Peripheral blood smear 2020

Medical laboratory science.
hematology
Blood smear preparation, staining and evaluation
Alyazeed Hussein, BSc-SUST

Slide
preparation
and staining
 Slide to slide blood smear is the most common smear
preparation in the hematology laboratory. The wright
stain, is the most common dye used to stain the peripheral
blood smear (PBS). Giemsa stain, field stain and leishman
stain also can be used.
 Diagnosis from the blood smear remains a crucial
diagnostic aid, which provides valuable information.
 Making a good smear:
1. A small drop of blood from an EDTA-anticoagulant specimen
or capillary blood is applied to the end of a cleaned standard
glass slide.
2. Place a second slide (spreader) in front of the drop of blood
at a 30 to 45 degree angle.
3. Draw the spreader slide into the drop of blood, and push the
spreader slide forward, spreading blood across the length of
the slide.
4. The slide is then stained either manually or on an automated
stainer.

Acceptable vs unacceptable smear

Steps in performing a differential at
different magnifications
Low-power scan (10x---100 magnification):
1. Determine the overall staining quality of the blood smear.
2. Determine if there is a good distribution of the cells on the
smear.
 Scan the edges and center the slide to be sure there are no
clumps of RBCs, WBCs or PLTs.
3. Find an optimal area (monolayer) for the detailed
examination and enumeration of cells.
 The RBCs should barely touch each other.
 There should not be areas containing large amounts of broken
cells or precipitated stain.

High-power scan (40x---400
magnification)
1. Determine the WBC estimate.
 Count the number of WBCs in 10 fields, and
divide by 10. Multiply this number by 2000.
For example, if the average number of WBCs
counted per (hpf) was 5, the WBC estimate
would be 5 x 2000 = 10,000/µL.
 Correlate the WBC estimate with the WBC
counts from the automated instrument.

Oil immersion scan (100x---1000
magnification)
1. Using a differential counter, count 100 WBCs, and
report in percentage. Evaluate RBC anisocytosis,
poikilocytosis, hypochromasia, polychromasia and
RBCs inclusions.
2. Perform a quantitative platelet estimate, and
evaluate platelet morphology.
 Count the number of platelets in 10 oil
immersion fields (OIFs).
 Divide by 10 to average. Multiply by 20,000.
Example, the average number of platelets in 10
fields is 13, the platelets estimate is 13 x 20,000
= 260,000/mm3.
3. Correct the total WBC count that has greater than 5
NRBCs per 100 WBCs counted.

Normal and abnormal cells in
the peripheral blood (PB)
1. Neutrophils (granulocytes): the segmented neutrophil is the
most common type of WBC in the PB smear (50% to 70%) of
cells in the differential. The nucleus of the seg. Neutrophil is
separated into two to five (usually 3) lobes, with a narrow
filament connecting the lobes. The cytoplasm is light pink, and
the granules are fine stain either pink or neutral color.
2. Vacuolated neutrophils in a patient with septicemia:
neutrophils are the first line of defense against bacterial
infection and have a phagocytic function. During septicemia
(bacteria in the blood), vacuolization in the cytoplasm of
neutrophils can be seen.
3. Neutrophils with toxic granulation: is often seen in patient
with bacterial infections, where the granules in the cytoplasm
stain very dark.
4. Pelgeroid neutrophils: the nucleus is nonsegmented or
bilobed, however, the cytoplasm is normal.
1
2 3
4 4Alyazeed Hussein, BSc-SUST

 Lymphocytes: lymphocyte are the second most
common WBC found in the PB (20% to 44%), most
lymphocytes are small. There are also intermediate
and some large lymphocytes. The cytoplasm is blue
scant, nucleus of small lymphocyte equal the size of
normal RBC.
 Large lymphocytes: has more cytoplasm, which is light
blue and can be indented by neighboring RBCs. In
large cells, there may be a few well-defined granules
that vary in size, unevenly distributed, and can be
counted. These granules are purplish-red and have
called azurophilic (Fig 4).
 Reactive lymphocytes: the margin of large reactive
lymphocytes is frequently indented by neighboring
RBCs, causing them to have (holly leaf) shape. Note the
increase in cytoplasm and the increase in basophilia at
the edges of the cytoplasm (Fig 5).
 Reactive (Atypical) lymphocytes: infectious
mononucleosis (IM), note the unusual shape of the
nuclei and the presence of nucleoli. Note the significant
indentations by neighboring RBCs (holly leaf) shape
6). 4 5 6

