Cell Survival Studies Research Articles

Techniques for measuring red cell, platelet, and WBC survival.

Blood cell survival studies yield valuable information concerning production and destruction of cells circulating in the bloodstream. Methodologies for the measurement of red cell survival include nonisotopic methods such as differential agglutination and hemolysis. The isotopic label may be radioactive or, if not, will require availability of a mass spectrograph. These methods fall into two categories, one where red cells of all ages are labeled (51Cr, DFP32, etc.) and those employing a cohort label of newly formed cells (14C glycine, 75Se methionine, etc.). Interpretation of results for methodology employed and mechanism of destruction, random or by senescence, are discussed. A similar approach is presented for platelet and leukocyte survival studies. The inherent difficulties and complications of sequestration, storage, and margination of these cells are emphasized and discussed.

Reduced Survival of Isotope-Labelled Rh(D)-Negative Donor Red Cells in a Patient with Anti-LW^ab

The serum of an 85-year-old Caucasian male with no history of blood transfusion contained an IgG3 antibody with anti-LW^ab specificity. The antibody failed to react with dithiothreitol-treated red cells, and there was a marked reduction in titre of the antibody with pronase-treated cells, findings consistent with an antibody having this specificity. High association values were obtained in a mononuclear phagocyte assay when LW-positive red cells, sensitised in vitro with the patient’s serum antibody, were incubated with peripheral blood monocytes from the patient. In vivo red cell survival studies demonstrated that 99mTc-labelled rhesus-negative (rr), LW-positive red cells had 53% survival at 1 h. The IgG subclass of the antibody, mononuclear phagocyte assay results and in vivo survival studies predicted a significant reduction in the posttransfusion survival of therapeutic volumes of rhesus-negative (rr), LW-positive red cells.

Inactivation of Chinese hamster V79/4(AH1) and HeLa cells by 24 keV neutrons.

Cell survival studies have been carried out with a filtered neutron beam providing a nearly pure, high intensity source of 24 keV neutrons. These suggest that 24 keV neutrons behave as high LET radiation. The RBE at 37 per cent survival was approximately 2.2 for V79 Chinese hamster cells while HeLa cells gave a value of 2.9.

The interaction between bleomycin and radiation on cell survival and DNA damage in mammalian cell cultures

Chinese hamster ovary (CHO) cells were exposed to various concentrations of bleomycin (BLM) for 0.5, 1, 2, 4, 6, or 24 hours, followed with graded doses (0 to 800 rad) or radiation. The response to this chemotherapy-radiation combination treatment was measured by cell survival studies and DNA damage as determined by alkaline elution assay. Isobolograms for 1 and 2 log cell kills showed that cytotoxicity from BLM and from radiation were additive at 4, 15 and 75 milliunit/ml (mu/ml) for 1 or 4 hour exposure. When the exposure time of BLM was extended to 24 hours, slightly supraadditivity of lethality was found for the combination treatment with pharmacologically pertinent concentrations of 4 or 15 mu/ml. Therefore 4, 15 and 75 mu/ml concentrations of BLM all interacted positively with all doses of radiation to give enhanced cell kill. The alkaline elution patterns from the BLM-radiation combination also showed an enhancing effect on single strand breaks of DNA from treated cells. A human oat cell carcinoma cell line (MEMAR cells) was also studied and found to be more sensitive to BLM than were CHO cells.

Stem cell studies of human malignant brain tumors. Part 2: Proliferation kinetics of brain-tumor cells in vitro in early-passage cultures.

The proliferation kinetics were studied in early-passage cultures of cells from 13 human malignant brain tumors and two specimens of normal brain under conditions similar to those used in clonogenic cell-survival studies. Autoradiography was performed in all but four cases to estimate the fraction of cells actively replicating deoxyribonucleic acid (DNA), the approximate cell cycle time, and the effect of low-dose tritiated thymidine on cell proliferation. The mean tumor cell doubling time (TD) was 53 hours for five glioblastomas, 46 hours for two ependymomas, and 83 hours for two medulloblastomas. A gliosarcoma grew fastest (TD = 22 hours) in culture and a pilocytic astrocytoma grew slowest (TD = 144 hours). The approximate cell cycle time ranged from 1 to 2.5 days for all tumors tested. This suggests that chemotherapeutic agents that predominantly kill proliferating cells should be administered in vitro for at least 2 to 2.5 days to achieve maximum cell kill. The approximate growth fraction ranged from 0.65 to 0.96 for all tumors except for the two medulloblastomas and the pilocytic astrocytoma, which had growth fractions of 0.34 and 0.35, respectively. Most laboratories investigating the chemosensitivity of primary or early-passage human tumor cells require that 40% to 70% of cells be killed to consider a drug active in vitro. The results of this study suggest that the cell-cycle-specific agents cannot achieve a high enough cell kill to be considered active for some tumors that grow slowly in culture. An estimate of the in vitro growth rate is necessary to reliably interpret cell-survival results with such agents. Tritiated thymidine appeared to slow cell proliferation in some of the cultures, presumably as a result of radiation-induced DNA damage caused by tritium that had been incorporated into DNA. The degree to which cell growth ws slowed in individual tumors correlated with the patient's clinical response to radiation therapy and postoperative survival time.

Thalassemia in southeast Asia: determination of different degrees of severity of anemia in thalassemia.

beta (0)-Thalassemia/Hb E in Southeast Asia varies greatly in severity, with hemoglobin levels ranging from 2.5 to 13.5 g/dl, averaging 7.7 g/dl. Results of systematic investigations to find out what determines different levels of severity are reviewed. Concomitant inheritance of alpha-thalassemia significantly decreases the severity. Different degrees of severity in the majority of cases, however, is not due to alpha-thalassemia. Concordance of hemoglobin levels among patients who are sibs prevails, suggesting polygenic factor determinants. Potential factors ruled out as determinants for different levels of severity are discriminating fetal hemoglobin production, erythrocyte superoxide dismutase activity, reticulo-endothelial function, and failure of erythropoiesis compensation. Red cell survival and globin synthesis studies indicate that different degrees of excess of alpha-chains leading to different red cell pathology and survival are responsible for variable severity. Degrees of excess of alpha-chains in this circumstance are probably mainly determined by erythrocyte proteolytic activity. The relationship between the hemoglobin levels and erythrocyte cytosol proteolytic activity in 15 beta(0) -thalassemia/Hb E disease patients in whom a deletional type of alpha-thalassemia had been ruled out by DNA mapping is striking, with a correlation coefficient of 0.78. This finding suggests that modulation of erythrocyte proteolysis is another approach for treatment of thalassemia.

Deoxyribonucleic acid crosslinking by 4-hydroperoxycyclophosphamide in cyclophosphamide-sensitive and -resistant L1210 cells

4-Hydroperoxycyclophosphamide, a synthetic, activated form of cyclophosphamide, has been used to study DNA crosslinking in L1210 cell lines sensitive and resistant to cyclophosphamide. The time course of crosslink appearance and the proportion of inter-strand to DNA-protein crosslinks support the belief that phosphoramide mustard is the ultimate alkylating agent derived from cyclophosphamide. Cell survival and DNA crosslinking studies with a cyclophosphamide-resistant L1210 cell line indicate that resistance is associated with a failure of 4-hydroperoxycyclophosphamide to produce DNA crosslinks. The ability to reverse this situation by exposure of resistant cells to disulfiram points to a role of aldehyde dehydrogenase in this mechanism of cyclophosphamide resistance.

