livrocitopapanicolau coletaemmeioliquido

Prévia do material em texto

obtained. This includes the original size, shape, and texture 
of the cytoplasmic and nuclear components present. Among 
It is important to match the name of the patients on the smears 
or LBP vials with the names in the requisition form, to prevent 
65
the desired results of a well-preserved cellular specimen is the 
ability to accurately assess quantitative and semiquantitative 
criteria including hyperchromasia, nuclear/cytoplasmic ratio, 
and chromatin patterns. The recognition of fine nuclear details 
such as grooves, notches, inclusions, and pseudo-inclusions 
are essential for the definitive diagnosis of certain subtypes of 
thyroid, breast, urinary, and soft-tissue tumors with concomi-
tant prognostic implications. Equally important is maximizing 
the number of true tissue fragments. In contrast to the screening 
nature of the Pap test, where an interpretation of the sample is 
followed by a biopsy to establish the final diagnosis, fine-needle 
aspiration (FNA) is used to obtain a definitive diagnosis and as 
more targeted therapies are developed, expectations on the diag-
nostic performance of cytopathologists will increase.1
Cervicovaginal Cytology
Specimen Type
Two types of specimen are available for cervicovaginal cytology: 
smear for the conventional Pap (CP) and liquid-based preparation 
(LBP), which emerged as an alternative sampling and prepara-
tion method in the 1990s.2 In the United States ThinPrep and 
mix-ups. The use of an automated processing system improves 
the accuracy of patient identification and ensures patient chain 
of custody with bar-coded labels and etched numbers on the 
glass slides.
Clinical Information
Clinical information needs to be integrated with the sample 
interpretation. Minimal information required is patient age, 
date of last menstrual period, history of previous abnormal Paps, 
the latter being more often available in the laboratory compu-
ter system. Age and menstrual status are particularly important 
for the interpretation of endometrial cells. A history of previous 
malignancies of the female genital tract will alert the laboratory 
of the possibility of a recurrent disease or changes secondary to 
treatment.
Microscopic Evaluation
In the United States most laboratories follow the 2001 Bethesda 
System for reporting cervicovaginal cytology.5 Other countries 
utilize national reporting systems. In most laboratory settings 
the evaluation of gynecologic specimens is performed by both 
Evaluation 
and Li
Introduction
Several factors play a role in the evaluation of the cellular sam-
ple. The method of sample collection and fixation, the labora-
tory procedure to process the sample, and the integration of the 
morphologic features observed in the sample with the clinical 
information may affect the quality of interpretation/diagnosis 
reached.
The ultimate goal in specimen processing is to preserve, 
as much as possible, in vitro or in vivo aspects of the sample 
Contents
Introduction
Cervicovaginal Cytology
Specimen type
patient Identification
Clinical Information
Microscopic evaluation
C h a p t e r 
of the Sample in Smears 
quid-Based Preparations
Marluce Bibbo and Joseph F Nasuti
SurePath are the first two LBP approved by the Food and Drug 
Administration. In several countries manual methods of LBP 
such as DNA Citoliq, Cyto-Screen, PapSpin, and Autocyte 
Manual are available. The overall quality of most LBP is surpris-
ingly good because cell preservation is enhanced in contrast to 
conventional smears, which may have thick and thin areas or 
air-drying artifacts.3,4
Patient Identification
5
Nongynecologic Cytology
Specimen type
Specimen Cross-Contamination
Specimen Mishandling
Concluding Remarks
66
General CytologyPART ONE
cytotechnologists, who screen all samples, and cytopathogists 
who are responsible for the final interpretation of all abnormal 
cases.
Sample Adequacy Assessment
Over the years there has been considerable debate about what 
constitutes an adequate sample. In the 2001 Bethesda con-
ference this issue was addressed and specific guidelines for 
assessing sample adequacy emerged. Applying the minimum 
squamous cellularity criteria a cellularity of 10,000 to 12,000 
squamous cells is considered adequate for conventional Paps. 
There is no need to count squamous cells but rather estimate 
the cellularity based on density cell patterns in reference 
images. In samples with cytolysis, atrophy, and cell clustering, 
when cell adequacy is borderline, professional judgment and 
hierarchical review is recommended. The presence or absence 
of endocervical cells/transformation zone component does 
not affect sample adequacy but should be mentioned as a 
quality factor. Specimens with more than 75% of squamous 
cells obscured should be termed unsatisfactory assuming that 
no abnormal cells are present. For liquid-based preparations 
5,000 squamous cells is considered adequate cellularity and 
estimates are reached by counting the number of cells in ten 
high-power fields across the main diameter of the preparation. 
Using a 40× objective and FN20 eyepiece the number of cells 
in each field should be 3 for the ThinPrep and 7 for the Sure-
Path for adequate cellularity.6
Unsatisfactory sample rates are variable. Centrifugation LBP 
have a lower rate of unsatisfactory samples than filtration-based 
preparations because in the latter red blood cells or inflam-
matory cells may plug the filter pores. Reprocessing is recom-
mended to lower the unsatisfactory rate.7,8
A meta-analysis of prospective studies comparing cytologic 
diagnosis and sample adequacy showed LBP improved sam-
ple adequacy and equal or superior results in diagnosing pre-
malignant cervical lesions when compared with conventional 
Papanicolaou test.9 In a comparison study of automated versus 
manual LBP few differences were found in the sample adequacy 
and cellular presentation: less uniform distribution of cells 
and more artifacts were noted in the manual methods such as 
DNA Citoliq and AutoCyte Prep compared with the ThinPrep 
method; sample adequacy and overall quality of all LBP were 
surprisingly good.3
Interpretation
Detailed criteria for the interpretation of gynecologic samples 
are described in Chapters 6–11 of this book. The following high-
lights some observations in LBP.6,10–14
For LBP fixed in ethanol-based fixative the interpretation is 
closer to conventional smears. When LBP are concentrated in 
smaller areas, with three-dimensional cell clusters above the 
plane of squamous cells, focusing may be required more often.
