Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy determined by in situ hybridization for J chain mRNA.

The origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed and in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7 +/- 4.6 cells/10(5) micron2) compared with controls (mean 36.9 +/- 3.5 cells/10(5) micron2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positive interfollicular IgA cells in patient tonsils (32 +/- 3.4%) compared with controls (21 +/- 2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain-positive Iga-negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.     

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.     

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Adenovirus pulmonary infections identified by PCR and in situ hybridisation in bone marrow transplant recipients.

AIMS--To investigate adenovirus pulmonary infections in bone marrow transplant (BMT) recipients. METHODS--Formalin fixed, paraffin wax embedded lung tissue was examined from 13 necropsy cases after BMT using PCR and in situ hybridisation to detect adenovirus DNA. The E1A region of the adenoviral genome was targeted for PCR. In situ hybridisation was performed only in the PCR positive cases. RESULTS--Of the 13 lung specimens analysed, nine cases were negative for adenoviral nucleic acid. Four (30%) PCR and two (15%) in situ hybridisation positive cases were found. In some of the patients there were clinical and pathological indications that some diseases might be associated with adenovirus infection--haemorrhagic cystitis (three cases); necrotising pneumonia (one case). In necrotising pneumonia in which no pathogenic agents had been shown by conventional histological study, the in situ hybridisation technique showed positive staining for adenovirus. In a patient who died of renal failure caused by adenovirus nephritis, both PCR and in situ hybridisation were positive in the lung as well as in the kidney, although no histological change was found. Two PCR positive cases lacked positive sites for adenovirus by in situ hybridisation. CONCLUSIONS--The combination of PCR and in situ hybridisation could be useful for diagnosing adenovirus infection of the lung in BMT recipients. These results provide a basis for exploring further the clinical use of PCR and in situ hybridisation to diagnose adenovirus infection.     

Immunoglobulin light chain mRNA detected by in situ hybridisation in diagnostic fine needle aspiration cytology specimens.

AIMS: To demonstrate expression of immunoglobulin light chain mRNA in diagnostic fine needle aspiration (FNA) cytology specimens using an in situ hybridisation (ISH) technique; and to evaluate ISH in a series of reactive lymphoid proliferations and malignant lymphomas. METHODS: Forty diagnostic FNA specimens showing a lymphoid cell population were examined for immunoglobulin light chain mRNA expression using ISH. Aspirates were obtained from lymph node (n = 34), salivary gland (n = 3), subcutaneous tissue, thyroid and breast (n = 1 each). The cases included 20 B cell lymphomas, five cases of Hodgkin's disease and 15 reactive lymphoid proliferations. Comparison with light chain immunoreactivity was made in 36 cases and histological correlation from biopsy material was available in 24. RESULTS: Immunoglobulin light chain restriction was demonstrated in 14 of 20 B cell lymphomas using ISH and in six of 17 B cell lymphomas using immunocytochemistry. A polytypic pattern of light chain expression was observed in four of five cases of Hodgkin's disease with both techniques, and in 12 of 15 and 11 of 14 reactive lymphoid proliferations using ISH and immunocytochemistry, respectively. CONCLUSIONS: The assessment of immunoglobulin light chain expression is a useful adjunct to morphology in the diagnosis of reactive and malignant lymphoid proliferations in FNA specimens. Light chain restriction can be shown using either immunocytochemistry or ISH, but the latter is more sensitive in the diagnosis of B cell lymphoma.     

In situ hybridisation and in situ polymerase chain reaction detection of parvovirus B19 DNA within cells

Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.     

Imbalances of chromosome 17 in medulloblastomas determined by comparative genomic hybridisation and fluorescence in situ hybridisation.

AIMS: To investigate the status of chromosome 17 in a series of medulloblastomas using comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). METHODS: Frozen tissue and formalin fixed, paraffin was embedded tissue from 27 medulloblastomas were analysed by CGH and FISH, respectively. CGH ratio profiles for chromosome 17 were compared with the results of FISH, for which loss or gain of 17p or 17q was assessed in two distinct ways using a combination of differentially labelled subtelomeric and centromeric probes and analysing 200 nuclei in each tumour. RESULTS: CGH revealed imbalances consistent with isochromosome 17q in eight of 27 tumours. Either loss of 17p or gain of 17q was identified in a further nine tumours, whereas 10 tumours were apparently balanced. Using control results from preparations of paraffin wax embedded tonsils, thresholds for the detection of abnormalities by FISH were established, either by determining the dominant pattern of signals in each case, or the mean ratio of subtelomeric to centromeric signals. Results by CGH and FISH were concordant in 21 of 27 tumours. In the remainder, most discrepancies related to methodological differences. CONCLUSIONS: CGH has a role in disclosing common, genome wide chromosomal gains or losses in tumours, the clinical relevance of which can then be studied in large archival series of paraffin wax embedded tumours using FISH.     

Detection of parvovirus B19 DNA in bone marrow cells by chemiluminescence in situ hybridization.

