Comparison Between Flow Cytometric and Cytogenetic Tumor Cell DNA Content Using a Simple Flow Sample Preparation Method: Ploidy Comparison of 86 Fresh Pediatric Tumors

A comparison of tumor ploidy by flow cytometry (FL) and cytogenetics (CYG) was made in 86 fresh pediatric solid (n = 47) and hematopoietic (n = 39) tumors using simple sampling and semiautomated proprietary FL preparation methods and defined histogram interpretive criteria. Tumor karyotypes with 14-48 chromosomes were regarded as CYG diploid and other chromosomal complements CYG aneuploid for comparison purposes. Five histograms were uninterpretable and nine cases failed to produce 15 or more metaphases for karyotyping. Mean G0/G1 peak coefficients of variation of all 86 cases were 2.7 and 3.0 for the diploid and aneuploid populations, respectively. Of the 72 eligible cases, 41 were concordant diploid and 16 concordant aneuploid with an overall concordance of 79%. The DNA index and karyotypic index correlation coefficient was 0.92 for the 16 concordant aneuploid cases. Analysis of the 15 discordant cases highlights the limitations of both methods and of the histogram interpretive criteria and indicates that FL is probably more sensitive for detection of tumor aneuploidy as defined and detected by these methods.     

Flow Cytometry DNA Applications in Pediatric Tumor Pathology

Received August 15, 1999; accepted December 3, 1999.     

Malignant Melanoma in Children and Congenital Melanocytic Nevi: DNA Content and Cell Cycle Analysis by Flow Cytometry

Malignant melanoma (MM) in children, although a rare neoplasm, can occur within a preexisting congenital melanocytic nevus (CMN). All the potential risk factors for this phenomenon are not well known, but increases in S phase and G₂+ M phase of cell cycle, DNA aneuploidy, and cell cycle abnormalities in precursor lesions might be among the risk factors. Using paraffin-embedded tissue, we performed a retrospective analysis of DNA content, aneuploidy, and cell cycle by flow cytometry. Two groups of patients were analyzed: 28 children with CMN who did not developed MM, and 6 patients who further developed MM. In this second group, three patients had four biopsies done before the appearance of MM and in two patients biopsies were done after the appearance of MM. All CMN not associated with MM exhibited diploid cells only, their S phase was 11.5% (± 3.8), and their G₂+ M phase was 2.5% (± 2.2). Among those patients who developed MM, 3/6 had an S phase > 15.5 and a G₂+ M phase > 2.3 prior to the appearance of MM. Two out of six patients had a tetraploid DNA when MM developed and died with a disseminated MM. They had an S phase > 15.5 and their G₂+ M phase was > 2.5. We propose that evaluation of DNA content and cell cycle by flow cytometry is a useful method to supplement biopsy findings in children with CMN who have lesions suspicious of developing a MM. Received August 12, 1999; accepted June 19, 2000.     

Cell Kinetic Analysis in Pediatric Brain and Spinal Tumors: A Study of 117 Cases with Ki-67 Quantitation and Flow Cytometry

We present cell kinetic data including Ki-67 quantitation and flow cytometry on 117 pediatric brain/spinal cord tumors and review the literature. Although, in general, these proliferation indices are in agreement with the histologic grade, they are useful in prognostication in some instances when the histological features of malignancy are equivocal. Specific examples in which flow cytometry may prove particularly useful in this context are childhood ependymomas, which do not show frank anaplasia but have cellular foci with focal increase in mitoses, and choroid plexus neoplasms, where elevated S phase fractions have been associated with an adverse outcome. Thus Ki-67 quantitation and flow cytometry not only serve as useful adjuncts to conventional histologic grading but also in specific instances may provide new information on tumor prognosis.     

流式细胞仪测定小儿恶性肿瘤细胞DNA倍体及细胞周期的临床意义

目的探讨小儿恶性肿瘤细胞核DNA倍性分布、细胞周期特征，并分析其临床意义。方法应用流式细胞仪测定20例小儿恶性肿瘤细胞核DNA倍体模式、DNA指数、增殖指数和S期细胞比例，并研究这些参数与肿瘤组织分期之间关系：结果二倍体肿瘤主要分布于细胞分化较好的肿瘤；随着肿瘤细胞病理分级的发展，异倍体肿瘤的检出率逐渐上升。相对于低分化组而言，高、中分化组的DI、SPF及PI有显著性差异：结论流式细胞仪测定小儿肿瘤细胞核DNA倍体模式及细胞周期特征在评估小儿肿瘤恶性程度上有重要意义，测定结果应与临床、病理资料相结合，为恶性肿瘤预后判断提供依据。     