There are many similarities in the evaluation of LBP and 
 conventional smears but there are differences:
 • Cell size;
 • Pattern; and
 • Background.
Because samples are collected in a liquid-based fixative solu-
tion, the presentation of the cellular material appears differ-
ent, concentrated and evenly distributed (Fig. 5.1). Cells are 
smaller, dispersed, and single, although cell clusters are also 
present. Cells in solution tend to round up and wet fixation 
enhances cytoplasm and nuclear morphology. Hyperchromasia 
as observed in abnormal cells on conventional smears is not 
always present in liquid-based preparations, especially in meth-
anol-based fixatives, and lack of hyperchromasia may render 
the interpretation of high-grade squamous intraepithelial lesion 
more difficult. Variations in nuclear size and shape and espe-
cially appreciation of nuclear contours play an important role 
in the evaluation on LBP.
The background is generally clean and debris more clumped. 
Blood, mucus, and inflammation are rarely obscuring, and 
inflammatory cells tend to cling to epithelial cells. Tumor dia-
thesis has a “ratty” appearance but this type of background can 
also be observed with cytolytic orinflammatory patterns.
Some cytologic entities have key features in LBP:
Key features of atrophy
 • Fewer bare nuclei;
 • Flat sheets of parabasal cells; and
 • Preserved nuclear polarity (Fig. 5.2).
Key features of trichomonads
 • Smaller;
 • Difficult to visualize; and
 • More visible nuclei, eosinophilic granules, or flagella.
Key features of lymphocytic cervicitis
 • Lymphoid cells in clusters; and
 • Confused with endometrial cells (Fig. 5.3).
Key features of repair
 • Cohesive cell groups;
 • More rounded cytoplasmic borders;
 • Less streaming; and
 • Prominent nucleoli.
Key features of metaplastic cells
 • Often single, smaller, and rounder;
 • Confused with HSIL; and
 • Paler chromatin pattern.
Fig. 5.1 Normal squamous and endocervical cells appear evenly 
distributed. Surepath (papanicolaou x Lp).
Evaluation of the Sample in Smears and Liquid-Based Preparations
5
atrophy. thinprep (papanicolaou x Mp).
Key features of low-grade squamous intraepithelial 
lesions (LSIL)
 • Large cell size;
 • Koilocytosis;
 • Multinucleation; and
 • Nuclei show decreased hyperchromasia (Fig. 5.4).
Key features of high-grade squamous intraepithelial 
lesion (HSIL)
 • Fewer abnormal cells;
 • Single cells more common than sheets;
 • Syncytial aggregates;
 • High nuclear cytoplasmic ratio; and
 • Nuclear membrane irregularities (Figs. 5.5, 5.6).
Key features of squamous cell carcinoma
 • Single cells and syncytial aggregates;
 • Pleomorphic and less hyperchromatic nuclei;
 • Rare nucleoli; and
 • Diathesis surround cells or cling to malignant cells 
(Figs. 5.7, 5.8).
Key features of endocervical adenocarcinoma in situ
 • Strips of cells with pseudostratification;
 • Nuclear crowding; and
 • Subtle appearance of feathering and rosettes.
Key features of endometrial cells
 • Tight or lose cell clusters;
 • Vacuolated cytoplasm;
 • Enhanced nuclear detail; and
 • Confused with low-grade endometrial adenocarci-
noma (Fig. 5.9).
Key features of endometrial adenocarcinoma
 • Papillary configurations;
 • 3D groups; and
 • Less prominent tumor diathesis (Fig. 5.10).
Fig. 5.3 Lymphoid cells appear in aggregates in chronic lymphocytic 
cervicitis. thinprep (papanicolaou x Mp).
Fig. 5.5 Single cells with high n/c ratio and hyperchromasia in HSIL. 
Surepath (papanicolaou x Mp).
Fig. 5.2 Flat sheet of parabasal cells with preserved nuclear polarity in 
Fig. 5.4 Koilocytes showing perinuclear cavitation and binucleation in LSIL. 
thinprep (papanicolaou x Mp).
67
General CytologyPART ONE
Nongynecologic Cytology
Specimen Type
The three major processing modalities for nongynecologic spec-
imens consist of direct smear, filtration (Millipore, SurePath, and 
ThinPrep) and cytocentrifugation-based preparations (Cytospin). 
All three techniques share the capacity to archive a portion of 
the specimen for the application of special and immunocyto-
chemical stains. Each modality subjects the cellular material 
to different degrees of physical forces and chemical influences. 
