Gene correction in hematopoietic progenitor cells by homologous recombination.

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. Corrected colonies were identified by their survival in selective medium after electroporation of correcting DNA into unfractionated mouse bone marrow cells and were confirmed by reverse transcription-PCR and sequencing. The observed frequency (4.4 +/- 3.3 x 10(-5) per treated clonogenic cell) is the same as in embryonic stem cells (2.3 +/- 0.4 x 10(-5)) with the same DNA and mutation. These data suggest that gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.

Quantitation of vital bleaching by computer analysis of photographic images.

BACKGROUND: The authors investigated the use of computer processing of photographic images to monitor changes in tooth brightness after nightguard vital bleaching, or NGVB. METHODS: Photographs of shade guides and clinical cases (patients' teeth) were taken on 35-millimeter film with electronic flash illumination and processed commercially. A slide scanner was used to digitize images as red, green and blue, or RGB, files, with constant brightness, contrast and linearity settings; the images were then analyzed with commercial software. Relevant image components (that is, teeth or shade guide tabs) were separated, and histograms of various numerical color descriptors were generated for each image component. RESULTS: Analysis of shade tab images showed that the mean pixel intensity for the RGB blue channel, or MPIb, was the most satisfactory brightness descriptor, with clear sequential MPIb increments from lighter to darker shades in each series of colors (A through D) and close correlation with the manufacturer's brightness scale (r = .83). Mathematical analysis of MPIb data for shade tabs in the same image yielded a brightness index that was reproducible and correlated well with the manufacturer's brightness scale. Sequential measurements of this index in three subjects whose teeth were bleached with carbamide peroxide for 14 days correlated well with assessments made by visual shade guide comparisons. CONCLUSIONS: The authors conclude that computer analysis of digitized photographic images with internal color controls provides an index of tooth brightness that is reproducible from image to image. CLINICAL IMPLICATIONS: A brightness index derived from computer analysis of digitized photographic images may be useful for monitoring the effectiveness of NGVB.

MUC1 expression in hematopoietic tissues.

The MUC1 gene encodes the core protein of episialin, which is recognized by several antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) detection of MUC1 transcripts has been proposed for the detection of micrometastases from breast cancers. MUC1 expression in hematopoietic tissues has been reported but not confirmed. Our preliminary RT-PCR studies confirmed MUC1 expression by MDA-231 breast cancer cells. Western blots of MDA-231 proteins stained with anti-MUC1 core gave one 68-kd (core protein) band, with an additional high molecular weight (HMW) band in blots stained with anti-epithelial membrane antigen (EMA). MUC1 expression was detectable by RT-PCR in 4 samples each of peripheral blood, bone marrow, and lymph node. MUC1 expression was detectable by Western blot analysis using anti-MUC1 core and anti-EMA in 2 peripheral blood samples and all bone marrow samples. Western blots from all lymph node samples stained positively with anti-EMA for the HMW product, but the 68-kd product was less prominent. Separated peripheral blood lymphocytes and granulocytes showed similar levels of MUC1 expression. RT-PCR studies demonstrated MUC1 expression in various hematopoietic cell lines. Western blots showed the 68-kd and HMW products in a granulopoietic line, with only the 68-kd product in 3 lymphoblastoid lines. MUC1 is expressed ubiquitously in hematopoietic tissues and is unsuitable for use as a marker for epithelial micrometastases.

Mice deficient in fas ligand (gld) or fas (lpr) show few alterations in granulopoiesis.

