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Biomedical subjects

H E Johnsen

Publications and source records attributed to H E Johnsen.

At least 19 recordsLinked to original sources
PubMed2003

Individual quality assessment of autografting by probability evaluation: a model estimated by analysis of graft-related end points in 204 patients with malignancies.

Haematological toxicity is considered a secondary end point important for graft evaluation - in today's practice graft evaluation focuses on the primary impact of health economic end points. This report illustrates the benefit of combining CD34 enumeration and demographic as well as disease-related variables in models for individual quality assessment of autografting following high-dose therapy. A total of 24 centres in Scandinavia enrolled 204 patients younger than 67 years who received high-dose therapy with autologous peripheral blood stem cell transplantation. Using the binary Logistic Regression Analysis, the prognostic value of diagnostic demographic variables, therapy and graft-related factors was entered into a multivariate analysis and the final significant models were used to estimate probabilities for acceptable or unacceptable outcome among different patient scenarios. The model that estimated post-transplant efficacy by selected primary end points (time on antibiotics and use of transfusions) includes six independent variables related to sex, age, disease, conditioning, growth factor administration, and graft CD34+ cell number. The model that estimated transplantation-related toxicity by selected secondary end points (time to blood cell recovery) included four independent variables related to age, disease, growth factor administration and graft CD34+ cell number.

Adolescent↗
PubMed2002

Assessing agreement between CD34 enumeration by flow cytometry and volumetric analysis.

Prior to replacement of an established method for CD34 enumeration by an alternative approach, evaluation of the agreement between the methods is essential. In this study, the comparison of two assays was evaluated according to the recommendation of Bland and Altman describing the agreement between two methods where the true value is not known. CD34 enumeration was performed on blood or leukapheresis product from 105 patients by flow cytometry (dual platform assay) and volumetric analysis (single platform assay). Both the flow cytometric and the volumetric analysis showed poor reproducibility for measures lower than approximately 9 CD34+ cells/mm3. For values higher than 29 CD34+ cells/mm3, evaluation of the agreement demonstrated a difference between the single and dual platform assay, where CD34 enumeration by the volumetric analysis demonstrated values 73-80% of the flow cytometric value. The difference between the two assays could be due to several technical pitfalls which are discussed.

Antigens, CD34↗
PubMed2002

Impact of high-dose chemotherapy on antigen-specific T cell immunity in breast cancer patients. Application of new flow cytometric method.

The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine-induced specific immunity in breast cancer patients. Peripheral blood was collected from five breast cancer patients at serial time points in connection with treatment and in a follow-up period of 1 year. The frequencies of CD8+ and CD4+ T cells responsive to cytomegalovirus (CMV), varicella zoster virus (VZV), and tetanus in antigen-activated whole blood were determined by flow cytometric analysis of CD69, TNF alpha, IFN gamma and IL-4 expression. Mononuclear cells were labelled with PKH26 dye and the CMV, VZV, and tetanus toxoid-specific proliferation of T cell subpopulations was analysed by flow cytometry. In none of the patients did the treatment result in loss of overall T cell reactivity for any of the antigens. Prior to chemotherapy 5/5 patients possessed TNF alpha expressing T cells specific for CMV, 4/5 for VZV, and 3/5 for tetanus. One year after stem cell transplantation all patients possessed TNF alpha expressing T cells specific for CMV, VZV and tetanus. The highest percentages of cytokine-responding T cells were seen after stimulation with CMV antigen. In general, the lowest reactivity (close to zero) was measured in G-CSF-mobilised blood at the time of leukapheresis. In spite of a continuously reduced CD4 to CD8 ratio after transplantation, recovery of CD4+ T cells usually occurred prior to CD8+ recovery and often to a higher level. The study demonstrates that natural as well as vaccine-induced specific immunity established prior to HD can be regained after stem cell transplantation. These data indicate that introduction of a preventive cancer vaccination in combination with intensive chemotherapy may be a realistic treatment option.

Antigens, Bacterial↗
PubMed2002

Detection of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies using end sequenced probes.

