use of destained cytology slides for the application of routine.pdf

Use of destained cytology slides for the application of routinespecial stains

R. Marcos, M. Santos, N. Santos, F. Malhao, F. Ferreira, R.A.F. Monteiro, E. Rocha

Cytology Diagnostic Services, Laboratory of Histology and Embryology, ICBAS - Institute of Biomedical Sciences Abel Salazar, UP, University

of Porto, Porto, Portugal

Key Words

Cytochemistry, cytology, destaining, dog,

special stains

Correspondence

Ricardo Marcos, Cytology Diagnostic

Services, Institute of Biomedical Sciences

Abel Salazar – ICBAS, Lg. Prof Abel Salazar, 2,

4099-003 Porto, Portugal

E-mail: [email protected]

DOI:10.1111/j.1939-165X.2008.00098.x

Background: Special stains to demonstrate microorganisms or intra- and

extracellular substances have not been evaluated in detail regarding their

applicability and usefulness in destained cytologic specimens.

Objectives: The aim of this study is to compare the results of routine special

stains on destained slides previously stained with Hemacolor and on fresh

(unstained) specimens.

Methods: Archival Hemacolor-stained fine needle aspirate specimens of

inflammation with infectious agents (bacterial, mycobacterial, and fungal

infections), neoplasia (melanoma, myxosarcoma, and mammary adeno-

carcinoma), and hemorrhage (pericardial effusion) from 14 dogs and 7 cats

were selected. Cells in a minimum of 4 fields were photographed and

5 slides from each case were then destained by different methods (alcohol

acid or microwave). Seven special stains were applied selectively to the

destained slides, depending on the cytologic findings: periodic acid Schiff,

Grocott–Gomori methenamine silver, Gram’s, Ziehl–Neelsen, Alcian blue,

Fontana–Masson, and Prussian blue. The same fields were rephotographed

and 2 observers evaluated the slides qualitatively, with comparison to fresh

cytologic specimens from similar lesions.

Results: Special stains applied to destained slides demonstrated the ex-

pected cellular and extracellular material or organisms independent of the

destaining method. Staining intensity, nonspecific staining (background),

cell morphology, and nuclear counterstaining results were similar to those

of special stains applied to fresh unstained slides.

Conclusions: Destaining does not appear to affect the results of routine

special staining for cytologic specimens. Destaining before special stains

may be a valuable diagnostic strategy when few slides are present or only

stained slides are available.

Introduction

Almost 150 years have passed since Bernard demon-

strated ferric iron in tissues with the Prussian blue

reaction, introducing histochemical methods in pa-

thology.1 Since then, different reactions and stains

have been described for demonstrating carbohydrates,

proteins, lipids, pigments, and microorganisms, includ-

ing bacteria and fungi.2 Histochemical methods play a

major role in diagnostic pathology for confirming or

reaching a diagnosis, especially in atypical infections or

undifferentiated neoplasia.2 Histochemical stains have

long been applied to cytologic specimens in medical

and veterinary pathology3,4; several recent reports

have been published that highlight the use and impor-

tance of special stains in veterinary cytology.5–11

One of the major drawbacks for the use of special

stains in cytology practice is the highly variable sample

quality among slides. Smears often differ in cellularity,

contaminants, individual cell populations, and mor-

phology.12,13 Sometimes, consecutive aspirations of the

same mass yield widely different samples, and acellular

samples and blood contamination frequently occur.

Typically, the use of special stains is done after the re-

view of a routine Romanowsky-stained smear, and

thus requires an additional unstained sample.14 If

94 Vet Clin Pathol 38/1 (2009) 94–102 c� 2009 American Society for Veterinary Clinical Pathology

Veterinary Clinical Pathology ISSN 0275-6382

i:/BWUS/VCP/98/[email protected]

additional samples lack representative material, false

negative or inconclusive results may occur.

