Judging specimen adequacy by Bethesda ’01 criteria
Estimating LBP Cellularity
George Birdsong, MD
Assessing and reporting specimen adequacy of Pap smears was
introduced in the original 1988 Bethesda System. The 1988 Bethesda
criteria stipulated that:
In the 1991 Bethesda System revision, the phrase “satisfactory
but limited by” replaced “less than optimal.” There
was no significant difference, however, in how adequacy was assessed.
- An adequate Pap should contain two groups of five endocervical
cells (EC) and/or squamous metaplastic cells.
- At least 10 percent of the slide area should be covered with
- Specimens obscured 50 to 75 percent by blood or inflammation
should be considered less than optimal, and those obscured greater
than 75 percent should be designated unsatisfactory.
Bethesda 2001’s revised adequacy criteria take both published
data and theoretical issues into account. The phrase “satisfactory
but limited by” has been deleted. The adequacy committee felt
the phrase was an oxymoron and that laboratory reports should indicate
specimen adequacy less ambiguously. Therefore, Bethesda 2001 designates
specimen adequacy as “satisfactory” or “unsatisfactory.”
Specimen quality indicators such as the presence or absence of a transformation
zone component, or of obscuring inflammation or blood, are reported
after the adequacy designation.
Bethesda 2001’s most significant change lies in the assessment
of squamous cellularity. The previous 10 percent coverage criterion
can be interpreted in at least two ways: that cellular material covers
at least 10 percent of the area under the coverslip (125mm2)
at a “normal” or “usual” cell density, or
that 10 percent or 125mm2 of the slide is actually covered
or obscured because of the presence of cells. The latter criterion
actually represents very high cellularity, and many, if not most,
Paps would fail to meet this criterion. Because of the 10 percent
criterion’s ambiguity, more specific criteria were developed.
For conventional Paps, an adequate smear contains between 8,000 and
12,000 well-preserved, well-visualized cells. Labs should not
attempt to count cells manually. Several images depicting low-power
(~4×) microscope fields with a known number of cells will be
available in the updated Bethesda System Manual and on a Web site,
so that cytologists can see what a field with.
By Bethesda 2001 criteria, adequate liquid-based preps (LBPs) contain
at least 5,000 well-visualized squamous cells. Optionally, labs can
elect to append a QIstatement indicating borderline cellularity for
cases that have 5,000 to 20,000 cells. LBP cellularity is set at a
lower level because the number of cells in an LBP can be estimated
more easily than the number in a conventional smear, since the cells
in most LBPs are distributed more evenly than they are in a conventional
prep. Estimate the number of cells in an LBP by counting the number
of cells in each of 10 40× fields taken across a diameter of
the preparation, and then averaging the results. Some LBPs will have
sparsely cellular areas, or “holes,” within the circle.
If such holes appear in the preparation, the 10 40× fields should
include a proportionate number of fields from the sparsely cellular
areas. Similarly, if portions of the preparation are obscured by inflammation
or blood, a proportionate number of the 10 fields should be from those
areas. Table 1
provides the number of cells you can expect to find in a 40×;
field of an LBP with 5,000 total cells for common microscope parameters
and prep diameters. With the information in the table, estimating
LBP cellularity is not difficult or time-consuming.
For example, with Cytyc ThinPreps, which have a 20-mm diameter circle,
and a microscope with field number 22 eyepieces with the 40×
objective, approximately four cells per field are necessary to have
5,000 cells on the whole slide. The 40× objective is recommended
because the number of cells to be counted is low. Most LBPs contain
many more than four cells per 40× field. Therefore, you can determine
at a glance whether cells must be counted to check for acceptable
cellularity, and only LBPs appearing to have borderline cellularity
must be counted.
Before the Bethesda 2001 conference, interested parties used an Internet
bulletin board to comment on issues pertinent to the project. Some
questioned the necessity of a more rigorous standard for squamous
cellularity. Admittedly, relatively little data on which to base a
specific quantitative guideline are available, but the committee felt
bringing consistency to cellularity assessment through a quantitative
standard was important.
Adequacy criteria for obscuring factors have not changed. The criteria
for cells from the transformation zone have been modified slightly
to require 10 EC or metaplastic cells instead of two groups of five
cells each. More studies on EC have been published than on any other
aspect of specimen adequacy. While cross-sectional studies show that
smears with EC are more likely to have abnormal cells, longitudinal
and retrospective case-control studies uniformly fail to correlate
absence of EC and subsequent diagnosis of squamous dysplasia or carcinoma.
The adequacy committee felt reporting the presence of EC was warranted,
however, because the topic remains controversial and because the significance
of EC in detecting adenocarcinoma is unknown.
a member of the CAP Cytopathology Committee, is associate professor
in the Department of Pathology and Laboratory Medicine at Emory University
School of Medicine, Atlanta.