Brief Scientific Reports
The Role of the Saccomano Technique in Sputum
Cytopathologic Diagnosis of Lung Cancer
ELIZABETH J. PERLMAN, M.D., YENER S. EROZAN, M.D., F.I.A.C, AND ARLINE HOWDON, CT(ASCP), C.F.I.A.C.
The Saccomanno technique of sputum preparation is widely used.
This study evaluates the role of this preparation in conjunction
with fresh smears in the diagnosis of lung cancer. All sputum
samples from September 1973 to July 1975 showing atypia were
randomized and negative controls added. The Saccomanno and
fresh smears were evaluated independently and blindly and classified as negative, atypical, suspicious, or cancer. When each
preparation was compared with the original diagnosis, the diagnostic accuracy for 55 squamous carcinomas was similar (fresh
95%, Saccomanno 86%) but significantly less in the Saccomanno
preparations of 22 small cell carcinomas (fresh 100%, Saccomanno 24%) and 26 adenocarcinomas (fresh 96%, Saccomanno
52%). Four cases negative on fresh smears were suspicious or
diagnostic of cancer on the Saccomanno slides. There were no
cases of small cell carcinoma in which the Saccomanno preparations added information not available on the fresh smears. The
authors conclude that in conjunction with fresh smears, the Saccomanno preparation may contribute to the diagnosis of nonsmall cell carcinomas but does not appear to aid in the diagnosis
of small cell carcinoma. (Key words: Sputum cytology; Saccomanno method; Lung cancer diagnosis) Am J Clin Pathol 1989;91:
57-60
SPUTUM CYTOLOGIC STUDY in experienced hands
has been demonstrated to be an accurate and convenient
method of screening and diagnosing primary epithelial
tumors of the lung.1"4,6 The most reliable method of processing sputum is the preparation of fresh smears from
unfixed, selected material. Fresh smears provide better
diagnostic cellular morphologic characteristics and retain
the background, which is often helpful. When fresh smears
cannot be processed, and when a significant time lapse
between collection and processing is expected, the sample
must be fixed. The best and most widely used method of
fixation and concentration of sputum has been described
by Saccomanno and colleagues.9 This method involves
collection and fixation of a single or pooled series of spu-
Department of Pathology, Johns Hopkins Hospital,
Baltimore, Maryland
turn in a solution of 50% ethyl alcohol and 2% Carbowax®,
followed by blending and smearing. The chief advantage
of the Saccomanno method is the ability to preserve the
specimen until preparation is possible, in some instances
enabling transportation of the specimen. The importance
of this technique is seen in the elegant studies of Dr. Saccomanno in which he follows the progression of epithelial
atypia to carcinoma in serial sputum samples of uranium
miners.7,8 In addition, this method enables concentration
and use of a representative sample of the entire specimen,
theoretically resulting in a higher diagnostic yield. However, few studies have been performed evaluating the Saccomanno method and none, to our knowledge, comparing
it with other techniques.
The aim of this study is to determine the role of the
Saccomanno method in conjunction with fresh smear
preparations. Comparison of the morphologic characteristics of the same tumor cells processed with each method
would provide a better understanding of the relative and
comparative diagnostic difficulties encountered in each
and evaluate the supportive role of the Saccomanno
method.
Materials and Methods
All sputum samples from September 1973 to July 1975
were reviewed. The specimens were received fresh (unfixed) shortly after collection. Smears were prepared from
suspicious areas (blood tinged, nonpurulent, nonsaliva,
tissue specks, etc.). These were immediately fixed in 95%
ethyl alcohol. The remaining material was placed in 50%
ethanol and 2% Carbowax. The sputum mixture was then
blended and centrifuged according to the Saccomanno
technique and at least two smears prepared from the sediment. After air drying, these were postfixed in 95%
ethanol and stained according to the modified Papani-
Received March 22, 1988; accepted for publication April 25, 1988.
Presented in part at the Fall Meeting of the American Society of Clinical
Pathologists, New Orleans, Louisiana, October 1987.
Address reprint requests to Dr. Erozan: Department of Pathology,
Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Maryland
21205.
