RED CELL

DISORDERS

DR. ASHUTOSH KUMAR

PROFESSOR, PATHOLOGY
TMMC&RC, MORADABAD
 yolk sac : 18th day after fertilization which include
RBC (For transport oxygen to developing tissue
early in gestation) and a few macrophages..

HEMATOPOIESIS
aorta-gonads-mesonephros (AGM) region
(primitive erythropoiesis): wide range of
hematopoietic cells including lymphocytes;

The hemoglobin in these

Embryonic red cells have cells consists of the
a megaloblastic embryonic varieties,
appearance with coarse
Gower 1 (ζ2ɛ2), Gower 2
clumped chromatin.
(α2ɛ2), and Portland(ζ2γ2)
 Liver (to a lesser degree in the spleen,
kidney, thymus, and lymph nodes also
later on): third month of fetal life,
beginning of adult pattern of
hematopoiesis with myeloid cell
predominant…

Cont…
Bone marrow : predominant site by
sixth month of gestation..

For T cells thymus and for B cells

Lymph nodes and spleen continue as
an important site for differentiation.
Hematopoiesis:
Chronology
HEMATOPOIESIS-
PRODUCTION OF BLOOD CELLS

SITE
• EMBRYO-YOLK SAC,AGM:UPTO 10TH
-12TH WK
• HEPATIC-6TH TO 30TH WK
• MYELOID-4TH MONTH,CHILDHOOD-FLAT
BONES,LONG BONES; ADULTS-ENDS OF
LONG BONES AND FLAT BONES
 • includes tissues and organs involved in
the proliferation, maturation, and
destruction of blood cells. which
ADULT includes
HEMATOPOIETIC – Bone marrow
TISSUE – thymus
– Spleen
– Lymph nodes
 BONE MARROW: COMPOSITION
Trabecula: projection of calcified bone extending from cortical
bone into the marrow space; it provides support for marrow cells.)

Bone Marrow: Blood-forming tissue located between the

trabeculae which is composed of two major compartments:

Hematopoietic compartment: is the site

Vascular compartment: composed of of formation and maturation of blood
the nutrient artery, periosteal arteries, cells. It includes
central longitudinal vein, arterioles, and • hematopoietic cells and
sinuses. • stromal cells.
Bone marrow maturation
Bone marrow
Vascular compartment
 The stroma is composed of
three major cell types:
reticular cells
macrophages, (fibroblasts), adipocytes.
and

Stroma
Function: it provides Favorable
microenvironment, 3D
scaffolding, Cytokines, for
sustained proliferation of
hematopoietic cells.
 Macrophages

• Functions as
– Phagocyte:
• clears dead debris and extruded nucleus etc,
• ? supply the developing erythroblasts with iron and
cytokines
– secretes hematopoietic cytokines:
• colony-stimulating factors .
Macrophages stain acid phosphatase positive.
 Reticular cells

• located on the abluminal surface of the vascular sinuses and

send long cytoplasmic processes into the stroma.
• These cells also produce reticular fibers (visible on silver
staining), which contribute to the three-dimensional supporting
network.
Reticular cells are alkaline phosphatase positive.
 Adipocytes

most likely develop from reticular cells that

synthesize fat.

These cells mechanically control the volume of

bone marrow in which active hematopoiesis takes
place.

They also provide steroids that influence

erythropoiesis and maintain osseous bone integrity.
 Osteoblasts

• Lining endosteum : involved in the formation of calcified bone

and provide a niche for resting hematopoietic stem cells.
• large cells (up to 30 μm in diameter) that resemble plasma cells
except
– paranuclear halo appears away from the nucleus.
– Cytoplasm less basophilic,
– Nucleus has finer chromatin pattern.
• Normally found in groups.
• The cells are alkaline phosphatase positive.
Osteoblasts
 Osteoclasts

Related to macrophages that are involved in

resorption and remodeling of calcified bone.

They are multinucleated and have granular

cytoplasm up to 100 μm in diameter.

