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Romanowsky, Automated, and Pearls Staining
Romanowsky, Automated, and Pearls Staining
The purpose of examining peripheral blood smear include assessing various elements of
peripheral blood cells such as erythrocytes, leukocytes and platelets and look for parasites such
as malaria, trypanosoma, microfilaria and others 1. Smear preparations that are well made and
daubed are an absolute requirement for getting good examination results.
The basis of Romanowsky staining is the use of two different dyes, namely Azur B
(Trimetiltionim) which is alkaline and eosin y (tetrabromoflurescein) which is acidic. Azur B will
color cell components that are acidic such as chromatin, DNA, and RNA. Whereas eosin y will
color basic cell components such as eosinophil granules and hemoglobin. The eosin y bond on
the aggregated Azur B can produce a purple color, and this condition is known as the
Romanowsky Giemsa effect. This effect occurs very significantly in DNA but not in RNA giving
rise to a colored nucleus with a cytoplasm that coloring is blue.2
The best examination material is fresh blood from the capillaries or veins, which are
blotted on the slide. In certain circumstances EDTA blood can also be used. 3
1. Wrights Stain4
Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue.
As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining.
However, fixation helps to reduce water artefact that can occur on humid days or with aged
stain.
Methanol fixes the cells to the slide. Eosin Y is an acidic anionic dye and methylene blue is
basic cationic dye. When diluted in buffered water, ionization occurs. Eosin stains the basic
components such as hemoglobin and eosinophilic granules an orange to pink color. Methylene
blue stains acidic cellular components such as nucleic acid and basophilic granules in varying
shades of blue. The neutral components of the cells are stained by both components of the dye,
producing variable colors.
2. Leishman Stain5
4. Giemsa Stain
Automatic staining machines are available that enable large batches of slides to be
handled. They may be either stand-alone staining machines or a part of a large automated
blood counting instrument. In many instances, the instrument spreads, fixes, and stains blood
films. Some automated instruments incorporating staining can only be programmed to prepare
and stain a single film per sample. Others can prepare and stain multiple films from a single
blood sample; this is useful in a teaching programme with a large number of students.
Some systems apply staining solutions to slides lying horizontally (flat-bed staining),
whereas others either immerse a slide or slides in a bath of staining solution (“dip-and-dunk”
technique) or spray stain onto slides in a cytocentrifuge. Problems include increased
background staining, inadequate staining of neutrophil granules, degranulation of basophils,
and blue or green rather than pink staining of erythrocytes. These problems are usually related
to the specific stains and staining protocols used rather than to the type of instrument,
although flat-bed stainers are more likely to cause problems with stain deposit. However, as a
rule, staining is satisfactory provided that reliable stains are used and there is careful control of
the cycle time and other variables. 11 Flat-bed stainers may not stain an entire film (e.g., a bone
marrow film) if the film exceeds the standard length.
Perls Staining
Prussian blue (Perls’) reaction is a method for staining non-haem iron in normoblasts
(siderocytes), macrophages (haemosiderin), and other cells containing particulate iron. The
granules are formed of a water-insoluble complex of ferric iron, lipid, protein and carbohydrate.
The method allows assessment of both the amount of iron in reticulo-endothelial stores and
availability of iron to developing erythroblasts. Principle of perls staining is The granules
(containing ferric iron) react with pottassium ferrocyanide [K4Fe(CN)6] to form a blue compound
ferriferrocynanide), Prussian blue reaction.12
Fixation of perls staining is Avoid the use of acid fixatives. Chromates will also interfare
with the preservation of iron.
Procedures: