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

 

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

drumsticks.

The mature eosinophil: 16 mm in size, granules are

acidophilic and larger. Nucleus is bilobed and is not

masked by granules.

The mature basophil: It usually has a bilobed nucleus,

but the nucleus is masked by about 10 large basophilic

granules.

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.

230 Concise Book of Medical Laboratory Technology: Methods and Interpretations Large lymphocyte: 12–16 μm in size, has abundant pale sky

blue cytoplasm with a few purplish red granules seen in

about 33% of the cells.

Small lymphocyte: 9–12 μm in size, has scanty cytoplasm.

Nucleus is usually round and shows heavily clumped

chromatin.

Monocytic Series (Fig. 9.14)

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

granules.

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.

Thrombopoiesis (Fig. 9.14)

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

denser.

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

circulating platelets.

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.

Examination of a Blood Film

Method

1. Mount: Cover the slide using a neutral mounting

medium.

2. Low power field examination: Look for:

– Quality of film

– Number, distribution and staining of WBCs

FIG. 9.15: Lymphocytic series

– 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

• Size

• Shape

• Hemoglobin concentration.

4. Oil immersion examination: Assess atypical cells and

note fine details, e.g. inclusion bodies.

Always Note

RBCs

Size: Normocytes, microcytes, macrocytes, anisocytosis

(variation in size).

Shape: Abnormal shape oval, pencil, tear, pear, oat

and sickle-shaped cells, fragmented cells, target cells,

Clinical Hematology 231

spherocytes, crenated cells, burr cells, acanthocytes,

stomatocytes (poikilocytes).

Hemoglobin: Normochromic, hypochromic.

Immature forms: Polychromatic, stippled or nucleated red

cells.

Inclusion bodies: Howell-Jolly bodies, Cabot rings,

Pappenheimer bodies, malarial parasites, etc.

Arrangement: Autoagglutination, excess rouleaux formation.

WBCs

Number: Normal, increased, decreased.

Abnormal or immature forms: Immature forms, hypersegmented macropolycytes, abnormal forms.

Platelets

Number: Normal, increased, decreased.

Form: Abnormalities of size and shape, present in groups

or lying scattered.

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

the smear.

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

(Fig. 9.16).

Bone Marrow Examination

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:

In Adults

¾ Sternal aspiration

¾ Anterior iliac crest

¾ Vertebral spinous processes

¾ Posterior superior iliac spine.

In Children

¾ Tibia, superior medial surface of the tibia, inferior to

the medial condyle and medial to the tibial tuberosity

¾ Posterior iliac crest

¾ Calcaneum.

Marrow Film Preparation

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

smear.

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

rapidly by waving.

Crush Preparation

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

obtained.

1. Good fixation is essential.

2. Any of the previously mentioned stains can be used

for staining bone marrow films.

Examination

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

of each type of cell.

The procedure given below is recommended for studying the

marrow films:

1. Naked eye inspection of the slides to select the smear

containing particles.

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

Normal Values

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

oncologist is required.

Response to staining: Iron stain for hemosiderin: 2+.

Periodic Acid-Schiff (PAS) glycogen reactions: Negative.

Sudan black B (SBB) granulocyte: Negative.

Differential cell count

Adult (%) Child (%) Infant (%)

Basophils 0.1 0.06 0.07

Eosinophils 3.1 3.6 2.6

Hemocytoblasts 0.1–1.0

Lymphocytes (all stages) 2.7–24 16 49

Megakaryocytes 0.03–0.5 0.1 0.05

Plasmacytes 0.1–1.5 0.4 0.02

Promyelocytes 0.5–8.0 1.4 0.76

Reticulum cells 0.1–2.0

Undifferentiated cells 0.0–0.1

Neutrophils, total 56.5 57.1 32.4

Metamyelocytes 9.6–24.6 23.3 11.3

Neutrophilic 10–32

Eosinophilic 0.3–3.7

Basophilic 0–0.3

Monocytes (all stages) 0–2.7

Myeloblasts 0.1–5.0 1.2 0.62

Myelocytes 4.2–15 18.4 2.5

Neutrophilic 5.0–20

Eosinophilic 0.1–3.0

Basophilic 0–0.5