Download The Authors` Reply: Nonspecificity of PNA Staining

CORRESPONDENCE AND CORRECTIONS
Vol. 89 • No. 2
whose stromal M - H envelop the tumor
cells. (An example is shown in Figs. 2A
and 2B of Ree and Hsu's article, 2 in
which RCA staining revealed M-H encircling tumor cells of malignant histiocytosis; PNA staining of this same tumor
showed an identical pattern.) Therefore,
we suspect that the large cells in their
Figure 4 are indeed tumor cells, but unstained, that are encircled by positively
stained cytoplasmic extensions of M-H.
In paraffin-section lectin histochemistry, it is important, especially with PNA,
to distinguish cell surface ( or membranous) staining from the diffuse cytoplasmic pattern because in lymphoid
tissues the cell surface staining pattern,
when it occurs, marks the cells of lymphoid origin, whereas the diffuse cytoplasmic pattern marks M-H. 1 We did
not encounter a single case of nonHodgkin's lymphoma that showed the
The Authors'
Nonspecificity
291
diffuse cytoplasmic staining pattern of
tumor cells in a series of cases we studied
(Cancer 1983,52:2089, Lab Invest
1984;50:48A nor in subsequently diagnosed cases which have been stained
routinely with a panel of lectins.
On the other hand, the diffuse cytoplasmic staining pattern of PNA has so
far been observed only in stromal M-H,
and the number of PNA-binding stromal M-H varies markedly in malignant
lymphomas (Cancer 1983,52:2089,
Cancer 1985;56:333). It should be noted,
however, that as a lineage marker for
M-H in paraffin sections, PNA staining
is not as effective as other lectins. 4
Rather, we have found PNA staining to
be most useful in detecting interdigitating reticulum cells, histiocytosis-X cells,
and Reed-Sternberg cells in paraffin sections 3 (see also Hum Pathol 1985;
18:309).
H. J. REE, M.D.
Department of Pathology
Rhode Island Hospital and
Brown University Program in Medicine
Providence, Rhode Island
but wonder how Ree and Kadin could
be certain that the histiocytes staining
for RCA and PNA are benign, and not
well differentiated malignant histiocytes.
The notion that a lectin or antibody
can distinguish between benign and malignant cells2"4 is not valid; the distinction between the two is based on morphologic features, specifically nuclear
morphology and/or derangement/effacement of architecture. Marker studies
are useful to determine lineage by virtue
of cytoplasmic or membranous staining
in order to categorize or classify the malignant neoplasm, not to determine malignancy per se. The only exception to
this rule is monotypic staining of some
types of lymphoid lesions for kappa or
lambda light chains; even in this instance, there are benign lesions with
clonal restriction that demonstrate the
limitations of this rule.
The staining patterns of benign or
background histiocytes in Hodgkin's
disease and large cell lymphoma are
clearly mentioned within our manuscript. The discussion also states the importance of distinguishing where the immunostaining is occurring—in benign
or malignant cells. This distinction is
easier in paraffin-embedded tissues be-
cause there is less endogenous peroxidase activity to obscure morphology
than in frozen tissue sections and there is
less artefactual distortion of the individual cells.
Fine details of staining patterns, such
as paranuclear, membranous, or cytoplasmic, are subject to inherent limitations of the immunoperoxidase technique, both in frozen section and paraffin-embedded tissue. There are several
uncontrollable variables, such as the
time interval between surgical removal
of the tissue and freezing or fixation, rapidity of the actual freezing process,
length of fixation in a particular fixative,
uniformity of fixation, length and type
of enzyme digestion to unmask antigens,
length and temperature of antibody incubation, and purity and specificity of
the antibodies or lectins used. All of
these variables differ between laboratories and possibly even within laboratories. In view of these variables, is it surprising that discrepancies in staining
patterns are reported? In fact, there is a
remarkable similarity between our study
and that of Ree and Kadin with regard
to the staining pattern of benign histiocytes with PNA. The differences between our studies are primarily ones of
References
1. Ree HJ, Hsu SM: Lectin histochemistry
of malignant tumors, 1: Peanut agglutinin (PNA) receptors in follicular
lymphoma and follicular hyperplasia:
An immunohistochemical study.
Cancer 1983;51:1631-1638.
2. Ree HJ, Kadin ME: Lectin distinction of
benign from malignant histiocytes.
Cancer 1985;56:2046-2050.
3. Ree HJ, Kadin ME: Peanut agglutinin: A
useful marker for histiocytosis X and
interdigitating reticulum cells. Cancer
1986;57:282-287.
4. Roholl PJM, Kleyne J, Pijpers HW, et al:
Comparative immunohistochemical
investigation of markers for macrophage-histiocytes. Hum Pathol 1985;
16:763.
Reply
of PNA Staining
To the Editor:—On histologic review,
the slides of Figure 4 ("A Comparative
Marker Study of Large Cell Lymphoma,
Hodgkin's Disease, and True Histiocytic
Lymphoma in Paraffin-Embedded Tissue"), 1 show a classical B immunoblastic
sarcoma (BIBS) in which there is a
monomorphic proliferation of morphologically malignant cells with abundant
cytoplasm which were monotypic
lambda in both frozen section and paraffin-embedded material. The cells staining with PNA are clearly malignant; as
seen in Figure 4, cells with large nucleoli
have intense cytoplasmic staining with
peanut agglutinin (PNA).
