematopoiesis in the spleen and liver. The onset is insidious
with symptoms of anemia. About ≥
⅓
of the patients have previous history of PV. Massive
splenomegaly is the main physical sign.
Laboratory findings:
1. Anemia is usual.
2. The WBC and platelet counts are frequently high at presentation but with advanced
disease, leucopenia and thrombocytopenia are common.
3. A leukoerythroblastic blood film is found and the red cells show characteristic 'tear-
drop' poikilocytes.
4. BM is usually unobtainable by aspiration. Trephine biopsy shows hypercellular marrow;
granulocytic proliferation and increased numbers of atypical megakaryocytes are
frequently seen with often decreased erythropoiesis in pre-fibrotic phase with extensive
marrow fibrosis in fibrotic phase.
Course & prognosis
MF has the poorest prognosis of the MPNs; the median survival is 3-5 years (range 1-15 years).
Causes of death include: heart failure, infection and in 10-20% of cases transformation to AML.
CHRONIC MYELOID LEUKEMIA (CML)
CML is characterized by proliferation of a population of differentiated cells that leads to a
greatly expanded total myeloid mass. CML represents about 15 % of leukemias.
CML has 3 phases during its course:
A. Chronic Phase (CP),
B. Accelerated Phase (AP),
C. Blastic Phase (BP).
The Chronic phase (CP) usually lasts 2-7 years and in 50% of cases it is transformed to BP
directly. In up to 50% of cases the diagnosis is made incidentally from a routine blood count
(asymptomatic). There may be features of anemia (pallor, dyspnoea, and tachycardia) and of
abnormal platelet function (bruising, epistaxis, and menorrhagia). Splenomegaly is nearly always
present and is frequently massive.
Laboratory findings
Anemia; usually normochromic normocytic.
Leukocytosis: usually in the range of 20-200 ×10
9
/L.
Blood film shows a full spectrum of granulocytic cells, ranging from blasts (usually 2-10%)
to mature neutrophils, with intermediate myelocytes and neutrophils predominating.
Eosinophils and basophils are usually increased.
Platelet count is usually increased.
BM Aspirate:
Markedly hypercellular marrow
Blast cells < 10% of all nucleated cells (ANC).
Megakaryocytes are small, hypolobed and increased in numbers.
9
BM Biopsy shows complete loss of fat spaces due to dense hypercellularity.
In the Advanced disease (AP & BP) the clinical features are quite variable:
Asymptomatic; the diagnosis is based entirely on blood and marrow findings.
Patients may develop fever, excessive sweating, anorexia and weight loss or bone pain.
Occasionally, patients present with generalized lymphadenopathy; where LN biopsy shows
nodal infiltration with blast cells that may be myeloid or lymphoid.
Localized skin infiltrates may be seen. Discrete masses of blast cells may develop at almost
any site; these are sometimes referred to as "Myeloid Sarcomas".
Laboratory findings
In AP: Blasts range (10-19%) in peripheral blood and/or BM, basophils ≥20%. Platelet
count is < 100 × 10
9
/L or persistently >1000 × 10
9
/L, increasing spleen size and WBCs
unresponsive to therapy. There may be megakaryocytic proliferation in sizable sheets and
clusters, associated with marked fibrosis.
In BP: Blasts >20% in peripheral blood and/or BM, or extramedullary blast proliferation
(LN, skin, elsewhere), or detection of large foci or clusters of blats in BM biopsy.
Course & prognosis
CML patients in chronic phase usually show an excellent response with the use of imatinib,
the 5-year survival is around 90%.
The 5-year survival after SCT is approximately 50-70%, providing that SCT done within the
first year following diagnosis.
The average survival of patients in AP is 1– 2 years.
Transformation to acute leukemia that ends with death within 2-6 months.
Death occurs from terminal blastic transformation or intercurrent hemorrhage or infection.
MYELODYSPLASTIC SYNDROME (MDS)
MDS is characterized by increasing BM failure with quantitative and qualitative abnormalities of
megakaryocytes, erythroid and myeloid cells. MDS is either primary or it is secondary to
chemotherapy ± radiotherapy.
Pathogenesis
There is increased stem cell proliferation with ineffective differentiation and maturation,
resulting in a hypercellular BM with peripheral blood pancytopenia; this is the hallmark of the
disease.
Clinically; patients may present with anemia (transfusion-dependent), recurrent infections and
easy bruising or bleeding (neutrophils and monocytes, and platelets are often functionally
impaired).
Laboratory findings
A. Peripheral Blood:
Pancytopenia is frequent
Anemia; is usually macrocytic.
Granulocytes are often decreased in number and frequently lack granulation.
Pelger abnormality (neutrophil with single or bilobed nucleus) is often present.
Platelets may be improperly large or small and are usually decreased in number.
Blasts in variable numbers are present in poor prognosis cases.
10
B. Bone Marrow:
Usually hypercellular.
Multinucleate normoblasts and other dysplastic (dyserythropoietic) features are seen.
Ring sideroblasts may be seen (>4 perinuclear iron granules/normoblast or covering ≥ ⅓
of the nuclear circumference).
Granulocytes and megakaryocytes are dysplastic with abnormal morphology.
At least 10% of the cells in a lineage should be dysplastic to consider the diagnosis of MDS.
THE CHRONIC LYMPHOID LEUKEMIA
A
number of lymphoproliferative disorders (LPD) are included in this group characterized by
accumulation in the blood of mature lymphocytes of either B- or T- cell type. In general the
diseases are incurable but tend to run a chronic and fluctuating course.
Diagnosis
This group is characterized by a chronic persistent lymphocytosis. Subtypes are distinguished by:
1. Morphology.
2. Immunophenotype.
3. Cytogenetics
4. DNA analysis may be useful in showing a monoclonal rearrangement of either Ig (for B-cells)
or T-cell receptor (TCR) genes (for T-cells).
CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
CLL is a low grade clonal LPD characterized by progressive accumulation of usually well-
differentiated CD5
+
lymphocytes in the marrow with an accompanying
peripheral
lymphocytosis. Involvement of LN, spleen and liver invariably occurs sometimes during the
disease course. The etiology is unknown. There is seven-fold increased risk of CLL in the close
relatives of the patient. CLL is the most common of the chronic lymphoid leukemia, accounting
for 60% of cases, and it is the most common in the West representing about 25% of all leukemias
in adults > 50 years.
