Page 293 - Textbook of Pathology, 6th Edition

prepared smears. While commercial preparations (e.g. After that, all except the bottom 200 ml is discarded. The 277
cytospray) are available, a standard hairspray with a high retained fluid is then processed. In specimens containing
alcohol content is also a suitable alternative. Smears while excessive amounts of blood, erythrocytes may be lysed by
still wet, are placed face-up on a table and sprayed with the the addition of 1 ml of glacial acetic acid for every 50 ml of
nozzle held at a distance of 10 to 12 inches. A period of 10 specimen.
minutes is sufficient for drying of coated smears, which may Commonly employed techniques for processing of fluids
then be wrapped or put in a box for transport to the are as under: CHAPTER 11
laboratory. Coating fixatives are ideal when unstained smears Sediment smears. The sample is poured into 50 ml centrifuge
are to be transported over long distances. tubes and centrifuged at 600 g for 10 minutes. (To achieve a
3. SPECIAL PURPOSE FIXATIVES. Buffered neutral relative centrifugal force of 600 g, the speed of the centrifuge
formalin, Bouin’s fluid and picric acid are used for specific in revolutions per minute varies with the rotating radius of
purposes when required. Formalin vapour fixation is also the centrifuge. With a rotating radius of 25 cm the required
employed for some staining procedures. speed is 1500 rpm).
Following centrifugation, the supernatant is decanted and
4. PRESERVATION OF FLUID SAMPLES. Samples of
fluids are best submitted to the laboratory in a fresh state for smears prepared from the sediment or cell button by
recovering the material with a glass pipette or a platinum
immediate processing. Where a delay is anticipated in wire loop. Smear preparation from samples collected in a
despatch to the laboratory or in processing, the sample is preservative require albuminised slides as cell adhesiveness Basic Diagnostic Cytology
collected in a suitable preservative for ‘prefixation’ so that is reduced by prefixation. Smears are wet-fixed in 95%
cellular morphology is preserved. When the samples have ethanol (or air-dried in some instances).
been processed and smears prepared, ‘fixation’ is effected
as described earlier. Cytocentrifuge and membrane filter preparations. These
Fluid samples with a high protein content (e.g. peri- methods are most useful for small volume fluids of low cell
cardial, pleural and peritoneal fluids) may be preserved content. The interested reader is referred to specialised texts
under refrigeration for up to 12 hours without prefixation. for descriptions of these methods.
However, fluids with a low protein content (e.g. urine and
cerebrospinal fluid) deteriorate in 1 to 2 hours even if D. Staining of Smears
refrigerated. Fluids with low pH (e.g. gastric lavage samples) Three staining procedures are commonly employed:
must be collected in pre-cooled preservative since cells Papanicolaou and H&E stains are used for wet-fixed smears
deteriorate extremely rapidly in such specimens. while Romanowsky stains are used for air-dried smears.
The best preservative for general use is 50% ethanol in
volumes equal to that of the fluid sample. Ninety-five per 1. PAPANICOLAOU STAIN. Papanicolaou staining is the
best method for routine cytodiagnostic studies. Three
cent ethanol precipitates proteins and hardens the sediment solutions are used comprising a nuclear stain and two
making smear preparation difficult; it is used only for gastric cytoplasmic counter-stains. Harris’ haematoxylin is the
aspirates. Pericardial, pleural and peritoneal fluids may be nuclear stain. Orange G (OG-6) and eosin-alcohol (EA-65 or
pre-fixed with an equal volume of 10% formalin if ethanol is EA-50) are the two cytoplasmic counterstains which impart
not available. Solutions containing ether and acetone are not the orange and cyanophilic tints to cytoplasm respectively.
used as preservatives as they cause extreme hardening of
the sediment making smear preparation virtually impossible. 2. H & E STAIN. The stain is essentially the same as that
used for histological sections. Harris’ haematoxylin is the
C. Processing of Samples in the Laboratory nuclear stain, and eosin is the cytoplasmic counterstain.
1. PREPARED SMEARS. Smears prepared at the bedside 3. ROMANOWSKY STAINS. Romanowsky stains used in
and wet-fixed in ethanol need no further processing in the haematological preparations may also be used for cytological
laboratory prior to staining. smear preparations. Leishman’s, May-Grünwald-Giemsa
(MGG) and Wright’s stains are most commonly used.
2. SPUTUM. The sample is prepared as under:
i) The sample is placed in a petridish and inspected against
a dark background. INTERVENTIONAL CYTOLOGY
ii) Bloody, discoloured or solid particles are removed with In interventional cytology, samples are obtained by
wooden applicator sticks and placed on glass slides. Strands aspiration or surgical biopsy. This branch includes fine needle
of ropy mucus are also selected (exfoliated cells adhere to aspiration cytology, imprint cytology, crush smear cytology
mucus strands). and biopsy sediment cytology.
iii) Clean glass slides are used to crush the particles/mucus
and spread the material evenly. I. FINE NEEDLE ASPIRATION CYTOLOGY
iv) Four such smears are prepared and immediately placed Interventional cytology is virtually synonymous with Fine
in 95% ethanol for fixation.
Needle Aspiration Cytology (FNAC). The technique has
3. FLUID SPECIMENS. Large volumes of fluid received gained wide acceptance in the last four decades and is
are allowed to stand in the refrigerator for half to one hour. increasingly being used to sample a wide variety of body