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TERMINOLOGY
HEMATOCRIT (Hct)
Hct is the percentage blood volume which is red cells.
Coulter instruments measure both the number of red cells per volume of blood, and the mean cell
volume (MCV) of the red cells (i.e. size of the RBCs).
Hct calculated as decimal fraction (litres RBC (red cell volume) / litre blood) { x 100 = %Hct }
Normal adult range for males: 40 50%. Females: 35 45%.
MEAN CELL VOLUME (MCV)
Mean volume of RBC (femtolitres) = the size of the RBC.
Normal adult range: 82 96 fl.
MCV < 82 = microcytosis
MCV > 96 = macrocytosis.
Because evaluation of the RBC size is key to the diagnosis of an anemia, the MCV is the most
important of the RBC indices.
MEAN CELL HAEMOGLOBIN (MCH)
Average weight (picograms) of haemoglobin per RBC.
Normal range: 27 32 pg
MCH = Hb x 10 / RBC .
MEAN CELL HAEMOGLOBIN CONCENTRATION (MCHC)
Concentration (g / dl) of hemoglobin in the RBC.
Normal range: 32 36 g / dl
MCHC = Hb / Hct .
RED CELL DISTRIBUTION WIDTH (RDW)
Indicates variation in size (measure of anisocytosis) in RBC population.
Normal range: 10 15% (standard deviation of the MCV as percentage of the MCV).
ABNORMALITIES IN SHAPE:
Poikilocytosis: This term indicates a variation in shape. It can result from abnormal
erythropoeisis or damage to the red cells in circulation. It is seen in a variety of anemias and
other conditions.
Tear drop cells: Often seen in conditions where the bone marrow is replaced by nonhaemopoeitic tissue. E.g. myelofibrosis and malignant infiltrates. They are also seen in iron
deficiency, thalassemia and are often associated with the pitting function of the spleen.
Spherocytes: These cells have lost their bi-concavity and thus their centre pallor. They
usually have a smaller diameter compared to their volume and stain very densely. Due to the
loss of surface area they usually have a short survival. Spherocytes are caused by abnormal
membrane proteins, lipid loss and excessive Na+ flux. They are often seen in hereditary
spherocytosis and post splenectomy. They can also be seen in a number of hemolytic
anemias.
Ovalocytes / elliptocytes: They are oval in shape and appear in a variety of disorders. The
most common is hereditary ovalocytosis, but also found in thalassemia, sickle cell anemia,
iron deficiency and megaloblastic anemia. In iron deficiency these cells could be cigar
shaped or pencil cells. They are caused by the inability of the red cell to return to normal
after being deformed by the shear stress in the microcirculation.
Target cells: Cells show an area of central staining. Caused by an increase in surface area
without change in volume. They are seen in thalassemia, post splenectomy and liver disease.
In liver disease it is thought they are caused by an increase in cholesterol and lecithin in the
cell membrane.
Sickle cells: Resemble crescents. They appear when HbS is present in high concentrations.
Often associated with deoxegenation of the blood and the condition is reversable. The
sickles are formed by the precipitation of polymerized hemoglobin.
Fragmentation: Formation of a variety of shapes, resulting from severe mechanical or
physical stress and are often seen in disorders like DIC and in patients with mechanical heart
valves (microangiopathic anemias). They are also observed in burns patients.
Stomatocytes: This term describes a red cell with a slit-like central pallor. They can be
induced by reducing the pH and may be caused by defects of the Na+ - K+ pump. They are
seen in hereditary spherocytosis, stomatocytosis and liver disease caused by alcohol abuse.
Burr cells: These cells have blunt projections. They are thought to be damaged or
fragmented cells and are seen in uremic patients, cancer and microangiopathic anemia.
Acanthocytes: Cells which are described as 'thorny'. They are due to abnormal lipid content
in the membrane. Can be inherited, and are seen in splenectomised patients and chronic
liver disease.
Crenation: These cells have puckered out edges. Generally have no clinical significance but
rather are artefacts produced by drying of the slide and long storage of the blood. They can
however occur in patients who have an electrolyte imbalance.