Clinical features of CLL
1. Asymptomatic; most cases are diagnosed when routine blood test is performed.
2. Lymphadenopathy: Symmetrical enlargement of cervical, axillary or inguinal LNs is usually
discrete and non-tender.
3. Features of anemia & thrombocytopenia may be present.
4. Splenomegaly and less commonly hepatomegaly are common in intermediate & later stages.
5. Early bacterial infections predominate but with advanced disease viral and fungal infections
such as candidiasis and herpes zoster are also seen.
Laboratory findings
Lymphocytosis; the absolute lymphocyte count is > 5 × 10
9
/L. The predominant cells are
small lymphocytes with compact dark-staining round nuclei, scanty cytoplasm, and little
variation in size. The CLL lymphocytes are fragile and are frequently disrupted during the
preparation of smears, which produces characteristic smudge cells.
Anemia and Thrombocytopenia are seen in later stages due to BM failure, or hypersplenism
or autoimmune process.
BM examination: BMA shows lymphocyte infiltration >30 % of all nucleated BM cells. BM
biopsy reveals early interstitial and late diffuse pattern of involvement.
11
Immunophenotype shows pan-B-cell markers (CD19
+
& CD22
+
) with CD5
+
& CD23
+
, weak
expression of surface membrane immunoglobulin with weak or negative FMC7 and CD79b.
Karyotype. The most common cytogenetic abnormalities are deletion of 13q14 which is
associated with good prognosis. Triosomy 12, deletion at 11q23 and structural abnormalities
of 17p involving the p53 gene have bad prognosis.
Staging of CLL
It is useful to stage patients at presentation both for prognosis and for deciding on therapy. The
stage is determined by several variables such as peripheral lymphocyte count, BM lymphocyte
percentage, presence or absence of lymphadenopathy, hepatosplenomegaly. The presence of
anemia <10 gm/dL and/or thrombocytopenia <100,000 /µ L indicates advanced disease stage.
Course & prognosis
Many patients, in early stage, never need therapy. Survival ranges from 12 years for early stage
to < 3 years for advanced stage. CLL may transform to:
CLL/PL or frank prolymphocytic leukemia (PL) that is resistant to treatment
Richter's transformation (Immunoblastic lymphoma, localized high grade NHL)
PROLYMPHOCYTIC LEUKEMIA (PLL)
The prolymphocyte is around twice the size of a CLL lymphocyte and has a larger central
nucleolus. PLL typically presents with splenomegaly without lymphadenopathy and with a high
and rapidly rising lymphocyte count. Diagnosis is made by the appearance of > 55%
prolymphocytes in blood film. Response to treatment is poor.
HAIRY CELL LEUKEMIA (HCL)
HCL patients typically present with infections, anemia or splenomegaly. Lymphadenopathy is
very uncommon. Pancytopenia is usual. The blood film reveals a variable number of unusually
large lymphocytes with villous cytoplasmic projections. BM biopsy; shows a characteristic
appearance of mild fibrosis and a loose diffuse cellular infiltrate.
PLASMA CELL NEOPLASMS (PCNs)
PCNs originate from a clone of B cells that differentiates into plasma cells and secretes a single
complete and/or partial immunoglobulin (Ig). These disorders are also called monoclonal
gammopathies, due to the presence of usually excessive amounts of serum Igs, referred to as an
M-protein or paraprotein. However, the presence of an M-protein is not necessarily an indication
of an overt B-cell malignancy as it is fairly common in otherwise normal elderly persons.
The plasma cell neoplasms can be divided into many variants:
1. Multiple Myeloma (MM)
MM is the most common of the malignant plasma cell dyscrasias. It is a clonal neoplastic
proliferation characterized by plasma cell accumulation in the BM or plasmacytoma, the
presence of monoclonal protein in the serum and/or urine and related tissue damage that is
usually associated with multifocal lytic lesions throughout the skeletal system.
The etiology of the disease is unknown. Dysregulation or increased expression of cyclin D is an
early unifying event. IL 6 is a potent growth factor for myeloma cells and is often active by
autocrine mechanism.
Hyperploidy is present in about half of the tumors whereas non-hyperploid cases have a high
incidence of translocations involving the Ig heavy-chain gene (IGH) on chromosome 14.
12
Monoallelic loss of 13q is frequent in both categories and all these genetic abnormalities are also
seen in MGUS. The characteristic immunophenotype is CD38
high
, CD138
high
and CD45
low
.
The most common M component is IgG (60%), followed by IgA (20% to 25%). In the remaining
15% to 20% of cases, the plasma cells produce only κ or λ light chains. Because of their low
molecular weight, the free light chains are rapidly excreted in the urine, where they are termed
Bence-Jones proteins (BJP). Even more commonly, malignant plasma cells produce both serum
M-proteins & BJP in urine. About (3%) of patients have non–secretory myeloma cells with no
paraprotein in serum or urine
Gross pathologic features of MM
Multiple myeloma presents most often as multifocal destructive bone lesions throughout the
skeletal system. The affected bones are; vertebral column (65%), ribs (45%), skull (40%),
pelvis (30%), and femur (25%). There are often pathological fractures and vertebral collapse.
These focal lesions generally begin in the medullary cavity, erode the cancellous bone, and
progressively destroy the cortical bone. The osteolytic lesions are caused by osteoclast
activation resulting from high serum level of RANKL (receptor activator of nuclear factor-
κB ligand), produced by plasma cells and BM stroma, which binds to RANK receptors on the
osteoclast surface, which promotes the differentiation and activation of osteoclasts.
Microscopic features of MM
BM examination reveals an increased number of clonal plasma cells.
The neoplastic cells can resemble normal mature plasma cells, but they more often show
abnormal features, such as prominent nucleoli or abnormal cytoplasmic inclusions
containing immunoglobulin.
Plasma cell infiltrations of soft tissues (plasmacytoma) can be encountered in the spleen,
liver, skin, kidneys, lungs, and lymph nodes early or with disease progression.
Terminally, a leukemic picture may emerge (plasma cell leukemia or acute leukemia).
Myeloma nephrosis refers to renal involvement; it is a distinctive feature of MM.
a. Proteinaceous casts are prominent in the distal convoluted tubules and collecting
ducts. Most of these casts are made up of BJPs.
b. Some casts have tinctorial properties of amyloid.
c. Multinucleate giant cells created by the fusion of infiltrating macrophages usually
surround the casts.
d. Very often the epithelial cells lining the cast-filled tubules become necrotic or
atrophic because of the toxic actions of the Bence-Jones proteins.
e. Pyelonephritis can also occur as a result of the increased susceptibility to bacterial
infections. Less commonly, interstitial infiltrates of abnormal plasma cells are seen.
Metastatic calcification stemming from bone resorption and hypercalcemia may be
encountered.
The clinical manifestations of the plasma cell dyscrasias result from:
A. The destructive effect of the infiltrating neoplastic cells in various tissues and,
B. The abnormal immunoglobulins secreted by the tumors.
1. Bone pain especially backache.
2. Features of Anemia results from marrow replacement as well as from inhibition of
hematopoiesis by tumor cells.
3. Recurrent bacterial infections are serious clinical problems. They result from severe
suppression of normal Ig secretion, abnormal cell-mediated immunity and neutropenia.
13
4. Features of renal failure and/or hypercalcemia: Renal insufficiency occurs in 50% of patients
as a result of proteinaceous deposit from heavy BJ proteinuria, hypercalcemia, uric acid,
amyloid and pyelonephritis.
5. Bleeding tendency. Myeloma protein may interfere with platelet function and coagulation
factors; thrombocytopenia occurs in advanced disease.
6. Amyloidosis develops in 5% to 10% of patients.
7. Hyperviscosity syndrome may occur in 2% of MM cases due to excessive production and
aggregation of myeloma proteins. Purpura, hemorrhages, visual failure, CNS symptoms,
neuropathies and heart failure may be present but these are much more characteristic of LPL.
Diagnosis of Multiple myeloma:
Diagnosis can be made with reasonable certainty if two of the following three criteria are
met:
1. BM clonal plasma cells >10 % of all nucleated marrow cells.
2. A paraprotein in serum and/or urine and,
3. Complications related to organ or tissue infiltrations such as bone disease (osteolytic
bone lesions or
osteoporosis), renal impairment, anemia, hypercalcemia,
hyperviscosity, amyloidosis or recurrent infection.
Note: If the serum paraprotein > 30g/L and/or BM clonal plasma cells are >10% but there is
no evidence of tissue damage the disease is termed asymptomatic or smouldering myeloma.
Electrophoresis of the serum and urine is an important diagnostic tool. In 97% of cases a
monoclonal spike of complete Ig or Ig light chain can be detected in the serum and/or
urine.
Anemia is usually normochromic normocytic or macrocytic. Rouleaux formation is
marked. Neutropenia and thrombocytopenia occur in advanced disease.
Few plasma cells may appear in the blood film in 15% of cases.
High ESR and C-reactive protein.
Radiological; the diagnosis is strongly suspected when the characteristic focal, osteolytic
punched-out lesions in the bone are present (in 60% of cases) especially when located in
the vertebrae or calvarium. Generalized osteoporosis (20%) can also be seen and no bone
lesions in (20%). In addition, pathological fractures or vertebral collapse are common.
S. Calcium increased in 45% of patients. Typically, the S. alkaline phosphatase is normal
(except following pathological fractures).
S. Creatinine is raised in 20% of cases.
S. Albumin decreases with advanced disease.
S. β
2
-microglobulin is often raised (level < 4 mg/L imply a relatively good prognosis).
Prognosis
Multiple myeloma is a progressive disease. Patients with serum β
2
-microglobulin > 5.5 mg/ L
and serum albumin level < 35 g/L have poor survival as do those with frequent circulating
plasma cells. The median survival with non-intensive chemotherapy is 3 - 4 years.
2. Monoclonal Gammopathy of Undetermined Significance (MGUS)
A serum paraprotein may sometimes be detected in asymptomatic individuals without any
evidence of MM or other underlying disease and is termed MGUS. It has a high prevalence
(3.2% and 5.8% in individuals over 50 and 70 years of age, respectively), making this the most
common plasma cell dyscrasia. There is no related organ damage or tissue impairment
14
(such as bone lesions or renal imparment). The proportion of plasma cells in the BM is
normal (<4%) or only slightly increased (<10%). S. paraproteins are <30 g/L, and there is
no BJP in urine. Also there should be no evidence of other B-lineage lymphoproliferative
disorder. However, patients with MGUS develop a well-defined plasma cell neoplasm (e.g MM)
at a rate of 1% per year. Moreover, MGUS often show the same chromosomal translocations that
are found in full-blown MM. Thus, the diagnosis of MGUS should be made with caution and
only after careful exclusion of all other forms of monoclonal gammopathies, particularly
multiple myeloma.
3. Localized Plasmacytomas (solitary plasmacytoma)
These are isolated plasma cell tumors involving the skeleton or the soft tissues. Extraosseous
lesions occur mainly in mucosa of the upper respiratory tract, GIT or the skin. The associated
paraprotein disappears following radiotherapy to the primary lesion. Most of those with solitary
skeletal plasmacytomas develop full-blown MM over a period of 5 to 10 years.
4. Plasma cell leukemia (PCL)
PCL occurs either as a late complication of MM or as a primary disease characterized by the
presence of ≥ 20% plasma cells in the peripheral blood. The outlook is poor.
1
Bleeding Disorders: (Hemorrhagic Diatheses)
Excessive bleeding can result from:
1. Increased fragility of vessels.
2. Platelet deficiency or dysfunction.
3. Derangement of coagulation.
4. Combinations of these.
Tests used to evaluate different aspects of hemostasis are the following:
•Bleeding time: This measures the time taken for a standardized skin puncture to stop
bleeding and provides an in vivo assessment of platelet response to limited vascular
injury. The reference range depends on the actual method employed and varies from 2 to
9 minutes. Prolongation generally indicates a defect in platelet numbers or function.
•Platelet counts: These are obtained on anticoagulated blood using an electronic particle
counter. The reference range is 150 to 400 × 10
3
/µL.
•Prothrombin time (PT): This assay tests the extrinsic and common coagulation
pathways. A prolonged PT can result from deficiency or dysfunction of: factor VII,
factors X, V, prothrombin, or fibrinogen.
•Partial thromboplastin time (PTT): This assay tests the intrinsic and common clotting
pathways. Prolongation of the PTT can be due to deficiency or dysfunction of: factors
VIII, IX, XI, or XII, factors X, V, prothrombin, or fibrinogen.