Translocations involving the immunoglobulin loci are recurring events of B cell oncogenesis. The majority of translocations involve the immunoglobulin heavy chain (IGH) locus, while a minor part involves the immunoglobulin light chain loci consisting of the kappa light chain (IGK) located at 2p11.2 and the lambda light chain (IGL) located at 22q11.2. We characterised BAC clones, spanning the IGK and IGL loci, for detection of illegitimate rearrangements by fluorescence in situ hybridisation (FISH). Within the IGL region we have identified six end sequenced probes (22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7) covering the variable (IGLV) cluster and two probes (165G5 and 31L9) covering the constant (IGLC) cluster. Within the IGK region four probes (969D7, 316G9, 122B6 and 2575M21) have been identified covering the variable (IGKV) cluster, and one probe (1021F11) covering the IGK constant (IGKC) cluster. A series of 24 cell lines of different origin have been analysed for the presence of translocations involving the immunoglobulin light chain loci by dual-colour FISH where the split of the variable cluster and the constant cluster indicated a translocation. Probes established in this study can be used for universal screening of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies.

DNA Probes↗
PubMed2001

Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes.

Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B-cell oncogenesis. The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH). In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster. Clones of the constant (IGHC) cluster were found in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH. We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV cluster, which, together with probes covering the IGHC cluster (11771 and 998D24), could be used in interphase nuclei and metaphase chromosome analysis. A visual split of the IGHV and IGHC clusters indicating a translocation was analyzed by dual-color FISH in a series of 21 cell lines of different origins. Translocations were found, as expected, in eight of eight myelomas, four of four lymphomas, none of five leukemias, and none of four Epstein-Barr virus-transformed B-lymphoblastoid cell lines. To summarize, we have established a set of IGHV and IGHC probes that can be used for universal screening of illegitimate rearrangement within the IGH locus in B-cell malignancies. These probes allow for routine FISH analysis to detect this early central oncogenic event.

Chromosome Banding↗
PubMed2001

Kinetic studies during peripheral blood stem cell collection show CD34+ cell recruitment intra-apheresis.

A sufficient number of CD34+ cells in the peripheral blood stem cell product is important to achieve a rapid and sustained engraftment. The purpose of the present work was to study CD34+ cell kinetics during leukapheresis. Blood samples before and after leukapheresis were analysed for CD34+ cells in 205 procedures. The number of CD34+ cells after plus the number of CD34+ cells harvested was 1.5-fold greater than the number available at the beginning of the procedure, indicating recruitment of CD34+ cells during leukapheresis. In a subgroup of 66 procedures, granulocytes and platelets were measured. In contrast to CD34+ cells, these cell fractions were not recruited to the blood stream during leukapheresis. An additional nine patients were studied with serial blood measurements during leukapheresis, showing an initial decline that was followed by an increase in CD34+ cells during leukapheresis. In conclusion, CD34+ cells are recruited to the blood during the leukapheresis procedure in contrast to granulocytes and platelets.

Adolescent↗
PubMed2001

A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts.

Recently, the regulatory authorities have begun to show interest in haematopoietic stem cell products. On a professional rather than a regulatory basis, the International Society for Hematotherapy and Graft Engineering (ISHAGE) has established the Foundation for the Accreditation of Haematopoietic Cell Therapy (FACHT), which has drawn up guidelines for standards and accreditation of such activity. In Europe, the regulatory environment with regard to haematopoietic stem cell grafts, processing and storage are currently less stringent. However, in 1998 the European Joint Accreditation Committee Euro-ISHAGE/EBMT (JACIE) prepared a regulatory document 'Standards for Blood and Marrow Progenitor Cell Collection, Processing and Transplantation' which was approved by the EBMT General Assembly. The major objectives were to promote quality of medical and laboratory practice in haematopoietic progenitor cell transplantation. The standards extend and detail the pre-existing activity of EBMT centres including all phases of collection, processing and administration of these cells. This is the platform for the proposed reference protocol for CD34(+) cell enumeration and clinical validation of quality assessment to ensure that appropriate standards of work and product quality are established and will be maintained.

Antigens, CD34↗
PubMed2000

Frequency and kinetics of polyclonal and clonal B cells in the peripheral blood of patients being treated for multiple myeloma.

Recent studies concerning the numbers of circulating clonal B cells in patients with multiple myeloma (MM) have reported conflicting data regarding the exact level and phenotype of clonal B cells and their response to treatment. In this report we document that the peripheral blood tumor burden at presentation was reduced by induction therapy to a low level, regardless of the initial tumor burden. However, the residual clonal compartment persisted before and after transplant. The level of clonal cells showed no correlation with CD19(+) cell levels. In a single patient with MM, high numbers of phenotypically aberrant clonal cells with altered CD19 expression were identified. (Blood. 2000;96:4357-4359)

Antigens, CD19↗
PubMed2000

Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: a population-based study. Nordic Myeloma Study Group.