The haematopoietic response to burning: Studies in an animal model

Changes in haematopoiesis which occur in humans after burning injury may have important effects on morbidity and mortality. Because of the heterogeneity of burn patients we studied the regulation of blood cell formation which occurs in an animal using an established mouse model. Mice received a 20 per cent third degree scald injury on the back. Serial studies of a variety of haematopoietic parameters including stem cell, bone marrow and peripheral blood findings were done post burn. Although anaemia occurred frequently after injury red blood cell survival studies and examination of the stool for occult blood showed that neither haemolysis nor blood loss were primary causes of the anaemia. Bone marrow erythroid stem cells fell markedly post burn and this was associated with the development of a substance in serum capable of inhibiting red cell colony formation but not white cell colony formation of normal marrow cells. Reticulocytosis occured but was mild and the anaemia was primarily of the aregenerative type. Partial compensation for the depressed marrow erythropoiesis occurred in the spleen with an increase in erythroid colony-forming cells and erythroblasts. Marked granulocytosis occurred in the peripheral blood and bone marrow. There was an increase in splenic granulocytic stem cells post burn. Megakaryocytosis was striking in the bone marrow and spleen and there was an increase in peripheral blood platelet count. Evidence of immune stimulation included an increase in the size of the spleen and an increase in peripheral blood and splenic lymphocytes. Correlations of many of these findings suggested that the events were not occurring at random but that the changes in haematopoiesis were linked together. We speculate that the anaemia was the result of the increase in granulopoietic and thrombopoietic effort seen post burn.

Coulter Volume Cell Sorting to Improve the Precision of Radiation Survival Assays

A new method for measuring cell survival at low doses of ionizing radiation has been developed through the use of flow cytometric cell sorting on the basis of Coulter volume signals. The cell sorter is capable of deflecting a precisely known number of cells directly into culture dishes, thus eliminating any errors associated with cell dilution and volume sampling. The use of Coulter volume signals as the sorting parameter is shown to be noncytotoxic for a variety of cell lines. Comparison of radiation survival curves measured above the 10% survival level by either the cell sorter or standard dilution assay demonstrates the increased precision of the cell sorter technique . Because of these advantages of cell sorting over conventional methods of plating cells, this technique has many applications in the field of radiation biology and other studies of cell survival.

The Life Span and Osmotic Fragility of Erythrocytes in Mice Bearing Benzo(a) Pvrene — Induced Fibrosarcoma

Red cell survival studies in mice with benzo(a) pyrene [B(a)p]-induced fibrosarcoma indicate marked reduction in RBC 51Cr t1/2. In contrast, the t1/2 of erythrocytes from B(a)p injected but tumor-free mice was not declined. Increasing tumor burden resulted in increment in the severity of this disorder, and preincubation of normal red cells with tumor supernatant caused significant reduction (p less than 0.05) in 51Cr t1/2. It is suggested that the observed alteration in RBC life span may be due to the effect of tumor. Apparently, both intrinsic and extrinsic cellular defects could be implicated for this abnormality. However, the shortened survival cannot be attributed to altered osmotic fragility, since the erythrocytes of the fibrosarcomatous mice were more resistant to hypotonic hemolysis in vitro.

Clinical Evaluation of a 51Cr-Labeled Red Blood Cell Survival Test for In Vivo Blood Compatibility Testing

Modified red blood cell survival studies with use of 51Cr were performed in three groups of subjects. Group 1 consisted of normal subjects who were given labeled autologous blood, group 2 were subjects in need of blood transfusions and given labeled ABO and Rh crossmatch-compatible blood, and group 3 were patients in need of blood transfusion but in whom problems arose in finding compatible blood. The results of the studies suggest that for patients with blood compatibility problems, normal red blood cell survival values at 1 hour do not exclude the possibility of severe hemolysis 24 hours later. Thus, if a 1-hour test result is normal, the procedure should be extended routinely to 24 hours. Moreover, the test can be used to evaluate the clinical importance of antibodies. We showed that anti-Yka and anti-Lan were clinically significant, but high-titer, low-avidity antibodies, anti-Kna, anti-I, and anti-HI were clinically insignificant in the cases studied. This finding emphasizes the importance of an in vivo test for the final compatibility evaluation in complicated blood replacement problems.

A study of cell survival in mammalian cells exposed to spatially correlated triads of protons.

