INTRODUCTION TO

ANEMIAS
DEFINITION
 A decrease in the oxygen-carrying capacity of the
blood
 A reduction, from the baseline value, in the
following:
 Total
number of RBCs
 Amount of circulating hemoglobin

 RBC mass

 A decrease of RBCs, hemoglobin and hematocrit

below the reference range.
CLINICAL FINDINGS
 Patient history
 Diet

 Ingestion
of certain drugs
 Exposure to chemicals

 Pica

 Physical Examination
 Reaction to physical activity
 Appearance

 Heart rate and blood pressure.

PHYSIOLOGIC ADAPTATION TO
ANEMIA (HYPOXIA)
 Release of erythropoietin
 Increased heart rate, respiratory rate, and cardiac
output
 Increase in 2,3-BPG.
MECHANISM OF ANEMIA
 Erythropoiesis-associated
 Acute Blood Loss and Hemolysis
REQUIREMENTS FOR ADEQUATE,
FUNCTIONAL RBC PRODUCTION
 Normal progenitor cells
 Nutritional factors such as iron, vitamin B12 and
folate
 Normal globin synthesis
 Normal heme synthesis
 Normal erythropoiesis.
ERYTHROPOIESIS-ASSOCIATED
 Ineffective Erythropoiesis
 Erythroid progenitor cells are defective
 Defective progenitor cells are destroyed in the bone marrow

 Megaloblastic anemia, thalassemia, sideroblastic anemia.

 Insufficient Erythropoiesis
 Decrease in the number of precursor cells
 Decrease in RBC production

 Decrease in iron supply, EPO

 Autoimmune reaction or infection

 Malignant cells.
ACUTE BLOOD LOSS AND HEMOLYSIS

 Traumatic injury
 Premature hemolysis
 Intrinsic factors
 Defects in RBC membrane
 Defects in RBC enzyme system

 Hemoglobin defects

 Extrinsic factors
 Antibody-mediated process
 Mechanical fragmentation

 Infection-related
 LABORATORY DIAGNOSIS
 Automated Complete Blood Count
 RBC count
 Hemoglobin
 Hematocrit
 Blood indices
 Red Blood Cell Distribution Width (RDW)
 WBC count
 Platelet
 Reticulocyte count
 RBC Histogram
 Peripheral Blood Film Examination
 Bone Marrow Examination
IMPORTANCE OF MCV
RED CELL DISTRIBUTION WIDTH
 The coefficient of variation of RBC volume
 It measures the variation in volume of RBCs
 Expressed in percentage:
INTERPRETATION OF RBC HISTOGRAM
Microcytic Anemia
Macrocytic Anemia
RDW and MCV
RETICULOCYTE COUNT
 Assess bone marrow’s ability to increase RBC
production
 Production of RBC is increased in response to
anemia due to RBC destruction or blood loss
 Increased reticulocyte count can be observed in:
 Acuteblood loss
 Hemolytic anemias.
PRINCIPLE
 Whole blood is incubated with supravital stain (new
methylene blue). The vital stain causes the ribosomal and
residual RNA to coprecipitate with the few remaining
mitochondria and ferritin masses in living young erythrocytes to
form dark-blue clusters and filaments (reticulum).
 Smears of this mixture are then prepared and examined. The
number of reticulocytes in 1000 red blood cells is determined.
This number is divided by 10 to obtain the reticulocyte count in
percent.
SPECIMEN

 Whole blood that is anticoagulated with either

EDTA or heparin is suitable.
 Capillary blood drawn into heparinized tubes or
immediately mixed with stain may also be used.
 Red blood cells must still be living when the test is
performed therefore it is best to perform it
promptly after blood collection.
 Blood may be used up to 8 hours after collection.
 Stained smears retain their color for a prolonged
period of time.
REAGENTS, SUPPLIES AND EQUIPMENT

