Research About Active Human Cytomegalovirus Infection Of Myeloid Tissue In Aplastic Anemia

Objective RT-PCR in situ technique is applied into plastic embedded bone marrow sections,we hope the histological evidence of active human cytomegalovirus (HCMV) infection in functional disorder of haematopoiesis could be ensured through this new way. The relationship between the active human cytomegalovirus infection and the ratio of the bone marrow CD34+ cell/ karyocyte is aquired by flow cytometry, through the way above we expect to know about the influence of active human cytomegalovirus infection in bone marrow on the haematopoiesis function of the aplastic anemia children.Methods 26 children with aplastic anemia were included into our research through a clinical screening evaluation. The exclusion criteria were anemia caused by HBV,HCV,HIV,Parvovirus B19,EBV,bacteria infection and drug,connective tissue disease and geno-metabolic disease.①The myeloid tissue biopsies from the 26 children were embedded in methyl methacrylate(MMA)plastics and cut into sections. Then these sections were put into Eppendorf tubes for in situ reverse-transcriptase proliferation. Expression of HCMV mRNA was detected by RT-PCR in situ technique under the microscope.②Meanwhile the myelo-fluid were collected and distributed to two parts, one part was used to extract RNA which went through RT-PCR, HCMV mRNA detection is done to set a contrast for RT-PCR in situ.③Another part of the myelo-fluid was handled by flow cytometry to detect the ratio of CD34+ cell/ karyocyte.④In addition, five myelo-fluid samples were collected from patients who suffered other diseases as normal contrast. Results①HCMV mRNA positive expression was detected in 9(34.6%) plastic embedded section; most of the positive cells are myelo-parenchyma cells which presented focal or flake disposition, bulk positive signal appeared inside of the endochylema and a few appeared inside of the nucleus.②HCMV mRNA positive expression was detected in 11(42.3%)myelo-fluid samples through routine RT-PCR. The difference of positive rates between two methods was not significantly(P > 0.05). In addition, nine myelo-fluid samples from patients who had positive HCMV mRNA expression through RT-PCR in situ are all detected positive through routine RT-PCR. HCMV mRNA positive expression are not detected in five myelo-fluid samples from normal contrast group.③The average ratio (CD34+ cell/ karyocyte) of aplastic anemia patients is (0.26±0.21)%,which is lower than the that of normal contrast group obviously. Moreover, the ratio (CD34+ cell/ karyocyte) of HCMV mRNA positive group from aplastic anemia patients is (0.13±0.07)%,which is lower than the ratio of HCMV mRNA negative group from aplastic anemia patients (0.34±0.23)%(P < 0.05)。Conclusion①Modified RT-PCR in situ technique can be applied into plastic embedded sections. This method is expected to provide level diagnosis of active HCMV infection in bone marrow, which can provide histological evidence of hematologic change influenced by HCMV infection. It is important for the pathogenesis research of functional disorder of haematopoiesis in bone marrow.②HCMV can infect the bone marrow cells of aplastic anemia patients, active HCMV infection could induce the reduction of the ratio (CD34+ cell/ karyocyte), which imply active HCMV infection has inhibitory action to hematopoiesis of bone marrow. This might be one reason lead to genesia of aplastic anemia.