Cellular survival results for late-S Chinese hamster V79 cells exposed to triads of protons of variable mean separations are reported. The data are analyzed within the framework of the Theory of Dual Radiation Action [8]. This analysis provides a new estimation of the function gamma (chi) representing the probability of two energy transfers separated by the distance chi to produce a lesion. In agreement with an earlier study [6] involving pairs of deuterons, it is concluded that: a) the process of lesion formation depends strongly on the relative separation of energy deposition events, and b) intratrack and intertrack lesions correspond to substantially different separations of pairwise energy transfers.

A New Low Ionic Strength Test for Assessment of Pretransfusion Compatibility. Studies In Vitro and In Vivo

A new simple low ionic strength antiglobulin test (LIS-AGT) is presented for use in the antibody screening phase of pretransfusion tests. The ionic strength during the incubation phase of LIS-AGT is held between 15-17% of that of indirect AGT, and 8 mM EDTA is added to serum to inhibit false-positive tests. The prevalence of false-positive LIS-AGT was determined to be approximately two times higher than that observed with the indirect AGT. The new test was superior to the indirect AGT in detecting antibodies specific to Rh, Duffy, Kidd, and MNSs antigens, while the indirect AGT was superior in detecting antibodies specific to K and Lewis antigens. On the basis of three 51Cr red blood cell (RBC) survival studies, it was shown that antibodies reactive with LIS-AGT only decreased the long-term survival of incompatible erythrocytes, although the one-hour recovery was not affected. It appeared that antibodies reactive by LIS-AGT only can cause delayed rather than acute hemolytic transfusion reactions. The data shown indicate that the LIS-AGT is a simple and valuable addition to the pre-transfusion antibody screening test.

Heat-Induced Thermotolerance Expressed in the Energy Metabolism of Mammalian Cells

Measurements were made of the effect of heat treatment on ATP levels in control and thermotolerant populations of murine lymphoma (L5178YS) and Ehrlich ascites cells to investigate whether the development of thermotolerance is associated with an increased ability of cells to maintain energy metabolism when challenged with heat treatment. For the L5178YS cells a single heat treatment produced a rapid reduction in ATP levels. However, previously heat treated L5178YS cells showed an increased ability to maintain ATP levels when challenged with a second heat treatment, and this ability to maintain ATP levels varied in a manner which correlated with the appearance and decay of thermotolerance seen in the parallel cell survival studies. In contrast both single and fractionated heat treatments did not reduce ATP levels in Ehrlich ascites cells.

Improved treatment of a brain-tumor model. Part 1: Advantages of single- over multiple-dose BCNU schedules.

Clonogenic cell and animal survival studies were used to determine the most effective BCNU therapy schedule in the 9L rat brain-tumor model. Survival of tumor cells following a single LD10 dose of BCNU (13.3 mg/kg intraperitoneally) was compared to cell survival after one to four daily 0.5 X LD10 doses. The posttreatment kinetics of surviving clonogenic cells were investigated at various times after BCNU was given in single doses of 0.25 to 1 X LD10 and in two daily doses of 0.5 X LD10. The cell kill was greater, time to reinitiation of cell growth was later, posttreatment rate of clonogenic cell proliferation was slower, and the interval to total repopulation of the clonogenic cell pool was longer with a single LD10 dose as compared to the multiple-dose schedules. Animal survival studies confirmed that a single LD10 dose of BCNU was at least as effective as a cumulative level of up to 1 1/2 times that amount when treatment was administered in smaller doses, regardless of the fractionation schedule. Clinical experience with patients harboring malignant brain tumors has shown that a single BCNU dose of 185 to 200 mg/sq m is tolerated well. Results of these animal experiments suggest that this therapy should have anti-tumor activity at least equivalent to the more commonly employed schedule of 80 mg/sq m/day given for 3 days. Although direct comparison of treatment efficacy using the two schedules is not possible, no adverse clinical effects have been observed with the recently adopted single-dose schedule. Furthermore, the duration of patient hospitalization for chemotherapy has decreased.

Immunological tolerance induced by in utero injection.

