Mobilization of peripheral blood progenitor cells by cyclophosphamide and rhGM-CSF in multiple myeloma.

Fifteen consecutive patients with multiple myeloma (MM) scheduled for peripheral blood progenitor cell (PBPC) transplantation, were randomly selected to receive cyclophosphamide (CY) (4 g/m2) alone (group I) or associated with recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (5 micrograms/kg/day) (group II). The mean time of neutropenia after CY administration was 9.8 +/- 4.3 days in group I and 6.4 +/- 1.2 days in group II (P = 0.0228). One hundred and eight aphereses were performed (7.1 +/- 1.8 aphereses per patient in group I and 6.4 +/- 2.8 aphereses per patient in group II). rhGM-CSF administration after CY allowed a higher collection of CD34+ cells in apheresis products (1.42 +/- 1.68 x 10(6) CD34+ cells/kg) in comparison to without factor administration (0.47 +/- 0.52 x 10(6) CD34+ cells/kg) (P = 0.0165). The mean number of cells infused per patient was 6.56 +/- 4.02 x 10(8) MNC/kg and 7.64 +/- 3.00 x 10(4) CFU-GM/kg in group I and 6.25 +/- 4.03 x 10(8) MNC/kg and 8.16 +/- 9.73 x 10(4) CFU-GM/kg in group II. The mean time to recover 0.5 x 10(9) granulocytes/I, 20 and 50 x 10(9) platelets/I in peripheral blood (PB) was 17.2 +/- 7.4, 13.4 +/- 3.7 and 16.5 +/- 6.9 days respectively, in group I and 13.3 +/- 1.7, 11.6 +/- 1.6 and 15 +/- 6.3 days, in group II. rhGM-CSF administration after CY treatment for PBPC mobilization in MM patients reduces the neutropenic period after CY and enhances apheresis CD34+ cell collection.

Relevance of forward scatter and side scatter in aneuploidy detection by flow cytometry.

The study of the nuclear DNA content by flow cytometry (FCM) has been classically accomplished by selecting the nuclear population on the biparametric forward scatter (FS)-DNA fluorescence or FS-DNA fluorescence peak histograms to determine ploidy and DNA index (DI). Different cellular factors such as nuclear morphological heterogeneity of the neoplastic cells, intratumoral variability, histological origin, displasia grade, necrosis, and size of the tumoral piece analyzed constitute important problems in ploidy studies and, consequently, residual or underrepresented clones with different ploidy levels can be masked by populations with a large cell number. In the present report, an alternative methodology is proposed for aneuploidy detection, since populations coinciding with DNA content may be different with respect to morphological criteria. The discrimination of aggregates and background noise by using peak or logarithmic fluorescence signal, and backgating in side scatter (SS)/FS histograms, permits the establishment of specific bounds through complete scatterplot mapping and to distinguish between scarce or minor populations in association with small or abnormal DNA peaks. Moreover, variations in the DNA modal channel value and the peak coefficient of variation value remained unmodified, also maintaining the quality of cytometric measurement data.

Changes in reticulocyte fractions during peripheral stem cell harvesting: role in monitoring stem cell collection.

We prospectively evaluated the changes in immature reticulocyte fractions during peripheral blood stem cell mobilization to determine any possible relationship with mobilization of stem cells into the peripheral blood. Circulating neutrophils, immature reticulocyte fractions (% of HFR + MFR) (HMFR) (measured by flow cytometry), circulating CD34+ cells (measured by flow cytometry) and CFU-GM (measured by semisolid media assay in the apheresis fluid) were closely monitored following priming with chemotherapy and colony-stimulating factors in 15 patients with hematological or solid tumors (group I). Day 0 was defined as the day on which the neutrophil count fell below 0.5 x 10(9)/l. In a second group of nine patients (group II) reticulocyte fractions and CD34+ cells were measured directly on the days on which they were predicted to increase using the data from group I. Reticulocyte counts and HMFR were also monitored in 18 patients who were mobilized with G-CSF alone. In group I, a significant rise in HMFR and CD34+ cells occurred on days 2, 4 and 6, and a linear correlation between HMFR on day 2 and CD34+ cells on day 4 was demonstrated (P = 0.0068, r = 0.74). In group II similar patterns of recovery were found. During mobilization with G-CSF alone HMFR significantly increased on days 2, 4 and 6 of treatment with respect to baseline values, and a multiplicative relationship between the increase of HMFR and neutrophils was observed (r = 0.707, P < 0.00001). Unfortunately, patients who did not mobilize CD34+ cells (one in groups I and II and three in the G-CSF group) showed similar HMFR kinetics to those who mobilized CD34+ cells. An increase in immature reticulocyte fractions precedes the presence of circulating CD34+ cells by about 2 days in patients mobilized with chemotherapy and growth factors, and it could thus serve as an indirect surrogate marker for monitoring the timing of stem cell harvesting. An unexpected increase of HMFR during treatment with G-CSF alone was found, indicating an effect of this factor on erythropoiesis in vivo.