Flow Cytometry of Fetal Adrenal Glands with Adrenocortical Cytomegaly

Adrenal glands from four autopsied fetuses of 18 to 36 weeks gestation showed varying degrees of cortical cytomegaly. Formalin'fixed, paraffin-embedded sections from these four pairs of glands were studied by flow cytometry to analyze their DNA content and cell cycle parameters. Flow cytometry of Case 1, which had diffuse bilateral cytomegaly, demonstrated a major diploid peak, an increased percentage of tetraploid cells, and a decrease in S phase compared to an age-matched control with no evidence of cytomegaly (Case 2). Cases 3, 4, and 5 showed focal and/or unilateral adrenocortical cytomegaly and were diploid by flow cytometry with no differences in synthetic or tetraploid fractions compared to the control tissues. The focal distribution of the lesions or the limits of resolution of the instrumentation could account for some of these results. However, the findings in Case 1 suggest that the cytomegalic cells are tetraploid in DNA content and may have decreased DNA synthetic activity. A current hypothesis that these cells have undergone a period of sustained hyperactivity followed by exhaustion in reaction to an unknown stimulus is supported by our observations.     

Case of a Child with Paroxysmal Nocturnal Hemoglobinuria Diagnosed by Flow Cytometry

A case of a child with paroxysmal nocturnal hemoglobinuria (PNH) is characterized by an increased sensitivity of the erythrocyte to hemolytic action of complement. The widely used Ham test may not always be reliable. Recently, a panel of monoclonal antibodies has become available to detect various glycosylphosphatidylinositol (GPI)-linked proteins by flow cytometry (FCM)¹ and the deficiency of GPI-anchored proteins on the various kinds of cell membranes is implicated as the pathogenesis of PNH. We diagnosed a case of a child with PNH by FCM and complement lysis sensitivity (CLS) test, which showed the increased sensitivity of PNH erythrocytes to complement. His diagnosis was delayed because of Ham test negativity and rarity of PNH cases in children.     

Flow cytometric analysis of DNA ploidy in 62 patients with Wilm's tumor

Flow cytometric analysis of DNA ploidy was done on nuclei extracted from paraffin-embedded tissue blocks from 62 patients with Wilms' tumor, the largest series reported to date. Thirty-eight tumors were aneuploid and 24 were diploid; there were no tetraploids. Sixty-seven percent of the diploids and 44.7% of the aneuploids survived; the difference was statistically significant (P <0.01). The 2 congenital mesoblastic nephromas were diploid; the only 2 adult patients were aneuploid. Aneuploids comprised 75% of those presenting in stage III/IV and 75% of the anaplastic tumors. Genetic heterogeneity, never before reported in Wilm's tumor, was found in 2 patients. Aneuploidy was an ominous prognostic factor, particularly when combined with tumor stage and histological grade. Offprint requests to: M. Rohatgi     

Effects of Copper and Hydrocortisone on Cell Cycle of Cultured Fibroblasts from the Macular Mouse as a Model of Menkes Disease: DNA Analysis by Flow Cytometry

Abstract Effects of copper and hydrocortisone on the proliferation of cultured fibroblasts from macular mouse, a model of Menkes kinky hair disease, were analyzed by flow cytometry. In fibroblasts from the macular mouse, copper induced cytotoxicity and S-phase accumulation of cell cycle were observed at lower concentrations of copper than in fibroblasts from the controls. However, the fibroblasts cultured with a combination of copper and hydrocortisone revealed recruitment of cell cycle. These results suggest that the proliferative activity of the cells from a macular mouse are more vulnerable to an excess amount of copper than normal, and that supplementation of hydrocortisone is effective for the Menkes kinky hair disease.     

Flow cytometric evaluation of the DNA profile and cell cycle of zinc supplemented human Chang liver cells

Zinc, an essential trace element, is important for normal cell growth. Growing children, especially at puberty, require increased zinc (2.8 mg/day for males and 2.65 mg/day for females). The DNA profile and cell cycle of human Chang liver cells grown in 0–900 μmol/L zinc chloride supplemented serum-free media for 24 h were analyzed using a Coulter flow cytometer. There was no significant difference in the G₁, S and G₂/M phases between zinc treated cells and control cultures except at 90 and 900 μmol/L zinc chloride. At these two higher dosages, fragmentation of genomic DNA into sub-2N DNA (sub-G₁ DNA), generally considered a hallmark of programmed cell death (PCD), was noted. Results of the present study seem to suggest that growth regulation by zinc during growth spurts such as at puberty, could also be influenced by other factors besides its direct effect on DNA synthesis. In addition, high dosages of zinc could be cytotoxic.     