Bite cells: When aggregates of hemoglobin form in red cells (e.g. Heinz bodies), as they
pass through the spleen mononuclear phagocytes remove the aggregates leaving cells that
look like a bite was taken out of them. They are seen in patients with unstable hemoglobins,
G6PD deficiency and drug induced hemolytic anemias.
Helmet cells: Cells shaped like a helmet. They are derived from repeated mechanical trauma
and found in conditions like DIC and hemolytic anemia.
VARIATIONS IN COLOR:
Hypochromasia: This term indicates cells which have pale staining. This is due to thin cells
and diminished hemoglobin formation. It is often associated with microcytosis and is seen in
iron deficiency, thalassemia and sideroblastic anemia.
Polychromasia: Indicates varying shades of staining. This is due to an increase in cells
which contain RNA and take up the basic (i.e. blue) stain. They are usually immature red
cells such as reticulocytes. This is most often seen in a variety of anemias such as hemolytic
OTHER ABNORMALITIES:
Rouleaux: Rouleaux formation when red cells arranged in stacks like coins. It is often seen
when there is an increase in plasma protein concentrations such as in Myeloma.
Agglutination: Describes the clumping of red cells. Seen in autoimmune hemolytic
anemias.
Howell Jolly Bodies: These are small, well-defined spherical bodies, usually acentrically
situated in the red cell. They stain deep purple and vary in size. It is generally accepted that
they represent chromosomes or nuclear remnants. Since they contain DNA, they give a
positive Feulgen reaction. Howell Jolly bodies are found after splenectomy or when splenic
function is diminished. May also be seen in the presence of a normal spleen, when there is
erythroid hyperplasia or dyserythropoeisis.
Pappenheimer Bodies: Small, dense blue granules which appear at the periphery of
reticulocytes or erythrocytes. They stain blue with Wrights stain and giemsa stain. They can
also be demonstrated using the Perl's Prussian Blue reaction which is used to demonstrate
iron. Pappenheimer bodies consist of ferric iron complexed with protein. They usually
occur when splenic function is absent, e.g. post splenectomy.
Basophilic stippling / Punctuate Basophilia: Appear as rounded granules which are evenly
distributed throughout the red cell. They stain blue-black color and consist of aggregated
ribosomes which do not contain iron. It is important to note that slow-drying of blood films
and subsequent staining may cause ribosomes to aggregate; so this could be an artefact.
They are demonstrated by romanowski stains. Basophilic stippling is seen in certain
conditions such as thalassemia and lead poisoning, so does have diagnostic significance. In
Pyrimidine-5-nucleotidase deficiency, an inherited hemolytic anemia, basophilic stippling is
a notable feature. It is interesting to note that the stippling seen in lead poisoning is related
to the deficiency of the same enzyme.
Siderotic Granules: Siderocytes are red cells containing aggregates of non-heme iron
which can only be demonstrated by the Prussian Blue stain or by electron microscopy.
Usually found in normoblasts in the bone marrow then called sideroblasts; and in
reticulocytes and erythrocytes termed siderocytes. Siderotic granules in reticulocytes
usually disappear as the red cell matures, whereas siderotic granules found in the red cell are
located in the mitochondria and are removed in the spleen. They are often seen in anemias
like sideroblastic anemia where they form a ring around the nucleus of the normoblast and
are called ringed sideroblasts. Siderocytes are seen in decreased splenic function.
Heinz Bodies: Usually single round structures, situated at the cell margin. They cannot be
seen using normal romanowski stains, but are demonstrated with supravital stains such as
methyl violet and in particular methyl green. They consist of denatured, precipitated
hemoglobin bound to the red cell membrane by disulphide bonds. They are produced
through oxidative stress caused by drugs or chemicals in patients with inherited Glucose-6Phosphate Dehydrogenase (G6PD) deficiency. The number of heinz bodies depends on the
nature of the hemoglobin defect and splenic function. They sometimes occur in premature
babies with hemolytic anemia.
Hemoglobin H: These inclusions appear as multiple evenly distributed blue staining bodies
giving a 'golf ball' appearance to the red cell. They are best demonstrated using the
supravital stain Brilliant cresyl blue. Hb H inclusions are caused by the precipitation of beta
globin chains in the absence of alpha chains as seen in alpha thalassemia.