Evidence exists that the life span of mature, circulating neutrophils is influenced by apoptosis induced by interactions between Fas ligand (FasL) and Fas (CD95). However, the role of FasL/Fas-mediated apoptosis in granulopoiesis has not been explored. The present study assessed differences in granulopoiesis between normal (B6) mice and mice carrying mutations in the genes for FasL (B6/gld) or Fas (B6/lpr). Granulopoiesis was examined by quantitating mature granulocytes in the blood, committed myeloid progenitor cells (or colony-forming units; CFU) in the bone marrow (BM), and granulocyte lineage cells in the BM. The present study also characterized through flow cytometry the ability of GR-1(+) granulocyte lineage cells from B6, B6/gld, and B6/lpr mice to undergo spontaneous apoptosis in vitro. In comparison to B6 mice, B6/gld mice (but not B6/lpr mice) showed small, but significant increases in the number and percentage of blood granulocytes and in the number of myeloid CFU. However, the number and percentage of GR-1(+) granulocyte lineage cells in the BM were similar in the three strains. The rate of spontaneous apoptosis of GR-1(+) granulocyte lineage cells also did not differ between B6, B6/gld, and B6/lpr mice. In B6 and B6/gld mice, Fas expression on granulocyte lineage cells was downregulated in conjunction with a decrease in forward-angle light scatter (fsc) and externalization of phosphatidylserine (PS), two measures of apoptosis. These results suggest that FasL-Fas interactions play only a minor role in modulating numbers of committed myeloid progenitor cells and the size of the peripheral pool of mature granulocytes. Interactions between FasL and Fas do not influence the size of the BM pool of granulocyte lineage cells or the ability of those cells to undergo spontaneous apoptosis.

Enumeration of CD34-positive stem cells: evaluation and comparison of three methods.

Accurate enumeration of CD34+ stem cells is important in assessing the need for continued mobilization and subsequent apheresis collections. We compared two new analysis systems, ProCOUNT (Becton Dickinson Immunocytometry Systems) and IMAGN 2000 STELLer (Biometric Imaging, Inc.) with our current (3-Color) flow cytometry-based method. The ProCOUNT system uses an absolute counting tube, which contains reference beads and a specific (multiple) gating strategy to determine an absolute count. The STELLer assay combines microvolume fluorimetry and automated analysis software to determine an absolute count. To evaluate linearity and reproducibility, peripheral blood was spiked with CD34+ cells (KG1a cell line). Three dilution series (measured at approximately equal to 0, 5, 10, 25, 50, and 100 CD34+ cells/microliter) were analyzed by each method. Analysis of predicted versus actual CD34+ concentration showed excellent correlation with all methods (r2 > or = 0.97, slope 0.98-1.04). To further assess precision, two PBSC samples, at approximately 200 and 800 CD34+ cells/microliter, respectively, were analyzed 10 times by each method. Coefficients of variation for the precision analysis of these samples were 5.1%-6.4% and 5.4%-12.3%, respectively. To assess overall performance, 75 patient specimens were analyzed. Excellent correlation (r2 values of 0.89-0.98) was observed among all three methods. We conclude that the three methods provide comparable linearity and reproducibility.

A method for the production of CD4+ chronic myelogenous leukemia-specific allogeneic T lymphocytes.

The graft-versus-leukemia effect is critical to the maintenance of remission in patients transplanted for the treatment of chronic myelogenous leukemia (CML). A pivotal issue in transplantation for CML is whether donor lymphocytes are specific for host tumor or myeloid cells or a subset of the lymphocytes that cause graft-versus-host disease. We have enrolled seven patients in an experimental trial to evaluate the specificity of HLA-matched donor lymphocytes in vitro. We have produced 11 CD4+ cytotoxic and proliferative T-cell clones from five of the donors that only lyse or proliferate to leukemic myeloid cells. These T lymphocytes do not react with interleukin (IL)-2-stimulated blasts, natural killer-sensitive targets, donor neutrophils, or bcr-abl+ EBV-lymphoblastoid cell lines. We show that the addition of the cytokines IL-7 and IL-12 during the production of T-cell clones enhances the recovery of myeloid-specific clones in vitro. Five of the myeloid-specific clones that we produced maintained specificity over 12 weeks in culture. Adoption of this method should allow for the expansion and in vivo testing of CML-specific CD4+ T-cell clones in adoptive immunotherapy.

Long-term engraftment failure after marrow ablation and autologous hematopoietic reconstitution: differences between peripheral blood stem cell and bone marrow recipients.