Flow cytometry analysis of pituitary adenomas.

UNLABELLED: Using flow cytometry, DNA content and index, and/or proliferative capacity (measuring proliferating cell nuclear antigen PCNA) in operated pituitary tumors, control pituitaries obtained at necropsy, and experimental pituitary hyperplasia induced in rats were analyzed. Simultaneous measurement of cell ploidy and proliferation differentiated normal pituitary (diploid DNA index and negative PCNA) from pituitary hyperplasia (diploid DNA index with intensely positive PCNA, between 30 and 72% of cells). In the tumors 83% (19/ 23) were positive for PCNA (between 3 and 84%) and 73% (17/23) aneuploid; only 1 tumor was diploid and negative for PCNA. CONCLUSIONS: Differentiation between normal and abnormal (neoplastic or hyperplastic) pituitary is possible by flow cytometry, but in the adenomas no correlation with postoperative clinical outcome was observed.

In vivo and in vitro flow cytometry comparative analysis of somatostatin-positive cells in the pineal gland of the neonatal rat.

Flow cytometry was used for comparative in vivo and in vitro analysis of cell populations staining positively for somatostatin. Experiments were carried out with pineals obtained from neonatal, 8- and 15-day-old rats. Pineal cells were obtained by dispersion with collagenase and then processed in a flow cytometer or maintained in culture for 1 or 2 weeks. Identification of somatostatin-immunopositive cell populations was performed using a polyclonal somatostatin antibody and confirmed by indirect immunostaining of cytospun smears with the avidin-biotin-peroxidase method. In vivo, the percentage of somatostatin-positive cells was 60.6 +/- 4% in neonatal pineals and declined to 22.2 +/- 11% in 15-day-old animals (p < 0.04). The density of peptide immunostaining decreased in 8-day-old animals but recovered to the neonate levels in 15 day-old animals; homogeneity in the immunopositive population increased with age. Maintenance in culture for 1 week resulted in an increase in positive somatostatin staining in animals of 8 and 15 days with no changes in neonates; however, after 2 weeks of culture, the percent of immunopositive cells decreased from 53.3 +/- 6 to 12.2 +/- 4% in the older animals and remained unchanged in neonates. We conclude that somatostatin is found in pinealocytes and shows a declining pattern during the perinatal period; this probably implies that the peptide plays a paracrine role important for cell differentiation in these young animals, since maximal cellularity and a high mitotic index occur within the first 3 days of life, and pineal cell differentiation is completed before the end of the third week of extrauterine life.

[Intensive chemotherapy with support of peripheral hematopoietic progenitor cells, mobilized with high-dose cyclophosphamide and G-CSF: fast hematologic recovery]. / Quimioterapia intensiva con soporte de células progenitoras hematopoyéticas periféricas, movilizadas con ciclofosfamida a dosis altas y G-CSF: rápida recuperación hematológica.