Flow Cytometric Analysis for the Evaluation of the Rat Sperm Viability and Number in the Male Reproductive Toxicity Studies

Abstract We investigated the application of flow cytometric analysis to evaluate the rat sperm viability and number in the male reproductive toxicity studies. Flow cytometric procedure has been developed to evaluate sperm number and viability that uses fluorescent dye (propidium iodide, PI) to distinguish between viable (negative staining) and dead (positive staining) sperm. Sperm samples were collected from the caudal epididymides of SD rats (13–21 weeks old). PI staining patterns/viabilities were compared among several ranges of sperm concentrations, and several kinds of sperm viability. Viabilities determined by flow cytometry (FC) were also compared with motility by direct microscopical observation in several kinds of sperm viability. No notable changes in the PI staining patterns/viabilities were observed in the range from 1 × 10⁵ to 6 × 10⁶ sperm/ml. Essentially similar results were obtained from both FC and microscopical analyses for three degrees of viability sperm: live sperm (general preparation as a control), weakly viable sperm (mixed by vortex mixer for 30 seconds), and dead sperm (treated with 90°C or Triton X-100).
Viabilities of normal rat samples were 95.0 ± 4.0% in FC and 93.7 ± 4.6% in microscopical observation, indicating good correlation in both analyses. Sperm numbers with FC analysis were approximately 0.8 × 10⁶ to 1.8 × 10⁶ sperm per mg indicating good correlation with those by microscopy.
It was concluded that the present flow cytometric procedure was objective, rapid and reliable, and that it was one of the useful methods for measuring the number and evaluating the viability of sperm collected from the caudal epididymides of rats in the male reproductive toxicity studies.     

Barriers to Cure for Children with Cancer in India and Strategies to Improve Outcomes: A Report by the Indian Pediatric Hematology Oncology Group

The survival of children with cancer in India is inferior to that of children in high-income countries. The Indian Pediatric Hematology Oncology Group (IPHOG) held a series of online meetings via www.Cure4kids.org to identify barriers to cure and develop strategies to improve outcomes. Five major hurdles were identified: delayed diagnosis, abandonment, sepsis, lack of co-operative groups, and relapse. Development of regional networks like IPHOG has allowed rapid identification of local causes of treatment failure for children with cancer in India and identification of strategies likely to improve care and outcomes in the participating centers. Next steps will include interventions to raise community awareness of childhood cancer, promote early diagnosis and referral, and reduce abandonment and toxic death at each center. Starting of fellowship programs in pediatric hemato-oncology, short training programs for pediatricians, publishing outcome data, formation of parent and patient support groups, choosing the right and effective treatment protocol, and setting up of bone marrow transplant services are some of the effective steps taken in the last decade, which needs to be supported further.     

Hematogones as an Internal Control in Flow Cytometric Analysis of Suspected Acute Lymphoblastic Leukemia

Hematogones are benign immature B cells that commonly populate the bone marrow of children. Their presence has been noted to interfere with the flow-cytometric analysis of cases of suspected acute lymphoblastic leukemia (ALL) because their immunophenotype (positive for CD19, CD10, CD34, and terminal deoxynucleotidyl transferase) is similar to that of pre–B cell lymphoblasts. Here we report a case in which the presence of a discrete population of hematogones, characterized by low-intensity CD10 cell-surface staining compared with pre–B cell lymphoblasts, actually aided in the recognition of early relapsed ALL and disease progression over a 4-day period. We also review our experience with flow-cytometric immunophenotyping in pediatric cases of suspected leukemia to evaluate the frequency of this occurrence. Received May 22, 1998; accepted August 31, 1998.     

Comparison of conventional to intensity modulated radiation therapy for abdominal neuroblastoma

OBJECTIVE: To compare three different techniques of irradiating abdominal neuroblastoma. PATIENTS AND METHODS: Six children with a median age of 4.1 years underwent radiotherapy (RT) to the primary site as part of treatment for high-risk neuroblastoma. Four had midline disease while two had well-lateralized lesions. Three different RT techniques were compared. Technique A used parallel-opposed AP/PA fields prescribed to the midplane of the patient. For Techniques B and C, intensity modulated radiation therapy (IMRT) plans were developed using inverse treatment planning with a sliding window or dynamic multileaf collimator approach, seven coplanar beams, and a 0.25 x 0.5 cm minimum beam resolution. The clinical target volume (CTV) included the tumor present prior to second look surgery but after induction chemotherapy with a 1.5 cm margin. The planning target volume (PTV) was the CTV with a 0.5 cm margin. The CTV was planned to receive 100% of the prescribed dose. For Technique C, the vertebral bodies adjacent to the tumor were included in the PTV to minimize heterogeneity of dose. Six MV photons were used for all techniques. Bilateral kidneys, liver, spine, spleen, stomach and bilateral iliac crests were contoured. RESULTS: Dose to the PTV and CTV were not significantly different using the three techniques. In comparison to Technique A, Techniques B and C delivered a lower mean dose to the bilateral kidneys in the four children with midline tumors but not the two children with a lateralized tumor where the contralateral kidney received a higher mean dose. Dose to the spine was less homogeneous with Technique B compared to Techniques A and C. The spleen, liver and stomach mean doses were higher using Techniques B and C compared to Technique A. CONCLUSION: Although Technique C was the best method of RT delivery in midline tumors with respect to kidney doses, this was at a cost of a higher mean dose to the liver, stomach, and spleen. This, together with the theoretical increase in secondary malignancies, should be considered when treating a child with IMRT techniques. IMRT was not found to be better than the conventional AP/PA field for lateralized tumors.