Morphometric evaluation and nonclassical criteria for the diagnosis of HPV infection and cytological atypia in cervical samples

Morphometric Evaluation andNonclassical Criteria for theDiagnosis of HPV Infection andCytological Atypia in CervicalSamplesDandara Emery Morais Sana, M.SC.,1

Priscila Mayrink de Miranda, M.SC.,2

Bruna Caroline Vieira Pitol, M.SC.,1 Mariana Soares Moran,1

Nayara Nascimento Toledo Silva,1

Ismael Dali Cotrim Guerreiro da Silva, PH.D.,3

Rita de C�assia Stocco, PH.D.,2 Willy Becak, PH.D.,2

Ang�elica Alves Lima, PH.D.,1 and Cl�audia Martins Carneiro, PH.D.4,5*

Herein, we evaluated cervical samples from normal tissue orHPV-infected tissue, to determine if the relative nuclear/cyto-plasmic ratio (NA/CA) and the presence of nonclassical cytolog-ical criteria are a novel cytological criterion for the diagnosisof HPV. Significantly, larger NA/CA ratios were found for theHPV2ATYPIA1 and HPV1ATYPIA1 groups compared withHPV2ATYPIA2 group, regardless of collection method. Forthe samples collected with a spatula, only three samples fromthe HPV2ATIPIA2 group showed four or more nonclassical

parameters (i.e., were positive), while a larger number of thesamples in the HPV2ATYPIA1, HPV1ATYPIA2, andHPV1ATYPIA1 groups were positive (13, 4, and 13 samples,respectively). Among those collected with a brush, no sampleshowed four or more nonclassical criteria in theHPV2ATYPIA2 group, while a number of samples were posi-tive in the HPV2ATYPIA1, HPV1ATYPIA2, andHPV1ATYPIA1 groups (4, 3, and 4 samples, respectively).HPV infection was associated with significant morphometricalchanges; no increase in the NA/CA ratio was found in theHPV1ATYPIA- samples, compared with the HPV2ATIPIA2samples collected with either a spatula or a brush. In conclusion,by including nonclassical cytological criteria into the patientdiagnosis, we were able to reduce the number of false negativeand false positive HPV diagnoses made using conventionalcytology alone. Diagn. Cytopathol. 2013;41:785–792. VC 2013

Wiley Periodicals, Inc.

Key Words: HPV; cervix-vaginal cytology; morphometry; non-classical criteria; molecular biology

A cytopathological diagnosis is based on the recognition

of morphological criteria within different cell types.

Despite the fact that most criteria are well established, an

embodiment of literature has demonstrated the influence

of marked interobserver and intraobserver variability.

Estimates have attributed up to 20% of false-negative

results to microscopic analysis error, with interobserver

discordance ratios varying from 10 to 100%.1,2 Koilocyto-

sis is described in the literature as a pathognomonic

1Departamento de An�alises Cl�ınicas, Escola de Farm�acia,Universidade Federal de Ouro Preto (UFOP), Ouro Preto, MG, Brasil

2Laborat�orio de Gen�etica, Instituto Butantan, S~ao Paulo, SP, Brasil3Departamento de Ginecologia, Universidade Federal de S~ao Paulo

(UNIFESP), S~ao Paulo, SP, Brasil4Laborat�orio de Imunopatologia, N�ucleo de Pesquisas em Ciencias

Biol�ogicas, ICEB II, Universidade Federal de Ouro Preto (UFOP), OuroPreto, MG, Brasil

5Laborat�orio de Pesquisas Cl�ınicas, CiPharma (Programa deP�os-Graduac~ao em Ciencias Farmaceuticas), Escola de Farm�acia,Universidade Federal de Ouro Preto (UFOP), Ouro Preto, MG, Brasil

Contract grant sponsor: Fundac~ao de Amparo �a Pesquisa do Estadode Minas Gerais (FAPEMIG); contract grant sponsor: Conselho Nacionalde Pesquisa (CNPq); contract grant sponsor: Universidade Federal deOuro Preto, Brazil.

*Correspondence to: Cl�audia Martins Carneiro, PH.D., Escola deFarm�acia, Rua Costa Sena, 171, Centro, Ouro Preto, MG, Brasil, 35400-000. E-mail: [email protected]

Received 14 July 2011; Revised 27 September 2012; Accepted1 January 2013

DOI: 10.1002/dc.22955Published online 30 April 2013 in Wiley Online Library

(wileyonlinelibrary.com).