The objective of this study was to evaluate the use

of special stains relevant in the diagnosis of pathologic

lesions in cytologic samples from dogs and cats, spe-

cifically to determine whether destaining slides after

Romanowsky staining was a reliable option. We also

evaluated 2 different destaining methods. The results

using destained slides were compared with the results of

special stains applied to fresh (unstained) samples from

similar lesions.

Materials and Methods

Slides were retrieved from the archives of the Cytology

Diagnostic Services at ICBAS, University of Porto, from

September 2002 to June 2007. Twenty-one cases (from

14 dogs and 7 cats) with the following lesions or diag-

noses were selected: inflammation with infectious

agents (bacterial, mycobacterial, and fungal infections),

neoplasia (melanoma, myxosarcoma, and mammary

adenocarcinoma), and hemorrhage (pericardial effu-

sion). Cases were included only if a minimum of 5 rep-

resentative slides was present and if the diagnosis was

later confirmed by histopathology or microbiological

methods (except for the pericardial effusion).

The slides had been stained with Hemacolor

(Merck, Darmstadt, Germany) with extended postfix-

ation and staining times, as described by Jorundson

et al.15 The slides were mounted with DPX mounting

medium (Merck). All slides were photographed with

a digital camera (Camedia 4050, Olympus, Tokyo,

Japan) fitted to a microscope (BX-50, Olympus), in re-

corded positions on the x and y axes of the microscope

stage (minimum of 4 fields per slide, using �10, � 40,

and �100 objectives). Afterwards, to remove the cov-

erslip, the slides were immersed in xylene for 1–3 days.

After coverslips were removed, the slides were left for

1 additional day in xylene to ensure complete removal

of the mounting medium.

The 5 representative slides of each case were

destained by one of 2 main methods: microwave at

600 W, placing the slides either in citrate buffer

(pH = 6.0) or in distilled water; and alcohol acid, in

which we tested 1%, 5%, and 10% solutions (ie, etha-

nol 70% with 1, 5, and 10 mL of concentrated HCl).

Afterwards, the slides were hydrated in a decreasing

series of alcohols, tap water, and distilled water (2 min-

utes in each solution) followed by cytochemical stain-

ing using conventional techniques and appropriate

counterstains (Table 1).1,16

The following special stains were applied to the

destained slides, depending on the type of lesion

(Table 1): periodic acid Schiff (PAS), Grocott–Gomori

methenamine-silver (GMS), Gram’s (including the

Twort’s variant), Ziehl–Neelsen, Alcian blue, Fontana–

Masson, and Prussian blue. Positive and negative con-

trols (paraffin tissue sections) were run for all the

special stains. A minimum of 15 slides from 3 different

cases were evaluated per special stain. The slides were

rephotographed in the same positions as for the Hema-

color-stained specimens and both specimens were eval-

uated qualitatively by 2 observers (RM, MS) blinded to

the diagnosis. The observers assessed staining inten-

sity, the presence of nonspecific (background) staining,

cell morphology, and nuclear counterstaining. In addi-

tion, Ziehl–Neelsen, Alcian blue, and PAS stains were

applied directly to 3 extra Hemacolor-stained slides

from the mycobacterial and fungal lesions and the my-

xosarcoma (which were hydrated in a decreasing series

of alcohols and distilled water, but not destained). In

the case of fungal specimens, we also tested the con-

secutive use of PAS and GMS stains.

Fresh material from one case of each inflammation

with infectious agents (bacterial, mycobacterial, fungal),

neoplasia (melanoma, myxosarcoma, mammary adeno-

carcinoma), and hemorrhage (pericardial effusion) was

Table 1. Special stains and counterstains applied to destained cytologic specimens in this study.