Bleeding Disorders Caused By Vessel Wall Abnormalities:
Disorders within this category, sometimes called nonthrombocytopenic purpuras, are
relatively common but do not usually cause serious bleeding problems. Most often, they
induce small hemorrhages (petechiae and purpura) in the skin or mucous membranes,
particularly the gingivae. The platelet count, bleeding time, and results of the coagulation
tests (PT, PTT) are usually normal.
The varied clinical conditions in which hemorrhages can be related to abnormalities in
the vessel wall include the following:
•Many infections induce petechial and purpuric hemorrhages, but especially implicated
are meningococcemia, other forms of septicemia, infective endocarditis, and several of
the rickettsioses. The involved mechanism is presumably microbial damage to the
microvasculature (vasculitis) or disseminated intravascular coagulation (DIC).
•Drug reactions sometimes induce cutaneous petechiae and purpura without causing
thrombocytopenia. In many instances, the vascular injury is mediated by drug-induced
antibodies and deposition of immune complexes in the vessel walls, leading to
hypersensitivity (leukocytoclastic) vasculitis.
•Scurvy, Cushing syndrome and Ehlers-Danlos syndrome are associated with
microvascular bleeding resulting from impaired formation of collagens needed for
support of vessel walls.
2
•Henoch-Schönlein purpura is a systemic hypersensitivity disease of unknown cause
characterized by a purpuric rash, colicky abdominal pain (presumably due to focal
hemorrhages into the gastrointestinal tract), polyarthralgia, and acute glomerulonephritis.
All these changes result from the deposition of circulating immune complexes within
vessels throughout the body and within the glomerular mesangial regions. It is an Ig A-
mediated vasculitis.
•Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder characterized
by dilated, tortuous blood vessels with thin walls that bleed readily.
Bleeding Related to Reduced Platelet Number:
Thrombocytopenia: Reduction in platelet number constitutes an important cause of
generalized bleeding. A count below 100,000 platelets/μL is generally considered to
constitute thrombocytopenia. However, spontaneous bleeding does not become evident
until platelet counts fall below 20,000 platelets/μL. Platelet counts in the range of 20,000
to 50,000 platelets/μL can aggravate post-traumatic bleeding. Bleeding resulting from
thrombocytopenia is associated with a normal PT and PTT. Spontaneous bleeding
associated with thrombocytopenia most often involves small vessels. Common sites for
such hemorrhages are the skin and the mucous membranes of the gastrointestinal and
genitourinary tracts. The many causes of thrombocytopenia can be classified into the four
major categories:
●Decreased production of platelets: This can accompany generalized diseases of bone
marrow such as aplastic anemia and leukemias or result from diseases that affect the
megakaryocytes somewhat selectively. In vitamin B
12
or folic acid deficiency, there is
poor development and accelerated destruction of megakaryocytes within the bone
marrow (ineffective megakaryopoiesis) because DNA synthesis is impaired.
●Decreased platelet survival: This important cause of thrombocytopenia can have an
immunologic or nonimmunologic etiology.
▪In the immune conditions: platelet destruction is caused by circulating antiplatelet
antibodies or, less often, immune complexes. The antiplatelet antibodies can be directed
against a self-antigen on the platelets (autoantibodies) or against platelet antigens that
differ among different individuals (alloantibodies). Alloimmune thrombocytopenias arise
when an individual is exposed to platelets of another person, as may occur after blood
transfusion or during pregnancy. In the latter case, neonatal or even fetal
thrombocytopenia occurs by a mechanism analogous to
erythroblastosis fetalis.
▪Nonimmunologic destruction of platelets: may be caused by:
Mechanical injury: in a manner analogous to red cell destruction in microangiopathic
hemolytic anemia. The underlying conditions are also similar, including prosthetic heart
valves and diffuse narrowing of the microvessels (e.g., malignant hypertension).
●Sequestration: Thrombocytopenia, usually moderate in severity, may develop in any
patient with marked splenomegaly, a condition sometimes referred to as hypersplenism.
3
The spleen normally sequesters 30% to 40% of the body's platelets, which remain in
equilibrium with the circulating pool. When necessary, hypersplenic thrombocytopenia
can be ameliorated by splenectomy.
●Dilutional: Massive transfusions can produce a dilutional thrombocytopenia. Blood
stored for longer than 24 hours contains virtually no viable platelets; thus, plasma volume
and red cell mass are reconstituted by transfusion, but the number of circulating platelets
is relatively reduced.
Immune Thrombocytopenic Purpura (ITP): ITP can occur in:
•The setting of a variety of conditions and exposures (secondary ITP) or
•In the absence of any known risk factors (primary or idiopathic ITP).
There are two clinical subtypes of primary ITP: acute and chronic; both are autoimmune
disorders in which platelet destruction results from the formation of antiplatelet
autoantibodies.
Chronic ITP:
Pathogenesis: Chronic ITP is caused by the formation of autoantibodies against platelet
membrane glycoproteins. Antibodies reactive with these membrane glycoproteins can be
demonstrated in the plasma as well as bound to the platelet surface (platelet-associated
immunoglobulins) in approximately 80% of patients. In the overwhelming majority of
cases, the antiplatelet antibodies are of the IgG class. The mechanism of platelet
destruction is as follows: Opsonized platelets are rendered susceptible to phagocytosis by
the cells of the mononuclear phagocyte system especially of the spleen. About 75% to
80% of patients are remarkably improved after splenectomy, indicating that the spleen is
the major site of removal of sensitized platelets. Since it is also an important site of
autoantibody synthesis, the beneficial effects of splenectomy may in part derive from
removal of the source of autoantibodies.
Acute ITP: Like chronic ITP, this condition is caused by antiplatelet autoantibodies, but
its clinical features and course are distinct. Acute ITP is a disease of childhood occurring
with equal frequency in both sexes.
Drug-induced immune thrombocytopenia: An immunological mechanism has been
demonstrated as the cause of many drug-induced thrombocytopenias. Quinine, quinidine
and heparin are particularly common causes. An antibody-drug-protein complex is
deposited on the platelet surface. If complement is attached and the sequence goes to
completion, the platelet may be lysed directly. Otherwise, it is removed by
reticuloendothelial cells because of opsonization with immunoglobulin and / or the C3
component of complement. The platelet count is often less than 10 x 10
9
/L, and the bone
4
marrow shows normal or increased numbers of megakaryocytes. Drug dependent
antibodies against platelets may be demonstrated in the sera of some patients.