This will result in certain artifact profiles that can affect cytomor-
phologic interpretation. True tissue fragments with architectural 
features similar to those of histologic specimens are present in 
quality direct smears (Fig. 5.11). The use of Rapid Pap and/or 
Diff Quik stains in conjunction with the direct smears technique 
allows for on-site cellular adequacy of FNA. The importance of 
on-site specimen evaluation cannot be overstated since it has 
been shown conclusively to significantly reduce the inadequacy 
rate.15 The preservation of diagnostically important true tissue 
fragments and the three-dimensional microtopography of the 
Millipore cellulose filter produces an increased depth of field 
and subsequent exquisite cytologic detail (Fig. 5.12). The Thin-
Prep technique consistently produces the truest monolayer, thus 
minimizing the obscuring effects of background elements and 
cellular clumping (Fig. 5.13). SurePath and Cytospin prepara-
tions also present excellent cytomorphology (Figs. 5.14, 5.15).
When sufficient cellularity and technical support are available 
cell blocks can provide much in the way of additional diagnostic 
information. Included among the possibilities are architectural and 
staining properties that approach what is seen in conventional paraffin-
embedded hematoxylin and eosin (H and E) stained surgical biopsies. 
In this manner cell blocks often provide an immeasurable level of 
comfort to many pathologists more accustomed to conventional 
histologic diagnoses. In particular types of FNA specimens such 
as salivary gland cystic lesions and thyroid nodules convention-
ally prepared cells blocks may provide key information to clinch a 
definitive diagnosis. A common example is in distinguishing chronic 
Fig. 5.7 Syncytium of malignant cells in squamous cell carcinoma with 
variation in nuclear size and shape, irregular chromatin distribution and 
clinging diathesis. thinprep (papanicolaou x hp). Fig. 5.9 Normal endometrial cells in tight cluster and enhanced nuclear 
detail. thinprep (papanicolaou x Mp).
6
Fig. 5.6 Immature cells showing irregular nuclear outlines in HSIL. thinprep 
(papanicolaou x Mp).
8
Fig. 5.8 pleomorphic cells and clean background in squamous cell 
carcinoma. Surepath (papanicolaou x hp).
Evaluation of the Sample in Smears and Liquid-Based Preparations
5
sialadenitis from Warthin’s tumor in which characteristic large pap-
illary oncocytic lymphoepithelial tissue fragments are best appreci-
ated and often only appear in the cell block pellet derived from 
needle rinses (Fig. 5.16).16 Similarly, cell blocks have been reported 
to be extremely helpful in discerning malignant papillary tissue 
fragments containing fibrovascular cores from benign papillary 
tissue fragments that for the most part lack fibrovascular cores.17 
Also the characteristic Orphan Annie-eyed nuclear chromatin pat-
tern (felt by some to be a useful reproducible histologic artifact) 
is best seen in cytology specimens that have been conventionally 
processed into formalin-fixed paraffin-embedded cell blocks.18–21 
In a study by Sanchez and Selvaggi additional morphologic infor-
mation derived from cell blocks was found to be diagnostically con-
tributory in 31% thyroid FNAs.22 In addition to its unique ability to 
provide essential histologic clues cell blocks offer in theory and 
often in practice the opportunity to perform the same battery of 
ancillary studies as conventionally processed surgical biopsy tissue 
including molecular analysis and electron microscopy.23,24
A few published reports document the fact that immunocyto-
chemistry can be performed successfully on Cytospin, ThinPrep, 
Millipore filter, and direct smear slides.25–28 Options are some-
what limited in terms of the number and types of antibodies 
available due to the typically sparse and delicate nature of the 
cellular material present in these preparations. Many articles, on 
the other hand, describe the diagnostic usefulness of a variety of 
immunohistochemical stains on cell block material.29–38 In par-
ticular, paraffin-embedded cell blocks have proven efficacious 
by helping to resolve benign reactive lesions from both primary 
and metastatic tumors (Fig. 5.17). Such common dilemmas 
may often require a panel of immunohistochemical stains. See 
Chapter 35 for additional information.
Comparison of Nongynecologic Processing Techniques
There are many articles found in the contemporary literature 
which compare the efficacy of the processing modalities based 
on costs, cellular yield, unsatisfactory rates, artifacts, and diagnos-
tic accuracy. Most deal with specific types of specimens such as 
three-dimensional effect in papillary thyroid carcinoma FNa. Millipore 
filter (papanicolaou x hp).
Fig.5.13 true monolayer of benign urothelial cells in clear background 
in voided urine. thinprep (papanicolaou x Mp).
endometrial adenocarcinoma. thinprep (papanicolaou x Mp).
Fig. 5.11 Follicular architecture in follicular thyroid adenoma FNa. Direct 
smear (Diff Quik x Mp).
Fig. 5.10 Cluster of malignant cells and granular tumor diathesis in 
Fig. 5.12 Nuclear grooves, chromatin clearing, margination and 
69
7
General CytologyPART ONE
urine, pleural fluid, pancreatic/biliary tract, soft tissue, breast, and 
thyroid FNA. The recommendations of the various authors differ 
somewhat in favoring one processing technique over the other. A 
thorough review of the relevant literature, however, leads one to 
the conclusion that direct smears, cytocentrifugation, and filtra-
tion techniques are worthy of routine use with comparable diag-
nostic and cost parameters for most nongynecologic specimens. In 
the end the decision to utilize one or more of the processing tech-
niques depends upon weighting the service demands against the 
financial and labor resources available in a particular laboratory.