High-dose therapy has become a common treatment for myeloma. The objectives of this study were to estimate in a prospective, population-based setting the impact on survival of high-dose therapy in newly diagnosed, symptomatic patients less than 60 years old and to compare the results with those of conventionally treated historic controls. The prospective population comprised 348 patients. Of these, 274 were treated according to a specified intensive-therapy protocol (Nordic Myeloma Study Group [NMSG] #5/94) and constituted the intensive-therapy group. The historic population consisted of 313 patients identified from 5 previous population-based Nordic studies. Of these, 274 fulfilled the eligibility criteria for high-dose therapy stated in NMSG #5/94 and constituted the control group. The expected numbers of patients in the prospective population and the historic population were 450 and 410, respectively, estimated from previously established data on the incidence in this population and the population base for each study. Survival was prolonged in the intensive-therapy group compared with the control group (risk ratio for the control group 1.62; 95% confidence interval 1.22-2.15; P =.001). These groups represented more than 60% of the expected number of patients. When survival for all the registered patients in the 2 populations was compared, representing more than 75% of the expected number of patients, the advantage for the prospective population persisted (risk ratio for the historic population 1.46; 95% confidence interval 1.14-1.86; P =. 002). These results indicate that the introduction of high-dose therapy for newly diagnosed myeloma has resulted in prolonged survival for the total patient population aged less than 60 years. (Blood. 2000; 95:7-11)

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

CD68+/CD83+/CD1a- dendritic cell subsets from patients with multiple myeloma are not infected with human herpesvirus 8.

Recently, a subset of dendritic cells with the phenotype CD68+/CD83+/CD1a-, present in patients with multiple myeloma (MM), was reported to be infected with human herpesvirus 8 (HHV-8). Therefore we wished to clarify whether HHV-8 infection might be related to the pathogenesis of MM. In an attempt to identify HHV-8 infected cells in patients with MM, long-term bone marrow cultures from 8 MM patients and dendritic cell cultures from 11 MM patients were established. In addition, fresh bone marrow aspirates from 10 MM and 10 patients with monoclonal gammopathy of undetermined significance (MGUS) were included in the study. All samples were analysed by a sensitive semi-nested PCR assay and were found to be consistently PCR negative. Phenotyping of day 7 dendritic cell cultures demonstrated the presence of a sufficient number of CD68+/CD1a- and CD83+/CD1a- cells. However, to exclude the presence of infrequent HHV-8 infected cells, the CD68+/CD1a- subset from 3 dendritic cell cultures was sorted in numbers of 105 for each PCR test, and again a negative PCR result was observed. This study documents that the CD68+/CD83+/CD1a- dendritic cells in patients with MM are not generally infected with HHV-8 and, as a consequence, it is unlikely that HHV-8 plays a role in the pathogenesis of MM.

Antigens, CD↗
PubMed2000

Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay.

OBJECTIVE: To develop a real-time PCR method, based on the 5'nuclease TaqMan technology, for quantitation of clonal cells in multiple myeloma (MM). MATERIALS AND METHODS: The real-time quantitative PCR method incorporates both an allele-specific oligonucleotides (ASO) primer and an ASO dual-labeled fluorogenic probe (ASO TaqMan probe). The ASO primer and probe corresponded to the complementary determining region 3 (CDR3) of the rearranged immunoglobulin heavy chain gene (IgH). With the use of a sequence detector, PCR product accumulation was measured through the ASO TaqMan probe. The real-time PCR method was compared with flow cytometric quantitation of myeloma plasma cells. RESULTS: The application of the real-time quantitative ASO IgH PCR method is illustrated by a sequential analysis of minimal residual disease (MRD) in bone marrow (BM) samples from myeloma patients undergoing peripheral blood stem cell (PBSC) transplantation. The real-time PCR method was able to quantitate residual malignant cells in BM samples from patients who were considered to be in complete remission. Further, it was illustrated that a potential problem in determining tumor cell content in myeloma BM samples is the heterogeneous infiltration of the marrow. CONCLUSION: The application of the real-time PCR method provides a sensitive, highly specific, and reproducible quantitation of myeloma cells.

Adult↗
PubMed2000

Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

Adolescent↗
PubMed2000

Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery.

In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell factor (SCF), Flt3 ligand (Flt3) and fetal antigen 1 (FA1)) and three lineage-specific growth factors (EPO, G-CSF and thrombopoietin (Tpo)) were analyzed by ELISA in 16 patients with multiple myeloma (MM) and 16 patients with non-Hodgkin's lymphoma (NHL). The relative number of SCF, Flt3, Tpo and G-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need to be confirmed in prospectively designed studies.

