Apoptotic fraction in childhood ALL assessed by DNA in situ labelling is ploidy independent.

BACKGROUND: Apoptotic cell fraction and presence or degree of aneuploidy may both affect treatment outcome in childhood acute lymphoblastic leukaemia (ALL), which is largely defined by drug resistance. Independence of the variables is at present not established. Until the development of in situ labelling of cells committed to the apoptotic pathway, the fraction of cells in apoptosis could not be determined objectively. AIM: To determine the relationship between apoptotic cell fraction and karyotype in childhood ALL using in situ labelling. METHODS: 1.3.1. Study Groups and Samples. Diagnostic, pretreatment bone marrow trephine and aspirate samples of 24 consecutive, unselected cases of childhood ALL were included in the study: Normal karyotype (n = 11, 5M,6F), high hyperdiploid aneuploidy (DNA index > 1.5, n = 7, 1M,6F), complex karyotypic anomalies (n = 6, 5M,1F). 1.3.2. Apoptotic Cell Labelling. In situ labelling of the 3'-OH ends of the apoptosis specific DNA (Klenow) fragment (Frag-EL, CalBiochem, USA). 1.3.3. Quantitation. Apoptotic cell fraction was established using 10 systematically random fields of > 20 nuclei. Results were tabled per group. After calculations of means, differences between groups were assessed using t-test. RESULTS: Apoptotic cell fraction, ranging from < 1 to 95%, did not differ statistically significant between the three study groups. CONCLUSION: Apoptotic cell fraction in childhood leukaemia is independent of ploidy status and euploid karyotypic anomalies.

PCNA bearing structures are retained in apoptotic phase of childhood ALL cell cycle.

BACKGROUND: Drug resistance to DNA directed therapy may depend on proliferative as well as apoptotic cell fraction. PCNA/Ki67 ratio excess, possibly reflecting DNA excision repair, is of additional interest to drug resistance in MTT testing. The cell cycle phase/antigen expression pattern in childhood acute lymphoblastic leukemia (ALL) is not known. AIMS: To study the relationship between nuclear expression of PCNA, Ki-67 and Frag-EL positivity in childhood ALL. METHODS: 1.3.1. Study Groups. Diagnostic bone marrow trephine biopsies of 32 consecutive unselected cases of childhood ALL were included in the study. 1.3.2. Immunohistochemistry. Commercially available Moab PCNA (PC10, DAKO, USA), Ki-67 (MM1, NovaCastra, UK) were used to label cycling cells in routinely processed 5 microns paraffin sections. 1.3.3. In-Situ Labelling of Apoptotic Cells. The 3'-OH ends of apoptosis specific DNA fragments were labelled in-situ on subsequent 10 microns sections (Frag-EL, CalBiochem, USA). 1.3.4. Quantitation. After blinding and randomisation, 10 systematic random fields of > 20 nuclei and nuclear size bias correction was used to determine positive nuclei fraction. RESULTS: While the sum of apoptotic and proliferative cell fraction (Ki-67 + Frag-EL%) equalled 100% in 5/32 cases, PCNA expression into at least the early phases of apoptosis ([%PCNA-%K-67] > [100-%Frag-EL] was found in 17/32 cases. CONCLUSIONS: PCNA/Ki67 ratio excess may not reflect DNA excision repair activity but rather slow degradation of antigen bearing structures limiting relevance to drug resistance study.

Apoptosis corrected proliferation fraction in childhood ALL is related to karyotype.