VC 2013 WILEY PERIODICALS, INC. Diagnostic Cytopathology, Vol. 41, No 9 785

criterion for HPV diagnosis, as it is a marker of viral ac-

tivity. Many authors stress, however, that this is not the

most frequent marker found in cervix-vaginal smears.3,4

New nonclassical or secondary morphological criteria

for HPV diagnosis have been proposed to increase the

sensitivity of the standard Papanicolaou test.5

Additionally, computerized image analyses, with their

high precision and reproducibility, have been employed in

the last decades to eliminate the diagnostic variability in

cytological and histopathological samples. Such methods

target the bidimensional measurement of parameters, such as

cellular and nuclear area (NA), perimeter, and mean diame-

ter, beyond the recognition of their shapes or staining inten-

sity.6,7 Thus, digital morphometry provides the observer

with precise, quantitative data that allow the construction of

a database of diverse parameters to characterize the cells as

normal, preneoplastic, or neoplastic. In addition, the system

is capable of detecting subtle variations in cell shape, size,

and/or texture that are mostly invisible to the human eye,

even by light, and electron microscopy.8

This study investigates novel criteria: the quantitative

assessment of NA, cytoplasmic area (CA), the nuclear/

cytoplasmic ratio (NA/CA), and a series of nonclassical

cytological criteria for HPV diagnosis. These criteria have

the potential of providing information not detected by

conventional cytology and, as a result, may be able to

explain the discordant results between the molecular and

morphological approaches.

Methods: Patient Cohort and Sample Collection

In total, 569 cervix samples were evaluated from 18- to

75-year-old women living in and around Ouro Preto, Bra-

zil. The samples for identification and typing of HPV

were collected between April 2008 and July 2010 by

physicians and nurses from the family health team

(Federal Government Health Care Program) of Ouro

Preto. Sample material was either collected with the aid

of a spatula to collect squamous cells, or with a brush to

collect cells of the squamous-columnar junction.

After collection, the brushes with endocervix material

were placed in tubes containing 1 mL of collection buffer

and disconnected from the handle. The samples were

refrigerated between 2 and 10�C, stored at 280�C, and

sent to the Laboratory of Genetics and Viral Oncogenesis

of the Butantan Institute, S~ao Paulo, for viral research.

The cervical smears were immediately fixed in 95% etha-

nol and sent to the Laboratory of Clinical Cytology of the

School of Pharmacy. They were analyzed using the Papani-

colaou method,9 dehydrated in xylene, and mounted in

Entellan. Microscopic evaluation was done using the 103

and 403 objectives, and the samples were classified accord-

ing to the Bethesda System for Cervix-Vaginal Cytology.10

Considering the results of virus research and inclusion/

exclusion criteria, the samples were divided into four

groups of 20 samples per group (Fig. 1).

For the HPV2ATYPIA2 and HPV1ATYPIA2

groups, cervix smears were selected based on the most

frequent microorganisms identified during the Papanico-

laou test (e.g., Lactobacillus sp., Gardnerella vaginalis,

Candida sp., and Trichomonas vaginalis). In the

HPV2ATYPIA1 and HPV1ATYPIA1 groups, the sam-

ples were selected randomly.

Morphometric Analysis

After cytological analysis, selected samples with satisfac-

tory technical conditions were used for image capture.

Fig. 1. Distribution of the groups analyzed in this study, based on viral research and results from the Papanicolaou test (n520 per group). TheHPV2ATYPIA2 group is represented in white boxes; the HPV2ATYPIA1 group is in light gray; the HPV1ATYPIA- group is in dark gray; theHPV1ATYPIA1 group is in black.

Diagnostic Cytopathology DOI 10.1002/dc

SANA ET AL.

786 Diagnostic Cytopathology, Vol. 41, No 9

Included samples had to be homogeneous, well estab-

lished, and with good representation of the squamous-co-

lumnar junction. Samples with inadequate color,

overlapping, and/or crowded cells were excluded. Sixty

microscopic fields per sample were captured (30 by each

sampling procedure) using conventional bright field mi-

croscopy, a 103 ocular, and a 403 objective, by a Leica

DFC340FX digital microcamera attached to a Leica

DM5000B microscope.

