Band or stab form: Cell becomes still smaller, nucleus
has a deep indentation, chromatin is coarsely clumped.
Cytoplasm is pink with purplish granules.
Segmented neutrophil: 12–14 mm in size, nucleus shows
2–5 lobes, chromatin in dark purple clumps, cytoplasm
FIG. 9.13: Erythropoiesis FIG. 9.14: Leukopoiesis
has numerous, fine, evenly distributed purplish granules.
In female at least 6 neutrophils or 500 should show
The mature eosinophil: 16 mm in size, granules are
acidophilic and larger. Nucleus is bilobed and is not
The mature basophil: It usually has a bilobed nucleus,
but the nucleus is masked by about 10 large basophilic
Lymphocytic Series (Fig. 9.15)
Lymphoblast: 15–20 mm in size, resembles myeloblast,
cytoplasm is agranular and moderately basophilic.
Nuclear chromatin gives fine reticular appearance with up
to 2 nucleoli. It is peroxidase negative.
blue cytoplasm with a few purplish red granules seen in
Small lymphocyte: 9–12 μm in size, has scanty cytoplasm.
Nucleus is usually round and shows heavily clumped
Monoblast: It resembles myeloblast.
Promonocyte: Up to 20 μm in size, has a large convoluted
nucleus, chromatin is seen in skein like strands. Cytoplasm
is dull gray-blue and may contain a few azurophilic
Monocyte: 15–20 μm in size, has abundant dull gray-blue
cytoplasm with a ground glass appearance and may show
vacuolation and fine azurophilic granules. It has a kidneyshaped nucleus.
Megakaryoblast: 20–30 μm in size, has a large, oval or
kidney-shaped nucleus with several nucleoli. It possesses
relatively small amount of agranular cytoplasm.
Promegakaryocyte: 30 μm in diameter, cytoplasm is
intensely basophilic with fine azurophilic granules.
Nucleus may show mild lobulation and chromatin appears
Megakaryocyte: 30–90 μm in diameter, it contains a single
multilobulated or indented nucleus. Nuclear lobes may
vary from 4–16 in numbers. Cytoplasm is bulky, light blue
with fine azurophilic granulation. The margin is irregular
and may show fragmentation or budding, precursor of
Mature platelet: 1-4 μm. It is formed by fragmentation of
megakaryocytic pseudopods. In circulation, they acquire
a discoid shape. Cytoplasm stains light blue and contains
purple reddish granules, which may be clumped centrally.
Control of platelet production: Perhaps by a humoral factor
called thrombopoietin, acts by a feedback mechanism.
1. Mount: Cover the slide using a neutral mounting
2. Low power field examination: Look for:
– Number, distribution and staining of WBCs
– RBCs examination, select an area where they
just touch each other without overlapping, i.e.
between tail and body of the film.
3. High power field examination: Assess RBC
4. Oil immersion examination: Assess atypical cells and
note fine details, e.g. inclusion bodies.
Size: Normocytes, microcytes, macrocytes, anisocytosis
Shape: Abnormal shape oval, pencil, tear, pear, oat
and sickle-shaped cells, fragmented cells, target cells,
spherocytes, crenated cells, burr cells, acanthocytes,
Hemoglobin: Normochromic, hypochromic.
Immature forms: Polychromatic, stippled or nucleated red
Inclusion bodies: Howell-Jolly bodies, Cabot rings,
Pappenheimer bodies, malarial parasites, etc.
Arrangement: Autoagglutination, excess rouleaux formation.
Number: Normal, increased, decreased.
Abnormal or immature forms: Immature forms, hypersegmented macropolycytes, abnormal forms.
Number: Normal, increased, decreased.
Form: Abnormalities of size and shape, present in groups
Differential Leukocyte Count (DLC)
For differential leucocyte counts, choose an area where
the morphology of the cells is clearly visible. Ensure that
there is no tailing of the WBCs or else a false DLC may
be obtained. Do differential count by moving the slide as
shown in order to include central and peripheral areas of
While doing DLC, look for vacuolation, toxic
granulation, size and maturity of the WBCs. Count at least
100 cells and give percentage of the cells seen. Counting
becomes easier if 100 squares are made on a paper and the
letters P for neutrophil, L for lymphocyte, M for monocyte,
E for eosinophil and B for basophil can be entered in each
square. Another easier way is using Laboratory cell counter
Bone marrow can be obtained by using Salah’s, Klima’s
or Jamshidi’s marrow aspiration needles. Not more than
0.2 mL of bone marrow should be taken out at one time. If
blood or dry taps have occurred on 2 different occasions,
a trephine biopsy should be performed.
