J Clin Pathol 1989;42:101-105

Use of microwave oven improves morphology andstaining of cryostat sectionsA KENNEDY, A K FOULIS Department ofPathology, Royal Infirmary, Glasgow

SUMMARY The quality of microscopic image of cryostat sections that had been subjected tomicrowave assisted fixation was compared with that resulting from conventional air drying of thesections. The role of microwaves in producing rapid special stains on cryostat sections was alsoassessed. Methods used permitted stains such as periodic acid Schiff, alcian blue, Gordon andSweets's reticulin, Masson Fontana, Elastica, Prussian blue and Van Gieson to be performed withinthree minutes ofcutting a cryostat section. The cytological detail of nuclei was much clearer using themicrowave technique, allowing more accurate determination ofcell type. The microwave oven seemsto have major potential in improving the diagnostic accuracy of surgical frozen sections.

The use of cryostat sections in rapid microscopicdiagnosis has become a routine part of the histopath-ological service in most laboratories. The technique isnot, however, without its problems. The quality of themicroscopic image, particularly when using a highpower objective lens, is very poor, and identification ofindividual cell types, which often relies on goodcytological detail of nuclei, is correspondinglydifficult. A second problem is that there is usuallyconstraint placed on the time available to make thediagnosis and this by and large precludes the use ofhistochemical stains other than haematoxylin andeosin.

It has been reported that the microscopic image of afrozen section can be substantially improved by usingmicrowave assisted fixation.' We evaluated this tech-nique to see whether we could confirm this.Microwave ovens have also been used to allow more

rapid staining techniques for formalin fixed paraffinwax embedded sections.2 We also assessed the pos-sibility of using a microwave oven to produce rapidspecial stains on surgical frozen sections.

Material and methods

The method of Kok et al' was modified as follows.Tissue was mounted on a cryostat chuck with OCTembedding compound (Miles Scientific, Slough,England) and frozen in a slush of methanol and solidcarbon dioxide. Five micron sections were cut andpicked up on clean glass slides. The cut sections wereimmediately plunged into 20 ml of 5% acetic acid inindustrial alcohol 990P (Wolman's fixative) in a plastic

Accepted for publication 21 July 1988

five-slide mailer (Histo-Lab, Hertfordshire, England).The slide mailer was placed at the centre of themicrowave oven turntable and subjected to eightseconds of microwave irradiation at full power (level10, 600W) (Network model NW610, Network Elec-tronic Industries, London). The oven was operated at2 45 GHz and had been designed for domestic use. Thelid of the mailer was left open during this period toprevent a build-up of pressure in the container. Thesections were then immediately washed in water andrapidly stained with haematoxylin and eosin in theconventional manner used for frozen sections. It wasessential that the slide should not dry out before,during, or after the microwave assisted fixation as thiswas shown to have deleterious effects on nuclearmorphological detail.

Section adhesives were not required even for thespecial staining methods, but the occasional "fattybreast", with a tendency to float, benefited from areduction in both the power level and the duration ofmicrowaving (to level 1 and five seconds, respectively).The benefit was complemented by transferring thesection directly from the microwaved fixative to thehaematoxylin solution.

SPECIAL STAINING METHODSAll of the methods listed below could be performedsuccessfully after the acetic-alcohol-microwave fixa-tion protocol. Not all of the steps in any methodrequired microwave stimulation; where this was neces-sary, the method refers to a standard (50 ml) glassCoplin jar containing the relevant solution. The use ofa glass lid to prevent spillage or spurting of solutions isrecommended. Glass Coplin jars were preferred tocurrently available plastic alternatives as they permit-

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ted direct viewing of the staining solution, providingearly warning of any solution boiling over. If asolution showed a tendency to boil the method couldbe changed either by reducing the power level of themachine or reducing the irradiation time.

It should be noted that with reuse of a hot stainingsolution, much shorter microwave treatments wouldbe required. Fresh solutions used at room temperatureworked better, but with the exception of the Perls'spotassium ferricyanide-hydrochloric acid reagent, allthe staining solutions used in this study were reusedseveral times without apparent deterioration in theirstaining properties.

