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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
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as the presence of blood parasites, Rouleaux formation in the case of multiple myeloma, and
estimation of cell counts (RBC, WBC, platelets) in order to check on the results of automated
enumeration. Morphological studies of red cells are able to recognize some of the important
types of anaemias and double check indices.
Normal values
Adult (male or female):
Neutrophils 40–75% (mean 57%)
Segmented 54%
Bands 3%
Eosinophils 1–6%(mean 4%)
Basophils 1% or Less
Lymphocytes 20–45% (mean 37%)
Monocytes 2–10% (mean 6%)
Specimen
EDTA-anticoagulated blood or blood from skin punctures (non-anticoagulated). Other
anticoagulants do not give the desired results. Prepare blood smear within 1–2 h after blood
drawing. The blood lm must be immediately xed in methanol. Fixed smears can be stained
and examined later.
Principle
Three major steps are involved in dierential count— (1) preparation of the blood smear,
(2) staining of the blood smear and (3) microscopic observation. The smear is taken directly
from the skin puncture which gives the true picture of blood morphology; however, EDTAanticoagulated venous blood is satisfactory if smears are made within 1–2 h of collection.
Staining is done with a polychromie stain that includes methylene blue and eosin in its
preparation (e.g., Leishman, Giemsa). The polychromie stain induces multiple colours when
applied to cells. The stains are dissolved in methanol and buered to pH 7.0–7.2 (close to
neutral). Methanol (must be acetone-free) acts as a xative and also as a solvent. The xative
does not allow any further change in the cell. Thus, methanol-based stains, stain and x
simultaneously.
Following staining, the basic components of the white cell (e.g., cytoplasm) are stained by
the acidic eosin dye and they are described as eosinophilic or acidophilic; while the acidic
components of the cell (e.g., nucleus with nucleic acid) take blue to purple shades by the basic dye methylene blue and they are called basophilic. The neutral components of the cell are
stained by both the dyes. May–Grünwald with Giemsa is another popular polychromie stain;
however, unlike Leishman’s stain, the nal solution is prepared fresh and staining time may
have to be changed. Field stain is ideal for fast results and is popular in physicians’ oces.
Procedure
After preparing the thin blood smear and staining it, cells are subjected to microscopic
examination. This allows one to determine the relative distribution of various types of white
blood cells (WBCs), which is known as dierential count. During dierential count, the
technician also studies the abnormal morphology of red cells as well as white cells.
Medical Laboratory Technology: Volume 1
Examination of stained blood smears (leishman)
1. First examine the stained blood smear under low power for screening. This allows
you to quickly scan the entire slide. Note the background colour and distribution of
the white cells. In a perfectly stained smear, three zones can be identied visually
(Figure 10.16) which includes the thick area or the ‘head’ of the smear, following by
the ‘body’ and nally the thin end of the smear (‘tail’). At the tail end the red cells lie
Routine Haematological Tests
279
singly and neutrophils and monocytes predominate, while in the body, the red cells
overlap each other to a certain extent and lymphocytes predominate. If the scanning
indicates non-uniform distribution of white cells, with larger white cells (neutrophils
and monocytes) concentrated towards the tail or edges, the smear gives an inaccurate
dierential count. In this case, make a new smear. The scanning of the entire slide also
gives the opportunity to identify Rouleaux formation, to estimate white cell count, to
detect the presence of large and abnormal looking cells, etc.
2.
Choose the portion of the blood smear usually slightly before the ‘tail end’ of the
smear (Figure 10.16) where there is only slight touching of the red cells. Place a drop
of immersion oil on the slide (do not put on a cover slip), directly on the smear. Now
switch to the oil-immersion objective, check whether the objective has made contact
with the oil, look through the microscope and increase the light by opening the iris
diaphragm as needed.
3. Identify various types of white cells on the basis of the following characters as a result
of staining with Leishman stain. Even if the smear is not properly stained, the shape and
size of various cells give sucient clues for their identication (Figures 10.16–10.18).