Bleeding Disorders Related To Defective Platelet Functions: Qualitative defects of
platelet function can be congenital or acquired. Several congenital disorders characterized
by prolonged bleeding time and normal platelet count have been described.
Congenital disorders of platelet function can be classified into three groups on the basis
of the specific functional abnormality:
1. Defects of adhesion.
2. Defects of aggregation.
3. Disorders of platelet secretion (release reaction).
Acquired defects of platelet function:
▪Ingestion of aspirin and other nonsteroidal anti-inflammatory drugs which significantly
prolongs the bleeding time.
▪Aspirin: Is a potent, irreversible inhibitor of the enzyme cyclooxygenase.
▪Uremia: Several abnormalities of platelet function are found.
Hemorrhagic Diatheses Related To Abnormalities In Clotting Factors: A deficiency
of every clotting factor has been reported to be the cause of a bleeding disorder, with the
exception of factor XII deficiency, which does not cause bleeding.
The bleeding in factor deficiencies differs from platelet deficiencies in that spontaneous
petechiae or purpura are uncommon. Rather, the bleeding is manifested by large post-
traumatic ecchymoses or hematomas, or prolonged bleeding after a laceration or any
form of surgical procedure. Bleeding into the gastrointestinal and urinary tracts, and
particularly into weight-bearing joints, is common.
Hereditary deficiencies have been identified for each of the clotting factors. Deficiencies
of factor VIII (hemophilia A) and of factor IX (Christmas disease, or hemophilia B) are
transmitted as sex-linked recessive disorders. Most others follow autosomal patterns of
transmission. These hereditary disorders typically involve a single clotting factor.
Deficiencies of Factor VIII-vWF Complex: Hemophilia A and von Willebrand disease,
two of the most common inherited disorders of bleeding, are caused by qualitative or
quantitative defects involving the factor VIII-vWF complex. Plasma factor VIII-vWF is a
complex made up of two separate proteins (factor VIII and vWF). Factor VIII; is an
intrinsic pathway component required for activation of factor X. Deficiency of factor VIII
gives rise to hemophilia A. Circulating factor VIII is noncovalently associated with very
large vWF multimers. The most important function of vWF in vivo is to promote the
adhesion of platelets to subendothelial matrix. The two components of the factor VIII-
vWF complex are encoded by separate genes and synthesized in different cells. vWF is
produced by endothelial cells and megakaryocytes and can be demonstrated in platelet α-
5
granules. Endothelial cells are the major source of subendothelial and plasma vWF. vWF
gene is located on chromosome 12. Factor VIII is made in several tissues; sinusoidal
endothelial cells and Kupffer cells in the liver and glomerular and tubular epithelial cells
in the kidney appear to be particularly important sites of synthesis. Factor VIII gene is
located on X chromosome.
Von Willebrand Disease: With an estimated frequency of 1%, von Willebrand disease is
believed to be one of the most common inherited disorders of bleeding in humans.
Clinically, it is characterized by spontaneous bleeding from mucous membranes,
excessive bleeding from wounds, menorrhagia. In this disorder there is either a reduced
level or abnormal function of VWF resulting from a point mutation or major deletion.
Patients with von Willebrand disease have defects in platelet function despite a normal
platelet count.
Lab findings:
Patients with von Willebrand disease typically have:
•A prolonged bleeding time.
•A normal platelet count.
•The plasma level of active vWF is reduced.
(Because vWF stabilizes factor VIII by binding to it, a deficiency of vWF gives rise to a
secondary decrease in factor VIII levels); this may be reflected by a prolongation of the
PTT in von Willebrand disease types 1 and 3. In most cases, it is transmitted as an
autosomal dominant disorder, but several rare autosomal recessive variants have been
identified. Because a severe deficiency of vWF has a marked affect on the stability of
factor VIII, some of the bleeding characteristics resemble those seen in hemophilia.
Hemophilia A (Factor VIII Deficiency):
Hemophilia A is the most common hereditary disease associated with serious bleeding. It
is caused by a reduction in the amount or activity of factor VIII. Hemophilia A is
inherited as an X-linked recessive trait, and thus occurs in males and in homozygous
females. However, excessive bleeding has been described in heterozygous females,
presumably due to extremely unfavorable lyonization (inactivation of the normal X
chromosome in most of the cells).
Approximately 30% of patients have no family
history; their disease is presumably caused by new mutations. Hemophilia A exhibits a
wide range of clinical severity that correlates well with the level of factor VIII activity.
•Those with less than 1% of normal activity develop severe disease.
•Levels between 2% and 5% of normal are associated with moderate disease.
•Patients with 6% to 50% of activity develop mild disease.
The variable degrees of factor VIII deficiency are largely explained by heterogeneity in
the causative mutations.
Several genetic lesions (deletions, nonsense mutations that
create stop codons, splicing errors) have been documented.
6
Lab findings:
Patients with hemophilia A typically have:
•A normal bleeding time.
•A normal platelet count, and a normal PT.
•A prolonged PTT.
(These tests point to an abnormality of the intrinsic coagulation pathway).
►Factor VIII-specific assays are required for diagnosis.
Hemophilia B (Christmas Disease, Factor IX Deficiency):
Severe factor IX deficiency produces a disorder clinically indistinguishable from factor
VIII deficiency (hemophilia A). This should not be surprising, given that factor VIII and
IX function together to activate factor X. Wide spectrums of mutations involving the
factor IX gene are found in hemophilia B. Like hemophilia A, it is inherited as an X-
linked recessive trait and shows variable clinical severity. In about 14% of these patients,
factor IX is present but nonfunctional.
Lab findings:
Patients with hemophilia B typically have:
•A normal bleeding time.
•A normal platelet count, and a normal PT.
•A prolonged PTT.
►Factor IX-specific assays are required for diagnosis.
Disseminated Intravascular Coagulation (DIC):
DIC is an acute, subacute, or chronic thrombohemorrhagic disorder occurring as a
secondary complication in a variety of diseases.
●It is characterized by activation of the coagulation sequence that leads to the formation
of microthrombi throughout the microcirculation of the body, often in a quixotically
uneven distribution.
●Sometimes the coagulopathy is localized to a specific organ or tissue.