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.     

骨髓间充质干细胞在IgA肾病大鼠肾脏中的定位与分布

背景：由于骨髓间充质干细胞缺乏特异性表面抗原,常根据其贴壁生长、成骨成脂分化等生物学特点来鉴定。 目的：观察骨髓间充质干细胞在IgA肾病大鼠肾脏中的定位与分布情况。 方法：以牛血清白蛋白+葡萄球菌肠毒素B+皮下注射四氯化碳的改良法建立SD大鼠IgA肾病模型。造模成功分别经尾静脉移植大鼠骨髓间充质干细胞或生理盐水,并设立不造模的正常对照组。移植后1,4周观察各组大鼠的尿蛋白、肾功能、肾脏病理变化、IgA荧光沉积变化,并通过免疫组织化学观察BrdU标记的骨髓间充质干细胞在肾组织中的分布情况。 结果与结论：移植后1周,骨髓间充质干细胞组尿蛋白、血肌酐明显低于生理盐水组;移植后4周,骨髓间充质干细胞组肾脏病理变化、IgA荧光沉积与生理盐水组比较差异有显著性意义。提示骨髓间充质干细胞输注可促进大鼠IgA肾病的修复,并可定位于肾小球系膜区及少数肾小管间质区,但随时间延长,BrdU标记的髓间充质干细胞在肾组织中分布却逐渐减少。     

Distribution of IgA1 and IgA2 subclasses in normal bone marrow trephines and in trephines infiltrated by IgA producing multiple myeloma.

A series of 20 bone marrow trephines biopsy and necropsy specimens were strained for IgA1 and IgA2 activity, together with total IgA, by means of an indirect immunoperoxidase technique using murine monoclonal antibodies applied to paraffin sections. The specimens showed normal histology and had been taken from patients not known to be suffering from haematological or related systemic disease. The IgA1, IgA2, and total IgA containing cells were counted and expressed as a percentage of all nucleate cells in the marrow cavities. Remarkably constant percentages of and ratios between these cell types were found. The same was true for a further 10 trephines taken from patients undergoing staging procedures for epithelial malignancies, where the marrow histology was normal. The pooled mean percentage of IgA1 containing cells from both groups was 1.18% of all cells, that for IgA2 containing cells 0.18%, and for total IgA positive cells 1.41%. In addition, 12 trephines containing known IgA producing myeloma were examined. Of these, 11 contained IgA1, the remainder contained IgA2 subclass.     

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

AIMS--To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. METHODS--A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. RESULTS--All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. CONCLUSION--When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.     

Short-term culturing influences the number of bcr/abl-fused cells detected by fluorescence in situ hybridisation in bone marrow aspirates of CML patients.

Fluorescence in situ hybridisation (FISH) has been proven as a helpful tool in diagnosis and monitoring of bcr/abl fusion in chronic myelogeneous leukaemia (CML). Since long-term cultures are known to decrease the number of bcr/abl-fused cells, it is questionable whether similar effects are detectable in short-term cultures, a technique often preceding FISH analysis. Therefore, we evaluated bone marrow aspirates of 10 CML patients at biopsy and after culturing for between 24 and 144 h by FISH. The percentage of bcr/abl-fused cells in FISH varied between 15 and 70% at biopsy. In samples with 15 and 30% of aberrant cells at biopsy, an increase of about 20% per day was seen within the first 48 h. In longer lasting cultures, the percentage of leukaemic cells then asymptotically approached a value of 60-70%. In patients with 38 and 50% of bcr/abl-fused cells at biopsy, an increase of about 20% could be detected in the first 24 h. Then 65-70% of the cells already bore the bcr/abl fusion, and the percentage of leukaemic cells was almost constant for longer lasting cultures up to 144 h. In patients with a percentage of about 70% before culturing, no increase in positive cells was detected. These results emphasize the impact of short-term culturing on the number of bcr/abl-fused cells. In particular, the importance for monitoring CML patients is obvious. Therefore the effect described should be taken into account in order to avoid misinterpretation and incorrect therapy decisions in CML patients.     

Absence of human papilloma virus in cervical adenocarcinoma determined by in situ hybridisation.

A few studies using DNA technology have suggested that human papillomavirus (HPV) may be an aetiological factor for adenocarcinoma of the uterine cervix. Twenty one cases of cervical adenocarcinoma were studied by in situ hybridisation using biotinylated DNA probes for HPV types 6, 11, 16 and 18 and a streptavidin, biotinylated alkaline phosphatase detection system. Intranuclear HPV DNA was detected in none of the adenocarcinomas, while positive controls gave a clear intranuclear signal. Adjacent areas of normal, koilocytic, and dysplastic squamous epithelium also gave positive results. It may be that squamous epithelium contaminates adenocarcinomas reported as positive by Southern blotting. Our results showing absence of detectable HPV DNA within adenocarcinomas suggest that HPV infection may not have a major role in the aetiology of adenocarcinoma of the uterine cervix.     