The following highlights some observations in the literature 
concerning these processing modalities:
Cellular yield was found to be superior by the ThinPrep method 
which retained three times as many cells as cytocentrifugation.39 
There were no statistically significant differences in unsatisfac-
tory rates, sensitivity, specificity, or positive predictive value in 
both FNA and body cavity fluid specimens processed by Thin-
Prep and direct smears methods.40 Overall technical quality was 
reported to be improved by ThinPrep processing when compared 
to direct smears on split FNA specimens due to cleaner back-
ground and better monolayer formation.41,42 Some authors, 
however, advised caution to avoid diagnostic errors when inter-
preting ThinPrep slides due to the increased incidence of follow-
ing cytologic artifacts (Table 5.1): disruption of tissue fragments, 
formation of cell clusters, aggregation of lymphocytes, cellu-
lar shrinkage, attenuation of nuclear details, and exaggeration 
nucleolar prominence.42 In comparison to SurePath processed 
specimens ThinPrep slides demonstrated increased cellular 
shrinkage, flattening, and fragmentation of large cellular sheets 
and nuclear chromatin patterns were reportedly more difficult 
to evaluate.43 SurePath slides were also found to contain larger 
branched three-dimensional tissue fragments.43
Separate studies involving FNAs of thyroid nodules, breast 
and salivary gland lesions, and pancreatic and soft-tissue tumors 
reported somewhat conflicting results in terms of unsatisfactory 
rates, quality of nuclear details, diagnostic accuracy, and rela-
tive prevalence of artifacts when ThinPrep-processed slides were 
compared to direct smears.44–53 Among the artifacts attributed 
Fig. 5.15 Metastatic ovarian carcinoma in peritoneal fluid showing clean 
background and cells with good nuclear detail. Cytospin (papanicolaou x Mp).
Fig. 5.17 Metastatic lung adenocarcinoma in pleural fluid. Cell block 
(ttF1 immunostain positivity x Mp).
0
Fig. 5.14 true tissue fragment of papillary urothelial carcinoma in 
voided urine. Surepath (papanicolaou x Mp).
Fig. 5.16 Warthin’s tumor in parotid FNa. Cell block (h&e x Mp).
Evaluation of the Sample in Smears and Liquid-Based Preparations
5
to the ThinPrep method for FNA specimens are the inability 
to assess cellularity of individual passes; diminished/distorted 
extracellular and stromal elements such as mucin, stroma, adi-
pose tissue, and colloid that also appeared as dense droplets (Fig. 
5.18); crowded tight tissue clusters with loss of cellular preser-
vation; increased cellular and tissue fragment disruption; artifi-
cially increased single epithelial cells; numerous naked nuclei; 
pronounced nucleoli; decreased nuclear details; and attenu-
ation of nuclear grooves and pseudo-inclusions. Authors did 
note that significantly more conventional smears were limited 
by air-drying artifact. Additionally, ThinPrep slides had greater 
cellularity, improved nuclear detail, and more easily recogniz-
able myoepithelial cells relative to direct smears (Fig. 5.19). An 
added benefit of greater suitability for immunoperoxidase stain-
ing was also documented for ThinPrep processing.48
Specific examples in which artifact-associated ThinPrep-
 processed FNA slides compromised diagnostic accuracy were cited: 
Four out of 21 fibroadenomas were correctly diagnosed on Thin-
Prep-processed breast FNA due to artificially increased single 
ductal epithelial cells and a lack of background stroma.47 Three 
benign pancreatic lesions were interpreted as atypical/suspicious 
due to presence of single atypical cells with distinct nucleoli, 
and one mucinous pancreatic neoplasm was incorrectly diag-
nosed due to lack of background mucin.51
The diagnostic value for pleural fluid specimens of ThinPrep 
versus Cytospin was compared by examining a large spectrum 
of cytologic features that would distinguish malignant meso-
thelioma (e.g., peripheral cytoplasmic skirt, bubbly cytoplasm, 
cyanophilic cytoplasm, and scalloped border of cell balls) from 
pulmonary adenocarcinoma (e.g., two-cell population, inspis-
sated cytoplasmic material, cytoplasmic vacuole, angulated 
and indented nuclei, and smooth border of cell balls).54 Based 
on statistical analysis most cytologic features examined in this 
study can be seen in both preparation techniques. The authors 
therefore concluded that ThinPrep preparation of pleural effu-
sions does not appear to provide additional diagnostic value 
when compared to Cytospin preparation.
A comparative analysis of urine specimens processed by 
both ThinPrep and Cytospin techniques found that the cyto-
morphology and screening time were comparable with both 
techniques.55 However, in cases of transitional cell carcinoma 
Cytospin was superior in terms of better preservation of archi-
tectural features and produced less artificial empty spaces and 
air-drying artifact (Fig. 5.20). A contrasting study compared 
Fig. 5.18 Dense colloid droplet in thyroid FNa in goiter. thinprep 
(papanicolaou x Mp). Fig. 5.20 high-grade urothelial carcinoma. Cytospin (papanicolaou x Mp).
Cellularity Nuclear Background
Cellular shrinkage 
 
Increased naked 
nuclei 
Loss of adipose tissue, 
stroma, mucin, and 
colloid
Disruption of tissue 
fragments
Decreased nuclear 
chromatin details
Colloid that appears 
as dense droplets
Flattening and 
fragmentation of large 
cellular sheets
attenuation of nuclear 
grooves and pseudo 
inclusions
aggregation 
of lymphocytes 
Formation of cell 
clusters
exaggerated nucleolar 
prominence
 
artificially increased 
single epithelial cells
 
Table 5.1 artifacts more commonly seen with nongynecologic LBp
Fig. 5.19 Benign ductal cells in association with myoepithelial cells. 
thinprep (papanicolaou x Mp).