Vs megakaryocytes : discrete and not

multilobulated nucleus.
Osteoclasts
Hematopoietic • Hematopoietic cells are arranged in distinct niches
within the marrow cavity.
Compartments: • Erythroblasts: 25–30% of the marrow cells, are
Erythroid produced near the sinuses, in erythroblastic islands
ORIGIN AND MATURATION OF BLOOD
CELLS
ERYTHROCYTE MATURATION

hemoglobin
synthesis:
numerous free
ribosomes

increasing
hemoglobin,
decreasing
ribosomes,
decreasing
nuclear nucleus
activity extruded
Downloaded from: StudentConsult (on 29 January 2009 03:46 PM)
© 2005 Elsevier
DEFINE AND
CLASSIFY
ANEMIA
DEFINITION…

Anemia is defined as a reduction in the oxygen-

transporting capacity of blood, resulting from a
decrease in the red cell mass to subnormal levels.

i.e. Hb < 2 S.D.  below the mean of an age- and

sex-matched reference range
 Decreased tissue oxygen tension in anemia

Increased
erythropoietin (kidney)

Compensatory Extramedullary
erythroid hyperplasia hematopoesis
of bone marrow (liver, spleen, kidney)

Increased
reticulocytes in
peripheral smear
 EXTRAMEDULLARY
HAEMATOPOIESIS
When hyperplasia of the marrow cannot
compensate the physiologic demand of tissues,

Production starts from hematopoietic tissue

other than bone marrow which are organs that
were active in the fetus, principally the liver and
spleen.

Results in Organomegaly +
WHY ?

erythrocyte loss or destruction

exceeds the maximal capacity of bone
marrow erythrocyte production or

the bone marrow erythrocyte

production is impaired.
C/F:
 • Ramlal a 50/M, c/of yellowish discoloration
CASE of eyes and had dark urine. His CBC
revealed a hemoglobin of 3.1 g/dl. Given this
clinical description, consider what
laboratory tests should be ordered to
assist in diagnosis……
LABORATORY
APPROACH…
Hemoglobin estimation

Morphology of RBCs.: size, color, and shape

RBC Indices:

• Mean cell volume (MCV): the average volume per red cell, expressed in
femtoliters (cubic microns)
• Mean cell hemoglobin (MCH): the average 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
• Red cell distribution width (RDW): the variation of red cell volume

Reticulocyte count
 RETICULOCYTES:
Immature, anuclear erythrocytes containing organelles and residual
ribosomes for hemoglobin synthesis are known as reticulocytes.

Usually spend 2–3 days in the bone marrow and an additional day in the
peripheral blood before losing their RNA and becoming mature
erythrocytes.

Indicates the degree of effective bone marrow activity

Can be used for monitoring anemia and response to therapy, and also in
classifying the pathophysiology of anemia.

Can be identified as polychromatophilic erythrocytes(due to the presence

of basophilic ribosomal RNA)
IDENTIDY THESE CELLS
IDENTIFY THE STAIN…

Normal value: 0.5–2.5% or 18–158 X 109/L.

 • RBC count is 0.71X1012/L and his
reticulocyte count is 22%.
Q:1 – Calculate his absolute reticulocyte
count.
– Is this count increased, decreased, or
normal?
ANS: 1
• Calculate the absolute reticulocyte count?
WHAT IS RPI ?
• In a stimulated marrow, Hematocrit is inversely proportional
to reticulocyte maturation time in peripheral blood

• If HCT= 0.35L/L :: retic maturation time = 1.5days

• If HCT= 0.25L/L :: retic maturation time = 2days
• If HCT= 0.15L/L :: retic maturation time = 2.5days
• Normal is 1 day..

RPI(Retic Patient’s hematocrit X Reticulocyte %

production --------------------------------------------------------
index 0.45 L/L X Reticulocyte maturation time in days
MEAN CORPUSCULAR VOLUME (MCV)

• Indicative of volume of red cell

• Expressed as femtoliter
• single most useful measurement
Hct (%)
MCV = ------------ x 10
RBC (million/ul)

Normal range = 86 – 98 fl

A patient has a hematocrit of 0.45 L/L and an RBC count of 5.0 X

1012/L.
MEAN CORPUSCULAR
HEMOGLOBIN (MCH)
• Indicate the amount of hemoglobin per red cell
• Expressed as Pico gram

Hb (g/dl)
MCH = ----------------- x 10
RBC (million/ul)

Normal range = 28 – 33 Pg
• A patient has a hemoglobin concentration of 15 g/dL and an RBC
count of 5.2 X1012/L.
 MEAN CORPUSCULAR HEMOGLOBIN
CONCENTRATION (MCHC)
• Denotes the average concentration of hemoglobin
in the red cell
• Expressed as % or g/dl

Hb (g)/dl
MCHC = -------------x 100
Hct (%)

• Normal range = 32 – 36 g/dl

• A patient has a hemoglobin concentration of 15.0 g/dL and a hematocrit
of 0.45 L/L.
 R D W:

1. GIVES AN IDEA ABOUT DEGREE OF

ANISOCYTOSIS
2. CALCULATED BY DIVIDING THE
STANDARD DEVIATION OF ERYTHROCYTE
SIZE BY MCV.
3. RDW MAY COME TO BE NORMAL IF
BOTH THE S.D. AND MCV ARE HIGH,
 Urinalysis
• Bilirubin / urobilinogen
• Haemoglobin in urine
• M/E for red cells – Haematuria

Stool
NON –
HAEMATOLOGIC • Occult Blood
INVESTIGATIONS • Examination for OVA / cysts
• (N. Americana / A. Duodenale)

Others
• BUN, S. CREATININE
• S. BILIRUBIN, SGOT, SGPT, S. ALK
PHOSPHATASE
• BARIUM STUDIES FOR GIT LESIONS
• USG ABDOMEN – KIDNEYS, LIVER,
SPLEEN
• CT / MRI – SUSPECTED MALIGNANCIES.
FURTHER INVESTIGATIONS…

Serum iron indices :

transferrin ferritin
serum iron, TIBC,
saturation, and concentrations

plasma unconjugated bilirubin, haptoglobin, and lactate

dehydrogenase levels, which are abnormal in hemolytic anemia;

serum and red cell folate and vitamin B12 concentrations,

hemoglobin electrophoresis, which is used to detect abnormal

hemoglobin; and

Coombs test,
FUNCTIONAL
CLASSIFICATION

Survival Proliferation Maturation

defects, defects, and defects
F U N C T I O N A L C L A S S I F I C A T I O N O F A N E M I A
ETIOLOGICAL CLASSIFICATION
OF ANEMIA

• Blood Loss
– Acute: trauma
– Chronic: gastrointestinal tract lesions,
gynecologic disturbances

• Increased Destruction (Hemolytic

Anemias)
– Intrinsic (Intracorpuscular) Abnormalities
– Extrinsic (Extracorpuscular) Abnormalities
INTRINSIC (INTRACORPUSCULAR) ABNORMALITIES
Hereditary
Acquired
Membrane abnormalities
• Membrane skeleton proteins: Membrane defect:
spherocytosis, elliptocytosis PNH
• Membrane lipids:
abetalipoproteinemia
Enzyme deficiencies
• Enzymes of hexose monophosphate shunt:
G6PD, glutathione synthetase
• Glycolytic enzymes: pyruvate kinase,
hexokinase

Disorders of hemoglobin synthesis

• Structurally abnormal globin synthesis
(hemoglobinopathies): sickle cell anemia, unstable
hemoglobins
• Deficient globin synthesis: thalassemia syndromes
EXTRINSIC (EXTRACORPUSCULAR)
ABNORMALITIES

Isohemagglutinins:
Antibody-mediated transfusion reactions, immune hydrops
(Rh disease of the newborn)
Autoantibodies: idiopathic
(primary), drug-associated, SLE

Mechanical trauma MAHA, TTP, DIC, HUS

to red cells Defective cardiac valves

Infections: Malaria, kala a zar

 Impaired Red Cell Production
Disturbed proliferation and differentiation of stem cells:
• Aplastic anemia, pure red cell aplasia
Disturbed proliferation and maturation of erythroblasts
• Defective DNA synthesis: deficiency or impaired use
of vitamin B12
• and folic acid (megaloblastic anemias)
• Anemia of renal failure (erythropoietin deficiency)
• Anemia of chronic disease (iron sequestration,
relative erythropoietin deficiency)
• Anemia of endocrine disorders
• Defective hemoglobin synthesis
• Deficient heme synthesis: iron deficiency,
sideroblastic anemias
• Deficient globin synthesis: thalassemias
Marrow replacement: MDL, Leukemias
Marrow infiltration (myelophthisic anemia): metastatic
neoplasms, granulomatous disease
 Morphological
classifcation

Anaemia

Microcytic Normocytic Macrocytic

Normal /Increased ferritin Decreased ferritin

chronic disease iron deficiency

thalassemia
hemoglobinopathies
sideroblastic anemia
lead intoxication
porphyrias

MICROCYTIC ANEMIA
MICROCYTIC
• RDW normal • RDW increased
1. Heterozygous 1. Iron deficiency
thalassemia (minor) 2. Homozygous
2. Chronic disease thalassemia
3. Hemoglobin E trait 3. Hb S/ Beta Thal
4. Hb H disease
5. Hemolytic anemia
with schistocytes
Normocytic anemia
>2 RPI/Corrected <2
retic count