Lesion

Structure/Cell/Material

Stained Special Stain

Color of a Positive

Reaction Nuclear Counterstain

Bacterial infection (septic

purulent inflammation)

Bacteria Gram’s and

Twort’s Gram

Blue (gram-positive) or

red (gram-negative)

Basic fuchsin and neutral red1

fast green

Mycobacterial infection Mycobacteria Ziehl–Neelsen Red Methylene blue

Fungal infection Fungal cell wall PAS, GMS Purple, black Gill’s hematoxylin, light green

Melanoma Melanin granules Masson–Fontana Black Light green or neutral red

Myxosarcoma Mucin Alcian blue Sky blue Gill’s hematoxylin

Mammary adenocarcinoma Mucin (secretory material) PAS Purple Gill’s hematoxylin

Hemorrhagic effusion Iron (ferric) Prussian blue Blue Neutral red

PAS indicates periodic acid-Schiff; GMS, Grocott–Gomori methanamine silver.

Vet Clin Pathol 38/1 (2009) 94–102 c� 2009 American Society for Veterinary Clinical Pathology 95

Marcos et al Use of destained cytology slides

obtained from 4 dogs and 3 cats by fine-needle aspira-

tion at the Veterinary Clinic at ICBAS - University of

Porto. A minimum of 2 unstained slides were obtained

per case. Cytochemical stains were applied directly

(without the destaining step) and the staining results

were compared, by the same observers, with those of

the same special stains on destained samples.

Results

Destaining was achieved using both acid alcohol and

microwave techniques; no major differences in cell

morphology were noted between the destaining meth-

ods. The time needed to completely destain the slides

ranged from 1 to 15 minutes, depending on the thick-

ness of the smear. Nevertheless, destaining was faster

(taking half the time) with the 5% and 10% alcohol

acid solutions compared with 1% alcohol acid solution

and the microwave. The use of citrate buffer vs distilled

water resulted in no difference in destaining time or

cell morphology in the microwave method.

In destained slides of bacterial infections, rod-

shaped and filamentous bacteria were stained red with

Gram’s stain (gram-negative), whereas cocci stained

blue (gram-positive) (Figure 1). Gram’s stain and

Twort’s stain rendered comparable results, although

the distinction of bacteria tended to be slightly easier

with the latter, because of the greenish background.

Ziehl–Neelsen clearly stained acid-fast bacilli of Myco-

bacterium lepraemurium in destained slides of a subcu-

taneous mass in a cat. Direct application of this stain to

Hemacolor-stained slides tended to increase the stain-

ing intensity (Figure 2).

PAS stained fungal elements positively in de-

stained slides containing Microsporum and Trichosporum

Figure 1. Bacterial infection in a cutaneous nodule from a dog. (A) Mod-

erately karyolytic neutrophils with intra- and extracellullar coccoid

bacteria (Staphylococcus sp.). Hemacolor. (B) Twort’s Gram stain applied

after destaining the Hemacolor slide with 5% alcohol acid. The bacteria

are blue (gram-positive) and the erythrocytes are light green.

Bar = 22.5 mm.

Figure 2. Mycobacterial infection (Mycobacterium lepraemurium) in a

subcutaneous nodule from a cat. (A) Large numbers of macrophages

filled with negative linear rod-shaped bacteria. Hemacolor. (B) Ziehl–Neel-

sen staining of the Hemacolor-stained slide. In A note that in some cells

(arrows) and in the background, the acid-fast bacteria that can be seen in

B are not apparent. Bar = 22.5 mm.


Marcos et alUse of destained cytology slides

(Figure 3). Like Ziehl–Neelsen, PAS applied directly to

Hemacolor-stained slides (ie, without destaining) tended

to increase the staining intensity. GMS stained hyphae

and fungal spores positively in 2 cases of dermatophyto-

sis (Figure 4); however, staining intensity was variable

between slides. Despite the use of light green counter-

stain, it was difficult to recognize the nuclei of inflam-

matory cells. Good results were obtained with the

consecutive use of PAS and GMS (Figure 3); slides ap-

peared similar when stained with PAS, destained a

second time, and restained with GMS. In contrast, the

silver precipitate in GMS-stained slides could not be

removed by destaining (data not shown).