●As a consequence of the thrombotic diathesis, there is consumption of platelets, fibrin,
and coagulation factors and, secondarily, activation of fibrinolytic mechanisms. Thus,
DIC can present with signs and symptoms relating to:
▪Tissue hypoxia and infarction caused by the myriad microthrombi or
▪A hemorrhagic disorder related to depletion of the elements required for hemostasis
(hence, the term "consumption coagulopathy" is sometimes used to describe DIC).
Activation of the fibrinolytic mechanism aggravates the hemorrhagic diathesis.
Etiology and Pathogenesis: At the outset, it must be emphasized that DIC is not a primary
disease. It is a coagulopathy that occurs in the course of a variety of clinical conditions.
Two major mechanisms trigger DIC:
7
1. Release of tissue factor or thromboplastic substances into the circulation: Tissue
thromboplastic substances can be derived from a variety of sources, such as the placenta
in obstetric complications and the granules of leukemic cells in acute promyelocytic
leukemia. Mucus released from certain adenocarcinomas can also act as a thromboplastic
substance by directly activating factor X, independent of factor VII. In gram-negative
sepsis (an important cause of DIC), bacterial endotoxins cause activated monocytes to
release interleukin-1 and TNF, both of which increase the expression of tissue factor on
endothelial cell membranes and simultaneously decrease the expression of
thrombomodulin. The net result is a shift in balance toward procoagulation.
2. Widespread injury to the endothelial cells: The other major trigger, can initiate DIC by
causing release of tissue factor, promoting platelet aggregation, and activating the
intrinsic coagulation pathway. TNF is an extremely important mediator of endothelial cell
inflammation and injury in septic shock. Even subtle endothelial injury can unleash
procoagulant activity by enhancing membrane expression of tissue factor. Widespread
endothelial injury may be produced by deposition of antigen-antibody complexes (e.g.,
systemic lupus erythematosus), temperature extremes (e.g., heat stroke, burns), or
microorganisms (e.g., meningococci, rickettsiae).
The initiating factors in these conditions are often multiple and interrelated. The
consequences of DIC are twofold:
●There is widespread deposition of fibrin within the microcirculation. This can lead to:
▪Ischemia of the more severely affected or more vulnerable organs
▪A hemolytic anemia resulting from fragmentation of red cells as they squeeze through
the narrowed microvasculature (microangiopathic hemolytic anemia).
●A hemorrhagic diathesis can dominate the clinical picture. This results from
consumption of platelets and clotting factors as well as activation of plasminogen.
Plasmin can not only cleave fibrin, but also digest factors V and VIII, thereby reducing
their concentration further.
Morphology: In general, thrombi are found in the following sites in decreasing order of
frequency: brain, heart, lungs, kidneys, adrenals, spleen, and liver.
However, no tissue is spared, and thrombi are occasionally found in only one or several
organs without affecting others.
Acquired disorders are usually characterized by multiple clotting abnormalities.
Vitamin K deficiency: Results in impaired synthesis of factors II, VII, IX, and X and
protein C.
Since the liver makes virtually all the clotting factors:
Severe parenchymal liver disease: Can be associated with a hemorrhagic diathesis.
Disseminated intravascular coagulation: Produces a deficiency of multiple coagulation
factors.
1
HEMATOPATHOLOGY
TRANSFUSION MEDICINE
Blood transfusion refers to the 'Safe' transfer of blood or blood components from a donor to a
recipient.
PRINCIPLES
Blood donation should always be voluntary.
Never give transfusion unnecessarily.
Blood transfusion should follow components policy.
BLOOD DONATION
Donor must be fit & healthy.
It should not harm the donor
It should not transmit any disease to the recipient
Before blood donation the donor should be subjected to:
1. Detailed Medical history (Questionnaire Form)
2. Limited physical examination
Questionnaire form:
1. Name of the donor
2. Sex
3. Age 18-65 year
4.
Weight > 50 Kg (vasovagal reactions become more common
in those who weigh < 50 kg, as the standard donation represents a greater proportion of
their total blood volume).
5. Occupation
Exclusion of any donor returning to occupations such
as fire fighter or driving bus, plane or train, heavy machine or crane operator,
scaffolding, , etc. because delayed faint would be dangerous
6. Last donation Not less than 2 months
7. Frequency of donation 2-3 times/y (Max 3 times/yr for females and 4times/yr for males
8. History of blood transfusion defer 6 months
9. Major surgery defer 6 months
10. History of heart disease, active pulmonary disease (active T.B), diabetics, hypertension,
hyperthyroidism. Those with one of the above diseases are generally deferred from
donation.
2
11. History of blood diseases such as leukemia, lymphoma, thalassaemia major, sickle cell
anemia and polycythemia should be deferred from donation.
12. History of abnormal bleeding tendency should also be deferred
13. History of epilepsy is generally a cause of deferral
14. History of infectious diseases
15. AIDS patients, AIDS contacts, homosexuals, drug abusers, those with multiple partners,
hemophiliacs receiving products of human origin all should be indefinitely deferred.
16. Hepatitis: history of jaundice or viral hepatitis A: deferred one year. Hepatitis B (HBs Ag
+) or C is deferred permanently.
17. Malaria: those infected are not accepted as blood donors.
18. Brucellosis: deferred for 2 years from last febrile episode.
19. EBV infected patients are deferred for 2 years.
20. Syphilis: patients with this disease are considered as permanent deferral
21. Drugs: patients on certain drugs (anticoagulants, antihypertensive, insulin) are not
accepted.
22. Pregnancy: not allowed. Accepted 3-6 ms postpartum to protect the donor from iron
deficiency.
23. Donor consent: written consent.
Physical Examination
This should be simple & brief and include
1. General appearance
2. Temp: Not more than 37°C
3. Pulse: 60-100 beats/ min
4. Blood pressure: within normal.
5. Weight: At least 50 Kg
6. Hb level: more than 13.5 g/dl for males & 12.5 g/dl for females
Anticoagulants:
ACD (Acid citrate dextrose)
Shelf life of blood
21 days (Now used only in automated plasmapheresis).
CPD (Citrate phosphate dextrose)
Shelf life of blood
28 days
CPD-A (Plus Adenine)
Shelf life of blood
35 days (used now)
3
Blood donation is taken by an aseptic technique into plastic bags designed to hold 450 ml + 45
ml of blood, mixed with 63 ml of anticoagulant. The ratio of anticoagulant to blood must be
maintained at the optimal level of 1:7.
The citrate anticoagulates the blood by combining with the blood calcium.
