Chapter 8

Routine red cell analysis: red cell indices and

morphological examination

Medical Laboratory Sciences

Dr. Ali Abdelfattah
 Headlines

➢ Complete blood count

• RBCs, WBCs, and platelets
• RBC indices
➢ Reticulocyte count
➢ Erythrocyte sedimentation rate
➢ Blood film analysis
• Blood smear preparation and staining
• Red blood cell morphology
• Abnormally shaped red blood cells
• RBC inclusions
• RBC arrangements
➢ BM examination
 Erythropoiesis assessment

❑ Under normal conditions the production and

survival of blood cells is a highly regulated process

❑ Quantitative and/or qualitative haematological

abnormalities may result when there is an imbalance
between cell production and/or survival

❑ Erythropoiesis can be assessed clinically by

1. Complete blood count (CBC) on peripheral blood
2. Blood film examination (peripheral blood smear),
morphological assessment
3. Bone marrow (BM) examination
 Complete blood count (CBC)

❑ CBC (haemogram) is a ▪ RBC count

▪ Haemoglobin
blood test used to evaluate ▪ PCV
blood cells that circulate in ▪ MCV
blood and gives ▪ MCH
information about overall ▪ MCHC
▪ RDW
health and detect a wide ▪ Platelet count
range of haematological ▪ MPV
disorders ▪ WBC count
▪ WBC differential count
• Neutrophils
• Lymphocytes
• Monocytes
• Eosinophils
• Basophil
 RBC Count, Hb, HCT
➢Red Blood Cell (RBC, Erythrocyte) count – total number of red
cells reported in 106/μL or 1012/L
Reference Interval: M: 4.2-6.0 X 106/μL, F: 3.8-5.2 X 106/μL
Newborn: 4.2-6.5 X 106/μL
➢Haemoglobin (Hb) – photometric measurement of Hb
concentration in red cells reported in g/dL
Reference Interval: M: 13.5-18.0 g/dL, F: 12.0-15.0 g/dL
Newborn: 16.5-21.5 g/dL

➢Haematocrit (Hct, packed cell volume (PCV))

– percentage (%) of red cells in a known volume
of whole blood
• Reference Interval: M: 40-54%, F: 35-48%
Newborn: 48-68%
 RBC Count, Hb, HCT
❑ Significance
➢ ↓ RBC, HGB and/or HCT values….Anaemia: Decreased
production, increased loss/destruction

➢ ↑ RBC, HGB and/or HCT values….Polycythaemia:

Increased production, fluid loss
 RBC Count, Hb, HCT

❑ The rule of three is applied to a quick visual check of the

results of the haemoglobin and hematocrit. This rule applies
only to specimens that have normocytic normochromic RBCs
➢ The value of the haematocrit should be three times the
value of the haemoglobin plus/minus 3

❑ If values do not agree with this rule, this may indicate

abnormal RBCs, or it may be the first indication of error,
and the blood film should be examined
 RBC indices
MCV, MCH, MCHC
These parameters are calculated to determine the average RBC volume and
haemoglobin content/concentration.
➢ Mean Corpuscular Volume (MCV) - RBC volume, reflects RBC size
Normal adult MCV: 80-100 fL = normocytic red cells
MCV <80 fL = microcytic red cells MCV= HCT
MCV >100 fL = macrocytic red cells RBC count
Normal newborn: 95-125 fL
➢ Mean Corpuscular Haemoglobin (MCH) – reflects average
haemoglobin content (weight) per erythrocyte
Normal adult MCH: 26 – 32 pg MCH= Hb
Varies with RBC size and Hb content RBC count
Adds little information
➢ Mean Corpuscular Haemoglobin Concentration (MCHC) - average
Hb concentration per RBC MCHC= Hb
Normal 32-36 g/dL = normochromic red cells Hct
MCHC <32 g/dL = red cells may be hypochromic
 RDW
Red Cells Distribution Width (RDW) - index of RBC size
variation
• Normal 11.5 - 14.5 %.
• Normal RDW indicates uniform RBC size
• A high RDW >15% is caused by variations in RBC size
called anisocytosis

RDW= Standard deviation of red cells volume

MCV
 WBCs count

❑White blood cell (WBC, Leukocyte) count - total number of

white cells reported in 103/μL or 109/L
does not distinguish WBC types
Reference Interval: Adult, 3.5-11 x103/μL
Newborn: 9.0-30 x 103/μL

➢Significance
↓WBC count….Leukopenia: Decreased production, increased
use
↑WBC count….Leukocytosis: Increased production,
shift/pseudo
Critical values in adult: WBC< 2.0 or > 30.0 x 103/μL
 WBC differential count

