Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal-to-ventral and temporal-to-nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Müller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Müller and bipolar cells. Finally, 100% levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Müller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.

Summary:Twenty-nine B cell follicular lymphoma (FL) patients had their BM (n ‫؍‬ 12) or PBPC (n ‫؍‬ 17) purged using a panel of monoclonal antibodies and immunomagnetic beads (IMB). The median recovery of nucleated cells (NC) and CD34 ؉ cells was 59.3% (40.5-74) and 56.1% (30.8-82.9) in BM and 77.2% (64.7-88.3) and 73.5% (61.5-98.6) in PBPC (P Ͻ 0.0005). A median of Ͼ1.62 and Ͼ1.02 log of target cell depletion was achieved as judged by flow cytometry analysis in BM and PBPC, respectively. Of 29% of initial harvests that had a bcl2 PCR-amplified signal, 37.5% became PCR negative in the final purged products. Absorbed cells containing IMB-target cell complexes gave bcl2 rearrangement signal in 20% of samples in which the start and final purged components were negative. Twenty-three of 26 patients receiving an autologous purged product are evaluable for engraftment. Median time to reach an ANC Ͼ 0.5 ؋ 10 9 /l and platelet count Ͼ20 ؋ 10 9 /l was 21 (11-43) and 41 days (13-70) for BM (n ‫؍‬ 9) and 14 (10-31) and 14 (8-37) for PBPC (n ‫؍‬ 14) autografted patients (P ‫؍‬ 0.01 and 0.001). One patient did not engraft and was rescued with a back-up BM. These data demonstrate that this indirect immunomagnetic technique is able to achieve a high grade of lymphoma cell depletion in BM and PBPC and that these purged products are capable of rapid engraftment after autologous transplantation.

Summary:A simplified cryopreservation method for bone marrow (BM) and peripheral blood progenitor cells (PBPC) was utilized in hematopoietic cell transplantation of 213 patients with hematological or solid neoplasms after ablative chemotherapy (187 with peripheral blood progenitor cells and 26 with bone marrow). Cells were cryopreserved, after addition of autologous fresh plasma with DMSO, without HES, by freezing to ؊80؇C in a methanol bath and non-programmed freezer. For the patients autotransplanted with PBPC, the median period necessary for recovery of more than 0.5 × 10 9 /l granulocytes was 11 days (range 6-44), and 15 (8-204) days were required to obtain more than 20 × 10 9 /l platelets. For the patients autotransplanted with BM, the median period necessary to recover Ͼ0.5 × 10 9 /l granulocytes was 12 days (range 9-33), and 24 (12-57) days to obtain more than 20 × 10 9 /l platelets. These results support this method as being very effective in achieving high-quality cryopreservation. The procedure, which uses a non-programmed freezer, simplifies and reduces enormously the cost of the technical measures currently in use, enabling its adoption in almost any clinical oncological institution. Keywords: hematopoietic cell transplantation; cryopreservation; plasma and DMSO; non-programmed freezing; methanolThe transplantation of hematopoietic progenitor cells, derived both from bone marrow and peripheral blood, is an ever more frequent procedure in the treatment of numerous neoplastic diseases. The technology for this type of therapeutic procedure requires, in the majority of cases, the freezing and storage of these cells for variable periods of time. The freezing procedure is traditionally carried out using machinery which incorporates pre-established, comCorrespondence: Dr F Hernández-Navarro, Servicio Hematología y Hemoterapia, Hospital La Paz, Paseo de la Castellana 261, 28046, Madrid, Spain Received 7 July 1997; accepted 21 October 1997 puterized freezing programs. 1-4 Experience using direct or non-programmed freezing systems for hemotopoietic progenitor cells and their transplant is less widespread. [5][6][7][8][9][10][11] In this article, we summarize our experience using a simple, non-programmed freezing method for the transplant of hematopoietic cells in patients with neoplastic disease. Two different in vitro experiments were designed to analyze the reproducibility and the effect of the methanol bath on freezing rates. Finally, a limited comparative study of in vitro cell viability following programmed and non-programmed freezing is also presented. Materials and methods Cooling ratesIn order to analyze the reproducibility of the cooling rates with the methanol system and the impact of this technique on the standardization of the freezing, two different in vitro experiments were performed.In the first (Figure 1), four different bags containing 160 ml of hematopoietic cells in 10% DMSO and autologous plasma were cryopreserved using a mechanical freezer at Ϫ80°C in a methanol bath as previously describe...

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