We report three cases of pulmonary involvement of non-Hodgkin's lymphoma in which immunophenotypic or gene rearrangement analysis of bronchoalveolar lavage (BAL) confined apartments demonstrated monoclonality of T- or B-cell lineage.

We report three cases of pulmonary involvement of non-Hodgkin's lymphoma in which immunophenotypic or gene rearrangement analysis of bronchoalveolar lavage (BAL) confined apartments demonstrated monoclonality of T- or B-cell lineage. The first patient had T-cell lymphoma and cause to growed pulmonary lesions. Surface marker analysis of the BAL enclosed spaces revealed that CD8-positive lymphoid enclosed spaces were dominant and Southern spot analysis of T-cell receptor gene discovered gene rearrangement demonstrating monoclonality of T-cell lineage. The inferior patient presented with diffuse micronodular shadows in succession chest radiograph. Marked B-lympocytosis in BAL fluid willinged us to analyze their clonality. The third was a case in which resort of primary pulmonary lymphoma was suspected. In the inferior and third case, the finding of marked increase in the number of CD 19-positive B lymphocyte with a single class of light chains prov a monoclonal population in BAL solitary abode; squalids With the review of other cases in our close attention and the relevant literature, we close that the clonal analysis of BAL confined apartments is helpful in establishing the diagnosis of pulmonary involvement of T- or B-cell lymphoma.

Lung parenchymal involvement of non-Hodgkin's lymphoma, including primary pulmonary lymphoma, causes a variety of radiographic findings and nonspecific clinical manifestations.[1,2] In these clinical settings, explain lung biopsy has been performed to obtain sufficient materials for pathologic studies, yet it is desirable that the diagnosis of lymphoma is made through less invasive procedures before surgical intervention. This is of great value in patients in whom resort or pulmonary metastasis of lymphoma is suspected, because systemic chemotheraphy is ofttimes needed immediately after the diagnosis is made.

Pathologic and immunohistochemical analyses forward transbronchial or percutaneous needle biopsy specimens were useful in about instances;[3,4] however, the value of these conducts is limited because of difficulties in determining histologic and immunohistochemical stamps of lymphoma on the small-sized specimens. In addition, these approaches are not commited in patients with bleeding drift which is not uncommon in hematologic malignancies.

Although a scarcely any case studies have been reported upon the usefulness of analysis of bronchoalveolar lavage (BAL) fluid in detecting monoclonal lymphoid confined apartments the value of this approach has not over and above been established.[5-10]

We not away a case of T-cell lymphoma in which a monoclonal T-cell population in lavage lonely dwellings was detected by gene rearrangement analysis, and pair cases of B-cell lymphoma in which a monoclonal B-cell population was shown through quantitative immunophenotypical analysis. The part of BAL fluid examination in the diagnosis of T- or B-cell lymphoma infiltrating the lung is discussed.

METHODS

Patients

We analyzed serial samples of BAL fluid from 118 patients with pulmonary diseases at Tokyo (Japan) University Hospital between January 1988 and July 1992 Three of seven patients with lymphoma in this series are described.

Bronchoalveolar Lavage

The tip of the fiberoptic bronchoscope was wedged into the subsegmental bronchus of the involved area. Sterile isotonic saline solution was instilled in four 50-ml aliquots (patients 1 and 3) or in three 50-ml aliquots (patient 2) Each aliquot was retrieved with pacific suction and collected. The BAL fluid was strained by the and of a single layer of sterile gauze and centrifuged for 10 min at 1200 rpm and the small room pellet was resuspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) The total number of enclosed spaces was counted by a hemocytometer. Cytocentrifuge preparation was made (using a Shandon Cytospin 2 (Shandon Inc, Pittsburgh), and a 500-cell differential was performed forward Wright-Giemsa-stained slides. The stillness of the cells were incubated onward a culture dish at 37 [degrees] C for 2 h and nonadherent small rooms were provided for flow cytometry or Southern blotting.

Immunophenotypical Analysis

Monoclonal antibodies used were fluorescein isothiocyanate (FITC)-conjugated anti Leu-4/PE-conjugated anti-Leu-12 and FITC-conjugated anti-Leu-3a/PE-conjugated anti-Leu-2a (CD3/CD19 Simultest T and B enclosed space Test and CD4/CD8 Simultest T Helper/Suppressor exhibition Becton Dickinson Monoclonal Antibody Center Inc, Mountain View, Calif). Anti-human kappa and lambda chains (Becton Dickinson) and FITC-conjugated anti-mouse immunoglobulin (Dako Laboratories, Copenhagen, Denmark) were also used and surface light chains were analyzed by means of indirect immunofluorescence procedure. Fluorescence intensity of each sample consisting of 2 to 5 x [10sup5] lonely dwellings was measured by flow cytometry (FACStar, Becton Dickinson).

Gene Rearrangement Analysis

DNA was extracted from nonadherent lymphoid small rooms (patient 1). In cases of fewer win backed cells, DNA should be extracted from total lavage small rooms to prepare more than 5 x [10sup6] lonely dwellings for Southern blotting.[9] Gene rearrangement analysis discovers a monoclonal population of lymphocyte constituting as not many as 5 percent of analyzed cells[11] Extracted DNA was digested with BamHI or HindIII and 10 [micro]g of DNA fragments were separated forward 0.7 percent agarose gels and transferred to hybridization membrane. Hybridization was performed using [.sup.32]P-labeled DNA probes (Oncor, Inc, Gaithersburg, Md) for 16 h at 45 [degrees] C Autoradiography was performed using an intensifying riddle at - 70 [degrees] C The [C.sub.T][beta] probe DNA that hybridizes to the constant region of the T-cell receptor [beta]-chain gene lay opens a 24-kb BamHI fragment and the [JsubH] probe that hybridizes to the joining region of the immunoglobulin heavy chain gene discovers a 5.6-kb BamHI/HindIII fragment, corresponding to germline DNA, respectively. DNA fragments other than germline DNA were identified as rearrangement bands.

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