 Monocytes: comprise 2% to 9% of the WBCs.
The cytoplasm is gray-blue in contrast to pink
cytoplasm of neutrophil. Numerous fine, small,
reddish or purplish stained, evenly distributed
granules in the cytoplasm give the cell a ground-
glass, cloudy appearance. Digestive vacuoles
may be observed in the cytoplasm. The nuclei of
monocytes frequently may be kidney-shaped,
deeply folded or indented, or occasionally
lobular. One of the special features of the
monocyte is the appearance of convolutions
(like those in the brain) in the nucleus. The shape
of monocytes is variable. Many cells are round,
other reveal blunt pseudopods.

 Band, staff, stab neutrophils: comprise 2% to 6% of the
WBCs. Band neutrophils have a nucleus with a
These cells do not have a lobulated nucleus.
 Eosinophils: comprise 1% to 4%. Eosinophils are usually
easily recognizable because of the large, round,
refractile, uniform in size granules that have an affinity
the acid eosin stain. Normal eosinophilic granules
become orange to reddish-orange. The nucleus is
bilobed.
 Basophils: comprise 0% to 2%. The large abundant,
violet-blue or purple-black granules aid in the
recognition of this cell. These granules are visible above
the nucleus as well as lateral of it, and they cover most
the nucleus. In cells that are poorly fixed during staining,
the center of the granule may disappear or the entire
granule may be washed away, leaving a small, colorless
cytoplasmic area.

Compare and
contrast
 Large lymphocyte versus monocyte: a
monocyte is often mistaken for a large
lymphocyte because the monocytic cytoplasm
may be blue, the granules may be indistinct,
the nucleus may be round, and the blunt
pseudopods and digestive vacuoles may be
missing. In the lymphocyte, these large
granules are prominent, usually red, and at
the periphery of the cytoplasm. The cytoplasm
of the lymphocyte has a relatively clear
nongranular background. Large lymphocyte
are often deeply indented by neighboring
RBCs.

Large lymphocyte vs reactive lymphocyte: note that the large lymphocytes (left) have
a lighter blue color to their cytoplasm, which is relatively clear. In the reactive
lymphocytes (right) the cell is much larger, the cytoplasm is more basophilic.

 Neutrophils vs band neutrophil: the nucleus of
segmented neutrophil is separated into 2 to 5
lobes, with a narrow segment or filament
connecting the lobes. Band neutrophil have a
nucleus with a horseshoe shape, not separated
lobes.
 Neutrophils vs eosinophils: the nucleus of
segmented neutrophil have 2 to 5 lobes with
neutral to pink granules. The eosinophil usually is
bilobed with very large refractile reddish granules.
 Neutrophil vs basophil: the neutrophil have 2 to 5
lobes with small neutral to pink granules, the
basophil have large coarse violet-blue granules.
The nucleus is usually difficult to visualize totally.
 Neutrophil vs monocyte: the nucleus of neutrophil
is segmented with dark clumped chromatin with
pink cytoplasm. The nucleus of the monocyte is
large with an irregular shape and a lacy
The cytoplasm is blue-gray, cloudy, contains
vacuoles.

 Metamyelocte vs band: the nucleus of
metamyelocte is kidney shaped, the nucleus
of band is horseshoe shaped (nucleus
indentation).
 NRBC vs plasma cell: nucleus of NRBC is
completely condensed or pyknotic, the
cytoplasm is pink. The plasma cell has
granular chromatin, the cytoplasm is very
basophilic, the perinuclear zone (Golgi area)
is visible giving white appearance.