71
72
General CytologyPART ONE
cytocentrifugation and ThinPrep techniques for cost efficiency 
including wages, investments in instrumentation and con-
sumables, overall cytomorphologic quality, and suitability for 
molecular studies.56 Based on their examination of 224 split 
urine samples the authors reported that cytocentrifugation 
with disposable chambers resulted in a global cytomorpho-
logic quality superior to that of ThinPrep. In addition utilizing 
a 200-specimen per month calculation a greater cost efficiency 
was achieved with cytocentrifugation than with ThinPrep. In a 
similar subsequent study the same authors compared cytomor-
phologic quality of urine specimens prepared by ThinPrep, 
direct smears, Cytocentrifugation, and ThinPrep and Millipore 
filtration.57
The conclusions of the study were as follows:
 • Direct smears show good overall results;
 • Cytocentrifugationwith reusable chambers should be 
avoided;
 • Cytocentrifugation with disposable chambers (Cyto-
funnels or Megafunnel chambers) gives excellent 
results; and
 • Millipore filtration followed by blotting should be 
avoided due to its poor global quality.
Contrasting results were reported when voided urine speci-
mens processed via Millipore filter cytosieve technique were 
examined.58 True tissue fragments consisting of either flat 
sheets or three-dimensional structures were significantly more 
common in voided urine specimens with follow-up biopsies 
of TCC than in negative biopsies. A considerably lower rate of 
tissue fragments was reported when voided urine specimens 
were processed utilizing cytocentrifugation with no statisti-
cally significant correlation found between the incidence of 
cell groups in voided urine specimens and the presence of TCC 
in follow-up biopsies.59 It has been suggested that the reason 
for these discrepant findings is due to the stronger, more dis-
ruptive centripetal force imposed on true tissue fragments by 
cytocentrifugation relative to the weaker, gentler forces of grav-
ity and suction encountered by the Millipore filter cytosieve 
technique.58
Specimen Cross-Contamination
The potentially catastrophic problem of cross contamination of 
cytology specimens can occur with any processing modality or 
in any stage of the process from fixation to coverslipping (Fig. 
5.21). Fortunately steps can be taken in the specimen processing 
and staining to minimize its rate occurrence in cytopreparatory 
laboratories.60,61
Specimen Processing
 A. All test requisitions, specimen cups, tubes, and slides 
must be identified with their own unique accession 
number before processing.
 B. Laboratory technicians must carefully aspirate sample 
into pipette tip, making sure no sample gets sucked 
into the pipette barrel. If this happens the pipette is 
immediately removed from production. The barrel is 
cleaned with alcohol and water, dried, and tested for 
contamination before being placed back into produc-
tion.
 C. Pipette tips are changed for every sample.
 D. All cytochambers and holders, if not disposable, are 
soaked for at least 30 minutes and then washed in the 
dishwasher and dried before being used the following 
afternoon.
 E. All staining solutions must be either filtered or 
changed after every staining run.
 F. All microscopists shall inform the supervisor of sus-
pected contamination. Immediate corrective action 
shall follow with appropriate documentation of each 
occurrence.
Specimen Staining
 A. All stains and staining solutions must be checked, 
filtered, and changed daily with documentation 
supporting this. Discard all water rinses after 
usage.
 B. Special staining is to be performed in separate Coplin 
jars with all reagents made fresh daily.
 C. Hacker coverslipping xylene wells must be filtered 
after each use.
 D. Known positive cases should be stained separately in 
Coplin jars or stained in the regular staining dishes 
provided that the regular staining setup is then 
 discarded of all solutions.
 E. Any suspected floater (atypical cell contaminant seen 
on a different focal plane found on a slide) identified 
on a slide should be brought to the attention of the 
supervisor. For nongynecologic specimens, it is rec-
ommended that additional slides be made with the 
leftover sediment. Again no staining should continue 
until all stains, dishes, and jars are either filtered or 
changed and cleaned.
 F. If contamination is suspected and no residual speci-
men sediment is available, consideration must be 
given to cancellation of the test. If the test is canceled 
a phone call must be placed to the requesting clini-
cian and a hard copy of the report with appropriate 
explanation shall be issued. All inquires will be docu-
mented in the reconciled report logbook.
Fig. 5.21 Cross-contamination from ovarian carcinoma (see Fig.5.15) in 
pancreatic cyst FNa. Cell block (h&e x Mp).
Evaluation of the Sample in Smears and Liquid-Based Preparations
5
o
l
c
r
C
e
h
r
m
h
1
A
c
s
 
K
f
 
:
 
o
 
 1. Bibbo M, Akerman M, Alves VAF, et al. 
How technology is reshaping the practice 
of nongynecologic cytology. Acta Cytol 
2007;51:123-152.
 2. Austin MR, Ramzy I. Increased detection 
of epithelial cell abnormalities by liquid-
based gynecologic cytology preparations. 
A review. Acta Cytol 1998;42:178-184.
 3. Alves VAF, Bibbo M, Schmitt FCL, et al. 
Comparison of manual and automated 
methods of liquid-based cytology: A mor-
phologic study. Acta Cytol 2004;48:187-
193.