Intrauterine injection of human whole blood into rabbit and rhesus monkey fetuses was found to result in long lasting unresponsiveness to human serum albumin. Intrauterine injection of viable allogeneic bone marrow cells into rabbit fetuses was without any apparent harmful effect and also resulted in permanent unresponsiveness demonstrated by donor red cell survival studies. The implication of these findings in respect of using this approach towards the correction of certain inherited diseases in man is discussed.

Intraoperative autotransfusion during abdominal aortic reconstruction

Intraoperative autotransfusion by a continuous-flow centrifuge system was used during more than 300 abdominal aortic reconstructions at the Cleveland Clinic since May 1979. Fifty consecutive patients undergoing such operations were studied prospectively. Volumes of blood lost, salvaged, and transfused during each operation were tabulated. Autologous autotransfused blood was compared with homologous bank blood with respect to oxygen-carrying capacity, coagulation factors, microaggregate levels, red cell mass, pH, and free hemoglobin concentration. Chromium-51 red cell survival studies were performed in autotransfused blood in random patients and in control subjects. Renal, hepatic, and coagulation functions were determined during the first postoperative week. Each patient received a mean volume of 1,203 ml of autotransfused blood and 1,682 ml of bank blood to replace a mean operative blood loss of 2,386 ml. Red blood cell survival of both salvaged autologous and unshed autologous blood in the control group was nearly identical. Salvaged blood had superior oxygen-carrying capacity, a lower microaggregate level, and better buffering capacity than bank blood. Although transient elevations in liver function values and free hemoglobin levels were noted, no clinically important aberration of coagulation, hepatic, or renal function was demonstrated.

Biological availability of nickel arsenides: Toxic effects of particulate Ni 5As 2

To determine if fugitive nickel arsenides from an oil shale retort could pose a threat to living organisms, we studied the effects of particulate Ni 5As 2 on cultured mammalian cells. Culture growth rate was greatly reduced at the lowest suspension concentration tested (10 μ M), even though much of the Ni 5As 2 powder remained insoluble. FCM analysis indicated Ni 5As 2 arrested cell-cycle traverse in G 1 and G 2. Cell survival studies indicated cells could overcome this toxicity if exposure levels were low ( <25 μ M in suspension) and restricted to <24 h. At higher powder levels, survival was greatly reduced. Transmission electron microscopy (TEM) demonstrated that cells exposed to <100 μ M powder did not phagocytize the Ni 5As 2 particles. At higher concentrations, TEM X-ray microanalysis demonstrated that As was preferentially extracted from the Ni 5As 2 particle surface and free Ni was deposited inside the cell. These observations suggest that the toxicity of Ni 5As 2 particles may be caused by some soluble product of Ni 5As 2.

Radiation and drug response of the rat glioma RG2

A clonogenic cell assay was developed for the chemically induced rat glioma RG2 that allows in vivo, in vitro, and in vivo to in vitro studies of cell survival after experimental therapy. RG2 monolayer cells were resistant to BCNU up to high concentrations. The radiation survival curves were characterized by a D 0 of 2.4 gray and n = 2.2 for monolayer cells, a D 0 of 3.5 gray and n = 1.3 for cells irradiated as brain tumors in air-breathing rats, and a D 0 of 5.9 gray and n = 1.2 for cells irradiated as brain tumors in nitrogen-asphyxiated rats. There was no evidence of a radiobiologically hypoxic fraction of cells in the brain tumors, but their radiosensitivity was definitely smaller than that of monolayer cells.

Book reviewsRadionuclide Tracer Techniques in Haematology. By BowringC. S., pp. vii+140, 1981 (Butterworths, Sevenoaks),£8.50. ISBN 0–407–00183–2

It is no mean task to attempt to cover basic physics of radionuclides, instrumentation, the measurement of red cell and plasma volume, cell survival studies, ferrokinetics, external counting, imaging and absorption studies in one pocket-sized book. It is also somewhat optimistic to hope that a book of this size and nature will be useful to clinicians, scientists and technicians, all of whom have different requirements from reference books. However, the author is to be congratulated on making good attempt to fulfil his objectives.