BACKGROUND: Tumour doubling time, a parameter in drug sensitivity testing, reflects both cell proliferation and apoptosis. Variable apoptosis fractions may explain the poor correlation of S-fraction and drug response. DNA aneuploidy (reflecting intrinsic DNA instability) may, by increasing apoptosis, affect drug response. AIM: To assess the relationship between apoptosis corrected proliferation fraction and DNA ploidy in childhood acute lymphoblastic leukemia (ALL). METHODS: 1.3.1. Study Groups. Thirty two consecutive, unselected diagnostic cases of childhood ALL were included in the study. 1.3.2. Karyotype. A normal karyotype was found in 15 cases (7M, 8F, age 8 m-12 yrs); high hyperdiploid aneuploidy (DNA index > 1.5) was found in 7 patients (1M, 7F, age 3-12 yrs) whereas complex karyotypic anomalies, but with 2n or near 2n DNA were present in 10 patients (7M, 3F, age 1 y 7 m -16 yrs). 1.3.3. Proliferation Fraction Assessment. Immunocytochemical demonstration of S-phase associated nuclear expression of the Ki-67 antigen (MM1, NovaCastra, UK). 1.3.4. Apoptosis Fraction Assessment. Binding of a horse radish peroxidase labelled DNA probe for the 3'-OH ends of apoptosis derived Klenow fragments (Frag-EL, CalBiochem, USA). 1.3.5. Quantitation. Computer assisted image analysis (Quantimet 570C), of 10 systematically random fields of a minimum of 20 nuclei each. A nuclear size bias correcting counting frame and rule were used to correct for cell proliferation associated nuclear volume increase and for the expected nuclear volume reduction resulting from apoptosis. RESULTS: Corrected for apoptosis, proliferation fraction was highest (mean 57.5%, range 1-100) in poor prognosis, complex karyotype anomalies. Good prognosis, high hyper diploidy showed significantly lower proliferation rates (mean 24.7%, range 12-40) (p < 0.01, t-test). CONCLUSION: Apoptosis corrected cell proliferation rate in childhood ALL is not independent of karyotype abnormality which may partly explain a relation to therapy response and prognosis.

Proliferation and apoptosis does not affect presenting white cell count in childhood ALL.

BACKGROUND: Treatment response/drug resistance in childhood acute lymphoblastic leukaemia (ALL) is related to presenting white cell count. This relationship might be explained by high proliferation fraction or by absence of significant apoptosis, but is presently unknown. AIMS: To study the relationship between proliferation and apoptosis in childhood ALL. METHODS: 1.3.1. Study Groups. Thirty consecutive, unselected cases of childhood ALL (15M,15F). White cell count varied from 1-200 at presentation. 1.3.2. Proliferation/Apoptosis Fraction. Immunocytochemical detection of Ki-67 (MM1, NovaCastra, UK) on 5 microns paraffin slides, Immunocytochemical detection of apoptosis specific DNA (Klenow) fragments by labelling of 3'-OH ends in 10 microns paraffin sections (Frag-EL, CalBiochem, USA). 1.3.3. Quantitation. Image analysis (Quantimet 570C) using nuclear size bias correcting counting frame and rule. Calculation of proliferation (%Ki-67) fraction and of apoptosis corrected proliferation fraction (%Ki-67/100-%apoptosis). RESULTS: Using both linear, logarithmic regression as well as power analysis, no relationship between variables was detected. CONCLUSION: Presenting white cell count is not related to apoptotic or cell proliferative activity or to net tumour growth defined by the balance between these two processes. The relationship to treatment resistance still requires explanation.

Lipid-laden macrophages in the tracheal aspirate of ventilated neonates receiving Intralipid: A pilot study.

Lipid-laden macrophages (LLM) in tracheal aspirates are reported to be pathognomonic findings in exo- and endogenous lipoid pneumonia in adults. A pilot study was carried out to evaluate the effect of lipid infusion on the LLM index of the tracheal aspirates from ventilated neonates. All intubated infants were eligible for the study. Infants receiving parenteral nutrition had intravenous (IV) lipid introduced by 4-7 days of age; most samples after 7 days were from infants receiving IV lipid. Four infants received minimal gastric feeding; none had evidence of aspiration pneumonia. Tracheal aspirates from 28 infants were analyzed for the LLM index. Alveolar macrophages were graded 0-4 in direct relation to the amount of lipid per cell. One hundred macrophages were graded; the maximum possible LLM index was 400. Two hundred forty-five of 387 tracheal aspirate samples were acceptable for analysis. LLM indices increased during the first week after birth; the mean LLM index then continued in the same range, but with a wide distribution of individual values. The mean LLM index from infants receiving an IV lipid infusion during days 4-7 was 87.9 (SD = 44.8), and was significantly higher compared to 58.7 (SD = 40.8) in infants receiving no IV lipid (P < 0. 003). Tracheal aspirates from infants with and without IV lipid infusion yielded many LLM index values >100. These observations invalidate the use of the LLM index >100 as proof of aspiration pneumonia in this group of infants.