The morphometric analysis of cells was performed

using Image J software for Windows Vista 1:43. One cell

presenting clear nuclear and cytoplasmic boundaries per

field was evaluated for each sample. The outlines were

traced using a mouse, yielding the NA and the CA. The

resulting NA/CA ratio was then calculated for each cell,

amounting to 60 cell measurements for each of the 20

samples, or 1,200 cell measurements per group.

The same images acquired for morphometric analysis

were evaluated for the presence of HPV on the screen of

a computer with Pentium Dual Core Intel 2.0 GHz, 3.0

GB RAM with a 15.4 inch monitor. Nonclassical cytolog-

ical criteria were used to identify the presence of HPV

and included binucleation or multinucleation, mild dys-

keratosis, keratinization, hyperchromatic nucleus, smudge

nucleo, spindle nucleo, mild koilocytosis, perinuclear

halo, karyorrhexis, keratohyalin-like granules, condensa-

tion of filaments, anucleate squames, ghost cells, spindle

cells, macrocytes, and koilocytotic parabasal cells.

Statistical Methods

The normality of the data was tested according to the

Kolmogorov-Smirnov, D’Agostino and Pearson, and

Shapiro-Wilk methods, followed by a Student’s t-test for

parametric samples and the Mann Whitney test for non-

parametric samples. These were analyzed by GraphPad

Prism 5 software with a confidence interval of 95%.

Results

Morphometric Evaluation

Figure 2 shows the results obtained for the NA/CA ratio

for the different categories of microrganisms (Lactobacil-lus sp., G. vaginalis, Candida sp., and T. vaginalis). This

set of data did not show a normal distribution and the

applied tests gave a significant difference of P<0.05

between the samples collected with a spatula or a brush

between T. vaginalis and Lactobacillus sp., or T. vagina-lis and G. vaginalis.

The mean and standard deviations of the NA measure-

ment, the CA measurement, and the NA/CA ratio

obtained for the squamous cells (collected with either the

spatula or the brush) are shown in Tables I and II.

For the cells found in the region collected with a spat-

ula, the statistical tests pointed to a normal distribution

and a homogeneous variance between groups. The

HPV2ATYPIA1 and HPV1ATYPIA1 groups had a

higher NA compared with the HPV2ATYPIA2 and

Fig. 2. Morphometrical analysis of the nuclear/cytoplasm area ratio insquamous cells sampled with the aid of a spatula or with a brush in theHPV2ATYPIA2 group, considering the different microorganisms foundin the cervix flora. Letters “a,” “b,” “c,” and “d” represent significantdifferences between the groups of Lactobacillus sp., G. vaginalis, Can-dida sp., and T. vaginalis.

Table I. Nuclear Area, Cytoplasm Area, and Nuclear/Cytoplasm Area Ratio Observed in Squamous Cells After Sampling with a Spatula in the Differ-ent Groups.

HPV2ATYPIA2 HPV2ATYPIA1 HPV1ATYPIA2 HPV1ATYPIA1

Nuclear Area (NA) 81.73 6 10.59 97.64a,c 6 19.95 78.20 6 1921 88.39a,c 6 14.26Cytoplasm Area (CA) 235.86c 6 44.30 217.58 6 41.47 202.64 6 37.49 230.73c 6 49.36NA/CA 0.04 6 0.01 0.05a 6 0.02 0.04 6 0.02 0.05a 6 0.01

The letters “a”, “b”, “c” and “d” represent significant differences between HPV2ATIPIA2, HPV2ATIPIA1, HPV1ATIPIA2 HPV1ATIPIA1groups, respectively.


HPV DIAGNOSES USING CONVENTIONAL CYTOLOGY

Diagnostic Cytopathology, Vol. 41, No 9 787

HPV1ATYPIA2 groups. With respect to CA, the

HPV2ATYPIA2 and HPV1ATYPIA1 groups had

higher CA measurements than the HPV1ATYPIA2

group. Overall, the NA/CA ratio was significantly higher

in the HPV2ATYPIA1 and HPV1ATYPIA1 groups

compared with the HPV2ATYPIA2 group.

The cells from the samples collected with a brush also

showed a normal distribution and homogeneous variance

between the groups. The NA measurements were higher

for the HPV2ATYPIA1 group compared with the

HPV2ATYPIA2 and HPV1ATYPIA2 groups, and for

the HPV1ATYPIA1 group compared with the

HPV2ATYPIA2 and HPV1ATYPIA2 groups. With

respect to CA, no significant differences were found

between groups. Accordingly, the NA/CA ratio was

higher in the HPV2ATYPIA1 and HPV1ATYPIA1

groups, compared with the HPV2ATYPIA2 group.