In destained melanoma specimens, Fontana–

Masson stained melanin granules black within macro-

phages, melanocytes, and in the background (Figure 5).

Counterstaining with light green provided better con-

trast than with neutral red, making recognition of cells

easier. Alcian blue stained the background blue and

the cytoplasm of neoplastic cells in myxosarcomas blue

(Figure 6). In these lesions, Alcian blue applied directly

to Hemacolor-stained slides (without destaining) also

gave acceptable results (data not shown). PAS stained

the cytoplasm of neoplastic cells (isolated and in clus-

ters) in destained specimens of mammary gland tu-

mors (Figure 7).

In the hemorrhagic pericardial effusion, Prussian

blue stained the cytoplasm of macrophages that con-

tained hemosiderin and exhibited erythrophagocyto-

sis, whereas the mesothelial cells were not stained

(Figure 8).

Results of cytochemical stains in destained slides

were similar to those in fresh material. The only differ-

ence was in nuclear detail (membrane outline and

chromatin pattern), which was slightly poorer in the

destained samples. However, in the cases of neoplasia,

this did not compromise the ability to diagnose malig-

nancy.

Discussion

The special stains used in this study have been applied

successfully for decades in human and veterinary cy-

tology, where they have been applied directly to fresh

air-dried8,10,11 or alcohol-fixed6 smears. Destaining,

however, is not commonly used and only isolated re-

ports recommend destaining slides when special stains

are required, namely when no unstained smears re-

main or when a particular feature has been observed

in a single slide.9,17 For the stains tested here, we

confirmed that destaining could be a useful procedure

when limited material or only previously stained ma-

terial is available. Thus, the recommendation of

leaving unstained smears for special stains may be

Figure 3. Organisms in a lymph node aspirate from a dog. (A) Numerous round to oval budding yeasts with a thin halo (later diagnosed as Trichosporum

sp.) can be seen among the lymphocytes. Hemacolor. (B) Periodic-acid Schiff (PAS) stain applied to the Hemacolor slide in A after destaining with 5%

alcohol acid. (C) Grocott–Gomori methenamine silver stain applied to the destained PAS slide in B. Bar = 22.5 mm.



unwarranted because it does not guarantee substan-

tially better quality staining and because false negative

results may occur if representative material is absent in

the unstained slides.12 In fact, using special stains in

unstained slides may be risky if we consider that it is

not unusual for cytologists to evaluate multiple slides

from a lesion, collected by the hands of experienced

clinicians, in which only a single slide is representative

and diagnostic.13

Although both destaining methods worked

equally as well in our hands, we recommend destain-

ing with 5% alcohol acid solution because it is faster,

especially in thick specimens. Based on the evaluation

of limited Hemacolor-stained samples that were di-

rectly stained with PAS, Ziehl–Neelsen and Alcian

blue, this destaining step can be obviated with good

results. With PAS, the periodic acid partially destains the

slides, whereas with Ziehl–Neelsen and Alcian blue, the

special stains actually highlight previously unstained ar-

eas. Our use of these stains directly on Hemacolor-

stained cytologic samples yielded good results.

The special stains used in this study have multiple

applications and are used routinely in veterinary cy-

tology. The Gram’s staining characteristics of bacteria

are important for selecting initial antibiotic therapy.16

Although often used in histopathology, this method

has been applied less frequently in cytology sam-

ples10,14,18–20 and, to the best of our knowledge, it has

not been evaluated in destained cytology samples.

