Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues.

Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate. Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin. For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue. Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively. Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases. Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43). In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized. Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies. B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.

Growth fraction in centrocytic and follicular center cell lymphomas: assessment in paraffin sections with a proliferating cell nuclear antigen antibody and morphometric correlates.

Measurement of growth fraction is an important way to provide an objective assessment of non-Hodgkin's lymphomas; however, many of the techniques used require fresh tissue and/or special instrumentation. Recently, antibodies to the proliferating cell nuclear antigen (PCNA)/cyclin reactive in paraffin-embedded sections have become available. To investigate the utility of one such antibody in the study of follicular center cell (FCC) lymphomas and cleaved cell lymphomas of centrocytic type (CC), paraffin sections from 40 cases that had been characterized in two previous morphometric studies were stained with a PCNA antibody. Strong correlations were found between PCNA staining in formalin- and B5-fixed tissues, between the overall proportion of PCNA-positive cells and the proportion in the area of greatest staining, and between strong and total staining. Proliferating cell nuclear antigen staining was significantly stronger in the noncleaved FCC lymphomas than in the cleaved cell lymphomas. The FCC lymphomas showed moderate to strong correlations between PCNA staining and morphometric features of transformation, but only nuclear area correlated with PCNA staining in the CC group. Proliferating cell nuclear antigen staining was not significantly different between CC lymphomas with and without the characteristic bcl-1/PRAD 1 gene rearrangement. In summary, PCNA staining of either B5- or formalin-fixed, paraffin-embedded tissue sections is a simple aid in the objective categorization of FCC lymphomas and may offer additional potentially prognostic information in some FCC subsets and in CC lymphomas. The findings further support the distinction between CC and true FCC lymphomas.

Differential antigen preservation during tissue autolysis.

Immediate fixation or snap freezing of tissue is ordinarily done to maximize antigen preservation for immunocytochemistry; however, delay in tissue allocation or spontaneous lymph node infarction can render tissue suboptimal for immunostaining. To test the effects of tissue autolysis/necrosis on the preservation of various lymphoid, epithelial, and mesenchymal markers, two lymph nodes (one with reactive lymphoid hyperplasia and one with metastatic ductal breast carcinoma) were evaluated for immunocytochemically demonstrated antigen preservation at 0-, 4-, 8-, 12-, 24-, 48-, and 72-hour intervals of autolysis at 37 degrees C. All specimens were stained by frozen section and formalin-fixed paraffin section immunocytochemical reactions with antibodies against CLA (CD45), UCHL-1 (CD45RO), L-26, kappa, lambda, anti-epithelial keratins (AE-1 and AE-3), epithelial membrane antigen, and vimentin. Frozen sections were additionally stained for Leu-1 (CD5), Leu-2a (CD8), Leu-3a+b (CD4), Leu-4 (CD3), and Leu-14 (CD22). The most resilient lymphoid antigen preservation was observed with CLA and UCHL-1, both exhibiting immunoreactivity at 72 hours in both frozen and fixed preparations. L-26 showed similar reactivity in frozen sections, but detectable antigen was observed only up to 24 hours in formalin-fixed tissue. Leu-2a proved to be the most labile antigen, persisting for only 12 hours in frozen sections. The epithelial markers epithelial membrane antigen and AE-1 exhibited excellent antigenic preservation in both frozen and fixed preparations; AE-3 persisted well in frozen section but was not demonstrated in fixed tissue. Vimentin immunoreactivity was vastly superior in frozen, as compared with fixed, tissue sections. Most antigens showed remarkable preservation despite morphologic degradation; however, differential antigenic resilience was demonstrated. Knowledge of this variation in antigen decay is critical for evaluation of immunoperoxidase phenotypic studies of autolyzed or necrotic tissue.

Marrow granulomas in coal workers' pneumoconiosis. A histologic study with elemental analysis.

The differential diagnosis of bone marrow granulomas is lengthy but has not previously included coal workers' pneumoconiosis. This report describes the first case in which noncaseating epithelioid granulomas containing anthracotic pigment and birefringent silica containing crystals were discovered in the marrow of a patient with progressive massive fibrosis of coal workers' pneumoconiosis. The silicotic nature of the crystals was confirmed using energy dispersive x-ray microanalysis. There was no evidence of a mycobacterial, fungal, or other etiology for the granulomas. This case demonstrates that coal workers' pneumoconiosis should be added to the differential diagnosis of bone marrow granulomas and that marrow examination may be a source of tissue for documentation of specific occupational exposures.

Diversity of organ site involvement among malignant lymphomas of mucosa-associated tissues.

Fifteen cases of low-grade B-cell lymphoma involving unusual extranodal sites have been studied in comparison to cases reported or observed arising in typical mucosa-associated lymphoid tissues. In every case, histopathologic features conformed to those characteristic for lymphomas of mucosa-associated lymphoid tissues, including the production of lymphoepithelial complexes. Immunoglobulin light chain restriction was demonstrated by immunocytochemistry in 14 cases. Sites of involvement included the breast (6), skin (5), kidney (1), prostate (1), gallbladder (1), and uterine cervix (1). In three cases there was simultaneous or previous lymphoma of mucosa-associated lymphoid tissues identified in a more common mucosal site. It is concluded that the unifying concept of lymphomas of mucosa-associated lymphoid tissues applies to extranodal organs less commonly associated with mucosa-associated lymphoid tissues, as well as to those mucosal organ sites described in earlier series.

Morphometric analysis of follicular center cells: a new approach.

Accurate and reproducible categorization of follicular center cells (FCC) on the basis of their nuclear size and shape is difficult. In addition, the relationship of cleaved to noncleaved FCC has been questioned. For these reasons, 2126 FCC (and 947 mantle cells) from five plastic embedded reactive lymph nodes were studied using a new morphometric approach. Using an image analyzer, the following nuclear features were studied: area (NA); our previously described shape factors which measure ellipticity (NCIe) and irregularity (NCIni); and a new objective measurement of relative chromatin dispersal (chromatin dispersal index, CDI). Definite clefts and nucleoli were visually identified and recorded. Compared to the mantle cells, FCC nuclei were significantly larger, more elliptical, more irregularly shaped, and had more dispersed chromatin. Among the FCC the only correlation that could be identified between the above parameters was that the larger FCC tended to have more dispersed chromatin (r = 0.46). Significantly more dispersed chromatin was, however, associated with nonclefted cells, less irregularly shaped cells having a NCIni less than the median NCIni, cells with nucleoli, and larger cells having a NA greater than the median NA. Clefted FCC had significantly greater nuclear irregularlity compared to nonclefted FCC but a similar degree of ellipticity. Distribution curves for the NA, NCIe, NCIni, and CDI revealed a continuous range of results rather than a limited number of distinct cell types. The addition of an objective quantitative measurement of chromatin dispersal permits a more complete morphometric description of FCC.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved nuclear contour indices for lymphoid morphometry.

The morphometric analysis of benign and neoplastic lymphoid proliferations relies heavily on nuclear shape factors to identify clefted, cerebriform or convoluted nuclei. Most studies employ the size-independent nuclear contour index (NCI = perimeter/square root area) or the closely related (nuclear) form factor to evaluate the degree of nuclear irregularity. These indices, however, cannot distinguish a truly irregular shape from a perfectly smooth elliptical one. A variation of the standard NCI is therefore proposed in which the NCI of the ellipse (NCIe) that best approximates the nuclear shape is determined to indicate the degree of nuclear elongation or ellipticity and the separate NCI of the nuclear irregularity (NCIni) is determined to indicate the true irregularity of the nuclear perimeter, independent of nuclear elongation. These two new shape factors were tested on a series of shapes and on mantle and follicular-center cells present in 3-micron plastic-embedded sections from three tonsils with reactive lymphoid hyperplasia. Whereas the standard NCI increased with increasing ellipticity or nuclear irregularity, the NCIe increased only with increasing ellipticity and NCIni with increasing nuclear irregularity. Mantle cell and, to a greater extent, follicular-center-cell nuclei showed mean NCIe and NCIni greater than what would be expected from a perfectly round nucleus. These nuclei, therefore, were both more elliptical and more irregular in outline than a perfect circle. The NCIe and NCIni were shown to vary independently in both mantle and follicular-center cells. These new and relatively simple indices should lead to a more accurate morphometric description of lymphoid cells.

Morphometric analysis of follicular center cell lymphomas.