Evaluation of Nonclassical Criteria for theCytologic Diagnostic of HPV Infection

Table III depicts the nonclassical criteria that were used

for the cytologic diagnosis of HPV infection, including

the number of samples in which the respective criterion

was observed, regardless of intensity. Mild diskeratosis,

smudge nucleus, condensation of filaments, spindle cells,

and koillocytotic parabasal cells were not found in either

group. Conversely, binucleation or multinucleation, peri-

nuclear halo, keratohyalin-like granules, anucleate

squames, and ghost cells were observed in most of the

samples in each group, so they are not isolated markers

of HPV infection.

A number of the criteria were associated with specific

groups. Keratinization was only found in the

HPV1ATYPIA1 group, whereas the detection of a spin-

dle nucleus was found in the groups with positive cyto-

logical diagnostics, regardless of presence of virus. The

detection of a hyperchromatic nucleus, mild koilocytosis,

and karyorrhexis were not found in the HPV2ATYPIA2

group. Macrocytes were only observed in samples from

the HPV1ATYPIA1 group.

Figure 2 presents the median (dashed line) obtained

from the sum of the number of nonclassical criteria

observed in the samples collected with either the spatula

or brush for all groups evaluated, independent of the in-

tensity for each criterion.

In the region collected by spatula, only three samples

in the HPV2ATYPIA2 group showed four or more non-

classical criterion (i.e., a positive reading), while the other

samples showed three (n58), two (n56), or only one

(n53) nonclassical criteria and were considered negative.

In the HPV2ATYPIA1, HPV1ATYPIA2, and

HPV1ATYPIA1 groups, 13, 4, and 13 samples showed

4 or more nonclassical criteria, respectively.

In the regions collected with a brush, all samples in the

HPV2ATYPIA2 group were negative for nonclassical

criteria. Conversely, in the HPV2ATYPIA1,

HPV1ATYPIA2, and HPV1ATYPIA1 groups, four,

three, and four samples, respectively, showed four or

more nonclassical criteria.

Discussion

The diagnosis of uterine dysplasias and neoplasias is

based on morphological changes, particularly in the cell

nucleus. These changes can include size variation, shape,

and chromatin distribution. In addition to these criteria, it

is also necessary to evaluate the amount of cytoplasm in

relation to the nuclear size, as a nucleus/cytoplasm ratio,

Table II. Nuclear Area, cytoplasm Area, and Nuclear/Cytoplasm Area Ratio Observed in Squamous Cells After Sampling with a Brush in the Differ-ent Groups

HPV2ATYPIA2 HPV2ATYPIA1 HPV1ATYPIA2 HPV1ATYPIA1

Nuclear Area (NA) 82.83 6 14.94 106.96a,c 6 29.55 83.42 6 19.10 109.37a,c 6 41.84Cytoplasm Area (CA) 204.23 6 41.20 207.64 6 51.98 200.91 6 36.09 216.07 6 60.84NA/CA 0.04 6 0.02 0.06a 6 0.03 0.05 6 0.02 0.06a 6 0.03

The letters “a”, “b”, “c” and “d” represent significant differences between HPV2ATIPIA2, HPV2ATIPIA1, HPV1ATIPIA2 HPV1ATIPIA1

groups, respectively.

Table III. Analysis of Nonclassical Criteria Per Sample in theHPV2ATYPIA2, HPV2ATYPIA1, HPV1ATYPIA2, andHPV1ATYPIA1 Groups, Irrespective of Intensity

Nonclassical criteriaHPV2

ATYPIA2

HPV2

ATYPIA1

HPV1

ATYPIA2

HPV1

ATYPIA1

Binucleation orMultinucleation

17 18 18 19

Mild diskeratosis 0 0 0 0Keratinization 0 0 0 2Hyperchromatic

nucleus0 2 3 9

Smudge nucleus 0 0 0 0Spindle nucleus 0 1 0 1Mild koilocytosis 0 6 1 8Perinuclear halo 19 20 20 17Karyorrhexis 0 1 1 4Keratohyalin-like

granules12 19 15 15

Condensation offilaments

0 0 0 0

Anucleate squames 5 7 8 4Ghost cells 3 10 3 6Spindle cells 0 0 0 0Macrocytes 0 0 0 1Koilocytotic

parabasal cells0 0 0 0

Total 56 84 69 86


SANA ET AL.