Ree and Kadin claim that the malignant histiocytes and monocytes seen in
malignant histiocytosis and monocytic
leukemia can be distinguished from benign "stromal" monocytes/histiocytes
based on staining with the lectins Ricinis
communis agglutinin (RCA) and PNA
in benign histiocytes and failure of malignant histiocytes to stain.2-3 This conclusion is based on the study of only two
cases of "well characterized" malignant
histiocytosis and one case of monocytic
leukemia.2'3 Since there is a spectrum of
pleomorphism and differentiation in
malignant histiocytosis, we cannot help
292
interpretation and conclusions. Interestingly, the results of a study by Roholl
and associates5 also conflict with those
of Ree and Kadin in that malignant histiocytes did stain with PNA.
We have not ascribed undue significance to cytoplasmic versus membranous or paranuclear staining unless
consistent patterns were seen, such as
punctate or globular paranuclear staining patterns in T-cell lymphomas and
Reed-Sternberg cells of Hodgkin's disease, respectively. In our study, the distinction between benign and malignant
histiocytes and lymphocytes was strictly
morphologic; it was based neither on
positive immunostaining nor pattern of
immunostaining. While in theory immunoperoxidase is a sophisticated and
scientific technique, in practice it can be
CORRESPONDENCE AND CORRECTIONS
mercurial and subject to countless variables which may be curtailed but not entirely eliminated. Though immunoperoxidase techniques remain useful in selected circumstances, one should be
cautious in drawing conclusions based
solely on this technique.
JEANNE M. MEIS, M.D.,
Department of Pathology
Henry Ford Hospital
Detroit, Michigan
JAMES J. BUTLER, M.D.
BARBARA M. OSBORNE, M.D.
M.D. Anderson Hospital and
Tumor Institute
Houston, Texas
References
1. Meis JM, Osborne BM, Butler JJ: A comparative marker study of large cell
A.J.C.P. • February 1988
lymphoma, Hodgkin's Disease, and
true histiocytic lymphoma in paraffin-embedded tissue. Am J Clin Pathol
1986;86:591-599.
2. Ree HJ, Kadin ME: Lectin distinction of
benign from malignant histiocytes.
Cancer 1985;56:2046-2050.
3. Ree HJ, Kadin ME: Peanut agglutinin: A
useful marker for histiocytosis X and
interdigitating reticulum cells. Cancer
1986;57:282-287.
4. Ree HJ, Hsu SM: Lectin histochemistry
of malignant tumors, 1: Peanut agglutinin (PNA) receptors in follicular
lymphoma and follicular hyperplasia:
An immunohistochemical study.
Cancer 1983;51:1631-1638.
5. Roholl PJ, Kleyne J, Pijpers HW, Van
Unnik JAM: Comparative immunohistochemical investigation of markers for malignant histiocytes. Hum
Pathol 1985;16:763-771.
Patient Samples as Controls
To the Editor:—This letter is in reference to the article, "Evaluation of the
Toa E-5000 Automated Hematology
Analyzer" by Payne and co-workers
(Am J Clin Pathol 1987;88:51-57).
The evaluation of the Toa E-5000 instrument included precision studies
using stabilized control blood and fresh
whole blood samples from volunteers.
These control practices are also used to
calibrate hematology instruments as a
routine quality control protocol in many
hospitals, with the exception that random patient samples are used rather
than samples obtained from volunteers.
In light of today's increased concern
with liability and transmission of potentially infectious agents to laboratory
workers, pathologists should consider
not using patient samples as secondary
quality controls. Reasons include:
1. The patient's blood chosen is
usually not tested for serologic
evidence of HIV or hepatitis.
Commercial preparations are
routinely tested for HIV antibody and hepatitis B surface antigen.
2. The use of patient tissue or
blood for purposes other than
1. Tangley L: Who owns human tissues and
cells? BioScience 1987;37:376.
should be made to decrease the risk of
exposure of laboratory workers to these
agents. We use samples collected from
blood donors. These donors are from
our local pool who have been repeatedly
tested and have a very low positive testing rate. This minimizes the problem
but does not solve it. One must run samples from patients who are unknown as
to their contamination and even from
those patients who are known to be positive for either virus. We cannot refuse to
handle such specimens. It is foolish to
handle such specimens in any different
manner than routine specimens, be-
cause the routine specimens can also be
contaminated. Therefore the approach
must be to pay scrupulous attention to
good laboratory handling practices, use
of gloves where the likelihood of contamination exists, and attempt to encourage manufacturers to develop instruments that sample from closed tubes
and use closed waste disposal. We must
also encourage manufacturers to develop blood collection devices that ensure that the outside of the container is
sterile.
In my opinion the introduction of a
patient control sample of unknown con-
diagnosis may not be acceptable
without the patient's permission
and theoretically could result in
legal action.1
THOMAS A. RUMA, M.D.
Immanuel Medical Center
Omaha, Nebraska
Reference
The Author's Reply
To the Editor:—I am enclosing my
reply to Dr. Thomas Ruma who wrote
relative to our article.
We wish to thank Dr. Ruma for his
comments. The first comment dealt
with the possible hazard of using a patient's blood for control material as opposed to using commercial preparations
which have been tested for HIV antibody and hepatitis B surface antigen and
are therefore safe to use; particularly if
routine patient samples are used instead
of blood from volunteers who are more
likely to be free of such contamination.
We agree that every reasonable effort