The Lukes/Collins classification of non-Hodgkin's lymphomas subdivides follicular center cell (FCC) lymphomas into four categories based on nuclear size, shape, and state of transformation. Because of the well-recognized difficulty in making these subdivisions in routine histologic sections, a morphometric analysis of typical small cleaved (SC), large cleaved (LC), small noncleaved (SNC), and large noncleaved (LNC) FCCs was performed to describe and compare these four categories using objective parameters. The following features, which had been previously tested on normal follicles, were measured and calculated: nuclear area (NA), nuclear contour index of ellipticity (NCIe), nuclear contour index of nuclear irregularity (NCIni), and a relative chromatin dispersal index (CDI). The presence of nucleoli also was recorded. Mean values for the NA, NCIe, NCIni, and CDI were significantly different among all four FCC lymphoma subtypes, except that the CDI, which reflects transformation, was similar for the SC and LC cell groups. Comparison using the proportion of cells with nucleoli in each case revealed significant differences between all but the SNC and LNC groups. The LC group had the highest mean nuclear ellipticity and nuclear irregularity values. Mean nuclear area was smallest for the SC group followed by the LC, SNC, and LNC groups. Despite these many differences, all parameters showed a broad spectrum of values when either mean values for individual cases of each FCC subtype or distribution curves for all cells within a certain subtype were compared. This morphometric data demonstrates that the four histologically recognized types of FCC lymphomas are distinctive using a more analytic technique. This study also provides further insight into the differences among them. Evidence of nuclear transformation (nuclear size, chromatin dispersal, and frequent nucleoli) is a more important criterion than nuclear contours in distinguishing LNC from LC lymphomas. Although LC lymphomas have some features intermediate between SC and the noncleaved FCC lymphomas, they more closely resemble SC lymphomas. Finally morphometric analyses such as these provide an objective morphologic foundation for future prospective investigations of transformation-related phenomena; these studies may facilitate comparison of morphologic data with immunophenotype and genotype in non-Hodgkin's lymphomas and comparison of various other B-cell neoplasms with follicular center cell neoplasms.

Hodgkin's disease in association with human immunodeficiency virus infection. Pathologic and immunologic features.

Six patients with Hodgkin's disease (HD) and demonstrable serum antibodies to human immunodeficiency virus (HIV) and two additional patients with HD belonging to HIV-associated high-risk groups but with negative HIV serology were studied. All patients were men and ranged in age from 21 to 45 years. The HIV risk factors included homosexuality (6), intravenous drug abuse (2), and hemophilia A (1). All patients had high pathologically determined stage (one Stage III and seven Stage IV), and bone marrow involvement was observed in five patients with the initial diagnosis of HD based on marrow biopsy in two cases. Four cases were histologically subclassified as mixed cellularity (MC) and three as nodular sclerosis (NS); one patient underwent only bone marrow biopsy and was not subclassified. Histologically all cases were characterized by numerous Reed-Sternberg cells and variants, and with the exception of one case, all had a distinctive decrease in the proportion of reactive background lymphocytes compared with what is usually expected in MC or NS Hodgkin's disease (relative lymphocyte depletion). Flow-cytometric immunophenotypic studies done on cell suspensions from diagnostic lymph node biopsies in four cases showed decreased CD4:CD8 ratios (mean = 1.4) compared with expected values of 4 to 6. The relative lymphocyte depletion observed histologically is probably a reflection of the decreased tissue CD4:CD8 ratios, and this impairment of host immune response may be related to the observed high stage in all eight cases. Patients with high stage HD and the described histologic and immunologic features should be evaluated for the presence of HIV infection.

Centrocytic lymphoma: a morphometric study with comparison to other small cleaved follicular center cell lymphomas and genotypic correlates.

Centrocytic lymphoma (ML,CC) is distinguished from other cleaved follicular center cell (FCC) lymphomas in the Kiel classification by the lack of noncleaved FCC and not by morphological differences between the centrocytes (cleaved cells). Immunophenotypic and genotypic studies, however, have shown that the centrocytes in ML,CC are distinct from those of other small cleaved FCC lymphomas (ML,FCC,SC). To morphologically compare the cells of ML,CC with nine previously studied ML,FCC,SC and to relate the findings in ML,CC to the varied descriptions of lymphomas of intermediate differentiation, a morphometric analysis of 22 ML,CC was performed. Nuclei in ML,CC were, on average, significantly larger, rounder, and had less frequent nucleoli than those in ML,FCC,SC; however, the proportion of small round lymphocytes did not differ. Among the ML,CC, the only apparent immunophenotypic/genotypic correlate that was identified was greater nuclear ellipticity for the biopsies lacking chromosome 11q13 bcl-1 or PRAD1 rearrangement. Repeat biopsies in four patients with ML,CC showed an increase in nuclear size. These data demonstrate that a lack of transformed cells is not the only morphological difference between ML,CC and ML,FCC,SC. The morphological distinction, however, is not based on the proportion of small round lymphocytes present. In addition, the morphometric parameters illustrate the nuclear variability among ML,CC and demonstrate how the disease may evolve over time.