as this criterion is markedly higher in dysplastic and neo-

plastic cells compared with their normal counterparts.11

The literature has shown that the quantitative analysis

of parameters, such as area, perimeter, roundness, elonga-

tion, major axis length, major axis angle, secondary axis

length, and secondary axis angle, provides reliable prog-

nostic factors and contributes to improving the diagnostic

parameters.12,13 Accordingly, the objective of the present

study was to define the NA, the CA and the NA/CA in

squamous cells of the uterine cervix collected with the

aid of either a spatula or with a brush, or in smears from

HPV-infected and noninfected patients and with or with-

out cellular atypia. Our goal was to identify significant

morphometric changes, which went unnoticed by conven-

tional cytological analysis. Because of its central role in

the etiology of virtually all cases of cervical cancer, HPV

is correctly called a “virtually necessary but generally not

sufficient” cause of cervical cancer. With the exception of

rare HPV-negative cases, cervical cancer arises via the

following distinct and sequential steps: (1) acute infection

with carcinogenic HPV type(s), (2) detectable viral per-

sistence, (3) the development of cervical precancer, and

(4) invasion.12

We compared the morphometric analyses of the differ-

ent categories of microrganisms selected as control

groups (HPV2ATYPIA2). With respect to the cytopa-

thogenesis of T. vaginalis, the literature agrees that as

this microrganism promotes a pronounced inflammatory

process that is difficult to distinguish from a pre-neoplas-

tic lesion. As expected, the samples infected with T. vagi-nalis showed high NA/CA ratios, compared with other

samples.

The smears showed nuclear changes, such as aniso-

karyosis, karyomegaly, nuclear membrane thickening

due to cell degeneration, clumps of chromatin material,

hyperchromacy, and frequent binucleation and

multinucleation.14,15

Regarding the mean values obtained for the NA, CA,

and NA/CA ratios for all of the groups collected with ei-

ther the spatula or the brush, the results showed the exis-

tence of significant morphometric changes in NA of

samples with positive cytology, compared with the sam-

ples with negative cytology, irrespective of the presence

of HPV. In addition, the groups with positive cytological

diagnoses also showed significantly higher NA/CA ratio

in the HPV2ATYPIA2 group.

These results are similar to those of Athanassiadou

et al.,16 who measured the NA/CA in 110 cervix smears

in women aged 18–55 years. These patients had cytologic

diagnoses of NIC I, NIC II, or NIC III associated or not

with the cytopathic effect resulting from HPV infection.

Statistical analyses showed that the increased severity of

the lesion was associated with an increased NA/CA ratio,

irrespective of the presence of HPV, that is, there was no

morphometric differences between the samples in groups

NIC I and NIC I with HPV, NIC II, and NIC II with

HPV, NIC III and NIC III with HPV, and NIC III and

NIC III with HPV and carcinoma.

It is worth stressing that this study addressed only sam-

ples with positive cytological diagnoses (e.g., NIC I, NIC

II, and NIC III), which excludes the possibility of observ-

ing significant morphometric changes between samples

diagnosed as either positive or negative by cytological

analysis. Furthermore, the presence of HPV was deter-

mined using a morphological approach (i.e., cytology),

which has inherent limitations.

The results from this study also confirm previous data

of Cavaliere et al.,17 who studied the NA/CA ratio in 44

smears with koilocytotic changes, classified according to

the World Health Organization (WHO, 1984) and then

evaluated for the presence of HPV antigen using the

peroxidase-antiperoxidase reaction. Statistical analysis in

this study showed that the NA/CA in the control group

was significantly smaller than in the inflamed and NIC

groups, irrespective of the presence of HPV. The inflamed

group also had a significantly smaller ratio than the HPV

antigen-positive and antigen-negative NIC groups.