For aspiration of bone marrow, Klima’s needle is better
as the guard has no chances of getting slipped and hence
dangers of puncturing substernal structures are less.
Before entering the site of puncture, take all aseptic
precautions, i.e. cleaning with spirit, iodine and spirit
again. The various sites for obtaining bone marrow are:
¾ Posterior superior iliac spine.
¾ Tibia, superior medial surface of the tibia, inferior to
the medial condyle and medial to the tibial tuberosity
Delay in marrow film preparation should not be there.
Get rid of blood by using a capillary pipette leaving the
grayish marrow particles behind. Make a smear as has
been described for peripheral blood. The smear should be
3 to 5 cm long and should not be more than 2 cm wide. The
particles should be dragged behind but not squashed. A
trail of cells is left behind. Criteria of a good preparation—
presence of both particles and free marrow cells in the
FIG. 9.16: Nine-unit laboratory cell counter
232 Concise Book of Medical Laboratory Technology: Methods and Interpretations Imprints
Another way is imprint method. The marrow particle is
picked and transferred immediately to a slide and made to
stick to it by a gentle smearing motion. The slide is air dried
Marrow particles in a small drop of aspirate are placed
on a slide near one end. Another slide is carefully placed
over the first. Slight pressure is exerted to crush the
particles, and the slides are separated by pulling them
apart in a direction parallel to their surfaces. Dry the
smears immediately. The appearance of fat (irregular
holes) on the smear implies that marrow has been
1. Good fixation is essential.
2. Any of the previously mentioned stains can be used
for staining bone marrow films.
The examiner should be informed of the clinical picture
before he or she examines the marrow films. An impression
can be formed by examining various fields on the different
stained slides prepared. Start with 10X through 40X, to oil
immersion, now actually make a differential count of large
number of cells (300 to 1000) and calculate the percentage
The procedure given below is recommended for studying the
1. Naked eye inspection of the slides to select the smear
2. With 10X objective, survey the particles whether they
are normoplastic, hypoplastic or hyperplastic.
3. Select a cellular area (usually in the tail portion of the
film around particles), study the cytologic details by
high power—40X and oil immersion—100X objectives.
Note the undermentioned points: Cellularity: Normo/
hypo/hyperplastic marrow particles. Cellularity is better
defined by studying histologic sections of aspirated
particles, though crude estimates can be given on films.
The normal cellularity of the marrow varies with age being
more in infants and least in elderly individuals.
Next, look for reaction of erythropoiesis, whether it is
normoblastic, megaloblastic or micronormoblastic. Look
for maturity of leukopoietic cells. The M:E ratio is based on
a count of 500 to 1000 marrow cells. In the normal adult,
the reaction is normoblastic, leukopoietic maturity is
normal and ME ratio is about 3 or 4:1. The ME ratio at birth
is 1.85:1; during the first 2 weeks, it reaches its peak of 11:1.
It then gradually drops to the (years 1 to 20) average of 3:1.
Look for megakaryocytes, their number, size, nuclear
lobulations, functioning capacity.
Megakaryocytes with budding or cloud-like appearance
at the periphery are functioning ones.
In addition, if suspected, look for metastatic cells,
increased number of normal or abnormal (myeloma)
plasma cells, cells containing unduly large amounts of
lipid, carbohydrate, etc.—storage disease. One may also see
hemoparasites in the marrow especially LD bodies of Kalaazar.
Bone Marrow Aspiration Analysis
Red marrow contains connective tissue, fat cells, and
hematopoietic cells. Yellow marrow contains connective
tissue and fat cells. Interpretation of cell count and
histopathology by a hematologist, pathologist, or
Response to staining: Iron stain for hemosiderin: 2+.
Periodic Acid-Schiff (PAS) glycogen reactions: Negative.
Sudan black B (SBB) granulocyte: Negative.
Adult (%) Child (%) Infant (%)
Lymphocytes (all stages) 2.7–24 16 49
Megakaryocytes 0.03–0.5 0.1 0.05
Promyelocytes 0.5–8.0 1.4 0.76
Undifferentiated cells 0.0–0.1
Neutrophils, total 56.5 57.1 32.4
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