Detection of parvovirus B19 DNA in fetal tissues by in situ hybridisation and polymerase chain reaction.

Attempts were made to detect human parvovirus B19-DNA by in situ hybridisation and the polymerase chain reaction in placental and fetal tissues from a case of intrauterine fetal death. In the in situ hybridisation experiments radioactive and non-radioactive (labelled with 2-acetyl-aminofluorene, AAF) DNA probes were used. B19-DNA was detectable in paraffin wax embedded fetal tissue from the liver, heart, lung, brain and thymus. The resolution with the AAF-labelled probes was higher than with the radiolabelled DNA. Parvovirus B19 DNA sequences were also detected in these tissues by an enzymatic in vitro amplification technique--the polymerase chain reaction. Amplification of a B19-DNA sequence before detection increases the rapidity and sensitivity of detection. The rapid, specific, and sensitive analysis of parvovirus B19 in normal and diseased tissues using these techniques may contribute considerably to determining the role of this virus as a risk factor in the outcome of pregnancy.     

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction (PCR) in the diagnosis of B cell lymphoma. METHODS: Analyses were applied to formalin fixed, paraffin wax embedded, routine diagnostic specimens from cases with a provisional diagnosis of reactive lymph node (n = 23), B cell lymphoma (n = 21), and T cell lymphoma (n = 4). Nonisotopic ISH for kappa and lambda immunoglobulin light chain mRNA was performed using both fluorescein and digoxigenin labelled oligodeoxynucleotide probe cocktails. PCR was carried out on DNA extracted from sections using primers to framework 3 (Fr3) of the V segments and to conserved sequences from the J regions of the immunoglobulin heavy chain genes. RESULTS: All reactive lymph nodes showed a polyclonal pattern of light chain mRNA by ISH, although one showed an excess of kappa positive cells. Nineteen of 21 (90%) cases of B cell lymphoma showed light chain restriction, and a further case showed a vast excess of kappa positive cells. By PCR, 20 of 23 reactive nodes (87%) showed a polyclonal pattern. In 13 of 21 B cell lymphomas (62%) a clonal band was detected. CONCLUSION: In the diagnosis of B cell lymphoma in routinely processed diagnostic material ISH for light chain mRNA was more sensitive (90%) than PCR for heavy chain gene rearrangement using Fr3 and J region primers (62%).     

The bone marrow as production site of the IgA deposited in the kidneys of patients with IgA nephropathy

Patients with primary IgA nephropathy have increased plasma levels of polymeric IgA1 and deposits of IgA1 in their kidneys. The origin of this material is unknown. The production of IgA and its subclasses was investigated in the bone marrow of 14 patients and 19 controls using two colour immunofluorescence and tissue culture. Patients had an increase in the percentage of plasma cells containing IgA (45.8 +/- 7.2 mean +/- s.d.) compared to controls (40.1 +/- 10.5) (P = 0.08). IgA plasma cells containing subclass IgA1 were significantly (P less than 0.01) increased in patients (89.9 +/- 2.7%) compared to controls (84.1 +/- 6.7%). Correspondingly IgA plasma cells containing subclass IgA2 were significantly decreased (P less than 0.01) in patients (7.4 +/- 3.0%) compared to controls (13.5 +/- 5.9%). Production of IgA in bone marrow culture in patients was increased (1,684 +/- 1,151 ng/culture) compared to controls (1,087 +/- 937), but this difference was not significant (P = 0.2). However, in patients the IgA1 subclass contributed significantly (P less than 0.01) more to the IgA synthesis in culture (ratio of IgA1 over IgA: 0.96 +/- 0.02) than in controls (ratio 0.90 +/- 0.06). These findings suggest that the bone marrow may well be the site of long-term overproduction of the IgA1 found in the circulation and mesangial deposits in IgA nephropathy.     

A role for mature B cells in bone marrow transplantation.

The proliferative potential of membrane Ig (mIg)-bearing B lymphocytes was assessed in an adoptive transfer system based on the use of non-inbred rabbits matched for major histocompatibility (MHC) antigens and mismatched for immunoglobulin (Ig) allotypes. Cell suspensions made from spleens (SP), mesenteric lymph nodes (LN), or bone marrow (BM) of allotype b4b5 rabbits were deprived of B cells with mIg of the b4 type by adherence to plastic dishes coated with affinity-purified anti-b4. When such b4-depleted cell populations were injected into newborn hosts of allotype b6b6, stable and lasting chimerism promptly resulted, in which donor-derived products were almost entirely of the b5 allotype. Chimeras formed by transfer of unfractionated cells from b4b5 donors, on the other hand, exhibited a predominance of the b4 allotype, as seen in the living donors. BM but not SP or LN contained precursors capable of differentiating into mIg+ lymphocytes in culture, but no evidence was obtained for engraftment and differentiation by such B-cell precursors or more primitive stem cells in vivo. These studies suggest a potentially significant role for mature B cells in reconstituting the immune system of human transplant recipients.