Cabot Rings: Thread-like inclusions which appear in erythrocytes as rings, figure-8 or other
shapes. They stain blue with romanowski dyes. They are thought to be remnants of the
mitotic spindle since they give a positive Feulgen reaction for DNA. They may also contain
histone protein and iron. Cabot rings are rare, but are seen in megaloblastic anemias.
Summary:
Inclusion
Nature
Demonstration
Howell-Jolly bodies
Pappenheimer bodies
Basophilic stippling
Ribonucleoprotein
(aggregated ribosomes)
Romanowski
Protozoa
Romanowski
Micro-organisms
Bartonella bacilliformis
Romanowski
Heinz bodies
Denatured hemoglobin
Supravital staining
Hb H inclusions
Precipitated Hb H
Siderotic granules
Fe+++ in ferritin or
mitochondria
Prussian-blue reaction
Reticulocyte inclusions
Ribonucleoprotein
(aggregated ribosomes)
Supravital staining
ANEMIA
Clinical features:
Breathlessness, pallor, fatigue, headaches, retinal hemorrhages, craving for unusual foods,
occasional enlarged spleen.
Peripheral Blood:
Microcytic / hypochromic red cells.
Anisocytosis
Poikilocytosis: oval and pencil cells, tear drops, occasional target cells
Basophilic stippling in severe anemia.
Bone Marrow:
Not routinely done in iron deficiency anemia.
Erythroblasts: ragged, vacuolated cytoplasm and pyknotic nuclei.
Smaller than normal macrophages with no iron.
Other:
Retic count: usually low for the amount of anemia, but can be raised in early stages. Increase after
treatment has commenced.
Serum iron: low
TIBC: raised
Treatment:
Oral or parenteral iron. Response measured by increase in retics and rise in Hb levels.
MEGALOBLASTIC ANEMIA
Megaloblastic anemia is usually caused by either: vitamin B12 deficiency or folate deficiency.
Pathogenesis:
Thymidylate is a DNA nucleotide. It is formed via two pathways:
(1) The Salvage pathway: Thymidylate is formed from the breakdown of old DNA. This alone
Clinical features:
Anemia often detected before clinical symptoms appear due to the increased MCV.
Main symptoms: weakness, tiredness, shortness of breath, angina, heart failure, weight loss,
glossitis, jaundice, occasional enlarged spleen.
Vit. B12 deficiency: occasional neurologic symptoms (role in myelin)
Laboratory findings:
Full Blood Count:
Hb: decreased
MCV: increased (> 95 fl)
WBC: often low
Platelets: often low
NB: Pancytopaenia (low RBC, WBC and platelets) is a feature of megaloblastic anemia.
Peripheral Blood smear:
RBC: macrocytes (oval macrocytes)
anisicytosis
poikilocytosis: tear drops and oval macrocytes
nucleated red cells
basophilic stippling Indicators of abnormal red cell development
Macrocytic anemias
Megaloblastic bone marrow: Vit. B12 deficiency
Folate deficiency
Disorders of DNA synthesis hereditary, acquired
Non-megaloblastic bone marrow: Accelerated erythropoeisis with polychromasia, normoblasts
in the peripheral blood.
: Increased cell membrane surface area e.g. hepatic disease
GLOBIN
IRON
AMINO ACIDS
PROTOPORPHYRIN
BILIRUBIN
LIVER
UROBILINOGEN (Urine)
STERCOBILINOGEN (Feces)
HEREDITARY SPHEROCYTOSIS
Pathogenesis:
Autosomal dominant
Cells have reduced spectrin content of the membrane skeleton
This causes loss of membrane integrity and therefore, sphere formation
The spherocytes are eventually phagocytosed by macrophages in the liver and spleen i.e.
Extravascular Hemolysis .
Clinical features:
Variable anemia
Jaundice
Pyropoikilocytosis
Stomatocytosis
another surface protein CD59. The underlying reason is the GP1 anchor on the membrane is
missing due to mutation of the PIG A gene.