Gram staining of destained cytologic preparations can

be particularly useful when it is difficult to distinguish

bacteria from granular precipitate or from a granular

proteinaceous background.18,21 Because both gram-

positive and gram-negative bacteria stain equally well

Figure 4. Dermatophytosis (later diagnosed as Microsporum canis) in a

dog. Degenerated neutrophils and fungal spores (arrows) localized

around a hair shaft (asterisk). (A) Hemacolor. (B) Grocott–Gomori me-

thenamine silver stain applied to the Hemacolor-stained slide in A after

destaining with 5% alcohol acid. Bar = 10 mm.

Figure 5. Cutaneous melanoma from a dog. (A) A fusiform melanocyte,

numerous erythrocytes, a lymphocyte, and a neutrophil are seen. Hema-

color, bar = 22.5 mm. Inset: round melanocyte with eccentric nucleus and

black-green cytoplasmic granules that also appear in the background.

Hemacolor, bar = 45 mm. (B) Fontana–Masson stain with light green

counterstain applied to the Hemacolor-stained slide after destaining with

5% alcohol acid. The melanin granules are positive (black) and the

erythrocytes appear green. Bar = 22.5 mm. Inset: Fontana-Masson count-

erstained with neutral red. Bar = 45 mm.



with Romanowsky-stains, Gram’s staining of cytologic

preparations is needed to determine gram-staining

characteristics.

In contrast with other bacteria, acid-fast bacilli do

not stain with Hemacolor; rather, negative staining oc-

curs, presumably because of hydrophobic interactions

between the water-based stain and the lipid cell wall of

the bacilli.22 Because these negative images may be

difficult to recognize, Ziehl–Neelsen has been recom-

mended to effectively diagnose mycobacterial infec-

tions in dogs and cats18; this stain has been mostly used

in unstained cytologic specimens.23–26 Based on the

results of this study, the application of Ziehl–Neelsen

stain to destained Hemacolor slides is a viable method

for the confirmation or diagnosis of mycobacterial in-

fections. Recently, Freeman described the use of

Ziehl–Neelsen superimposed on a Wright–Giemsa-

stained smear,18 a strategy that we also tested and rec-

ommend for this special stain.

PAS stains a myriad of carbohydrate-containing

substances like glycogen, fibrin, amyloid, certain

epithelial sialo- and sulphomucins, and fungal cell

walls,1,3 and is useful for demonstrating yeast-forming

fungi.16,18,27 PAS has been used previously in de-

stained cytologic samples from humans, where it is

recommended for the diagnosis of cryptococcosis,

when few or thin-walled organisms are present.28 To

our knowledge, PAS has not been evaluated previously

in destained samples from animals, although based on

our results, it should also be useful. As we showed,

slides stained with PAS can even be destained and

restained with GMS, a stain recommended for the

identification of fungi.17 This practice may be useful

for avoiding misinterpretation of PAS-positive material

Figure 6. Myxosarcoma from a dog. (A) Population of fusiform and stel-

late cells with mild anisokaryosis and slightly eosinophilic cytoplasm.

Amorphous, faintly eosinophilic material is visible in the background.

Hemacolor. (B) Alcian blue staining of the Hemacolor-stained slide

destained with 5% alcohol acid. The background and cytoplasm of some

neoplastic cells are stained blue, positive for mucin. Bar = 65 mm.

Figure 7. Mammary adenocarcinoma from a dog. (A) Acinar structure of

neoplastic cells with moderate anisokaryosis, vacuoles in the cytoplasm

(arrowheads), and basophilic material (black arrow), presumably secre-

tory. Additional peripheral material (white arrow) is present. Hemacolor.