Quantitation of nucleolar organizer regions by image analysis in glottic squamous cell carcinoma.

OBJECTIVE: To apply computer image analysis as a quantitative method for analyzing interphase nucleolar organizer regions (NORs) to determine whether this proliferative marker provides useful prognostic information in glottic squamous cell carcinoma. DESIGN: Retrospective testing of biopsy samples and resected tissue. SETTING: Nova Scotia Regional Cancer Centre and regional hospitals in Nova Scotia and Prince Edward Island, Canada. PATIENTS: Patients with primary glottic cancer presenting to the cancer centre between 1984 and 1991. INTERVENTIONS: Semiautomated image analysis was used to measure the nuclear area and the NOR area in formalin-fixed paraffin-embedded tumour samples. OUTCOME MEASURES: Mean nuclear area, mean NOR area and NOR percentage of nuclear area, calculated as the mean NOR area divided by the mean nuclear area, expressed as a percentage. RESULTS: Of 154 cases, 90 samples were received; however, 8 paraffin blocks were exhausted and 29 samples stained poorly due to extent of fixation. Analysis of the remaining 53 cases, all primary squamous cell carcinomas, showed no statistically significant association between, on one hand, mean NOR area or NOR percentage of nuclear area and, on the other hand, tumour grade, tumour stage, tumour recurrence or disease-related death. CONCLUSIONS: This study does not demonstrate a prognostic value of NOR measurement as a proliferative marker in primary glottic squamous carcinoma. However, given the small number of cases in this study, further research should be conducted using a larger number of cases from one centre and comparing NOR measures with other markers of cell proliferation.

Immunocytochemical characterization of the pancreatic islet cells of the Nile Tilapia (Oreochromis niloticus).

The cellular composition and topography of the pancreatic islet of Oreochromis niloticus, now known to be a donor source for islet xenotransplantation studies, were characterized. Whole tilapia islets were harvested using an enzymatic method and then further digested into single-cell preparations. Cell cytospin preparations of islet cells and paraffin sections of whole islets were stained using antisera against tilapia insulin, human glucagon, salmon somatostatin-25 (SST-25), human somatostatin-14 (SST-14), and salmon peptide tyrosine-tyrosine (PYY) using the immunoperoxidase method. Cell counts, performed on cytospin preparations using a Quantimet 570 computerized image analysis system, revealed that O. niloticus islets contained 78% endocrine cells and 22% immunonegative cells (i. e., mainly nucleated erythrocytes and rare tissue eosinophils). The proportions of immunopositive endocrine cell types were: 42.3% insulin immunopositive cells, 11.5% glucagon immunopositive cells, 23.1% SST-25 immunopositive cells, 21.8% SST-14 immunopositive cells, and 1.3% PYY immunopositive cells. Islet cell topography was evaluated using histologic sections of whole endocrine pancreata including large, medium, and small islets. Round to polygonal insulin immunopositive cells with round central nuclei were distributed in clusters throughout both the principal and the smaller islets. Elongate SST-14 immunopositive cells were closely associated with the clusters of insulin immunopositive cells; both were surrounded by SST-25 immunopositive cells, which were similar in shape to the insulin immunopositive cells. There were elongate glucagon immunopositive cells throughout the islets, whereas the PYY immunopositive cells were restricted to the periphery and to channels of fibrovascular connective tissue penetrating the islets.