57
58
PERLMAN, EROZAN, AND HOWDON
Table 1. Classification of Case Population According to
Type of Lung Cancer and Degree of Atypia
A.J.C.P.-January 1989
Table 3. Specimens Negative or Mildly Atypical on
Fresh Smears: Diagnosis on Saccomanno Smears
Classification
Number of Cases
Saccomanno Diagnosis
Number of Cases
Squamous carcinoma
Adenocarcinoma
Small cell carcinoma
Large cell undifferentiated carcinoma
Suspicious for malignancy, type unspecified
Atypical, probably reactive
Negative
55
26
22
3
9
19
70
Diagnostic of cancer
Undifferentiated carcinoma
Adenocarcinoma
Suspicious of cancer
Squamous carcinoma
Adenocarcinoma
Negative on follow-up
Negative or mildly atypical
1
1
83
204
Total
colou's procedure. All sputum samples prepared in this
way in the 22-month time period were blindly screened,
and those with any degree of atypia were randomized with
added negative controls. The Saccomanno smears were
separated from the fresh smears. Slides prepared according
to the Saccomanno and fresh smear techniques from each
specimen were evaluated independently and blindly by
at least two of the authors and classified as unsatisfactory,
negative, atypical, suspicious for cancer, or cancer. The
standard of comparison was considered to be the diagnosis
made at the time the specimen was originally submitted,
which took into account all methods of preparation. The
original diagnoses were tabulated and confirmed by available subsequent cytopathologic and histopathologic evidence and the diagnostic accuracy of each method determined. "Positive" specimens were those classified as cancer or strongly suspicious for cancer. The cellular
morphologic characteristics of malignant cells prepared
in each of the two methods were compared.
Results
Two hundred four sputum samples were randomized,
including specimens with atypia and negative controls.
After tabulation of original diagnoses and all subsequent
pathologic evidence, these samples were found to include
55 specimens diagnostic or suspicious for squamous carcinoma, 26 specimens diagnostic or suspicious for adenocarcinoma, 22 small cell carcinomas, and 3 large cell
undifferentiated carcinomas. Nine specimens contained
Table 2. Diagnostic Accuracy of Fresh and
Saccomanno Preparations Alone According
to the Types of Lung Cancer
Type of Cancer
Fresh
Saccomanno
Squamous carcinoma
95%
(52/55)
96%
(25/26)
100%
(22/22)
86%
(47/55)
52%
(17/26)
24%
(5/22)
Adenocarcinoma
Small cell carcinoma
cells suspicious for tumor of unspecified type, 19 specimens showed atypical cells reactive in nature, and 70
specimens showed no evidence of atypia (Table 1).
Comparing each preparation with the original diagnosis, the diagnostic accuracy of each tissue type is shown
in Table 2. The diagnostic accuracy of the squamous carcinomas was similar in the Saccomanno and fresh preparations but was significantly less in the Saccomanno
preparations of small cell and adenocarcinomas. Three
squamous carcinomas were diagnostic only on the Saccomanno slides. However, two of these were suspicious
for malignancy in the fresh smears.
Of 88 specimens diagnosed as negative, mildly atypical,
or unsatisfactory on fresh smears, 5 were suspicious or
diagnostic for cancer in the Saccomanno slides alone (Table 3). One specimen was diagnostic of undifferentiated
carcinoma and one was diagnostic of adenocarcinoma.
Three specimens were suspicious for carcinoma, including
one with subsequent development of squamous carcinoma and one with subsequent development of adenocarcinoma. One specimen was suspicious for carcinoma
on the Saccomanno smear and negative on the fresh
smear, and the patient subsequently showed no further
evidence of atypia, despite numerous specimens. This
likely represents a "false positive" diagnosis. There were
no cases of small cell carcinoma in which the Saccomanno
preparations added information or diagnostic material not
available on the fresh smears.