The second objective of this study was to evaluate the

samples using nonclassical criteria for the detection of HPV

in squamous cells from the uterine cervix. Koillocytosis

and diskeratosis are classical cytological features associated

with HPV effects in genital infections; these are considered

pathognomonic criteria for the diagnosis of HPV infec-

tion.18–20 However, many authors suggest that classical cri-

teria are not those most frequently observed in cytological

preparations and, in spite of being specific, are only moder-

ately sensitive for the HPV diagnosis. For instance, approx-

imately one third of HPV cases would go unnoticed if the

only criteria were koilocytosis and diskeratosis.21

Nonclassical cytological criteria have been suggested to

improve the sensitivity of the cytological detection of

HPV infection (Fig. 3). They are grouped as: (1) slight

nuclear changes (mild hyperchromacy, mild anisocytosis

and binucleation or multinucleation); (2) keratinization

disorders (mild diskeratosis and parakeratosis); (3) kerato-

hyaline-like granules and perinuclear halo, and (4) degen-

erative changes (macrocytes and condensation of

filaments).22 These nonclassical criteria are significantly,

but not independently, associated with HPV infection,

that is, the presence of four or more nonclassical criteria

in the same sample are required to consider it HPV-posi-

tive by cytological examination.23

From 40 samples collected with the aid of a spatula and

previously determined to be HPV-negative by conven-

tional cytological analysis, seven were further found to be

HPV-positive using nonclassical criteria. In turn, three

samples collected with a brush and previously determined

to be HPV-negative were found to be HPV-positive after




Fig. 3. The number of nonclassical criteria observed in individual samples collected either with a spatula or a brush, per group, irrespective of the indi-vidual criterion. Each circle represents one sample in each group. The dashed line corresponds to the median obtained from the sum of the number ofcriteria for all groups evaluated.


SANA ET AL.


consideration of nonclassical criteria. These cases repre-

sent an increased HPV diagnosis of 17.5 and 7.5%, after

either spatula or brush collection, respectively.

However, and irrespective of viral research, 40 samples

collected either using a spatula or a brush was previously

determined to be positive, according to conventional cyto-

logical analysis. Of these, 14 samples were found to be

HPV-negative after sampling with a spatula while 32

samples were found to be HPV-negative after sampling

with a brush, representing a decreased accuracy of 35 and

80% for spatula and brush collection, respectively.

These numbers differ slightly from those reported by

Schneider et al.,21 who studied 200 smears from asymp-

tomatic patients with no previous abnormal cytological

diagnostics. One hundred of these patients showed posi-

tive HPV infection, as determined by in situ hybridiza-

tion. The results showed that after inclusion of the

nonclassical diagnostic criteria and cytological examina-

tion, 84 samples were considered to be positive for HPV

infection. This means that from the 200 patients initially

characterized as negative by cytological examination, 92

of them were diagnosed as HPV-positive, yielding a 46%

increase in the frequency of HPV infection by the incor-

poration of nonclassical diagnoses.

In a retrospective study, Cavaliere et al.24 used the same

nonclassical morphological parameters described by

Schneider et al.21 to examine 40 cervix-vaginal smears

with a previous positive diagnosis (NIC, in situ carcinoma,

or invasive carcinoma) that had no evidence of HPV

cytophagy. In this study, it was possible to detect the pres-

ence of HPV in 17 cases by cytological examination, that

is, a 42.5% increase in HPV diagnostic frequency.

It is worth stressing that both the Schneider et al.21 and

Cavaliere et al.24 studies considered a partially limited list

of cytological criteria, and only samples with negative

diagnoses in the former and only positive diagnoses in

the latter. In both cases, the evaluation was unilateral,

and did not allow the confirmation of additional changes

in diagnostic frequency by either increased or decreased

frequencies within the counter-groups. Furthermore, no

attempt was made to distinguish the impact of the sam-

pling procedure, which potentiates the finding of four or

more nonclassical criteria.

This study showed that T. vaginalis promoted signifi-

cant morphometrical changes in squamous cells collected

by spatula or by brush in the control group. In addition,

HPV was partially capable of inducing significant mor-

phometrical changes, as no increase in the NA/CA ratio

was found in the HPV1ATYPIA2 group compared with

the HPV2ATYPIA2 group, irrespective of the sampling

procedure.

A higher frequency of nonclassical criteria was observed

in the samples collected with a spatula, compared with

similar samples collected with a brush. Finally, the inclu-

sion of nonclassical criteria for the diagnosis of HPV

resulted in an increased frequency of HPV infection in

groups initially categorized as negative by conventional cy-

tology. These cases were outnumbered by the decrease in

the diagnostic frequency of HPV in groups initially catego-

rized as positive by conventional cytology.

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Morphometric evaluation and nonclassical criteria for the diagnosis of HPV infection and cytological atypia in cervical samples