 4. Mintzer MP, Curtis P, Resnick JC, et al. 
The effect of the quality of Papanicolaou 
smears on the detection of cytologic 
abnormalities. Cancer (Cancer Cytopathol) 
1998;87:113-117.
 5. Solomon D, Davey D, Kurman R, et al. 
The 2001 Bethesda System: Terminology 
for reporting results of cervical cytology. 
JAMA 2001;287:2114-2119.
 6. Solomon D, Nayar R. The Bethesda System 
for Reporting Cervical Cytology, 2nd edn. 
New York: Springer-Verlag, 2004.
 7. Hellman DJ, Bolick DR. Improving the 
adequacy of the ThinPrep Pap Test*. Ref-
erence Pathology Services, Murray, Utah. 
Acta Cytol 1998;42:1241.
 8. Vassilakos P, Cossali D, Albe X, et al. Effi-
cacy of monolayer preparations for cervi-
cal cytology: Emphasis on suboptimal 
specimens. Acta Cytol 1996;40:496-500.
 9. Bernstein SJ, Sanchez-Ramos L, Ndubisi B. 
Liquid-based cervical cytologic smears 
study and conventi
smears: A metaana
studies comparing 
and sample adequa
2001;185:308-317.
 10. Douglas KL. ThinP
ence Manual. Cytic 
 11. ThinPrep Pap Test M
Atlas. Cytic Corp, 20
 12. AutoCyte Prep Refer
Imaging, 1998.
 13. Wilbur DC, Dubes
Use of Thin-layer p
logic smears with e
morphology of hig
lesions and carcino
1996;14(3):201-21
 14. Wood MD, Horst J
atypical glandular 
the true atypical le
pap tests: A review.
2007;35:12-17.
 15. Nasuti JF, Gupta P
tic value and cost-e
evaluation of fine n
specimens. Review
Cytopathol 2002;27
 16. Nasuti JF, Yu GH, G
aspiration of cystic
An institutional rev
histologic correlati
2006;90:111-116.
 17. LiVolsi AL. Surgical
roid. Major Problem
22. Philadelphia: W
nal Papanicolaou 
ysis of prospective 
ytologic diagnosis 
cy. Am J Obstet Gynecol 
ep Morphology Refer-
orp, 1994.
orphology Reference 
04.
nce Manual. TriPath 
ter B, Angel C, et al. 
eparations or gyneco-
phasis on the cyto-
-grade intraepithelial 
mas. Diagn Cytopathol 
.
, Bibbo M. Weeding 
ell look-alikes from 
ions in liquid-based 
Diagn Cytopathol 
, Bloch ZW. Diagnos-
fectiveness of on-site 
eedle aspiration 
of 5,688 cases. Diagn 
1-4.
upta PK. Fine needle 
parotid gland lesions. 
iew of 46 cases with 
n. Cancer Cytpathol 
Pathology of the Thy-
s in Pathology, vol 
B Saunders, 1990.
 18. Carcangiu ML, Zampi G, Rosai J. 
Papillary thyroid carcinoma. A study 
of its many morphologic expressions 
and clinical correlates. Pathol Annu 
1985;20:1-44.
 19. Deligeorgi-Politi H. Nuclear crease as a 
cytodiagnostic feature of papillary thy-
roid carcinoma in fine needle aspiration 
biopsies. Diagn Cytopathol 1987;3: 
307-310.
 20. Vickery AL. Thyroid papillary carcinoma. 
Pathologic and philosophic controversies. 
Am J Surg Pathol 1983;7:797-807.
 21. Vickery AL, Carcangiu M, Johannessen 
JV, et al. Papillary carcinoma. Sem Diagn 
Pathol 1985;2:90-100.
 22. Sanchez N, Selvaggi SM. Utility of 
cell blocks in the diagnosis of thyroid 
 aspirates. Diagn Cytopathol 2006;34: 
89-92.
 23. Schoedel KE, Finkelstein SD, Swalsky PA. 
Molecular profiling of primary and 
metastatic neoplasms in lung using mate-
rial obtained by fine needle aspiration. 
A report of two cases. DiagnCytopathol 
2004;30:342-346.
 24. Young NA, Naryshkin S, Katz SM. Diag-
nostic value of electron microscopy on 
paraffin-emdedded cytologic material. 
Diagn Cytopathol 1993;9:282-290.
 25. Fetsch PA, Simsir A, Brosky K, et al. 
Comparison of three commonly used 
cytologic preparations in effusion 
immunocytochemistry. Diagn Cytpathol 
2002;26:61-66.
It is also important to remember that the possibility of cross-
contamination is not limited to the cytopreparatory laboratory. 
This is particularly true when cell blocks are processed along with 
surgical pathology tissue utilizing the same formalin baths and 
automated processors. Contamination due to shedding of malig-
nant cells and tissue can occur from cell block to histology tissue block 
and vice versa. To minimize this type of cross-contamination the 
following steps should be taken:
 1. The histology laboratory should be notified when cell 
blocks are requested from cytology specimens likely 
to shed tumor cells such as ascites fluid from a known 
ovarian carcinoma patient (Fig. 5.21).
 2. Cassettes for cell blocks should be placed in separate 
formalin baths from histology tissue cassettes.