Comparison of the tumor cells within both methods of
preparation revealed important differences. Squamous
carcinomas in the fresh smears showed distinct nuclear
borders and chromatin, and the cytoplasmic keratin, when
present, was well defined. The same tumor cells in the
Saccomanno preparations showed some loss of nuclear
and the cytoplasmic definition, however, the characteristic
features were present, resulting in the similar diagnostic
accuracies of the two methods. Adenocarcinomas on the
fresh smears often showed large tissue fragments with crisp
nuclear features and cytoplasmic vacuoles. In the Saccomanno preparation, the tissue fragments were smaller
and in many cases the cytoplasm was poorly preserved.
Vol.91 •No. I
BRIEF SCIENTIFIC REPORTS
59
B
FIG. 1. Small cell carcinoma in a single sputum sample prepared fresh (/), left) and with the Saccomanno technique (B. right). The fresh smear
shows larger tissue fragments with well-preserved nuclear features and a thin cytoplasmic rim. The Saccomanno preparation demonstrates loss of
these features to a great extent with nuclear pyknosis and degeneration (X 1,550).
In addition, there was loss of chromatinic detail in the
nuclei, many of which were pyknotic. This often caused
problems in establishing definitive diagnoses in the Saccomanno preparations.
The greatest discrepancy in cytologic appearance was
found in the small cell carcinomas (Fig. 1). In fresh preparations, these tumors showed prominent streaming with
cellular molding and background necrosis. The nuclear
features were distinct, and a thin cytoplasmic rim was
evident. However, the Saccomanno preparations showed
scattered individual and small clumps of cells that were
shrunken and pyknotic and often lacked a cytoplasmic
rim. These features were difficult to differentiate from
certain benign cellular changes such as bare nuclei of degenerated columnar cells. In addition, the helpful background information, e.g., necrosis, was lost. This resulted
in a significant reduction in the diagnostic accuracy of
small cell carcinomas in the Saccomanno preparation.
Discussion
The Saccomanno technique remains an invaluable
method of preserving and concentrating sputum samples
that cannot be processed fresh. Risse and associates examined the reliability of the Saccomanno technique in
primary and metastatic lung tumors, studying sputum
specimens entirely prepared with the use of the Saccomanno method. They found the diagnostic accuracy of this
method in 362 patients with primary lung carcinomas to
be 87% and the cytologic typing accuracy to be 97% for
non-small cell tumors and 90% for small cell carcinomas,
providing five sputum specimens from each patient were
collected before a negative diagnosis was rendered.5 Although these authors have demonstrated a diagnostic accuracy comparable to that of fresh smears, our study suggests some diagnostic difficulties with the Saccomanno
method, particularly with small cell carcinomas. These
tumors have very fragile cells with a recognized tendency
to deform and show chromosomal diffusion in histologic
preparations, a property often used diagnostically by histopathologists. It is not surprising that a method relying
on fixation and blending would disrupt these cells and
significantly alter their morphologic characteristics.
Although there is no doubt that pathologists relying
solely on the Saccomanno preparation learn to compensate to some extent for many of its artifacts, attempts to
compensate for loss of diagnostic features may well cause
false results in either underdiagnosis or overdiagnosis of
cancer.
This study cannot be interpreted as a direct comparison
of Saccomanno's method with fresh smear preparations
because the most diagnostic material is being used in the
fresh smears before the Saccomanno preparation. This
thereby reduces the quantity of diagnostic material available for Saccomanno preparation. However, the difference
in diagnostic accuracy of the small cell carcinomas cannot
60
PERLMAN, EROZAN, AND HOWDON
be explained by a lack of diagnostic material alone and
likely results from loss of the diagnostic features we have
described.
The study directly addresses the role of the Saccomanno
method in conjunction with fresh smears. In that context
the Saccomanno preparations may contribute to the diagnosis of non-small cell carcinomas (four cases were diagnostic or suspicious for carcinoma in the Saccomanno
slides alone) but do not appear to aid in the diagnosis of
small cell carcinoma.