(a) A typical stained blood smear shows three zones, (b) Tendency of uneven distribution
of white cells can be minimized by making a good smear, (c) One of the methods of
moving the slide for dierential count, (d) Hand tally counter used in dierential count
Medical Laboratory Technology: Volume 1
Granulocytes These are cells (listed below) with granulated cytoplasm, which stain a faint
pink. These include neutrophils, eosinophils and basophils.
Neutrophils Pale pink cytoplasm with ne mauve-coloured granules, include band and
segmented forms (lobes); normally 3 to 4 lobed.
Band Nucleus sausage-shaped, not segmented; also called stab.
Segmented Nucleus with denite lobes.
Eosinophils Cytoplasm stains faint pink and contains large red and red-orange granules.
Basophil Cytoplasmic granules, large, dark and blue-black which ll the cells and obscure
the nucleus.
Lymphocytes Large-sized lymphocytes have clear blue cytoplasm on the margin of the
nucleus. In smaller lymphocytes, dark violet-coloured nucleus almost lls the entire cell
and has a rim of clear cytoplasm.
Monocytes Largest in size of all white cells, wavy margin of cytoplasm, grey-blue
cytoplasm; kidney-shaped nucleus.
Identication of various cells as seen on the blood smear
Routine Haematological Tests
(1) Normal white cells: (a) band neutrophil, (b) monocyte, (c) eosinophil, (d) young
lymphocyte, (e) basophil, (f) segmented neutrophil, (g) lymphocyte, (h) atypical
lymphocyte, (i) segmented neutrophil, (2) Band neutrophil, (3) Band neutrophil, (4)
Eosinophil, (5) Basophil, (6) Megaloblast, (7) Promyelocyte, (8) Lymphoblast, (9)
Monocyte, (10) Platelets, (11) Nucleated red cells, (12) Reticulocytes, (13) Heinz
bodies, (14) Neutrophil with appendage
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Medical Laboratory Technology: Volume 1
(15) R vivax causing Schuner’s dots (in malaria), (16) P. ovale (microgametocyte),
(17) Ring stage of P. vivax, (18) P. falciparum gametocyte (banana-shaped), (19) P.
malaria (microgaetocyte), (20) Ring stages of trophozoites (malaria), (21) Plasma
cells
4. Red cells and platelets: These are easy to identify, based on size and shape.
Red cells Stain pink-red; small-sized cells with haemoglobin.
Platelets Stain mauve-pink; cells of smallest size, often under high-power may be
confused with dirt and stain deposits.
5. Procedure for dierential count: Examine the smear under the oil-immersion
objective, moving the slide as shown in Figure 10.16, and counting individually each
type of white cells seen. Record the observation either on a piece of paper, tabulated
under dierent types mentioned above or using a dierential counter, which has
dierent keys for dierent types of white cells. Continue counting until 100 cells are
counted. If any nucleated red blood cell (NRBC) is seen, keep a separate account. The
NRBCs are not included in the dierential count.
Study of Abnormal Cell Morphology
While performing the dierential count, make a note of abnormalities in the morphology of
red cells and white cells (Figures 10.19 and 10.20). In case of any abnormal ndings, make
a separate observation and report abnormalities. Along with the report of RBC and WBC
Routine Haematological Tests
283
morphology (abnormality only), make a rough estimate of the number of platelets. If the
average number of platelets (study at least 10 microscopic elds) is less than 5, report as
‘platelet decient’. If blast cells or other juvenile cells are seen, mention them in the dierential
count report. A quantitative estimate of the platelet count from the blood smear is described
in the following section.
Erythrocytes
In certain diseases, especially anaemia, erythrocytes (Figure 10.19) may have an abnormal
shape, size or colour. To check for abnormal erythrocytes, look at the cells just before the
thin end of the lm; this is where they are spread out. Just touching one another but not
overlapping. Do not look at the thick end, where the cells are too closely packed, or the thin
end, where there are not enough cells.
Abnormal morphology of red cells (Figure 10.19) must be reported along with the dieren-
tial count. These reports should be made in technical terms so that the reporting is precise and
meaningful. The following are the common abnormalities noted in RBCs during the study of
the blood smear.