Periodic acid Schiff (PAS)3The standard procedure was adapted by treating thesections with 1% periodic acid in the microwave ovenfor 20 seconds at full power. The sections were thenrinsed in water and treated with Schiff reagent for 30seconds at full power. A haematoxylin counterstainwas applied after a wash in water, and the sectionswere dehydrated, cleared, and mounted.The microwave treatment caused a magenta

colouration of the upper layers of the staining solu-tion. This spontaneously disapppeared on standing atroom temperature and the solution could safely bereused. If the solution ultimately failed to lose thiscolour it was discarded.

Alcian blue-PAS3The conventional 1% alcian blue in 3% acetic acidsolution was used in the microwave oven at full powerfor 10 seconds. It was then possible to counterstainwith the PAS method described above.

Van Gieson3The following method did not require microwavestimulation at any stage. Sections were stained withcelestin blue for 30 seconds, rinsed in water, andstained in Gill's haematoxylin for 30 seconds. Thesections were then rinsed in tap water and blued inScott's tap water substitute. After a further rinse in tapwater the sections were rinsed in methylated spiritsand immersed in van Gieson solution for 30 seconds.The sections were then transferred to meths beforebeing dehydrated, cleared, and mounted.

Gordon and Sweets's reticulin3Only the sensitisation step in 2-5% iron alum requiredmicrowave stimulation (power level 3 for one minute).The acidified potassium permanganate step was

limited to 10 seconds before bleaching with oxalicacid. Gold chloride toning was not necessary. Treat-ment with sodium thiosulphate could also be shor-tened to 10 seconds without the need for microwaving.

Kennedy, Foulis

Perls's Prussian blue3The staining solution was microwaved for 30 secondsat full power. The solution was discarded after use.

Masson Fontana3The microwave frozen section method differed fromthe conventional one in that silver solution was used inthe microwave oven at power level 5 for one minute.Gold chloride toning was seldom necessary andsodium thiosulphate treatment was limited to 10seconds without microwaving.

Miller's elastic3Sections were treated with acidified permanganate for10 seconds and then bleached with oxalic ,cid. Afterrinsing in water the sections were transfeired to thestaining solution and microwaved for one minute atpower level 1. It was convenient to counterstain thesections with van Gieson stain, for which a 10 seconddip was adequate.To make an objective assessment of the quality of

image achieved using the microwave assisted frozensection technique, paired sections from 24 specimens(which included 11 different tissues) were processedand stained by the standard technique-frozen sec-tions were warmed, air dried, and then placed inWolman's fixative4-or by the microwave assistedfixation technique. The slides were then viewed blindby four pathologists: they were not told the identity ofthe pairs. They were asked to state which section ofeach pair they felt provided the better microscopicimage.

Results

At low power an obvious difference could be discernedbetween sections which had been treated convention-ally (air drying, followed by fixation in Wolman'ssolution) and those which had been processed usingthe microwave assisted technique (fig 1). The micro-waved sections tended to appear crisper and to have abetter definition between haematoxyphilic and eosino-philic areas. The difference in quality of the sectionswas, however, most obvious when using high powerobjectives: the cytological detail of nuclei was verymuch improved by microwaving (fig 2). This differenceapplied to all tissues tested but was probably mostobvious in lymphoid organs. A practical example ofthe benefit of such improved imaging was a thyroidbiopsy specimen for which the differential diagnosisgiven by the surgeon was Hashimoto's disease ortumour. The frozen section showed a monotonousinfiltrate of uniform cells in a collageneous back-ground. On high power examination with the conven-tional technique the nature of the cells could not bedefined and no diagnosis was possible, but the micro-

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Microwaves and cryostat sections

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(a)Fig 1 Cryostat sections ofan adenolymphoma ofparotid treated conventionally (a) and by microwave assistedfixationtechnique (b). Note improved low power image in (b). (Haematoxylin and eosin.)

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Fig 2 Cryostat sections ofnon-Hodgkin'sfollicular lymphoma treated conventionally (a) and by microwave assistedfixation(b). Note increased nuclear detail (b). Nucleoli are much more prominent and the chromatin pattern approximates more

closely to that seen in paraffin wax embedded sections. (Haematoxylin and eosin.)

waved section showed that virtually all the cells wereplasma cells and thus Hashimoto's thyroiditis was

diagnosed conclusively (fig 3).When the 24 paired sections were given to the four

pathologists to determine which sections they thoughtwere more useful diagnostically, 91 of the 96 possiblechoices favoured the microwaved sections.