In summary, the preparation of fresh smears enables
effective concentration of diagnostic material by selection
of suspicious areas. The cellular morphologic characteristics are more conducive to cytologic diagnosis and the
background diathesis is preserved. When fresh smears
cannot be prepared, the Saccomanno technique is a valuable method for fixing the specimen until processing is
possible. It also provides a concentrated sample representative of the entire specimen. However, when fresh smears
can be prepared, the Saccomanno method should not replace the fresh smear preparations.
A.J.C.P. • January 1989
References
1. Benbassat J, Regeu A, Slater PE: Predictive value of sputum cytology.
Thorax 1987;42:165-172.
2. Erozan YS, Frost JK: Cytopathologic diagnosis of cancer in pulmonary material: a critical histopathologic correlation. Acta Cytol
1970;14:560-565.
3. Johnston WW, Bossen EH: Ten years of respiratory cytopathology
at Duke University Medical Center. Acta Cytol 1981,25:103108.
4. Pilotti S, Rilke F, Gribaudi G, Revesi GL: Sputum cytology for the
diagnosis of carcinoma of the lung. Acta Cytol 1982;26:649-654.
5. Risse EKJ, van't Hof MA, Laurini RN, Vooijs PG: Sputum cytology
by the Saccomanno method in diagnosing lung malignancy. Diag
Cytol 1985;1:286-291.
6. Rosa U, Prolla JC, Gastel ES: Cytology in diagnosis of cancer affecting
the lung: results in 1,000 consecutive patients. Chest 1973;63:
203-207.
7. Saccomanno G: The contribution of uranium miners to lung cancer
mutagenesis. Recent Results Cancer Res 1982;82:43-52.
8. Saccomanno G, Archer VE, Auerbach O, Saunders RP, Brennan
LM: Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 1974;33:256-270.
9. Saccomanno G, Saunders RP, Ellis H, Archer VE, Wood BG, Becker
PA: Concentration of carcinoma or atypical cells in sputum. Acta
Cytol 1963;7:305-310.
Nucleoli of Blood Monocytes inMalignant Lymphoma
A Morphometric Study
ROBERT J. SOKOL, M.D., PH.D., F.R.C.PATH.
Morphometric methods were used to study the nucleolar ultrastructure of blood monocytes in 23 patients with Hodgkin's disease, 12 patients with non-Hodgkin's lymphoma, and 20 normal
subjects. Nucleolar volume (V„), surface area (S„), volume fraction within the nucleus (VV„), surface-to-volume ratio, and number
of nucleolar profiles per section were measured. The results were
examined with the use of multivariate and univariate analyses
of variance, and significant differences between the patient and
normal groups were found. Compared with the normals, values
for Vn, S n , VV„ and number of profiles per section were 16-20%
smaller in the Hodgkin's monocytes and 19-32% smaller in those
of the patients with non-Hodgkin's lymphoma. The changes in
nucleolar ultrastructure may be related to the known mononuclear
phagocyte dysfunction in patients with lymphoma. (Key words:
Hodgkin's disease; Lymphoma; Microscopy, electron; Monocytes; Morphometry; Nucleoli) Am J Clin Pathol 1989;
91:60-63
Received March 16, 1988; accepted for publication April 12, 1988.
Supported by the Yorkshire Cancer Research Campaign and the Trent
Regional Health Authority.
Address reprint requests to Dr. Sokol: Regional Blood Transfusion
Centre, Longley Lane, Sheffield, S5 7JN, United Kingdom.
Department of Hematology, University of Sheffield, Sheffield
S10 2RX, United Kingdom
MONONUCLEAR PHAGOCYTE DYSFUNCTION is
known to occur in patients with malignant lymphoma,24
and it has been suggested that ultrastructural changes
in peripheral blood monocytes26 and skin window
macrophages23,30 were the morphologic counterparts of
such dysfunction. Because nucleoli, by taking part in the
production and processing of ribosomal RNA, provide
an essential link between nucleus and cytoplasm5 and are
prominent in cells with active protein synthesis,1 it seemed
important to determine whether morphologic changes
were present in the nucleoli of mononuclear phagocytes
in patients with lymphoma. In the work presented here,
nucleolar measurements have been made on peripheral
blood monocytes of patients with Hodgkin's disease (HD)