| Objective： To explore the method of detecting Cyclin D1and BCL-2by flowcytometry, as well as feasibility and significance.Methods:1） Patients with hematologic diseases in final diagnosis werecollected and recorded with45cases in detail,22were male,23female,age8-84, including HL3cases, CLL15cases, DLBCL11cases, MCL3cases,MZL2cases,FL2cases， NK-T cell lymphoma4cases, T cell lymphoma5cases,including treatment, follow-up and evaluation of curative effect.According to the2008WHO lymphoma classification criteria, patients wereclassified. According to Zhang Zhinan 《blood disease diagnosis andtreatment guide》 to judge the curative effect, treated patients weredivided into complete remission (CR), does not determine the completeremission (CRu), partial remission (PR), stable disease (SD),recurrence (for CR/Cru) and disease progression (PD).2）Accordingto the age and gender matching principle, selected routine physicalexamination and all tests were within the normal range in20healthy personsas a healthy control group.3）Select the pathology confirmed Cyclin D1or BCL-2positive cases as positive control group (8cases), select thepathology confirmed Cyclin D1or BCL-2negative cases as negative controlgroup (7cases). Four color monoclonal antibodies directly labeled withimmunofluorescence were used to analyze the surface and cytoplasma antigens,such as CD5, CD19, CD23, CD20, Cyclin D1, BCL-2and so on, CD19+cells wereset as door, we used multi-parameter flow cytometry to investigate peripheral blood Cyclin D1and BCL-2subsets in lymphoma patients.All datawas analyzed by statistical software SPSS13.0; the measurement data wasexpressed as mean±standard deviation ((?)±s). We used t test when twosets of values in line with normal distribution and homogeneity of variance,or using non-parametric test; we used analysis of variance when multiplesets of comparisons between values in line with normal distribution andhomogeneity of variance, or we used non-parametric tests,P<0.05meant thedifference was statistically significant. We used paired chi-square testto compare differences in detection methods, P<0.05meant the differencewas statistically significant.Results：1）, The subject of the25normal adult volunteers Cyclin D1MFIwere analysed x±s values and95%confidence interval, so that the CyclinD1MFI>0.5is positive, thus establishing the Cyclin D1diagnosticcriterion; BCL-2MFI were also analysised x±s values and95%confidenceinterval in25cases of normal adult volunteers, so that the BCL-2MFI>2.7is positive, thus establishing the BCL-2diagnostic criterion;2）,Cyclin D1and BCL-2diagnosed by FCM criteria of positive and negative,simplely compared with immunohistochemistry results of pathological in MCLpatients, FCM analysis of Cyclin D1sensitivity and specificity of100%,the detection of patients with FL BCL-2sensitivity and the specificityis100%.FCM detection of Cyclin D1and BCL-2with the combination of immunephenotype clear5cases of pathology can not be classified by lymphomasubtype.3) Compared with the healthy control group, all the B-cell lymphomain patients with peripheral blood of Cyclin D1(1.824±0.312) and BCL-2MFI (4.259±0.541) was significantly much more increased expressionlevels than normal control group (0.356±0.159,1.938±0.324), bothstatistically significant difference (p <0.0001).4) Different subtypes of lymphoma in peripheral blood of patients express differences of CyclinD1MFI.In patients with Hodgkin’s lymphoma of Cyclin D1MFI (0.386±0.112)was significantly lower than non-Hodgkin’s lymphoma patients (1.623±1.987)(p <0.001).In non-Hodgkin’s lymphoma patients, mantle cell lymphomapatients with Cyclin D1-positive (100%), and the remaining subtypes werenegative; different subtypes of lymphoma in peripheral blood of patientswith BCL-2expression differences, in patients with Hodgkin’s lymphomaBCL-2(2.045±0.877) was significantly lower than non-Hodgkin’s lymphoma(4.045±0.499) patients (p <0.001), in non-Hodgkin’s lymphoma, T celllymphoma express lowest, positive rate of MCL and FL was the highest (100%),followed by MZL (50%), CLL (30%), DLBCL (45%).5) lymphoma patients withCyclin D1or BCL-2-positive, more than patients detected the clinicaljudgment of partial remission after treatment by flow cytometry and foundthat Cyclin D1(before treatment3.099±0.349; after treatment,1.008±0.279)or expression of BCL-2(before treatment7.814±1.030; aftertreatment,3.131±0.522) mean fluorescence intensity are significantlylower (p <0.001).Conclusion:1, the method of flow cytometry for detection of Cyclin D1andBCL-2is feasible;2, Establishment of Cyclin D1and BCL-2reference valuein flow cytometry;3, FCM analysis of cyclin D1and BCL-2Diagnosticclassification of B-cell lymphoma meaningful, with good sensitivity andspecificity.4. Cyclin D1or BCL-2of Lymphoma patients after treatmentdownregulation, and its expression change with treatment, can be used asa clinical observation on evaluate the treatment. |
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