Expressin Of BSAP And Detection Of Rearrangement Of IgH Gene Of H/RS Cell And Its Background Lymphocytes In Classical Hodgkin's Lymphoma

Objective The origin and the nature of the Hodgkin and Reed-Sternberg(H/RS) cell of Hodgkin's lymphoma (HL) has been attracting a lot of medical researchers engaged in studying it,for its character by scattered large atypical cells residing in a complex admixture of inflammatory cells.Great improvement has been made since a new method of isolation of single H/RS cells from a frozen section had been set up by KUppers in 1994,and many studies have approved of the B-cell derivation of H/RS cell.lt has been reported that H/R-S cells might partly be originated from B-cell in our research before,but at the same time,we also found that only 18.8% of H/RS cells express CD20,31.3% of immunoglobulin heavy chain (IgH) rearrangement have been revealed. It still remains a question whether the rearrangements of IgH come from H/RS cell or the background lymphocytes. In this study,we have detected the IgH clonal correlation between the H/RS cells and the background cells,from a new aspect to study the clonality of H/RS cell and its relation with the background cells. The expression of B-cell-specific activator protein (BSAP) was detected in HL.IgH gene rearrangements were analysed by the methods including gene analysis in neoplasms tissue and micropicked cells from paraffin-embedded sections,sequencing to test the PCR product,and in situ PCR.Methods Protein expressions of BSAP,CD30,CD 15,CD20 and CD45RO were detected in 33 cases of cHL;The rearrangements of IgH gene were detected by polymerase chain reaction (PCR);Sections of 6 cases which have been "revealed single clonal bands of IgH gene were used for the isolation of H/RS cell and background lymphocytes immunostained for BSAP by micromanipulation,then to detected the rerrangements of IgH gene by PCR to analyze the relations of H/RS cells and lymphocytes;Resulting PCR products of 3 cases were selected and sequenced,then synthesized Dig marked probes,with which we proceeded in situ PCR detection.Results (1) 30/33(90.91%) and 33/33(100%) were BSAP positive on H/RS cells and background lymphocytes respectively;10/33 (30.30%) were CD20 positive,1/33(3.03%) were CD45RO positive,31/33(93.94%) were CD30 positive,25/33(75.75%) were CD15 positive. (2) 16/33(48.48%)of paraffin-embedded tissue and 14/19(73.68%) tubes micropicked H/RS cells and 2/12(16.67%) background lymphocytes were revealed rearrangements of IgH gene by PCR. (3) The sequencing obtained is the rearranged V segment. (4) In situ PCR,we found brown staining particles not only in nucleoli of H/RS cells but also in those of surrounding background lymphocytes.Conclusion (1) In our research,frequent expression of BSAP in H/RS cell of classical Hodgkin's disease provides further evidence for its B-cell origin,and the expression of BSAP on H/RS cells also can be in favor of the identification of H/RS cells and can distinguish HL from ALCL. (2) Expression of BSAP is located in nucleoli of cells,while that of CD20 in membrane. The micropicked cells stained by BSAP were of B cells and only contained nucleoli of cells. (3) The detection rate of IgH gene rearrangement rises with the criterion of oligoclonality and clonal evolution. (4) Detection of IgH gene rearrangement by the means of paraffin-embedded tissue and micropicked cells,sequencing,and in situ PCR,to give a evidence that H/RS cells may be originated from B cell of distinct clonality. (5) The background lymphocytes may not be proliferative cells with parts of them revealed IgH gene rearrangements. A further study was need to decide whether the background lymphocytes is a part of neoplasms or not.