scholarly journals Immunocytochemical Phenotyping of Disseminated Tumor Cells in Bone Marrow by uPA Receptor and CK18: Investigation of Sensitivity and Specificity of an Immunogold/Alkaline Phosphatase Double Staining Protocol

1997 ◽  
Vol 45 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Heike Allgayer ◽  
Markus Maria Heiss ◽  
Rainer Riesenberg ◽  
Rudolf Babic ◽  
Karl Walter Jauch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Staining Method ◽  
Bone Marrow Cells ◽  
Double Staining ◽  
Staining Protocol ◽  
Double Staining Method ◽  
Marrow Cells

Phenotyping of cytokeratin (CK) 18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15–80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (106 bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1–280 in 106; double staining 808, range 0–253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant ( p<0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.

scholarly journals Effects of hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells

Cytometry ◽  
1982 ◽  
Vol 3 (1) ◽  
pp. 42-47 ◽  
Author(s):  
Jerrold Fried ◽  
Jeffrey Doblin ◽  
Shigeru Takamoto ◽  
Amaury Perez ◽  
Herbert Hansen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Normal Bone Marrow ◽  
Tumor Cell Lines ◽  
Hoechst 33342 ◽  
Normal Bone ◽  
Survival And Growth ◽  
Marrow Cells

scholarly journals Elimination of myeloma cells from bone marrow by using monoclonal antibodies and magnetic immunobeads

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1248-1254 ◽  
Author(s):  
C Shimazaki ◽  
D Wisniewski ◽  
DA Scheinberg ◽  
J Atzpodien ◽  
A Strife ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Magnetic Beads ◽  
Ex Vivo ◽  
Autologous Bone Marrow Transplantation ◽  
Optimal Conditions ◽  
Myeloma Cells ◽  
Marrow Cells

The efficacy of immunomagnetic beads to purge human myeloma cells from bone marrow ex vivo was evaluated. The optimal conditions for purging were studied first by using three myeloma cell lines: RPMI-8226, SKO- 007, and SKMM-2. Myeloma cells labeled with the vital fluorescent dye Hoechst 33342 were admixed with normal bone marrow cells, and two monoclonal antibodies reactive with the myeloma cells (PCA-1 and BL-3) were added alone or in combination with the cells. Magnetic beads coated with goat antimouse immunoglobulin G were then added, and the tumor cells to which beads were attached were separated from the mixture with a magnet. The efficacy of tumor cell removal was dependent on the bead-to-tumor ratio; a ratio of more than 500 was optimal in the presence of excess normal marrow cells. The combination of monoclonal antibodies PCA-1 and BL-3 increased the tumor cell removal as compared with either antibody alone. Two cycles of treatment were more effective than one cycle was. Under optimal conditions, 2.3 to 4 logs of tumor cells could be removed from the mixture containing 10% myeloma cells without a significant loss of normal hematopoietic progenitors as measured by CFU-GM, CFU-GEM, and BFU-E. When the efficacy of this procedure was tested on fresh bone marrow from patients with multiple myeloma (MM) by using the combination of PCA-1, BL-3, and J-5, 1.6 to 2.5 logs of tumor cells could be removed by one cycle of treatment, even from marrows containing less than 10% myeloma cells. These observations support the use of monoclonal antibody combinations and immunobeads as a reliable and nontoxic method to eliminate contaminating myeloma cells ex vivo in preparation for autologous bone marrow transplantation in patients with MM.

Superoxide- and NO-Dependent Mechanisms of the Reprogramming of Bone Marrow Cells by Tumor Cells

2014 ◽  
Vol 45 (11) ◽  
pp. 1261-1273 ◽  
Author(s):  
A. P. Burlaka ◽  
I. I. Ganusevich ◽  
S. N. Lukin ◽  
M. R. Gafurov ◽  
E. P. Sidorik
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Marrow Cells

scholarly journals PHYTOHORMONE INDUCED CHANGES ON BONE HISTOLOGY IN NORMAL AND DIABETIC MALE RAT

2017 ◽  
Vol 9 (2) ◽  
pp. 287 ◽  
Author(s):  
Velan Athithan ◽  
Kotteazeth Srikumar
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Bone Marrow Cells ◽  
Bone Histology ◽  
Male Rat ◽  
Diabetic Rat ◽  
Treatment Schedule ◽  
Marrow Cells

<p><strong>Objective: </strong>To study the effect of brasslinolide keto isoform 28-homocastasterone in diabetic male rat bone marrow cells, bone histological changes, electrolytes and alkaline phosphatase activity in rat blood.</p><p><strong>Methods: </strong>Diabetes was induced in the group (n=6) of rats with a single peritoneal injection of streptozotocin (60 mg/kg bwt). With a treatment schedule of 15 consecutive days, control (n=6) and diabetic rats received 666µg/kg bwt of 28-homocastasterone. Circulating blood cell count, cell indices, bone marrow cells, bone histology, electrolytes Na<sup>+</sup>, K<sup>+</sup>, Cl<sup>-</sup>, P, Ca<sup>2+</sup>and alkaline phosphatase activity was assessed.</p><p><strong>Results: </strong>Cytological examination showed an increased erythrocyte progenitor and megakaryocyte cell lineage along with improved osteoblastic and bone histomorphology in the treated group was noted. A significant reduction in electrolytes level (p&lt;0.05) and increased in alkaline phosphatase activity (p&lt;0.05) was noted in treated groups.</p><strong>Conclusion: </strong>It is suggested that brassinosteroid keto isoform 28-homocastasterone exhibits a hematopoietic effect in diabetic rat and improves bone histology while reducing hyperglycemic damage in bone.<p> </p>

Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad-spectrum antifolate resistance in murine bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2356-2363 ◽  
Author(s):  
Divyen H. Patel ◽  
James A. Allay ◽  
Judith A. Belt ◽  
Brian P. Sorrentino
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Combined Treatment ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Therapy Strategy ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of thees nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive eitransporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.

scholarly journals Histochemical Double Staining Method for Simultaneous Demonstration of Acid and Alkaline Phosphatases on Bone Marrow Smears

1968 ◽  
Vol 95 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Atsuo Suzuki ◽  
Akira Shibata ◽  
Seiju Onodera ◽  
Akira B. Miura ◽  
Shinobu Sakamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Staining Method ◽  
Double Staining ◽  
Alkaline Phosphatases ◽  
Acid And Alkaline Phosphatases ◽  
Double Staining Method

scholarly journals Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1925-1935
Author(s):  
MA King ◽  
DS Nelson
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cell ◽  
Tumor Cells ◽  
B Lymphocytes ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Pokeweed Mitogen

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad-spectrum antifolate resistance in murine bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2356-2363 ◽  
Author(s):  
Divyen H. Patel ◽  
James A. Allay ◽  
Judith A. Belt ◽  
Brian P. Sorrentino
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Combined Treatment ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Therapy Strategy ◽  
Marrow Cells ◽  
Myeloid Progenitors

Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of thees nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive eitransporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.

scholarly journals Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1925-1935 ◽  
Author(s):  
MA King ◽  
DS Nelson
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cell ◽  
Tumor Cells ◽  
B Lymphocytes ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Pokeweed Mitogen

Abstract Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic- idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

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