Standard routine cross matching is done by:
Saline tube
Mixing donor cells & recipient serum,
leave the tube at room temp (18-25°C)
Albumin tube
by adding albumin to the mixture of the donor cells & recipient
serum at 37°C to detect warm- reacting antibodies
Indirect antiglobulin test
at 37°C to detect antibodies in the recipient serum that coat or
cause sensitization of the donor red cells
Mandatory tests on blood units:
1. ABO &Rh grouping
2. Test for HIV Ab
3. Test for HBs Ag
4. Test for HCV Ab
5. Test for syphilis
6. Screening for atypical antibodies.
BLOOD TRANSFUSION
Before giving blood to the patient we should do Compatibility testing, which includes:
1. ABO & Rh typing of the donor and the recipient blood
2. Screening of the donor & the recipient sera for unexpected antibodies
3. Cross matching the donor & the recipient blood by cross matching the donor cells & the
recipient serum.
Objectives of cross matching are:
1. Assurance of the ABO compatibility
2. Recognition of clinically significant antibodies
4
The limits for infusions:
Start infusion
Complete infusion
Whole blood or red cells
Within 30 minutes of
removing pack from
refrigerator
Within 4 hours* (or less in
high ambient temperature)
Platelet concentrates
Immediately
Within 20 minutes
Fresh frozen plasma and
cryoprecipitate
As soon as possible
Within 20 minutes
*If a unit is not completed within 4 hours:
Discontinue its use and
Dispose of the remainder through the clinical waste system.
The Whole blood or Packed red cells should be kept refrigerated at 2-6 °C
The upper limit of 6°C is essential to minimize the growth of any bacterial contamination
in the unit of blood.
The lower limit of 2°C is essential to prevent hemolysis, which can cause fatal bleeding
problems or renal failure.
Complications of blood transfusion
Incidence of transfusion reaction is about 2-5%. It is mostly of mild degree.
Most of the cases are due to (clerical or administrative error).
Laboratory error, nursing service, anesthesia service, and clinical staff errors.
Fatal complications are uncommon (1 in 100,000 to 1 in 500,000 patients transfused),
mainly due to improper patient identification (the major cause of transfusion deaths).
Complications can be divided broadly into:
1. Immunological complications
2. Nonimmunological complications
IMMUNOLOGICAL COMPLICATIONS:
1. Sensitization to red cells antigens
Because the ABO & Rh D antigens are the only Ags matched between donor and recipient, there
is a possibility of sensitization to other red cells antigens.
In clinical practice this sensitization could lead to:
A. Hemolytic disease of the newborn if the recipient is a female
B. Difficulties in compatibility testing if the recipient required further transfusion
C. Hemolytic transfusion reaction
2. Hemolytic transfusion reaction
This reaction is caused by premature destruction, almost always of the donor cells by antibodies
present in the recipient plasma.
The hemolytic transfusion reaction could be: Immediate or Delayed
5
Immediate Transfusion reaction:
This is the most dangerous type
Usually caused by ABO incompatibility
The antibodies are IgM in type that bind to the red cells and cause complement activation leading
to intravascular lysis of the red cells with production of the anaphylatoxins the C3a & C5a
librated during complement activation. The C3a & C5a will cause smooth muscle contraction,
platelets aggregation, increased capillary permeability, release of vasoactive amines and
hydrolases from mast cells and granulocytes
Sign & Symptoms
Occur within minutes to I hour from the start of transfusion
Heat in the vein
Throbbing headache
Flushing of the face
Chest tightness
Nausea
Lumber pain
Hypotension & tachycardia
DIC, hemoglobinuria, acute renal failure, collapse & death in severe cases.
Less commonly the haemolysis is extra-vascular caused by removal of C3b & IgG coated red
cells by the macrophages in the liver and spleen. Symptoms are usually less rapid in onset occur
usually after 1 hour with fever, jaundice and unexplained decrease in Hb. Renal failure is rare.
Management of Hemolytic Transfusion Reaction
Stop transfusion immediately. Keep the IV line.
Give physiological saline to maintain the blood pressure >100 mg Hg.
Give diuretics to maintain urinary flow > 100 ml/hour
Collect blood sample from site a way from the site of infusion in 3 tubes
1. EDTA sample – for CBP.
2. Citrated sample-for coagulation studies
3. Clotted sample -for serological studies (Blood grouping, Coombs test, repeat antibodies
screening for the recipient, repeat the compatibility testing)
Collect the next urine sample and 24 hr urine post transfusion check for Hburia
Check the label and the number on the blood unit and check the cross match form for any
error.
Tests to be done in the lab
Check the ABO & Rh group of the recipient and the donor samples again
Examine the post transfusion sample for hemolysis & check the donor unit for hemolysis
Do Coombs test on recipient post transfusion sample
Repeat cross match with both pre- & post-transfusion samples
Screen pre- & post-transfusion samples and donor plasma for antibodies
Check the Hb
Coagulation screening test for the possibility of DIC
6
Bacteriological evaluation: inspect the donor unit hemolysis or clot. Blood from the
giving set and the blood unit should be cultured
Biochemical studies: test for hemoglobinemia and for bilirubin
Check the urine for hemoglobinuria.
Delayed Transfusion reaction
This is manifested usually 7-10 days after transfusion and is caused by antibodies, which are
present in low titer and are not detected at time of cross matching. So this reaction is neither
predictable nor preventable. The antibodies are caused by sensitization due to previous
pregnancy or transfusion.
Signs and symptoms: fever, jaundice and lowering of Hb.
3. Febrile reaction due to WBC & platelets Antigens:
Most common immunological reaction
Seen in patients having multiple blood transfusion or pregnancy
Caused by Ab to HLA Ags, WBC & platelets specific Ags (Usually WBC)
The onset of the reaction is delayed 30-90 min after start of transfusion
The main symptom is fever
Management
Slow the transfusion
Give antipyretic
No need to terminate the transfusion
If symptom recur in patients require repeated transfusions we should check the patient for
WBC or Platelets Abs &if these are present we should use WBC depleted blood (by using
WBC filter).