❑ WBC differential classifies WBC types. Reported in relative

cell count (%) and absolute number (relative cell count (%) x
total WBC count), % easier to evaluate but absolute # more
reliable
➢Significance:
–Deviations from normal may indicate disease
–No immature cells should be present
–Critical values: Blasts, absolute neutrophil # <500/uL
Cells relative absolute Increase/ Decrease
Neutrophils 50-70% 1.7-7.5 x 103/μL Neutrophilia/ Neutropenia
Lymphocytes 18-42% 1.0-3.2 x 103/μL Lymphocytosis/
Lymphocytopenia
Monocytes 2-10% 0.1-1.3 x 103/μL Monocytosis/ Monocytopenia

Eosinophils 1-3% 0.0-0.3 x 103/μL Eosinophilia

Basophil 0-2% 0.0-0.2 x 103/μL Basophilia
 Platelets and MPV

❑ Total number of Platelet (Plt, Thrombocyte) reported in 103/ μL

• Platelet count: 150-450 x103/ μL
• Platelet counts < 150 x103/ μL: thrombocytopenia
• Platelet counts > 450 x103/ μL: thrombocytosis

❑ Mean platelet volume (MPV)

The MPV value is used to assess the platelet volume
• Reference Interval: 7-12fL
• Normal MPV: Normal platelets diameter
• MPV >12: Increased platelets diameter
• MPV can recognise abnormally large platelets (giant
platelets)
 Reticulocyte Count

❑Reticulocyte Measures rate of RBC production by the bone

marrow. Reported in relative % and absolute number
(relative (%) x RBC count / 100). Absolute number (103/μL)
is more reliable than %

• Reticulocytes (%) = (Retics count / 1000 red cell) ×100

• In specimens with a low hematocrit, the percentage of
reticulocytes may be falsely elevated because the whole
blood contains fewer RBCs.

• Reference range varies with age

• Adult: 0.5-2.5%, Absolute number: 20-115 x 103/μL
• Newborn 2.0-6.0%
 Reticulocyte Count

❑ Significance
• ↓ absolute Retic count….Reticulocytopenia: Decreased
RBC production
• ↑ absolute Retic count….Reticulocytosis: Increased RBC
production (↑ EPO stimulus)
• Good indicator for haemolytic anaemias

❑ Retics appear as polychromic (multi-

coloured red cells) RBCs on a Giemsa-
stained blood smear.
• reticulocyte is stained with a supravital
stain Brilliant cresyl blue or New
methylene blue to form
microscopically visible dark-blue
filaments or granules (reticulum).
 Erythrocyte Sedimentation Rate

❑ ESR refers to the sedimentation rate of red cells

In normal persons, sedimentation or falling of red cells is slow
Reported in distance the red cells fall in mm/time….mm/hr
Reference range varies with age & sex
• Males 0-15 mm/hr, Males>50y 0-20 mm/hr
• Females 0-20 mm/hr, Females>50y 0-30 mm/hr

❑ The ESR is a non-specific indicator of disease.

Mainly used to monitor patients with chronic inflammatory
diseases, and their response to therapy.
 Erythrocyte Sedimentation Rate

- - Rouleaux
formation
- - -
Acute phase reactants - +
- - - -
Immunoglobulins + -
- - +
Albumin -
+
- + -
- - - + - -
-
- -
- - + - + -
- - -
- -
- -
Normally,
(-) charge on RBC Added (+) charge
repel them away from NEUTRALIZES
each other (results in Zeta potential
Zeta potential)
 Erythrocyte Sedimentation Rate

❑Significance
• Under normal conditions, red cells do not
form rouleaux….fall slowly
• High concentrations of certain plasma
proteins promote rouleaux = ↑ ESR
• Fibrinogen, a positive acute phase reactant,
is most responsible for an abnormal ESR
➢ Increased ESR level is associated with activated inflammation, anemia,
malignancies, and aging. We can distinguish between inflammation and
anaemia using corrected ESR formula.
▪ Corrected ESR= ESR value x 15 / 55 – PCV
ESR: 100 ESR: 100 ESR: 50
PCV: 40% PCV: 25% PCV: 25%
Corrected ESR: 100 Corrected ESR: 60 Corrected ESR: 30
Blood Film Analysis and
RBC Morphology
 Blood Film

❑ Accurate assessment of blood cell morphology

requires a properly prepared blood smear. Well-made
and well-stained Smear can provide:
• Estimates of cell count
• Proportions of the different types of WBC
• Cell Morphology