 3. Cassettes for cell blocks whenever possible should 
have their own separate runs in the automated proces-
sors without surgical pathology tissue cassettes.
Specimen Mishandling
Once errors in handling the specimens are detected, a root cause 
analysis should take place to help identify what, how, and why 
the error happened. Understanding why a mistake occurred is 
the key to develop effective quality control measures to pre-
vent it from occurring again. A review of skill-based activities, 
if appropriate, to ensure appropriate level of hands-on training 
should be provided in addition to the training development 
process to ensure adequate guidance. For errors related to sam-
ple interpretation see Chapter 4, Diagnostic Quality Assurance 
in Cytopathology.
Concluding Remarks
The parameters for evaluation of smears and liquid-based 
preparations have been described in this chapter. As we have 
seen, several factors play an important role in the evaluation 
of the cellular sample and for optimal results good fixation 
and processing techniques, availability of clinical informa-
tion, and expertise in interpretation are required. Criteria for 
interpretation of gynecologic and nongynecologic samples 
are described in all chapters on diagnostic cytology in this 
book but observations on liquid-based preparations were 
highlighted here to show differences in cell size, pattern 
background, and artifacts. A comparison of nongynecologic 
processing modalities and specimen cross-contamination 
as well as procedures to prevent the problem were also pre-
sented. The use of adjunct techniques in diagnostic cytology is 
already an important component of the specimen evaluation. 
In the future the diagnostic potential of cytology will increase 
with development of new fluorescent in situ hybridization 
(FISH) probes and more assays to identify genetic altera-
tions that serve as therapeutic targets in addition to the use 
of microarrays, allowing a global and integrative approach to 
diagnosis.
References
73
General CytologyPART ONE
 26. Lozano MD, Panizio A, Toledo GR, et al. 
Immunocytochemistry in the differential 
diagnosis of serous effusions. A compara-
tive evaluation of eight monoclonal anti-
bodies in Papanicolaoy stained smears. 
Cancer 2001;93:68-72.
 27. Fadda G, Rossi ED, Mule A, et al. Diag-
nostic efficacy of immunocytochemistry 
on fine needle aspiration biopsies 
processed by thin-layer cytology. 
Acta Cytol 2006;50:129-135.
 28. Baloch ZW, Cobb C, Roberts S, et al. 
Millipore filter cell block preparation. 
An alternative to cell block in non-
 gynecologic specimens of limited cellular-
 37. Lin F, Abdallah H, Meschter S. Diagnostic 
utility of CD10 in differentiating hepato-
cellular carcinoma from metastatic carci-
noma in fine needle aspiration biopsy of 
the liver. Diagn Cytopathol 2004;30:92-97.
 38. Shin SJ, Hoda RS, Ying L, et al. Diagnos-
tic utility of the monoclonal antibody 
A103 in fine needle aspiration biop-
sies of the adrenal. Am J Clin Pathol 
2000;113:295-302.
 39. Papillo JL, Lapin D. Cell yield verses 
cytocentrifuge. Acta Cytol 1994;38:33-36.
 40. Leung CS, Chiu B, Bell V. Comparison of 
ThinPrep and conventional preparations. 
Nongynecologic cytology evaluation. 
 50. Al-Khafaji BM, Afify AM. Salivary gland 
fine needle aspiration using the Thin-
Prep technique. Diagnostic accuracy, 
cytologic artifacts and pitfalls. Acta Cytol 
2001;45:567-574.
 51. de Luna R, Eloubeidi MA, Sheffield MV, 
et al. Comparison of ThinPrep and con-
ventional preparations in pancreatic fine 
needle aspiration biospy. Diagn Cytopathol 
2004;30:71-76.
 52. Siddiqui MT, Gokaslan ST, Saboorian 
MH, et al. Split sample comparison of 
ThinPrep and conventional smears in 
endoscopic retrograde cholangiopan-
creatography-guided pancreatic fine 
ity. Diagn Cytopathol 1999;20:389-392.
 29. Chandan VS, Faquin WC, Wilbur DC, 
et al. The utility of GLUT-1 immunolocal-
ization in cell blocks. An adjunct to the 
fine needle aspiration diagnosis of cystiv 
squamous lesions of the head and neck. 
Cancer 2006;108:124-128.
 30. Chhieng DC, Benson E, Eltoum I, et al. 
MUC1 and MUC2 expression in pancreatic 
ductal carcinoma obtained by fine needle 
aspiration. Cancer 2003;99:365-371.
 31. Morgan RL, DeYoung BR, McGaughy VR, 
et al. MOC-31 aids in the differentiation 
between adenocarcinoma and reactive 
mesothelial cells. Cancer 1999;87: 
390-394.
 32. Fetsch PA, Abati A, Hijazi YM. Utility 
of the antibodies CA19-9, HMBE-1 and 
thrombomodulin in the diagnosis of 
malignant mesothelioma and adenocarci-
noma in cytology. Cancer 1998;84:101-108.
 33. Levine PH, Joutovsky A, Cangiarella J, et al. 
CDX-2 expression in pulmonary fine 
 needle aspiration specimens. A useful 
adjunct for the diagnosis of metastatic 
colorectal adenocarcinoma. Diagn 
 Cytopatol 2006;34:191-195.
 34. Saad RS, Essig DL, Silverman JF, et al. 
Diagnostic utility of CDX-2 expression 
in separating metastatic gastrointestinal 
adenocarcinoma in fine needle aspira-
tion cytology using cell blocks. Cancer 
2004;102:168-173.