4. Reaction to platelets Ag (Post-transfusion Purpura)
Seen in women with history of multiple pregnancies or in those with history of multiple
transfusion
Caused by Abs to platelets Ag (PI)
The reaction occurs 7-10 days after transfusion
The main feature is purpura due to thrombocytopenia (caused by destruction of the
platelets by the Abs)
It is usually self limiting
5. Reaction due to plasma protein antibodies
Majority are due to Anti IgA antibodies
Main symptom is urticaria
Treatment is by antihistamine
Rarely more severe anaphylactic reaction occur which should be treated urgently with
adrenaline and any next transfusion should be IgA deficient blood
7
Important notes:
Severe reactions most commonly present during the first 15 minutes of a
transfusion.
All patients and, in particular, unconscious patients should be monitored during this
period and for the first 15 minutes of each subsequent unit.
NON-IMMUNOLOGICAL COMPLICATIONS:
1. Reaction due to bacterial pyrogens or bacteria:
Although rare complication, it has very high mortality rate characterized by sudden onset of high
fever, shock and bleeding due to DIC. Blood may be contaminated by cold-growing organisms
(pseudomonas or colon-aerogenes group). These microorganisms utilize citrate as the primary
source of carbon, which leads to citrate depletion and hence clotting of blood. Visual inspection
of the blood units may reveal clots and indicate the presence of contamination.
The infusion of large number of gram-negative microorganisms results in a serious reaction i.e.
endotoxic shock. The latter is accompanied by fever, marked hypotension, pain, vomiting and
the development of profound shock. The reaction may start with shaking chills following a latent
period of 30 minutes or more. As little as 10 ml of blood may contain sufficient microorganisms
to produce the reaction.
Management
Do direct examination and culture of the blood from the patient and the blood unit
Give antibiotic IV
This complication could be prevented by
Ensuring aseptic technique in the preparation of blood bags & anticoagulant
Aseptic condition in blood donation
Bags should not be opened for sampling and the unit should be transfused within 24 hr if
any open method has been used
Blood should be kept in accurately controlled refrigerator at 2-6 C
Avoid leaving blood at room temp.
Inspect all blood units for signs of contamination as clotting or haemolysis.
2. Circulatory overload
Transfusion generally increases blood volume except in those who are actively bleeding. This
increase in blood volume may be dangerous in the elderly with a compromised cardiovascular
function, pregnancy and in those with severe anemia
Prevention
Blood should be give n slowly over 4 hr.
Give diuretics at the start of transfusion-No more than 2 units should be given within 24
hr.
Blood should be given during the daytime and the patient should be followed carefully
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If signs and symptoms of overload and pulmonary edema occur
Transfusion should be stopped
Patient propped upright
Give diuretics IV
3. Thrombophlebitis; this is a complication of indwelling venous cannulae and is not specifically
related to blood transfusion.
4. Air embolism; this is now a rare complication of transfusion therapy due to the introduction of
plastic bags, which provide a closed system. Only large volumes of air, and not the entry of a
few bubbles, result in a clinically significant air embolism. Symptoms include pain, cough, and
sudden onset of dyspnoea. The treatment includes clumping off the administrating tube.
5. Hemosiderosis; each unit of blood contain approximately 200 mg of iron. Repeated transfusions
over many years, in the absence of blood loss, cause deposition of iron initially in the reticulo-
endothelial system. After 50 units in adults, and lesser amount in children, the liver, myocardium
and endocrine glands are damaged. This is a major problem in thalassemia major and other
severe chronic refractory anemias, and this could be prevented by giving chelating agent.
6. Complications of massive transfusion
These tend to occur in cases of replacement the total blood volume within 24 hr (For adult about
10 units/24 hr). This could lead to:
1. Dilution of platelets. As blood stored more than 48 hr has no functional platelets. Transfusion
of 8-10 units of blood to an adult will lead to thrombocytopenia (low platelets). It follows that
any patient receiving many blood units should be monitored through platelets count & judged on
his clinical condition. Some give one platelets unit for every 4 blood units. Others give platelets
transfusion if platelets count becomes less than 100,000 /cmm if there is bleeding or surgical
intervention
2. Dilution of coagulation factors
This occurs if blood stored more than 14 days is given. Blood stored less than 14 days has
adequate level of most of the coagulation factors except factor V & VIII, as they are the most
labile factors.
3. Metabolic changes
a. Citrate toxicity. This is not a problem except in a very rapid transfusion (unit every 5
minutes).
b. Hyperkalemia & hypocalcemia. These are usually transient & rapidly corrected.
7. Transmission of Infection:
A. Bacterial diseases
Syphilis
The agent is Treponema Pallidum
Donor is infective during the early spirochetemia phase i.e. before the development of
the antibodies
Blood products implicated: fresh blood & components
Viability in blood: the bacteria are unlikely to survive more than 3 days at 4-6 C, so
transmission of syphilis by blood is a rare complication.
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It is more likely to be transmitted by platelets concentrate because of its storage at room
temp and its short shelf life.
If blood is taken from seropositive donor (Showing positive serological tests for syphilis)
this cause passive transmission of the antibodies to the recipient and the recipient become
seropositive for 4-10 days
Prevention
Mandatory screening of all donor units by VDRL or TPHA
Exclusion of high-risk group.
Brucellosis
The agent is Brucella abortus
Viability in stored blood: months
Incubation period: 6 days- 4 months
Reports of transfusion related brucellosis: mainly in children, splenctomized or
immunocompromized.
Prevention: defer infected patient for 2 years after cure
B. Protozoal diseases
Malaria
The gent is Plasmodia Species (vivax, ovale, malariae, falciparum)
Viability: viable in stored blood at 4°C at least 1 week; in case of P. falciparum up to 2
weeks
Blood product implicated: products containing red cells
Incubation period: vivax & falciparum 1 week to 1 month; malariae: months
Prevention
In endemic areas: prophylactic treatment of donors with chloroquine 48 h before donation or
single dose of chloroquine to the recipient 24 before transfusion.
C. Viral diseases
AIDS (Acquired immune deficiency syndrome)
The agent is Human immunodeficiency virus HIV type I & II
Blood product implicated: whole blood (cellular& plasma blood components)
Incubation period: mean incubation period is 4.5 yr
Prevention
1. Education through the media
2. Self –exclusion of high risk group
3. Screening all donors for HIV antibodies
Hepatitis Viruses
Post transfusion hepatitis could be caused by the following viruses
1. Hepatitis viruses (A, B, C)
2. Cytomegalovirus (CMV)
3. Epstien-Bar virus (EBV)
Prevention
Tests to screen for Hepatitis B (HBsAg)
Tests to screen for HCV
Exclusion of high risk group