1. Commercially prepared liquid new methylene

blue solution. It should be stored in a brown
bottle. If precipitate is a problem on the
smear, the stain should be filtered prior to use.
2. Microscope slides
3. Microscope
4. 10 x 75 mm test tubes
5. Pasteur pipets (with bulb if pipets are glass)
6. Capillary tubes
PROCEDURE
Preparation of smears
1. Add 3-4 drops of new methylene blue solution to 3-4
drops of thoroughly mixed EDTA anticoagulated
blood to a glass test tube 10 x 75 mm.
2. Mix the contents by gently shaking and allow to
incubate at room temperature for a minimum of 10
minutes.
3. At the end of 10 minutes, gently mix the blood/stain
solution.
4. Using a capillary tube, place a drop of the mixture
on each of three slides near the frosted edge as you
would when making a peripheral smear.
5. Using the wedge smear technique, make acceptable
smears not too thick or thin.
6. Allow to air dry. (Do not blow to hasten to drying.)
counting The Reticulocytes Cells:
1. Place the first slide on the microscope stage and, using
the low power objective (10x), find an area in the thin
portion of the smear in which the red cells are evenly
distributed and are not touching each other. Carefully
change to the oil immersion objective (100x) and
further located an area in which there are
approximately 100 red cells per oil immersion field.
Do this by finding a field where the cells are evenly
distributed and mentally divide the field into 4
quadrants. Count the cells in 1 quadrant. If there are
about 25, you are in a good area. There will be a lot
of open space between the red cells.
2. Be sure to count all cells that contain a blue-staining
filament or at least 2 or more discrete blue
aggregates of reticulum in the erythrocyte.
3. Count 1000 red cells in consecutive oil immersion
fields. Record the number reticulocytes seen.
4. You may count 500 cells on two slides each. They
should agree within ± 15% of each other. If they do
not, repeat the reticulocyte count on the third smear.
5. Calculate the result as follow:
RETICULOCYTE
 Normal value :
 ADULT:
 0.5 to 1.5/100 red blood cells (or, 0.5 to 1.5%)
 Absolute count : 25 to 75 X 109/L
 Newborn (0-2 weeks):
 2.5-6.0%
 Normal Reticulocyte Index :
 1-3%
RETICULOCYTE EVALUATION
Retics (%)
Absolute Reticulocyte Count = X RBC Count
100

Retics (%) x Hct.

Corrected Reticulocyte Count =
45

Corrected Retic Count

Reticulocyte Production Index =
Maturation Time
MATURATION TIME TABLE

Maturation Hematocrit
Time %
1 day 45
1.5 days 35
2 days 25
3 days 15
REFERENCE RANGE
ABSOLUTE RETICULOCYTE COUNT 25 – 75 x 109/L

RETICULOCYTE PRODUCTION INDEX >3 IF ANEMIC

RETICULOCYTES AND MCV
PERIPHERAL FILM EXAMINATION
 Evaluation of RBC morphology
 Verification of automated analyser results (QC)
 Identification of abnormal RBCs

RBC DIAMETER = 6 – 8 μ
Normal RBC’s
 Round, elastic, non-
nucleated, bi-concave discs
 Many RBCs have an area of
central pallor which covers
about one-third of the cell.
 The pallor occurs as a result
of the disc-shaped cells
being spread on the slide.
 Normal RBC’s

 Average diameter of
7.2 microns with a
range of 6-9 microns,
almost the same size as
the nucleus of a small
lymphocyte,
Critical area 10x
 A view of the "critical
area" using the low
power (10X) objective is
shown here.
Critical area 100x
 Once the correct area
has been located on low
power, switch to oil
immersion
 Notice the red cells are
lying singly with
occasional doublets.
Too thin
 The area shown in this field
is too thin for accurate red
cell morphology evaluation.
 The cells have large spaces
between them, show no
central pallor and many are
somewhat square, showing a
"cobblestone effect."
Too thick
 These cells are in an area which
is too thick, and should not be
used for red cell morphology
assessment.
 Some of the cells appear to be
stacked like coins because of
the large number of cells
present in this section of the
slide.
 The morphology seen in the too thin and too thick
areas of the smear is referred to as artificial
morphology.
Size Variation
Size variation
 Red blood cells can vary in size from smaller than
normal, microcytes, to larger than normal,
macrocytes.
 When red cells of normal size, microcytes and
macrocytes are present in the same field, the term
anisocytosis is used.
Normal size

 Size of normal RBC is

almost the size of the
nucleus of the
lymphocyte.
Microcyte

 Smaller than a nucleus

of the lymphocyte,
central pallor is greater
than 1/3 of the cell
Microcyte, increased central pallor
Microcyte, normal Hb content
Microcytes
Macrocyte (megalocyte)
 Diameter of 9-14
microns (1.5 - 2 times
larger than normal red
cells)

 MCV is 100 cubic

microns or more.
Megalocytes
 Megalocytes are the result of decreased DNA
synthesis, frequently due to vitamin B12 and/or
folic acid deficiencies.
 Decreased DNA synthesis causes the nucleus in the
developing red cells to mature at a slower than
normal rate.
 Since hemoglobin production is not affected, the
mature red cell is larger than normal
Macrocytes
 Pseudomacrocytes

 Appears larger than the

lymphocyte but in contrast to
megalocytes has an area of
central pallor.
 Size is the result of
exaggerated flattening and
thus the presence of the central
pallor.
 In patients with cirrhosis of the
liver, obstructive jaundice, post
splenectomy.
Psudomacrocytes
 Summary
• True macrocytes (megalocytes). Increased MCV, MCH
• Pseudomacrocytes. Normal MCV, MCH
Anisocytosis
 Increased variation in size of the red cell population
present on a blood smear.
 Normal, small and large cells can be seen in one
field.
 Normal MCV, high RDW
 As the severity of the anemia increases, the amount
of significant anisocytosis present also increases.
Anisocytosis
Anisocytosis
RBC Color
RBC Color
 Erythrocytes, when spread on a glass slide, show
varying degrees of central pallor
 This central pallor is related to the hemoglobin
concentration present in the red cells.
RBC Color
 The central area (1/3 of the cell) is white, while buff-
colored hemoglobin is visible in the outer 2/3 of the cell.