Survival Defect
- hemolysis Non-Megaloblastic
- hemorrhage (Proliferation, maturation
and/or survival defects)
Nuclear Maturation Defect
- chronic liver disease
(megaloblastic)
- alcohol abuse
- B12 deficiency
- endocrinopathy
- folate deficiency
- aplastic anemia
- drug induced
- congenital
- myelodysplasia
 WHAT IS RPI ?
In a stimulated marrow, Hematocrit is inversely proportional
to reticulocyte maturation time in peripheral blood

• If HCT= 0.35L/L :: retic maturation time = 1.5days

• If HCT= 0.25L/L :: retic maturation time = 2days
• If HCT= 0.15L/L :: retic maturation time = 2.5days
• Normal is 1 day..

Patient’s hematocrit X Reticulocyte %

RPI(Retic
production --------------------------------------------------------
index 0.45 L/L X Reticulocyte maturation time in days
SO OUR CASE:

0.08X22%
• Corrected retic count =-------------- = 3.9
0.45
0.08X22%
• RPI= -------------- = 1.56
0.45 X2.5
 NORMOCYTIC
RDW NORMAL RDW INCREASED

1. Acute hemorrhage 1. Immune hemolytic anemia

2. Splenic pooling 2. Early iron, Vit B 12 or folate
3. Chronic disease 3. deficiency
4. Chronic leukemia 4. Sideroblastic anemia
5. Myelofibrosis
6. Sickle cell anemia/trait
7. Chronic liver disease
8. Myelodysplastic syndrome
Macrocytic anemia
RPI/Corrected <2
retic count
Proliferation Defect
A. Marrow damage or
replacement or
>2 suppression by
drugs/toxins
B. Stem cell defects
C. Chronic renal disease
D. Endocrine hypofunction
Survival Defect E. Chronic infections or
A. Hemolysis inflammation
B. Hemorrhage F. Liver disease
G. Malignancy
MACROCYTIC
RDW NORMAL RDW INCREASED

1. Chronic liver disease 1. Immune hemolytic

2. Aplastic anemia anemia with marked
reticulocytosis
3. Chemotherapy
2. B12 or folate deficiency
4. Alcohol ingestions
3. CLL with high lymph
5. Antiviral
count
medications
4. Cytotoxic chemotherapy
5. Chronic liver disease
6. Myelodysplastic
ABOUT THIS CASE:
morphologically classified as macrocytic, hyperchromic.

Functionally, it is classified as a survival defect.

Looks like the cells are being destroyed by an immune

hemolytic process. The bone marrow has increased
production of cells as the reticulocyte count is increased.
CASE SUMMARY….
• The patient Ramlal had a severe macrocytic anemia which
developed slowly over time, which gave his body the opportunity
to adapt to a low hemoglobin level. Otherwise the patient would
have gone into shock.
• The yellowness of the eyes suggests a high bilirubin
concentration, which is typical of a hemolytic anemia. The patient
had a test that revealed antibodies and complement on his red
blood cells. This supports the diagnosis of an immune hemolytic
anemia. The presence of spherocytes supports this diagnosis
because they are a sign that the spleen has removed
antibody/antigen complexes from the cell membrane. Note that
the MCHC is increased, which is typical of spherocytes.
• The high reticulocyte count and presence of polychromatophilic
erythrocytes indicate the bone marrow is responding
appropriately by increasing output of erythrocytes. The high
reticulocyte count is probably responsible for the increased MCV.
ANSWER:2
1. Calculate the erythrocyte indices. Does the
information given suggest acute or chronic blood
loss? What is the significance of the RDW?

Explanation: MCV 113 fL; MCH 43.7 pg; MCHC 38.8

g/dL. The case history suggests chronic blood loss.

The hemoglobin is very low, and the patient probably

would be in shock if he had lost this much blood
suddenly. The RDW suggests significant anisocytosis.
BLOOD SMEAR OF RAMLAL
REVEALED..

• marked
spherocytosis.
– Explain the
importance of this
finding.
– Explain his abnormal
indices.
 EXPLANATION….
• Spherocytes are cells that have
lost membrane. They are
significant here because they
indicate a hemolytic anemia.
• The indices are all elevated:
– MCV 113 fL: may be due to the
high reticulocyte count.
– MCH 43.6 pg: Is elevated due
to the presence of these large
cells that are able to hold more
hemoglobin than a smaller cell.
– MCHC 38.8 g/dL: elevated due
to the marked spherocytosis.
• What laboratory test is the least
invasive and most cost efficient to
evaluate erythrocyte production in the
presence of anemia?