❑ Blood should be collected in EDTA Tubes and smears

should be done within an hour of collection
Smear Preparation
Wedge Technique
1.Place a drop of blood, about 2-3 mm in
diameter approximately 1 cm from one end of
slide.
2.Place the slide on a flat surface and hold the
other end between your left thumb and forefinger.
3.With your right hand, place the smooth clean
edge of a second (spreader) slide on the specimen
slide, just in front of the blood drop.
4.Hold the spreader slide at a 30°- 45 angle, and
draw it back against the drop of blood
6.Allow the blood to spread almost to the edges of
the slide.
7.Push the spread forward with one light, smooth
moderate speed. A thin film of blood in the shape
of tongue.
8.Label one edge with patient name, lab id and
date.
9.The slides should be rapidly air dried by
waving the slides or using an electrical fan.
 Characteristics of A Good Smear

1. Good smear is tongue shaped with a smooth tail.

2. Does not cover the entire area of the slide.
3. Has both thick and thin areas with gradual transition.
4. Does not contain any lines or holes.
 Blood smear staining

❑ Blood film is stained with any Romanowsky stains, an example

of these stains involve: Wright stain, Leishman stain, and Giemsa
stain. It mainly composed of:
➢Eosin
It is acidic component of stain and stains basic component of cells
like haemoglobin. Gives radish pink color, eosinophilic color

➢Methylene Blue (Azure)

It is basic component and stains the acidic component of cells like
DNA and RNA (nucleus of WBC). Gives blue color, basophilic
color
 Critical Area of PBS

Thick Area Critical Area Thin Area

 Sources of Errors

(A) Chipped or rough edge on spreader slide.

(B) Hesitation in forward motion of spreader slide.
(C) Spreader slide pushed too quickly.
(D) Drop of blood too small
(E), Drop of blood not allowed to spread across the width of the slide
(F) Dirt or grease on the slide; may also be due to elevated lipids in the
blood specimen.
(G) Uneven pressure on the spreader slide.
(H) Time delay; drop of blood began to dry.
 Evaluation of peripheral blood smear

1. RBCs 2. WBCs
• Size • Estimate total counts
• Shape • Differential counts
• Color • Abnormal WBC
• Arrangement
• Inclusions

3. Platelets 4. Parasites
Estimate total counts
Abnormality
 Evaluation of peripheral blood smear
WBC & Platelet
➢ To perform a platelet estimate, the average counts of the
platelet in 10 fields per Oil-power field (100x) is multiplied by
20,000

➢ To perform a WBC estimate, the average counts of the

WBCs in 10 fields per high-power field (40x) is multiplied by
2000
➢ Corrected WBC counts
The Nucleated RBCs (NRBCs) are counted as WBCs because they are
indistinguishable by automation or hemacytometer. NRBCs are identified through
differential WBC count. If five or more NRBCs per 100 WBCs are observed on the
differential count on a stained peripheral blood film, the WBC count must be
corrected. Report the result as the “corrected” WBC count
 Red blood cell morphology

Abnormal erythrocyte morphology is found in

pathological states. The study of RBCs under the
microscope involves the investigation of three basic
features:

1. Size (anisocytosis)
2. Haemoglobin content
3. Shape (poikilocytosis)
4. Inclusions
5. Arrangements
 1. Size

➢ Circular, homogenous disc nearly of

uniform size(7–8 µm) deep pink
cytoplasm with central pallor <1/3rd
➢ These cells are called normocytes
➢ Size of normal RBC is almost the size
of the nucleus of the lymphocyte

➢ The mean corpuscular/cell volume (MCV) obtained in a CBC indicates the size
of RBCs which is roughly 7μm
➢Normal MCV is 80-100 femtoLiters (fL)
• If MCV value is normal the RBC called Normocytic.
• If MCV value is reduced the RBC called Microcytic.
• If MCV value is elevated the RBC called Macrocytic.
➢Anisocytosis- Variation in size of the red blood cells,
i.e. both microcytes and macrocytes.
Anisocytosis is a feature of most anaemias.
 2. Colour

RBC with normal intensity of staining (haemoglobin) are called

normochromic cells (central pallor that makes up 1/3 of the cell).

➢ The mean corpuscular/cell haemoglobin concentration (MCHC)

in CBC gives an indication of the haemoglobin content
(Concentration).

Normal MCHC is 32 – 36 g/dL

• If MCHC value is normal the RBC called Normochromic
• If MCHC value is reduced the RBC called Hypochromic
• If MCHC value is elevated the RBC called Hyperchromic??
 2. Colour
➢ Hypochromic: Decrease in haemoglobin
content of RBC leading to increase in central
pallor (>1/3rd)
• Seen in Iron Deficiency anaemia and
thalassaemia
➢ Polychromasia
➢ Blue grey tint of red cells due to the ribosomes
(RNA residual) in young cells
➢ Larger than normal and may lack
central pallor.
➢ Sign of active erythropoiesis
➢ Seen in haemolysis and acute blood loss
 2. Colour

Dimorphic blood picture

Anisochromia – presence of hypochromic cells and
normochromic cells in the same film. Also called dimorphic
blood picture. Seen in:
▪ Some weeks after iron therapy for iron deficiency anaemia
▪ Hypochromic anaemia after transfusion with normal cells.
 3. Shape

Variation in shape is called Poikilocytosis.