(B) Periodic-acid Schiff stain of the Hemacolor-stained slide in A, after

destaining with 5% alcohol acid. The cytoplasm, cytoplasmic vacuoles

(arrowheads), and peripheral material (black arrow) are positive for neu-

tral glycoprotein, in contrast with the cell debris (white arrow in A), which

is negatively stained. Bar = 22.5 mm.



in macrophages as fungi. In addition, predigestion with

diastase before PAS can reduce nonspecific staining of

nonfungal material or cells (eg, neutrophils).1

GMS stains different types of fungi in cytologic

samples,17,29 but the conventional technique is time-

consuming, taking about 2 hours.16 This time can

probably be reduced substantially, to minutes, by us-

ing a 80 1C water-bath for the oxidation and reduction

step30 instead of using only a 58 1C oven for the lat-

ter.16 Even with technical refinements, however, GMS

is still laborious, expensive, and requires well-trained

personnel. For that reason, and considering our re-

sults, we would recommend PAS as a confirmatory

stain for fungi, instead of GMS.

Fontana–Masson can also be used for the differen-

tial staining of fungi,27 but is most commonly used for

the detection of melanin. In our study, we found Fon-

tana–Masson useful in the detection of coarse melanin

granules within macrophages, in which it is often diffi-

cult to distinguish melanin from nuclear debris or from

other dark pigments like hemosiderin.31 To our knowl-

edge, Fontana–Masson has not been evaluated previ-

ously for use in destained cytologic specimens from

dogs and cats. Staining with Fontana–Masson is labo-

rious, requires great care to prepare the staining

solution in order to avoid background, and is time-

consuming.1,16 However, our results indicate it can be

used in destained slides, and the technique we fol-

lowed can probably also be optimized to reduce the

time needed.32,33

Acid and neutral glycoproteins in cells and in ex-

tracellular matrix are stained by Alcian blue and PAS,

respectively.1 The diagnostic use of Alcian blue has

been noted in a few reports in animals using fresh cy-

tologic samples from abdominal fluid,11 a myxosarco-

ma,34 and a liposarcoma.7 We demonstrated in this

study that Alcian blue can also be used in destained

fine-needle aspirate smears to highlight the mucinous

nature of a myxosarcoma. PAS has also been used in

unstained smears, for evaluating abdominal fluid,11

conjunctival impressions (goblet cells),6 and in aspi-

rates of cutaneous nodules in the dog.8 In the latter,

PAS was used to demonstrate mucus secretion, thus

helping in the diagnosis of metastatic adenocarcinoma.

In this study, we also applied PAS to destained smears

of neoplastic lesions in which mucinous cellular prod-

ucts could be demonstrated.

Prussian blue stain has been described previously

for use with destained human cytologic samples.3

Prussian blue stains ferric iron blue, and is helpful in

the differentiation between anthracotic pigment, mel-

anin, and hemosiderin.1,31 In samples from animals,

Prussian blue is used often in the detection of hemo-

siderophages.18,35,36 In our study, we verified the use

of Prussian blue for detecting iron-laden macrophag-

es in destained specimens from pericardial effusions.

Although we did not test other specimens, the stain

also can likely be used to demonstrate iron in other

types of destained samples.

In conclusion, we demonstrated that good results

can be obtained for routinely-used special stains ap-

plied to destained Hemacolor specimens from dogs and

cats. Staining quality was comparable to the quality

seen in unstained smears. Limited results in our labora-

tory with destaining May–Grunwald–Giemsa–stained

slides, using the destaining strategy presented here,

suggest that these results can probably be generalized

to other Romanowsky-type stains. Based in our results,

and depending on the available cytologic material, there

is no strong support for leaving slides unstained for la-

ter use or for not using special stains when limited ma-

terial or only previously stained slides are available.

Figure 8. Pericardial effusion from a dog. (A) Cluster of mesothelial cells

and macrophages with erythrophagocytosis (arrowheads). Hemacolor.

(B) Prussian blue stain of the same Hemacolor-stained slide after destain-

ing with 5% alcohol acid. The macrophages are positive for ferric iron, in

contrast with the mesothelial cells. Bar = 22.5 mm.



Acknowledgment

The authors deeply thank Prof. Mario Caniatti (Universita

degli Studi di Milano) for his generosity in supplying mate-

rial from 1 cat used in the evaluation of Ziehl–Neelsen stain.

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