All the special stain techniques given here werefound to work well on frozen section (figs 4 and 5). Allcould be performed within three minutes ofcutting thefrozen section.

Discussion

Frozen sections have found an established role in thehistopathology laboratory. Most pathologists,

however, find that when making the diagnosis theyhave to rely on a relatively low power image becausethe use of high power objectives adds little informa-tion. The poor cytological detail, particularly ofnuclei, which generally appear opaque, makes iden-tification of cell types very difficult if their nature was

not apparent on the lower power. We can confirm theresults of Kok et al': the microscopic image is verymuch improved when using microwave assisted fixa-tion, and this is bound to lead to greater diagnosticaccuracy.The main purpose of this report has not been to

investigate how the microwaving is effective, but itseems likely that microwave irradiation may help insection adherence to the slide. This may be facilitatedby microwaves permitting rapid diffusion of water

(a)

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Kennedy, Foulis

Fig 3 Cryostat sections ofplasma cells in Hashimoto 's thyroiditis. Microwave assistedfixation was used in both (a) and (b)but sections were allowed to air dry brieflyfollowingfixation (a). Notefine quality ofnuclear detail (b) and deleterious effectofair drying (a). (Haematoxylin and eosin.)

Fig 4 PAS stain on a cryostat section ofstomach. This wasperformed within one minute ofcutting the section.

molecules from between the section and the slide.Rapid uniform heat production may also be beneficial.The conventional technique relies on air drying toachieve section adherence before fixation. We haveshown that if a section is allowed to air dry, even if ithas been microwaved previously, then the quality ofthe image is poor (fig 3). Thus the improved micro-scopic image of the microwave assisted fixation tech-nique is probably at least partially due to the ability toavoid the deleterious effects of air drying. No doubt,prompt uniform fixation also helps.

Conventional "special" histochemical stains areseldom used when reporting surgical frozens. We haveshown that a wide variety can be performed satisfac-torily on frozen sections within three minutes of

Fig 5 Perls's Prussian blue on a liver biopsy specimen.Residual brown pigments include bile and lipofuschin. Adeposit ofcarcinoma lay adjacent to thisfield.

cutting the section, making their use a practicalproposition. Individual pathologists will easily be ableto imagine situations in which these techniques couldbe useful. An obvious example would be the use of thePAS method when assessing resection margins in agastric carcinoma.The range ofmethods presented has been restricted

to those for which, we believe, there is greatestapplication in frozen section diagnosis. Ourexperiments on paraffin wax sections and on frozensections, however, indicate that virtually any specialstain can now be completed within three minutes andcan thus be applied on a routine basis to frozen sectionrequests.

Since the techniques described here were developed

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Microwaves and cryostat sections 105

in our laboratory we have abandoned the previoustechnique of air drying sections. The microwaveassisted fixation technique is now also used by otherlaboratories in our area. Because ofthe huge domesticmarket microwave ovens are now cheap. The modelused in this study cost £150. Microwave ovens arefinding an increasing number ofuses in histopathologylaboratories.5 We feel that the improvement in qualityoffrozen sections alone is sufficient reason to purchasesuch a relatively inexpensive item.

Mrs I Main kindly typed the manuscript.

References

I Kok LP, Boon ME, Suurmeijer AJH. Major improvement inmicroscopic-image quality ofcryostat sections combining freez-ing and microwave-stimulated fixation. Am J Ciif ;Pathol1987;88:620-3.

2 Boon ME, Kok LP, Suurmeijer AJH. Microwaves and staining.In: Boon ME, Kok LP, eds. Microwave cookbook ofpathblogy.Leiden: Coulomb Press Leyden, 1987:97-114.

3 Bancroft JD, Cook HC. Manual of histological techniques.Edinburgh: Churchill Livingstone, 1984.

4 Culling CFA. Handbook of histopathological and histochemicaltechniques. Third edition. London: Butterworths, 1974:146-7.

5 Boon ME, Kok LP, eds. Microwave cookbook of pathology.Leiden: Coulomb Press Leyden, 1987:224.

Requests for reprints to: Mr A Kennedy, Department ofPathology, Royal Infirmary, Glasgow, Scotland.

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