RBC morphology
 Normal peripheral blood smear:
 Anisocytosis is the variation of RBCs size.
 The majority of laboratories use either
qualitative remarks (slight, moderate or
marked) or a numerical grading (1+ to 4+)
based on the percentage of variation in size.
Percentage of cells that differ in size from normal RBCs
5 to 10%Slight
10 to 25%1+
25 to 50%2+
50 to 75%3+
>75%4+Alyazeed Hussein, BSc-SUST

 Anisocytosis slight (SL): assuming each OIF has
approximately 200 RBCs, then 5-10% is about 10
20 RBCs differed in size.
 Anisocytosis 1+: assuming each OIF has
approximately 200 RBCs, then 10-25% would be
about 20 to 50 RBCs differed in size.
approximately 200 RBCs, then 25-50% is about 50
t0 100 RBCs differed in size.
approximately 200 RBCs, then 50-75% is about
to 150 RBCs differed in size.
approximately 200 RBCs, then more than 75%
would be greater than 150 RBCs differed in size.
SL 1+
2+
3+
4+

Poikilocytosis
 Most laboratories require the reporting of the specific shape and grading of the
number of these cells seen under OIF.
 For example:
3+ acanthocytes
1+ echinocytes
Then a 4+ poikilocytosis would be reported.

Hypochromia
 The red color of RBC is due to the amount of
hemoglobin present. The amount of central
pallor is inversely proportional to the amount
of hemoglobin in the RBC. The greater the
central pallor, the lower the hemoglobin
value.
Hypochromia
Area of central pallor is one-half the cell diameter in the RBC.1+
Area of central pallor is tow-thirds the cell diameter in the RBC.2+
Area of central pallor is three-fourths the cell diameter in the RBC.3+
Thin rim of hemoglobin on the periphery of the RBC.4+Alyazeed Hussein, BSc-SUST

Red blood cell
size
 Normocytic: normal size, MCV: 80-100fL
(hemolytic anemia). Fig.1
 Anisocytosis: variation in cell size, dimorphic
picture(severe anemia), note that increased
RBC distribution width (RDW) also correlate
with anisocytosis (IDA). RDW measures
distribution of RBC volume (11.5-14.5%). Fig.2
 Microcytic: small RBCs, MCV: <80fL (IDA,
thalassemia). Note that! The small lymphocyte
is often used as a micrometer to determine
size of the RBCs. Fig.3
 Macrocytic: large RBCs, MCV: >100fL
(megaloblastic anemia). Macrocytes should be
evaluated either oval or round, either red or
polychromatophilic. Oval macrocytes are seen
in megaloblastic anemia, round macrocytes
are seen in alcoholism or liver disease.
Polychromatophilic macrocytes are seen in
acute blood loss or in reticulocytosis. Fig.4
1
2
3
4
4

RBC Hgb concentration
 Normochromic: normal Hgb, normal pale
central area: one-third of the RBC (hemolytic
anemia).
 Hypochromic: central pallor > one-third of
RBC (IDA, thalassemia).
 Hyperchromic: RBC have no central pallor
(Hereditary spherocytosis, immune hemolytic
anemia).
 Polychromasia: slightly larger size than a
normal RBC, variation of color (blue to gray),
seen in: increased erythropoiesis. With
supravital stain is called a reticulocyte.

RBC shape
 Acanthocyte (spur, thorn cell): severe liver disease, hemolytic anemia, Fig.1
 Blister cell: glucose-6-phosphate dehydrogenase (G6PD) deficiency, Fig.2
 Target cell (codocyte, leptocyte): thalassemia, IDA, Fig.3
 Teardrop cell (dacrocyte): megaloblastic anemia, thalassemia, Fig.4
 Sickle cell (drepanocyte): sickle cell anemia, Fig.5
 Echinocyte (burr cell): uremia, renal disease, hepatitis, Fig.6
 Elliptocyte (pencil cell): hereditary elliptocytosis, IDA, Fig.7
 Ovalocyte: megaloblastic anemia, Fig.8
 Schistocyte (fragmented cell): disseminated intravascular coagulation (DIC), heart
valves, Fig.9
 Spherocyte: hereditary spherocytosis, immune hemolytic anemia, Fig.10
 Stomatocyte: hereditary stomatocytosis, liver disease, Rh null disease, Fig.11
1
2
3
4
5
6
7
8
9
10
11