It is of following types-
• Elliptocytes
• Spherocytes
• Target cells
• Teardrop
• Schistocytes
• Acanthocytes
• Echinocytes
• Bite cell
 ➢ Target cells
➢ Elliptocytes
Cells with central round-
Elliptical shape
stained area and peripheral
rim of cytoplasm ➢ Stomatocytes
Red cells appear like
coffee beans

➢ Spherocytes
Spherical shape, with a
diameter smaller than normal
and lack central pallor
➢ Teardrop cells
One side of cells is
tapered and other is blunt ➢ Sickle cell
Cells are crescent shaped
 ➢Schistocytes
Fragmented erythrocytes.
As red blood cells travel through
these damaged vessels, they are
fragmented resulting in
intravascular haemolysis.
➢ Bite cell (keratocytes)
cell with one or more semi-
circular portions removed
from the cell margin
➢ Acanthocytes
Red cells with small number of
sharp spicules of inconstant length,
thickness and shape, irregularly
disposed over the surface.
➢Echinocytes (crenated cells)
Numerous, short, blunt regular
projection
Commonly occur as an artifact
during preparation of film
 4. RBCs Inclusions
They are elements that may be present in RBCs due to retained
remnants of cellular components. Identification and reporting of
these inclusions are important because their presence may
indicate diseases

• Basophilic stippling
• Howell – jolly Bodies
• Pappenheimer Bodies
• Heinz bodies
• Cabot Rings
• Malarial parasite
➢ Basophilic Stippling
Presence of irregular basophilic granules
within RBC which are variable in size
and stain dark blue.
• Aggregates of Ribosomes ➢ Pappenheimer Bodies
• Indicative of disturbed erythropoiesis They are small single or multiple
peripherally sited angular basophilic
(almost black) erythrocyte
inclusions.
• Composed of haemosiderin.
• Can be confirmed by Perls’ stain

➢Howell-Jolly Bodies
Smooth single large
round inclusions which
are remnant of nuclear
chromatin.
➢ Heinz bodies
Seen on supravital stains only. Purple,
blue, large, single or multiple inclusions
attached to the inner surface of the red
blood cell. Represent precipitated
haemoglobins
➢ Malarial parasite
Plasmodium is an
intracellular parasite
• Causes anaemia

➢ Cabot Rings
These are reddish
purple ring shaped.
Remnant of the
nuclear membrane
 5. RBCs arrangements

❑ Rouleaux Formation
Alignment of red cells one upon
another so that they resemble stacks
of coins.
• Occurs in elevated plasma
fibrinogen or globulin level

❑ Agglutination
More irregular and round clumping
than linear rouleaux
• Autoagglutination is caused by
the presence of antibody in the
plasma.
BM examination
 BM specimens

➢ Bone marrow collection

sites include the
posterior superior iliac
crest.
➢ BM specimens
• Needle BM aspiration
• Trephine biopsy
 BM Specimens

BM aspirate
• Cellular detail

BM Biopsy/Trephine
• Marrow architecture
 BM examination
BM aspiration
• Physician collects 1.0 to 1.5 mL of marrow into the syringe
• Using spreader slides, spreads the drop into a wedge-shaped smear
3⁄4 the length of the slide, similar to a peripheral blood film.
• Marrow aspirate smears are stained with Wright or Giemsa stains
using the same protocols as for peripheral blood film staining.

BM core biopsy
• The core biopsy is 1 to 1.5 cm long and 1 to 2 mm in diameter. The
specimen is suspended in 10% formalin fixative
• The specimen is then placed in a paraffin-embedding cassette. A
histotechnologist sections the embedded specimen, applies
hematoxylin and eosin (H&E) stain, and examines the section.
 BM examination

➢BM Cellularity
Normal: Approximately 50% haemopoietic cells
Hypocellular: <30% haemopoietic cells
Hypercellular: >70% haemopoietic cells
➢M:E ratio, Normal M:E ratio is 1.5:1 to 5:1
➢Morphologic examinations, malignant cells ?
➢Cell maturity, maturation stages of the myeloid, erythroid,
lymphoid, and megakaryocytes.
➢Cytochemistry: cell types
➢Iron stores: Prussian blue stain
BM cellularity

Hypercellular

Normocellular

Hypocellular