We infused peripheral blood stem cells (PBSC) into 51 patients with various malignant disorders, after myeloablative conditioning. Twenty-four patients also received autologous bone marrow (PBSC + BM). In a multivariate analysis, the only statistically significant predictors of neutrophil engraftment were log-dose CFU-GM (P < 0.001) and the number of prior chemotherapy regimens (P = 0.004). The factors predicting RBC and platelet engraftment were log-dose CFU-GM (P = 0.002), PBSC + BM infusion (P = 0.007) and the absence of neoplastic bone marrow involvement (P = 0.009). Seven patients remained platelet and/or red cell transfusion-dependent for 100 days or more post-transplant after good neutrophil recovery. Six of these seven long-term engraftment failures, as well as five additional patients, received < 10(5) CFU-GM/kg. Of the 11 patients who received < 10(5) CFU-GM/kg (low-dose patients), seven were PBSC recipients, of whom six were long-term engraftment failures. In contrast, there were no long-term engraftment failures among the four low-dose autologous marrow recipients. This difference in long-term engraftment failure rate was significant (P = 0.015). The low-dose PBSC patients all had a diagnosis of lymphoma with bone marrow involvement. The low-dose PBSC + BM group was more heterogeneous, but no patient had malignant involvement of the marrow. The low-dose PBSC patients had also received significantly more prior chemotherapy regimens than the low-dose PBSC + BM patients and a significantly higher proportion received total body irradiation (TBI) as part of their conditioning regimen. We conclude that marrow damage resulting from a combination of neoplastic infiltration, chemotherapy and TBI may result not only in low PBSC yields but also in an impaired capacity of the marrow microenvironment to support transplanted stem cells.

North American Multicenter Study on flow cytometric enumeration of CD34+ hematopoietic stem cells.

Transplant centers often rely on CD34+ cell quantitation by flow cytometry to ensure adequacy of hematopoietic progenitor cell collection. Because of variation in interpretation, a lack of interlaboratory proficiency studies, and no generally accepted methodology, comparison of CD34 data from site to site is difficult. Twenty-one samples from marrow and peripheral blood stem cell collections were shipped to 10 participating North American laboratories for analysis. Duplicate samples were included to assess reproducibility. Participants were surveyed for methodology. Three centers had previously attempted to standardize their methodology among themselves. The variability observed in the CD34 values ranged from a max/min. reported value per sample of 2.9 to 749 (median 76). Exclusion of two outlying sites reduced the variability of results to 1.2 to 27 (median 3.1). Variation among the three standardized sites ranged from 1.2 to 4.4 (median 1.6). Overall reproducibility (excluding the outlying sites B and G) ranged from a minimum of 0-16.5 (percent mean difference) for site C to a maximum of 4.1-133 for site H. Strategies for gating were found to largely influence results. We observed an alarming variation among the CD34 cell counts reported from different laboratories. Standardization substantially reduced observed variation. The need for standardized methodology, reporting, quality control, and proficiency testing is underscored by these findings.

Correction of bone marrow nucleated cell counts for the presence of fat particles.

Although the use of bone marrow transplantation has increased greatly in recent years, the quality control procedures used in bone marrow processing laboratories remain less than ideal. Accurate marrow total nucleated cell (TNC) counts are essential for effective monitoring of bone marrow collection and processing. Aspirated marrow is variably contaminated by fat particles, resulting in overestimation of marrow TNC by automated analyzers. A recently-marketed hematological analyzer (Cobas-Helios; Roche Diagnostic Systems, Branchburg, NJ) offers the potential to correct marrow TNC counts for fat particles using available software. The authors investigated the accuracy of corrected TNC counts on 21 marrow samples, using a visual chamber count as the reference method. The correction methods studied were software correction, using the Cobas-Helios differential system, and replacement of the sample plasma with saline. Uncorrected automated marrow TNC counts (mean, 28.4 x 10(9)/L) were significantly higher than the visual reference counts (mean, 23.1 x 10(9)/L). Neither the mean corrected automated count (24.3 x 10(9)/L) nor the mean saline replaced count (24.6 x 10(9)/L) differed significantly from the mean visual reference count. For both the corrected automated and saline replaced counts, 20 of the 21 data points (95%) fell within a 95% confidence interval computed for the reference method. The authors conclude that both the corrected automated method, using the Cobas-Helios, and the saline replacement method are acceptable alternatives to the visual chamber count.