Compare & contrast,
RBC shape
 Echinocyte vs acanthocyte: echinocytes have
small, uniform, evenly spaced blunt projections.
Acanthocytes have small and large irregularly
spaced spiny projections.
 Elliptocyte vs ovalocyte: elliptocytes are more
pencil or cigar-shaped. Ovalocytes are more
egg-shaped.
 Elliptocyte vs sickle cell: elliptocytes have
elongated rounded ends. Sickle cells have
pointed ends. Sickle cells are full of hemoglobin.

RBC inclusions
 Basophilic stippling: dust-like granules, RNA remnants
(thalassemia, lead poisoning), Fig.1
 Cabot ring: nuclear membrane remnants (thalassemia,
megaloblastic anemia), Fig.2
 Heinz bodies: denatured Hgb, visualized by a supravital stain
such as crystal violet (G6PD, Hgb ZÜrich), Fig.3
 Hemoglobin C crystal: rod like, bar of gold (Hgb C, Hgb SC
diseases), Fig.4
 Hemoglobin SC crystal: (Hgb SC disease), Fig.5
 Hemoglobin H: accumulated beta chains, visualized only by
supravital stains, resembles a pitted “golf ball” (Hgb H disease,
alpha thalassemia), Fig.6
 Howell-jolly bodies: remnants of DNA (megaloblastic anemia,
hemolytic anemia), Fig.7
 Pappenheimer bodies (siderotic granules): beadlike, excess
available iron (sideroblastic anemia, thalassemia), Fig.8
1
2
3
4
5
6
7
8Alyazeed Hussein, BSc-SUST

Other
 Bite cell: result from removal the denatured Hgb
by the macrophage in the spleen (G6PD). Fig.1
 Folded (envelope) RBC: Hgb C, Hgb SC diseases.
Fig.2
 Dove or crossed RBCs: primary myelofibrosis,
vaso-occlusive crisis in SCD. Fig3.
 Auto agglutination: autoantibodies to RBCs
antigens react at temperature below 32°C (cold
agglutinin disease, mycoplasma pneumoniae), Fig.4
 Rouleaux: RBCs appearing as a stack of coins, high
concentration of fibrinogen and immunoglobulins
(multiple myeloma), Fig.5
1 2 3
4 5

 Chediak-higashi syndrome: giant lysosomal granules in cells, Fig.1
 May-hegglin anomaly: decreased platelets production, cells with blue-
cytoplasmic inclusion resemble dohle body, except that they are larger,
Fig.2
 Alder-reilly anomaly: disorder of mucopolysaccharidosis, granules stain
positive with metachromatic stain, Fig.3
 Pelger-huet anomaly: bilobed or non lobulated neutrophil 70-90% of
all neutrophils are affected, Fig.4
 Hypersegmented neutrophil: defect in DNA synthesis (megaloblastic
anemia), have six or more nuclear lobes, Fig.5
 Ehrlichiosis: light blue-colored Ehrlichia bacteria in the cytoplasm of
neutrophil, Fig.6
 Auer rods: rods in myeloblasts, promyelocyte (faggot cell), (AML), Fig.7
 Smudge cell: crushed lymphocytes (CLL), Fig.8
 Hairy cell: B-cell leukemia, HCL, Fig.9
 Flower T lymphocyte: adult T lymphocyte leukemia (HTLV), Fig.10
1
2
3
4
5
6
7
8
9
10

Platelet, (giant Fig.11), (bizarre Fig.12), (stress Fig.13), (Babesia Fig.14) (bacteria Fig.15), (fungi Fig.16),
(malaria Fig.17), (microfillaria Fig.18),
11 12 13 14 15
15 16 17 18

 This has been a presentation of Alyazeed
Hussein.
 Thanks for your attention and kind patience.

Peripheral blood smear 2020

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Peripheral blood smear 2020

Similar to Peripheral blood smear 2020 (20)

Recently uploaded

Recently uploaded (20)

Peripheral blood smear 2020