Clinical Features:
Often occurs after drug induced bone marrow aplasia.
Fatigue
There is usually increased hemolysis at night due to a fall in pH during sleep. Morning urine is
often discolored, hence the name of the condition.
Pallor, jaundice .
Laboratory Features:
Anemia with low Hb
Increased reticulocytes
Low white cell count and platelets, i.e. pancytopenia .
Peripheral Blood smear:
No abnormalities
Increased polychromasia
Other tests:
Increased plasma Hb
Increased methemoglobin
Increased plasma bilirubin
Increased hemosiderin
Coffee colored urine after hemolysis
Sucrose lysis test (concentration gradient test) and Hams acid test for increased lysis due to
complement: Positive .
Complications:
Thrombosis, Iron deficiency, and development of Leukemia.
Therapy:
Bone marrow transplant
Iron therapy
Corticosteroids
Anticoagulant therapy after thrombosis.
HEMOLYSIS CAUSED BY ANTIBODIES
Two main groups of antibodies: IgG and IgM .
The Coombs test investigates antibody-involvement in the destruction of red cells.
Direct Coombs: ? Antibodies bound to cell surface.
Indirect Coombs: ? Antibodies in the serum.
Alloantibodies:
ABO Reactions
Patients receiving incompatible blood.
Hemolysis occurs due to antibodies reacting with the donor red cells.
HDN
Pathogenesis:
Pathogenesis:
The red cells are coated with the antibody.
They are then removed by the spleen, liver and bone marrow.
Macrophages engulf pieces of the cell, resulting in the damaged cells forming spherocytes.
They can also fix complement resulting in hemolysis.
Clinical features:
Weakness, malaise, fever, jaundice, splenomegaly .
Laboratory Findings:
White cell count and platelets: Normal or slightly raised.
Smear: Polychromasia
Nucleated red cells
Spherocytes
Increased reticulocytes .
Coombs: Positive
Serum bilirubin: Increased
Treatment:
Transfusion
Steroids, immunosuppressives
Splenectomy
Cold antibodies
Usually IgM. Do not normally react with red cells in the body unless:
In case of certain infections like Cytomegalovirus and mycoplasma.
Cold agglutination disease: seen in older patients often with underlying lymphoma.
Pathogenesis:
As cells pass through cooler peripheral areas, IgM attaches to red cell surface and activates
complement. The red cells are usually removed by the spleen and liver.
Laboratory findings:
Agglutination is seen on the smear.
Polychromasia and spherocytes.
Cold agglutination test: Positive
Coombs test positive.
Cell count: High MCHC due to agglutination incubate sample at 37 deg.Celsius to remove cold
antibodies.
II. HEMOLYSIS DUE TO ABNORMAL RED CELL METABOLISM:
These anemias are caused by deficiencies of enzymes found in the glycolytic pathway and the
hexose monophosphate shunt.
Pathogenesis:
This is the most common enzyme deficiency of the glycolytic pathway. The others are extremely
rare.
Deficiency results in the failure of the red cell to produce ATP.
Hemolysis occurs because the sodium pump fails to control Na+ influx. This leads to osmotic lysis.
Clinical symptoms:
Inherited disorder
Anemia and jaundice are present from birth. The anemia worsens during an infection.
Often find enlarged spleen.
Laboratory findings:
This disorder does not have any distinctive feature.
Increased serum bilirubin, increased polychromasia and increased reticulocytes point to possible
hemolysis.
No spherocytes
Decreased pyruvate kinase assay is NB.
Therapy:
Transfusions and splenectomy.
G6PD DEFICIENCY
Hemolysis usually occurs after exposure to a drug or an infection, e.g. anti malaria drugs,
sulphonamides, analgesics, eating Fava beans.
This precipitates a hemolytic attack with jaundice, weakness and dark urine.
Laboratory diagnosis:
Anemia (low Hb)
Polychromasia with increased retics.
Heinz bodies and consequent bite cells.
Increased bilirubin and urobilinogen.
Decreased G6PD screen.
III. DRUG INDUCED HEMOLYSIS:
Antibodies can bind to a red cell / drug complex (e.g. Penicillin). Complex removed by spleen.