Abnormalities in the morphology of red blood cells (arrows indicate the abnormal
cell compared against the normal cells): (a) Target cell, (b) Howell Jolly bodies, (c)
Nucleated red cells, (d) Hypochromic red cells, (e) Macrocyte, (f) Microcytic red cell,
(g) Cabot’s ring, (h) Acanthocytes – speculated rbc, (i) Schistocytes – fragmented
red cells, (j) Elliptocytes, (k) Sickle cells, (I) Hyperchromasia and spherocyte, (m)
Basophilic stippling, (n) Pappenheimer bodies-siderotic granules, (o) Blister or pruse –
prekeratocyte, (p) Hypersegmentation, (a) Siderocytes, (r) Crystals in red cells – HbC,
(s) Rouleaux formation
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Medical Laboratory Technology: Volume 1
Normal erythrocyte They are 6–8 μM, round-shaped, discoid, and occasionally slightly
irregular. Periphery of the cytoplasm is deep pink, centre pale pink or colourless.
Target cells They are 6–8 μM in size, round or slightly irregular shape, centre and periphery
of the cytoplasm stain well, but between them there is a colourless ring. Seen in thalassemia,
vitamin В deciency, haemoglobinopathy, liver diseases, sickle cell anaemia and iron
deciency anaemia.
Colour variation Normal-coloured red cells (normochromic) have uniformly-coloured
haemoglobin inside the cell with a small paler region in the centre. The paler region occupies
a large volume of the cell in the case of hypochromic cells. Hypochromasia is often related to
iron deciency. Polychromie red cells are grey-coloured and may be slightly larger in size. It
is often associated with increased reticulocyte count (reticulocytosis). On the other hand, red
cells with a deep stain and nucleus are suggestive of megaloblastic anaemia.
Size variation Red cells of normal size (7 μM) are known as normocytic. Increased size
(9–10 μM) of the red cells (macrocytosis) suggests a clinical diagnosis of megaloblastic
anaemia, which is associated with deciencies of folic acid or vitamin B
and can also be
ļ2
seen in some liver diseases. These cells must be dierentiated from reticulocytes. Decreased
red cell size (microcytosis) to about 5 μM size, is associated with iron deciency. The term
anisocytosis refers to variation in the size of red cells in a blood smear. Anisocytosis is seen
in many blood disorders.
Shape variation The shape of erythrocytes, which is normally round, varies in case of several
congenital defects. Anisocytosis refers to a condition in which erythrocytes of dierent sizes
are present in the blood. This happens in various types of anaemia. In case of sickle cells,
the shape is elongated and narrow, often one or both ends curved and pointed. Patients
with sickle cell anaemia and sickle cell thalassemia may also have nucleated erythrocytes,
target cells and often macrocytes. Stomatocytes are red cells with a central biconcave area,
which appears like a slit in dried lms (the name is derived from the stomata which are
microscopic structures found in leaves). Elliptocytes are elliptical or oval-shaped red cells
close to normal in size (8 μM). The cytoplasm stains are darker at the periphery (especially
at the poles). These are found in cases of hereditary elliptocytosis, iron-deciency anaemia,
pernicious anaemia, sickle cell diseases, thalassemia and myelobrosis. Spherocytes are
slightly smaller red cells (6 μM), perfectly round or spherical, with darker stained cytoplasm
than the normal erythrocytes. In smears, spherocytes appear without any paler region in
the centre and slightly smaller in appearance. Spherocytosis may occur in case of inherited
disorders or in autoimmune haemolytic anaemia. Poikilocytosis refers to variation in the
shape of RBCs. Like anisocytosis, poikilocytosis is associated with many blood disorders.
Target cells are red cells with a darker central region instead of the paler region and give a
‘bull’s eye’ appearance to the red cell. Presence of target cells in the peripheral blood smear
is associated with thalassemia, iron-deciency anaemia and haemoglobin С disease. A few
target cells in the peripheral blood smear should be considered as normal. Schistocytes are
normal or slightly smaller than normal erythrocytes. They are fragmented red cells and are
often seen in intravascular haemolysis and disseminated intravascular coagulation. Burr cells
are crenated red cells, which may occur as an artefact or in uraemia and in other medical
conditions.
