Blastocystis hominis: neutral red supravital staining and its application to in vitro drug sensitivity testing

2000 ◽  
Vol 86 (7) ◽  
pp. 573-581 ◽  
Author(s):  
Alexandre A. Vdovenko ◽  
John E. Williams





2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18535-e18535
Author(s):  
Jing Chen ◽  
Glorymar Ibanez Sanchez ◽  
Paul Calder ◽  
Mark G. Frattini

e18535 Background: To individualize therapy for relapsed/refractory AML patients, optimal in-vitro culture conditions to support primary leukemic cells are essential for drug sensitivity testing. Our lab has validated a high throughput chemosensitivity assay with primary AML cells maintained by growth factors (cytokines); however, growth factors have not been shown to support long-term assays of primary AML cells. Stromal cells of the tumor microenvironment are crucial to maintain normal hematopoiesis and leukemic cells and have been shown to support long term in-vitro expansion of primary AML cells. However, there is little information characterizing these growth conditions. The aim of this study was to compare long-term proliferation and phenotypes of primary AML cells with growth factors or stromal support to best determine their utility as a platform for drug sensitivity testing in functional assays. Methods: Patient-derived AML cells were cultured in 96-well plates in: 1) cell culture medium only 2) Human HS5 or HS27 stromal cells 3) HS5 or HS27 stroma-conditioned media or 4) cytokine cocktail. Viability readout by Guava ViaCount and leukemic cell surface phenotypes by fluorescently-conjugated antibodies were performed weekly over 3 weeks. Results: Primary AML cells cultured with only cytokines maintained proliferation at 3 weeks. In comparison, AML cells cultured in HS5 stroma-conditioned medium also maintained proliferation at a similar rate at 3 weeks, while co-culture with HS5 stromal cells demonstrated significantly higher proliferation. Leukemic immunophenotypes were maintained for all growth conditions over 3 weeks. Conclusions: Contrary to known data, primary AML cells with cytokines continued to expand at 3 weeks, at a similar rate to HS5 stroma-conditioned medium, a finding that has not been reported. Consistent with previous studies, we confirmed that stromal cells such as HS5 can provide long-term in-vitro expansion of primary AML cells, which cannot be substituted by stroma-conditioned medium. The ability to maintain long-term expansion of primary AML cells by both cytokines and stromal cells sets up a platform for a direct comparison of high throughput drug sensitivity testing under these growth conditions.



1989 ◽  
Vol 41 (3) ◽  
pp. 201-205 ◽  
Author(s):  
S. K. Khoo ◽  
T. Hurst ◽  
M. J. Webb ◽  
G. Dickie ◽  
J. Kearsley ◽  
...  


Author(s):  
P. Staib ◽  
T. SchinköThe ◽  
S. Wiedenmann ◽  
T. Dimski ◽  
C. Schoch ◽  
...  


1989 ◽  
Vol 41 (3) ◽  
pp. 318-330 ◽  
Author(s):  
Joan E. Jackson ◽  
John D. Tally ◽  
Douglas B. Tang




Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2959-2965 ◽  
Author(s):  
Teruaki Hongo ◽  
Shuhei Yajima ◽  
Minoru Sakurai ◽  
Yasuo Horikoshi ◽  
Ryoji Hanada

Abstract It is vital to develop effective therapy for children with acute lymphoblastic leukemia (ALL), in whom no remission occurs or who suffer relapse with current protocols. Cellular drug resistance is thought to be an important cause of induction failure and relapse. We performed in vitro tests of bone marrow samples in 196 children with newly diagnosed ALL with a 4-day culture and a methyl-thiazol-tetrazolium assay. We tested 16 drugs and calculated the 70% lethal dose (LD70) for 14 drugs and the leukemic cell survival (LCS) rate for dexamethasone and prednisolone. For each single drug, patients were classified into two groups, sensitive or resistant, by median concentration of LD70 or LCS. When patients were classified into three groups by sensitivity to four drugs of DPAV (dexamethasone, prednisolone, L-asparaginase, and vincristine), 3-year event-free survival (EFS; 95% confidence intervals) of the super sensitive group (SS; sensitive to all 4 drugs) was 0.833 (0.690 to 0.976), that of the intermediate sensitive group (IS; sensitive to 2 or 3 drugs) was 0.735 (0.609 to 0.863), and that of the relatively resistant group (RR; sensitive to no drugs or to 1 drug) was 0.541 (0.411 to 0.670; P = .0008). We then investigated the relationship between the above four-drug sensitivity and the time of relapse. The SS and IS patients tended to maintain continuous complete remission, and RR patients tended to undergo induction failure and early and late relapse (P = .004). Initial white blood cell count, immunologic classification, and age were also predictive factors, but the patient numbers showed no statistical correlation between these factors and the four-drug sensitivity groups (SS, IS, and RR). When we took three groups SS/IS/RR and investigated the EFS for various clinical groups, DPAV sensitivity strongly influenced EFS in the standard-risk ALL (P = .016). In vitro drug sensitivity testing provides additional prognostic information about childhood ALL, and early detection of drug resistance at the time chemotherapy commences may provide a successful strategy for individualizing treatment, as the results indicate de novo resistance to front-line drugs and suggest alternative, second-line drugs.



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