IV. RED CELL FRAGMENTATION OR MICROANGIOPATHIC ANEMIA:
The anemia is caused by direct damage to the red cell.
Artificial heart valves
Passing through fibrin strands caused by DIC Anemia
Malignancies secreting mucin
Thrombotic thrombocytopenic purpura (TTP).
The main feature of the smear is the presence of Fragmentation.
GLOBIN CHAINS
Hb A
Hb Ac
b A2
Hb F (fetal Hb)
22
22
22
22
% OF TOTAL HB
95
< 3.5
< 3.5
< 1.0
Many abnormal hemoglobins have been discovered, e.g. Hb S in Sickle cell anemia.
Abnormalities are usually caused by amino acid substitutions / deletions / insertions in the globin
chain.
When the substitution causes clinical symptoms the patient has a hemoglobinopathy.
In cases where there is not amino acid substitution, but reduced synthesis the patient has a
thalassemia.
Seen in 5 20% of populations in West and Central Africa. Also in the Mediterranean, Middle East
and India.
These areas correspond to Malaria Areas and it is thought that Sickle cell anemia affords some sort
of protection from malaria (like G6PD deficiency).
Pathogenesis:
Hb S is formed by the substitution of glutamic acid with valine at position 6 of the globin chain.
Red cells wich contain increased amounts of Hb S form sickle cells when deoxygenation takes
place. This is caused by the polymerisation and gelation of the Hb molecule.
Sickle cells have a shortened lifespan.
They are removed by the spleen and liver.
SICKLE CELL TRAIT
These patients have just one abnormal chain.
They have no clinical disease.
Laboratory Diagnosis:
Sickle cells will form if the cells are incubated with Sodium Metabisulphite which deoxigenates the
blood.
Hb Electrophoresis: Hb A: 60 70%
Hb S: 30 40%
Hb F: normal .
SICKLE CELL DISEASE
These patients are homozygous for Hb S, i.e. both chains are abnormal.
Clinical features:
Chronic hemolytic anemia from birth.
Hepatosplenomegaly
Leg ulcers .
Sickle cell crisis:
Occurs after infection or other identifiable cause.
Sickle cells increase in circulation.
Patient suffers extreme pain.
Cells can get caught up in the spleen.
Aplastic crises:
The bone marrow is unable to respond to the anemia and does not produce any new reticulocytes.
Usually seen after parvovirus infection, toxins or drugs.
Laboratory:
FBC: low Hb
normal or slightly increased MCV
increased white cells
increased platelets.
Blood Smear: increased sickle cells (especially during crises)
polychromasia
Howell Jolly Bodies
increased retics.
Hb Electrophoresis: Hb S: 75 95%
remainder is Hb F
Serum bilirubin: slightly increased
X-ray: widening of marrow space.
Treatment:
Prevention of crises.
Transfusion in aplastic crises.
HB S CAN BE FOUND IN COMBINATION WITH OTHER ABNORMAL HB
Hb S / Hb C:
Most common.
Results in similar conditions to sickle cell disease.
Hb S / Hb D and Hb S / Hb E:
This results in a mild condition.
Hb S / thalassemia:
This combination increases the life span of sickle cell patients.
MCV: low
Hb S / thalassemia:
Increases expression of the Hb S gene.
No normal chains are produced.
Severity of disease similar to sickle cell disease.
MCV: low
Hb Electrophoresis: mostly Hb S.
HEMOGLOBIN C AND E
UNSTABLE HEMOGLOBINS
Pathogenesis:
The unstable Hb denature and form Heinz Bodies.
The cells are removed by the spleen causing a hemolytic anemia.
Patients have an enlarged spleen.
Laboratory findings:
Polychromasia and increased retics.
Heinz Bodies
Bite cells
Positive for unstable hemoglobins (precipitate when lysed red cells are incubated with isopropanol).
HEMOGLOBIN M
THALASSEMIA:
Results from abnormal globin chain synthesis.
THALASSEMIA
THALASSEMIA
Clinical:
Rare recessive disorder which is transmitted via the X-chromosome.
It is therefore seen predominantly in males.