 35. Bedrossian CW, Davilia RM, Merenda G. 
Immunocytochemical evaluation of liver 
fine needle aspirations. Arch Pathol Lab 
Med 1998;113:1225-1230.
 36. Yoder M, Zimmerman RL, Bibbo M. 
Two-color immunostaining of liver 
fine needle aspiration biopsies with 
CD34 and carcinoembryonic antigen. 
Potential utilization in the diagnosis of 
primary hepatocellular carcinoma versus 
metastatic tumor. Anal Quant Cytol Histol 
2004;26:61-64.
Diagn Cytopathol 1997;16:368-371.
 41. Dey P, Luthra UK, George J, et al. 
Camparison of ThinPrep and con-
ventional preparations on fine needle 
aspiration cytology material. Acta Cytol 
2000;44:46-50.
 42. Michael CW, Hunter B. Interpretation 
of fine-needle aspirates processed by the 
ThinPrep technique. Cytologic artifacts 
and diagnostic pitfalls. Diagn Cytopathol 
2000;23:6-13.
 43. Micheal CW, McConnel J, Pecott J, et al. 
Comparison of ThinPrep and TriPath 
PREP liquid-based preparations in 
nongynecologic specimens. A pilot study. 
Diagn Cytopathol 2001;25:177-184.
 44. Biscotti CV, Hollow JA, Toddy SM, et al. 
ThinPrep versus conventional smear 
cytologic preparations in the analysis of 
thyroid fine needle aspiration specimens. 
Am J Clin Pathol 1995;104:150-153.
 45. Afify AM, Liu J, Al-Khafaji BM. Cytologic 
artifacts and pitfalls of fine needle aspira-
tions using ThinPrep. A comparative 
retrospective review. Cancer 2001;93: 
179-186.
 46. Malle D, Valeri RM, Pazaitou-Panajiotou K, 
et al. Our experience from the use of 
ThinPrep techniquein thyroid fine needle 
aspiration. Acta Cytol 2006;50:23-27.
 47. Perez-Reyes N, Mulford DK, Ruthowski 
MA, et al. Breast fine needle aspiration. 
A comparison of thin-layer and con-
ventional preparation. Am J Clin Pathol 
1994;102:108-110.
 48. Biscotti CV, Shorie JH, Gramlich TL, et al. 
ThinPrep versus conventional smear 
cytologic preparations in analyzing 
fine needle aspiration specimens from 
palpable breast masses. Diagn Cytopathol 
1999;21:137-141.
 49. Bedard YC, Pollett AF. Breast fine needle 
aspiration. A comparison of ThinPrep 
and conventional smears. Am J Clin 
Pathol 1999;113:312-314.
needle aspirations. Diagn Cytopathol 
2005;32:70-75.
 53. Das K, Hameed M, Heller D, et al. Liquid-
based versus conventional smears in fine 
needle aspiration of bone and soft tissue 
tumors. Acta Cytol 2003;47:197-201.
 54. Ylagan LR, Zhai J. The value of ThinPrep 
and cytospin preparation in pleural effu-
sion cytologic diagnosis of mesothelioma 
and adenocarcinoma. Diagn Cytopathol 
2005;32:137-144.
 55. Nassar H, Ali-Fehmi R, Madan S. Use 
of ThinPrep monolayer technique and 
cytospin preparation in urine cytology. 
A comparative analysis. Diagn Cytopathol 
2003;28:115-118.
 56. Piaton E, Huntin K, Faynel J, et al. Cost 
efficiency analysis of modern cytocen-
trifugation methods versus liquid based 
(Cytcy ThinPrep) processing of urinary 
sample. J Clin Pathol 2004;57:1208-1212.
 57. Piaton E, Faynel J. Huntin K, et al. Con-
ventional liquid-based techniques versus 
Cytyc ThinPrep processing of urinary 
samples. A qualitative approach. BMC 
Clin Pathol 2005;5:9-14.
 58. Nasuti JF, Fleisher SR, Gupta PK. Signifi-
cance of tissue fragments in voided urine 
specimens. Acta Cytol 2001;45:147-152.
 59. Goldstein ML, Whitman T, Renshaw 
AA. Significance of cell groups in voided 
urine. Acta Cytol 1998;42:290-294.
 60. Holmquist DH, Keebler CM. Cytoprepar-
atorytechniques. In: Keebler CM, Somrak 
TM (eds) The Manual of Cytotechnology, 
7th edn. Chicago: American Society of 
Clinical Pathologists Press,1993:411-448.
 61. Bales EB, Durfee GR. Principles of opera-
tion of a cytopathology laboratory. In: 
Koss LG (ed) Diagnostic Cytology and 
Its Histopathologic Bases, vol 2, 4th edn. 
Philadelphia: JB Lippincott, 1992: 
1451-1531.
74
	Evaluation of the Sample in Smears and Liquid-Based Preparations
	Introduction
	Cervicovaginal Cytology
	Specimen Type
	Patient Identification
	Clinical Information
	Microscopic Evaluation
	Sample Adequacy Assessment
	Interpretation
	Nongynecologic Cytology
	Specimen Type
	Comparison of Nongynecologic Processing Techniques
	Specimen Cross-Contamination
	Specimen Processing
	Specimen Staining
	Specimen Mishandling
	Concluding Remarks