Routine Haematological Tests
Abnormalities in the morphology of white cells: (a) Peiger Hûet anomaly, (b) Auer body,
(с) Toxic granulation, (d) Dohle body, (e) Hypersegmentation of neutrophil, (f) Hyposegmentation of neutrophil, (g) Monocytes in infectious mononucleosis, (h) Smudge cells,
(i) Basket cell, (j) Atypical lymphocyte
285
Inclusions Under pathologic conditions red cells may contain various inclusion bodies. In
order to communicate the abnormality seen by you in the blood smear it is important to
report it using the appropriate technical terms.
• Nucleated Red Cells (NRBC): These are immature red cells, slightly bigger in size
(8–10 μm) and are called erythroblasts. The shape is round or irregular; the nucleus is
round often eccentric, with deep purple, dense chromatin. The cytoplasm is pink or
greyish blue. They are found in the peripheral blood smear in severe haemolytic condi-
tions, for example, haemolytic disease of the newborn or blood loss. Their maturation
could be normoblastic or megaloblastic. They can also appear in patients with megaloblastic anaemia or leukemia.
• Howell–JollyBodies: These are dark coloured, small nuclear remnants found in red
cells; mostly single but sometimes multiple. Howell–Jolly bodies are regularly seen in
blood lms of post-splenectomy patients. They may also appear in haemolytic anaemia
and in megaloblastic anaemia. Do not confuse them with a thrombocyte lying on the
top of the cell.
• Basophilic Stippling: Punctuate basophilia is recognized by the appearance of ne blue-
black dots scaered in red cells and this is often associated with thalassemia, lead poisoning, infections and many other blood disorders. Do not confuse these with stain deposits.
• Cabot Rings: These are purple staining, thread-like laments in the shape of a ring or
loop or gure- of-eight in the RBCs, which are often encountered in haemolytic anaemia
and megaloblastic anaemia.
• Haemoglobin СCrystals: These are seen within and outside red cells in the congenital
defect of homozygous HB C-C disease. Diagnosis made on the basis of blood smear
study should be conrmed by Hb electrophoresis.
• Heinz Bodies: These inclusion bodies are found in patients with glucose-6-phospha-
te deciency. The blood specimen of the suspected patient is subjected to supravital
staining with methyl violet (0.5% methyl violet in 0.85% NaCl). In supravital staining,
cells stay functional while absorbing the stain. Following the staining process, the blood
smear is made. Reticulocytes are also stained in the same way. Heinz bodies will not be
visible with Leishman stain.
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Medical Laboratory Technology: Volume 1
• Additional Information Supravital staining is a method of staining used in microscopy
to examine living cells that have been removed from an organism. The core concept in
supravital staining is that the cell being examined is still alive. The most common su-
pravital stain is performed on reticulocytes using new methylene blue or brilliant cresyl
blue, which makes it possible to see the reticulolamentous partem of ribosomes characteristically precipitated in these live immature erythrocytes by the supravital stains.
By counting the number of such cells the rate of red blood cell formation can be determined.
• Siderocytes: Siderocytes are mature red cells with deposits of iron (ferritin), which are
stainable by Prussian blue stains. If the iron deposits are seen in erythroblasts and found
in the bone marrow, they are called sideroblasts. The granules may be single or many.
Smears stained with Romanowsky stains show less vividly stained iron granules (called
Pappenheimer bodies). For conrmation of these granules, a separate smear should be
stained with an iron stain like Prussian blue stain.
Prussian Blue Staining of Blood Smear
Principle Prussian Blue or Perls’ reaction is used to demonstrate ferric iron and ferritin. This
is not a true staining technique; rather it is a histochemical reaction. The protein is split o by
the hydrochloric acid, allowing the potassium ferrocyanide to combine with the ferric iron.
• Siderocytes: Prussian blue reaction (hydrochloric acid—ferrocyanide) gives a bright
blue colour to the non-haemoglobin iron granules present in the abnormal red cells,
called siderocytes. Siderocytes are most commonly found in patients with haemolytic
anaemia and after splenectomy. Occasional siderocytes may be found in normal blood.
The siderocyte granules tend to occur in younger red cells.