Patients suffer from anemia which is prominent from the first year of life.
Laboratory findings:
FBC: decreased Hb
decreased MCV
decreased MCHC
Smear: Microcytic / hypochromic red cells
Often dimorphic picture present
Siderocytes are seen on smear when Iron stain is done
Decreased retics .
Bone Marrow: Erythroid hyperplasia
Ragged and vacuolated normoblasts
Macrophages have adequate iron
Iron stain shows many siderotic granules in normoblasts and many ringed sideroblasts
Serum iron is raised.
Primary or idiopathic:
This is the same disease also called Refractory Anemia with Ringed Sideroblasts.
It is classified under the Myelodysplastic Syndromes.
At least 15% of the normoblasts in the bone marrow must be ringed sideroblasts.
Secondary:
This usually occurs as a result of drugs or toxins.
Alcohol: megaloblasts and ringed sideroblasts.
Anti-tuberculosis drugs: inhibit pyridoxine metabolism which is a co enzyme to ALA synthase.
Chloramphenicol
Lead: impairs heme synthesis at a variety of steps. Formation of ringed sideroblasts. (Basophilic
stippling is another feature of lead toxicity.)
Vitamin B6 deficiency.
Therapy:
Pyridoxine, B12 and Folate.
Withdrawal of offending drug.
OTHER ANEMIAS
ACUTE BLOOD LOSS ANEMIA
Acute blood loss: Depletion of blood volume is important. The blood constitution remains
unaltered, until plasma volume is replaced, causing dilution of the hematocrit.(e.g. IV fluid given)
Chronic (gradual) blood loss like a bleeding ulcer: Body compensates for volume loss quickly but
loss of red cells will affect O2 delivery to tissues.
Clinical features:
Depend on amount of blood loss.
< 20% loss: no noticeable symptoms
30 40% : fall in cardiac function and onset of shock.
> 40% : organ damage and often death.
The body will attempt to replace the lost blood volume by increasing the plasma volume reduced
red cell mass and anemia.
Laboratory features:
Hematocrit : No change until plasma volume is replaced. Then it will drop.
White cells: Increase after a few hours;
Platelets: Dramatic increase.
Erythropoeitin: Increased
Erythropoeitic response: Slow process. Progenitor cells will mature over 2 3 days. By tenth day
retics will peak.
Retics appear earlier in response to erythropoeitin.
NB: Polychromasia is seen on the smear.
Therapy:
Maintain blood volume to prevent shock.
Blood transfusions, electrolyte solutions, saline.
Iron therapy if stores depleted.
APLASTIC ANEMIA AND PURE RED CELL APLASIA
Patients with aplastic anemia have:
pancytopenia,
hypocellular bone marrow .
Pathogenesis:
Two mechanisms:
1. Depletion of hemopoeitic stem cells by an agent or event which kill the stem cells, e.g.
radiation, chemo, benzene.
2. Suppression of proliferation of stem cells by an immune mechanism. (antibodies or T / Bcells)
Causes of this condition:
Genetic or congenital: Fanconis anemia. These children usually have other abnormalities like
stunted growth and limb deformities.
Toxins or radiation
Drugs: Those that damage resting stem cells and those that affect cells during their cycle. (cytotoxic
drugs)
Infection: Hepatitis, Infectious mononucleosis, parvovirus.
Pregnancy.
Clinical findings:
Weakness with cardiovascular and cerebral symptoms.
Infections: low white cell count
Bleeding: low platelets
Pallor, petechiae.
Laboratory findings:
FBC: Pancytopenia: low white cells, platelets, Hb
Smear: Normocytic / normochromic anemia
Very low reticulocytes
Few white cells and platelets.
Bone Marrow: Hypocellular
Trephine : Almost total replacement by fat spaces.
The remaining cells are usually plasma cells, lymphocytes and mast cells.
Treatment:
Very serious condition.
Bone marrow transplant only hope of cure.
Androgens, immunosuppression.
Notes from Cape Peninsula University of Technology (Fmr. Cape Technikon), 2002, ND., B.Tech.:
Biomedical Technology.
More notes at:
http://www.scribd.com/document_collections/2374607