 The MCHC (32-36 gm/dl) is the index value which is used

to verify the presence of adequate hemoglobin
concentration in the cells visible on the peripheral smear.
RBC Color
 A decreased amount of hemoglobin is referred to as
hypochromasia or hypochromia.

 MCHC values of 30% or less reflect this condition.

 Hyperchromasia and hyperchromia, refer to a

hypothetical situation rather than an actual occurrence.
RBC Color
 Cells located in the "too thin" portion of the smear
often appear to be "hyperchromic".
 Megalocytes (macrocytes) are normochromic.
Normochromic cells
Hypochromic cells
Hyperchromia
Hypochromia
Hyperchromasia
Polychromasia
Poikilocytosis
Poikilocytosis
 Variations in shape.
Grading system
 1+ = 2 – 4 /OIF
 2+ = 5 - 7
 3+ = 8 - 10
 4+ = >10
 The terms few, moderate, many, and marked may
be substituted for the 1+ - 4+ grading system.
 Acanthocytes

 3-12 thorn-like projections

irregularly spaced around the cell.
 Smaller than normal and have
little or no central pallor.
 Acanthocytes have an excess of
cholesterol
 Large numbers of these cells on a
smear can be of diagnostic
significance.
 Acanthocytes
 Abetalipoproteinemia
 Hereditary
acanthocytosis,50 – 100%
of blood cells.
 Alcoholic cirrhosis
 lipid disorders
 splenectomy
Acanthocyte
 Codocyte
 Target cells are thin-
walled cells showing a
darkly-stained centre
area of hemoglobin
which has been
separated from the
peripheral ring of
hemoglobin.
Codocyte
 Codocytes appear in conditions which cause the
surface of the red cell to increase
disproportionately to its volume.
 This may result from a decrease in hemoglobin, as in
iron deficiency anemia, or an increase in cell
membrane.
Codocyte
 Thalassemias, Hb C disease, post splenectomy,
obstructive jaundice.
Dacrocyte

 Dacryocytes are
pear-shaped or
teardrop shaped
cells.
 myelofibrosis/myeloi
d metaplasia,
Drepanocytes
 Drepanocytes or sickle cells
are formed as a result of the
presence of hemoglobin S in
the red cell.
 As the red cell ages, it
becomes less flexible or
deformable and becomes
rigid as it passes through the
low oxygen tension
atmosphere of the small
capillaries in the body.
 In the absence of oxygen,
hemoglobin S polymerizes into
rods, causing the sickle cell
shape.
 Sickle cells can be somewhat
pointed at the ends,
 Most sickled cells can
revert back to the
discoid shape when
oxygenated.
 About 10% of sickled
cells are unable to
revert back to their
original shape after
repeated sickling
episodes.
Echinocyte (Urchin)

 Echinocytes are
reversible,
 The projections are
rounded and evenly
spaced around the
cell.
 Acanthocytes have
irregularly spaced
thorn-like projections.
Echinocyte

 Uremia,
 Following heparin
injection,
 Pyruvate kinase
deficiency.
 Artificial
Elliptocytes

 Elliptocytes can vary

in appearance from
slightly oval to thin
pencil-shaped forms.
Less than 1% of red
cells in normal blood
are oval.
Elliptocytes

 Hereditary
Elliptocytosis
 Thalassemia,
megaloblastic
anemia, iron
deficiency.
Elliptocytes
Elliptocytosis
 Keratocytes

 Keratocytes are cells

which have been
damaged due to
contact with fibrin
strands.
 Intravascular
coagulation
 Microangiopathic
hemolytic anemia
 Glomerulonephritis
 Rejection of renal
transplants.
 Shistocytes
 Schistocytes are red cell
fragments
 Formed when fibrin
strands come in contact
with circulating red cells.
The strands cut a small
piece from the original
cell.
 Spherocytes
 Cells which have a
decreased surface-to-
volume ratio.
 Cell is thicker in diameter
than normal red cells
 They appear to be round,
darkly-stained cells without
central pallor.
 Hereditary spherocytosis
 Immune hemolytic anemia
 Severe burns
 In-vitro prolonged storage of blood
Stomatocytes
 Cup-shaped
erythrocytes which
have an elongated or
slit-like central pallor.
 Hereditary
stomatocytosis,
neoplastic disorders,
liver disease and rh
null disease, in-vitro
change in ph
Rouleaux & autoaglutination
 Forms of poikilocytosis describing a group of cells.
 May be true and false formation
True Rouleaux
True Rouleaux
 Most of the red cells, in the proper viewing area, are
stacked together like coins.
 Four or more cells make up each formation, leaving much of
the field empty of cells (increased white space).
 Rouleaux is clinically significant when increased globulins
are present, as in multiple myeloma.
False rouleaux
True rouleaux
Autoagglutination

 Cells clumping
together rather than
stacked like coins.
 Autoagglutination is
caused by the
presence of antibody
in the plasma.
THANK YOU!