Flow-cytochemical differential leukocyte analysis with quantitation of neutrophil left shift. An evaluation of the Cobas-Helios analyzer.

The Cobas-Helios (Roche Diagnostic Systems, Inc., Branchburg, NJ) is a new, fully automated hematology analyzer that performs a complete blood count and differential leukocyte count (DLC), classifying leukocytes by flow-cytochemical technology. The DLC component of the Cobas-Helios was evaluated according to the National Committee for Clinical Laboratory Standards H20-A protocol. Instrument performance was acceptable with respect to all parameters investigated, including imprecision, inaccuracy and clinical sensitivity for the identification of quantitative and qualitative leukocyte abnormalities. In a minority of samples with neutrophil left shift, neutrophils tended to overlap the monocyte domain, resulting in overestimation of monocytes and underestimation of neutrophils. This problem did not affect clinical sensitivity and was generally associated with a positive instrumental left-shift flag. Flags for the identification of specific qualitative abnormalities of the leukocyte population (atypical lymphoid cells, nucleated red cells, blast cells, immature granulocytes and neutrophil left shift) performed well. In addition to a conventional five-part DLC, the Cobas-Helios also identifies and quantitates atypical lymphoid cells and "large immature cells," the latter corresponding to bands and immature granulocytes. Counts of atypical lymphoid cells and large immature cells correlated well with the equivalent cell classes as enumerated by the reference method of the National Committee for Clinical Laboratory Standards. The Cobas-Helios offers the most reliable quantitative index of neutrophil left shift currently available in a commercial automated DLC analyzer.

Two color flow cytometric analysis of concomitant acute myeloid leukemia and chronic lymphocytic leukemia.

During evaluation for macrocytic anemia and thrombocytopenia, a 74-year-old female was found to have a leukocytosis with two apparent populations of malignant cells identified in her peripheral blood smear and bone marrow aspirate. Morphologic characteristics of the two cell types were unusual, and cytochemical assays yielded ambiguous results. Two-color flow cytometric analysis demonstrated two distinct cell populations with immunophenotyping patterns consistent with chronic lymphocytic leukemia (CD5+/CD20+) and acute myelocytic leukemia (CD33+/CD34+), detected concurrently. The concomitant appearance of CLL and AML has been reported only rarely. The use of two-color flow cytometry to differentiate the populations demonstrates the utility of this technology in resolving unusual hematological malignancies.

A parallel evaluation of four automated hematology analyzers.

A parallel evaluation was performed on four automated hematology analyzers: the Celldyn 3000 (Unipath Corp., Mountain View, CA), the Coulter STKS (Coulter Electronics Inc., Hialeah, FL), the Sysmex NE-8000 (Baxter Healthcare Corp., McGaw Park, IL), and the Technicon H*2 (Miles Corp., Tarrytown, NY). The protocol included evaluation of the complete blood count and differential leukocyte count (DLC) parameters. The DLC evaluation was performed using the National Committee for Clinical Laboratory Studies H20-A protocol. Based on this evaluation, the authors could not identify a single instrument that was clearly superior to the others. Overall, the four instruments were found to be safe and effective for diagnostic use; however, there were areas in which their performance was less than optimal. Particular questions were raised regarding the clinical usefulness of instrumental "flags" to identify qualitative leukocyte abnormalities. The results are discussed in relation to the selection of instruments for specific clinical applications.

Bone marrow histiocytic proliferation in association with colony-stimulating factor therapy.

A 6-year-old white male had a myelodysplastic syndrome (refractory anemia with excess blasts in transformation) and was treated with high dose chemotherapy. A combined esterase stain of the marrow blasts showed granulocytic differentiation. Subsequently, persistent pancytopenia with a severely hypocellular bone marrow developed, which was treated with a combination of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Sixteen days after the initiation of therapy, histologic examination of the bone marrow revealed a marked proliferation of histiocytes. To the best of the authors' knowledge, this is the first report of histiocytic proliferation in the bone marrow after colony-stimulating factor therapy.

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies.

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.