• Reticulocytes: These are immature red cells that pass into the blood stream from the
bone marrow. They contain granules (nuclear remnants) that stain dark blue with vital
staining such as brilliant cresyl blue. Under normal conditions, they disappear within
4 h after the release of erythrocytes into the blood. Their presence in circulation (reticulocytosis) indicates blood loss. The procedure for a quantitative report of reticulocyte
(reticulocyte count) has been presented later in this chapter.
Reagents
Hydrochloric acid, 4% v/v in water
Hydrochloric acid (concentrated) 4mL
Distilled water 96 mL
Potassium ferrocyanide solution, 4%, aqueous
Potassium ferrocyanide 4g
Distilled water (made to) 100 mL
Nuclear fast red counterstain
Nuclear fast red 0.1 g
Aluminium sulphate solution, 5% aqueous 100 mL
Dissolve with heat, then cool and lter. Add a crystal of thymol as preservative.
Working solution of Prussian Blue reagent: Mix equal parts of hydrochloric acid and
ferrocyanide solutions just before use.
Procedure
1. Fix the smear in methanol for 2–3 min.
2. Place the smear in staining jar (Coplin) containing Prussian blue reagent for 30 min.
Note The reagent must be fresh.
Routine Haematological Tests
3. Rinse in distilled water.
4. Counterstain with nuclear fast red for 5 min.
5. Rinse in distilled water.
6. Air-dry and examine.
287
Leukocytes
In contrast to erythrocytes, leukocytes contain a nucleus (Figure 10.20) that may vary in size
and shape. As already mentioned, there are ve main types of leukocytes—neutrophils,
eosinophils, basophils, lymphocytes and monocytes. Most of the abnormalities are seen in
neutrophils but other cells like atypical lymphocytes and megakaryocytes (parent cells of
thrombocytes) may also be involved.
• Neutrophils, Eosinophils and Basophils (Figures 10.17 and 10.18): These white cells are
grouped as polymorphonuclear cells because they contain a nucleus with several lobes.
The cytoplasm contains granules and they are also called granulocytes. The neutrophils
are approximately 12–15 μM, round- shaped and well dened. The nucleus usually
bears two lobes and the cytoplasm is barely visible. The cytoplasm contains numerous
large, round, densely packed orange-red granules. Sometimes the cells appear damaged
with scaered granules. The basophils are 11–13 μM, round-shaped; however, the nucleus is dicult to see because it is covered by granules. The cytoplasm of basophils is
not clearly visible. It contains deep purple granules, less densely packed than those of
eosinophils. Small colourless vacuoles are sometimes present.
Relative sizes of white cells and their nuclear structures.
• Lymphocytes and Monocytes have compact nuclei (Figure 10.21). They may or may not
have granules in the cytoplasm. Hence they are not granulocytes. Small lymphocytes
(7–10 μM) are close to the size of the red cells. They are round-shaped and the nucle-
us is large, occupying most of the cell with a densely packed dark purple chromatin.
The cytoplasm is barely visible. If seen, it is blue in colour, with no granules. Large
lymphocytes are 10–15 μm size, round or irregularly shaped; the nucleus is oval or
round and can lie on one side of the cell. The cytoplasm is abundant, pale blue in colour and contains several large, dark red granules. Monocytes are the largest of the
leukocytes (15–25 μm) irregular-shaped, with typical kidney-shaped or variable-shaped
nucleus, with pale mauve chromatin arranged in strands. The cytoplasm of monocytes
is pale blue in colour and contains ne, dust-like, usually reddish granules. Vacuoles
are usually present in the cytoplasm. In patients suering from malaria, the cytoplasm
often contains brownish-black masses. These masses are malaria pigment.
• Plasma Cells (Figure 10.19): Under certain clinical conditions (measles, tuberculosis, oth-
er viral and bacterial infections, multiple myeloma), plasma cells are visible in circula-
tion. They are 12–15 μM, with round nuclei, eccentric in location, bearing densely packed
chromatin and often in a wheel-like arrangement. The cytoplasm is dark blue in colour
with a pale-staining area around the nucleus. They are large in number and very small;
not easily seen vacuoles are present. Plasma cells produce antibodies to combat infection.