BACKGROUND: Hematopoietic progenitor cells mobilized to peripheral blood by a chemotherapy combined or not with hematopoietic growth factors and harvested with cyto-apheresis (CTA) provide a rapid hematological recovery when infused as a support step after intensive chemotherapy (IC). METHODS: Cyclophosphamide (CI) 4 g/m2 and G-CSF 5 mcg/kg/d were administered to 19 patients with a solid tumor or lymphoma. Daily CTA were performed during hematological recovery to harvest > 2.5 x 10(6) cells CD34+/kg. Seventeen patients received IC with infusion of peripheral hematopoietic progenitor cells (PHPC) and G-CSF. RESULTS: A total of 52 CTA were performed, with a median (M) of 2 per patient. A M of 4.4 x 10(8) mononucleated cells/kg and 9.8 x 10(6) CD34+/kg were harvested per patient. Hematological recovery after IC with support of PHPC and G-CSF was rapid in all cases, but the aplastic period was shorter in the ten patients who received > 5 x 10(6) CD34+/kg cells than in the seven patients with < 5 x 10(6) kg: The median of recovery to neutrophils > 0.5 x 10(9)/l was 8 days compared with 9 days (p = 0.0005), to platelets > 20 x 10(9)/l of 8 days compared with 12 days (p = 0.001), and to platelets > 50 x 10(9)/l of 11 days compared with 14 days (p = 0.001). CONCLUSIONS: Toxicity of IC (4 g/m2) with G-CSF is moderate and allows the harvesting of an adequate number of PHPC. Its infusion after IC provides a rapid hematological recovery, which was more marked in patients receiving 5 x 10(6) CD34+/kg cells, than with the same IC schedules of IC with autologous bone marrow transplantation.

Ploidy of 36 stromal tumors of the gastrointestinal tract. A comparative study with flow cytometry and image analysis.

DNA ploidy was investigated by flow cytometry (FC) and image analysis (IA) in paraffin-embedded tissue sections from 36 stromal tumors of the gastrointestinal tract. The results of both techniques were correlated with pathologic features of the tumors and survival. Ten (27.8%) tumors were aneuploid by FC and IA. Most of the diploid tumors were identified by both techniques, but FC appeared to be superior to tissue section IA for identification of aneuploid tumors (25% vs. 13.8%). Aneuploidy by FC correlated with pathologic grade and mitotic index (P < .05), and a trend to short survival was also detected (P < .1). No similar correlation was found by IA. Enlargement and variation of nuclei may explain the discrepancy between FC and IA.

Stromal tumours of the gastrointestinal tract: a clinicopathological and ploidy analysis of 33 cases.

The clinicopathological and DNA flow cytometric data of 33 patients with stromal tumours of the gastrointestinal tract (STGIT) were analysed to select pathological features of prognostic value. Tumours had been previously classified as benign (21 cases) or malignant (12 cases). Data relating to poor prognosis statistically were local invasion, pathological grade, size greater than 10 cm, mitotic index (MI) and necrosis. Pathological grade was related to local invasion. Aneuploidy did not correlate with poor survival although a common trend was detected between both. DNA content may help to predict prognosis of STGIT, but its real value has not yet been clearly established. Currently, stage (invasion), size, MI and pathological grade remain the most useful prognostic factors.

[Prognostic value of the changes in the DNA of blast cells from patients with acute lymphoblastic leukemia]. / Importancia pronóstica de las alteraciones en el ADN de las células blásticas en pacientes con leucemia aguda linfoblástica.

The cytogenetic characteristics of 37 patients diagnosed of acute lymphoblastic leukaemia are presented. The studies were performed by cytofluorometry (CFM) after DNA staining with propidium iodide (34 cases) and/or chromosome identification with trypsin G bands (13 patients). Hyperdiploid DNA index was present in 15% of the patients, whereas none of them had hypodiploid DNA index. Abnormal karyotype was found in 69% of the evaluated cases. Good correlation was observed between the ploidy attained by CFM and by karyotyping cells. The highest percentage of aneuploidy corresponded to the L2 morphological subtype (55%), followed by L1 (36%). Structural alterations were the commonest in L2 variant, while numerical ones were commonest in the L1 variant. The 4 L1 patients with aneuploidy had the common immunophenotype, whereas the 6 aneuploidic patients of the L2 variant had common, early pre-B and undifferentiated immunophenotype. The actuarial survival of patients with diploid DNA index was 48.5% (IC 95%, 25-73%), whereas pseudodiploid patients have relapsed and died before 16 months from diagnosis (p less than 0.005). None of the patients with hyperdiploid DNA index and lacking structural alterations has relapsed. Patients with structural abnormalities have the poorest prognosis, while patients with hyperdiploid DNA index showed several favourable risk factors and are in the first complete remission.