Adjuvants, Immunologic↗
PubMed2000

A comparative study of sequential priming and mobilisation of progenitor cells with rhG-CSF alone and high-dose cyclophosphamide plus rhG-CSF.

Stem cell mobilisation can be achieved either by administration of rhG-CSF alone or after high-dose cyclophosphamide (HDCy) plus rhG-CSF. We have compared both mobilisation procedures intra-individually in 43 patients with haematological malignancies. Furthermore, the toxicity data were registered. The CD34+ cell yield was higher after mobilisation with HDCy plus rhG-CSF than after rhG-CSF alone in 21 out of 22 patients who were actually harvested after both procedures. If a patient mobilised insufficiently after rhG-CSF alone, the yield of CD34+ cells after the following HDCy priming was lower compared to patients who mobilised sufficiently after rhG-CSF priming alone. In 12 patients with B cell malignancies a reduced number of B cells such as CD10+, CD19+, CD20+ cells in bone marrow as well as in leukapheresis products was observed after HDCy plus rhG-CSF compared to rhG-CSF alone. Toxicity data revealed HDCy as a relatively toxic priming regimen with all patients hospitalised and 74% experiencing neutropenic fever and administration of intravenous antibiotics. In two patients, seizure-like episodes were observed during cyclophosphamide bolus infusion. In conclusion, HDCy increased the yield of CD34+cell and reduced B cells in leukapheresis products indicating reduced tumour cell load compared with rhG-CSF priming alone. The efficacy of HDCy priming is limited by its profound toxicity and morbidity. Studies evaluating efficacy and safety of lower doses of cyclophosphamide are needed. Bone Marrow Transplantation (2000) 26, 717-722.

Adolescent↗
PubMed2000

The clonal hierachy in multiple myeloma.

In this report we evaluated the number and phenotype of blood circulating B-cell subsets at different stages of differentiation in 26 patients with newly diagnosed multiple myeloma (MM). In all patients, plasma cells and/or plasma blasts could be identified by flow cytometry with a mean frequency of 1.20% and 0.07%, respectively. In 76.9% of the patients these cells showed aberrant expression mainly of CD56, CD28 and CD117, none of these markers were found on the earlier B-lymphocytes. Clonal B-cells preceding the plasma blast stage were identified by patient specific IgH RT-PCR on sorted B-cell subsets. The clonal cells included the less differentiated CD38+ CD19+ and CD38-/CD19+ subsets. illustrating that the clonal cells are part of an ongoing differentiation process. Further, the presence of CD38-/CD19+ cells with somatically mutated Cgamma transcripts identical to the tumor-specific Calpha transcript, shows that the clonal hierarchy in myeloma may include memory B-cells.

ADP-ribosyl Cyclase↗
PubMed2000

Technical aspects and clinical impact of hematopoietic progenitor subset quantification.

As high-dose therapy for malignancies is now being applied to newly diagnosed patients as adjuvant therapy, it has become a requirement that quality and safety assessment of hematopoietic stem cell grafts be evidence-based. This process has developed a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation and safety assessment in stem cell handling naturally places this activity in transfusion medicine. Multiparametric flow cytometry can identify progenitor subsets in normal human bone marrow and peripheral blood, and such subset quantification has been used retrospectively to predict three-lineage engraftment following high-dose therapy for malignancies. Published single center data have suggested an impact on clinical outcome, and a standardized technique for subset enumeration needs to be established before prospective multicenter trials can be initiated to document the prognostic value of such quality assessment in autografting. Based on experiences of CD34 enumeration, which we consider to be the first step in quality assessment of hematopoietic stem cell grafts, this review discusses flow cytometry subset identification by lineage-specific differentiation markers, stromal-dependent adherence molecules, and regulatory growth factor receptors from a technical point of view. The aim of this review is:To recommend a simple method based on the experiences of the Nordic workshop III on subset identification; To present new molecular genetic-based methods for future use in quality assessment; and To propose new endpoints necessary for validation of the likely clinical impact of subsets in prospective trials. As sample differences between blood and marrow result in technical difficulties, this review only focuses on the methodology of identifying subsets in blood and leukapheresis products. Methods for subset analysis in diagnostic bone marrow samples will be covered in a forthcoming review.

Antigens, CD34↗