[Evaluation of bone-marrow cell cryopreservation using long-term cultures]. / Valoración de la criopreservación de células de medula ósea mediante los cultivos celulares a largo término.

PURPOSE: Autologous bone marrow transplantation (ABMT) is frequently used in the treatment of neoplastic diseases. It involves several manipulations of bone marrow cells in vitro that can damage the stem cells responsible for grafting. Long-term marrow cultures (LTBMC) support hematopoiesis in vitro for several weeks. We analyzed the effect of bone marrow cryopreservation on haematopoiesis when using this technique. PATIENTS AND METHODS: 9 bone marrow from healthy donors (Group A) and 15 from patients who were about to undergo ABMT (Group B) were assayed in LTBMC. In all cases, cultures were initiated after cell concentration and also after cryopreservation in group B patients. Adherent cell layer formation, supernatant nucleated cell counts and CFU-GM growth from the non-adherent fraction were assessed. Statistical analysis were evaluated using Wilcoxon test for paired results and Mann-Whitney test for unpaired results. RESULTS: No significant statistical differences were observed when the LTBMC from group A controls were compared to those from group B patients prior to cell cryopreservation. There was a significant statistical difference between cumulative cell recoveries among the cultures developed prior to and after cryopreservation of BM cells from group B patients. CONCLUSION: The results supports the use of LTBMC to obtain information on the extent of the injury of BM cells during manipulations.

Generation of LAK cells in vitro in patients with acute leukemia.

The in vitro stimulation of lymphocytes with interleukin-2 (IL-2) generates lymphokine-activated killer (LAK) cells with tumoricidal potential. In this work we studied the cytolytic capacity of LAK cells in 51 acute leukemia patients in complete remission (CR) after chemotherapy (CT), in 24 acute leukemia patients who had undergone autologous bone marrow transplantation (ABMT), and in a control group of 44 normal donors. In the normal donor control group the effect of non-IL-2-activated peripheral blood mononuclear cells (PBMC) against blast cells was always lower than 10% lysis, which we have taken as a lower limit for positive results. In 95% of post-CT patients, the lytic effect of PBMC was negative. LAK cells produced positive results in 82% of normal donors and in 37.5% of post-CT patients. The effect of PBMC against K562, i.e. natural killer (NK) activity, in post-CT patients as well as in post-ABMT patients was reduced in comparison with the average for normal donors. LAK cells from 25% of post-CT patients had no notable activity against K562 or Raji, nor was there any positive effect against autologous blast cells. In the rest (75%), one-half generated positive activity. We did not observe any correlation between lytic activity in PBMCs or in LAK cells, nor did we observe significant differences between lytic activity in patients with acute lymphoblastic leukemia (ALL) and those with acute myeloblastic leukemia (AML), or between patients who had undergone CT and those receiving ABMTs. These results support the use of IL-2 as a treatment against minimal residual leukemia.

A fully automated method for mononuclear bone marrow cell concentration.

We describe our experience in processing 40 bone marrow aspirates harvested for autotransplantation from patients with several hematological diseases using the CS-3000 blood cell separator. The bone marrow of the first 30 patients was processed by a semiautomated method, and a fully automated procedure was used for the remaining 10 cases. Both procedures were developed in our laboratory and yielded a similar average mononuclear cell recovery of 87.78% and 86.98%, respectively, and similar nucleated cell recovery (27.39% and 27.11%). The cloning efficiency of hematopoietic progenitor cells, measured as the total CFU-GM colony recovery in the in vitro cultures, did not differ between processed and recovered mononuclear cells. On the other hand, all the patients with transplants showed complete hematologic recovery, and the time to engraftment was similar to that described for other procedures. The automated procedure resulted in an average red cell removal of 97.81%, similar to the semiautomated procedure (94.19%), though with a narrower range (96.31-98.6% vs. 80.34-98.34%). The time taken to process a similar amount of bone marrow cell suspension was very different for each method: 1 hour for the fully automated vs. 2 1/2 hours for the semiautomated method to process 1,000 ml. Furthermore, the semiautomated procedure required the addition of homologous or irradiated plasma in a laminar air flow chamber, while the automated method is performed in a closed sterile system. We conclude that our procedure using the CS-3000 processor is an efficient method for fully automated large-scale processing of human bone marrow cells.

[Sudan black B-positive acute lymphoblastic leukemia]. / Leucemia aguda linfoblástica negro sudan B positivo.

Inasmuch as Sudan black B stain is highly specific for myeloid cells and since over 3% positive blast cells meet the needs of diagnosis for myeloblastic leukaemias, we had the opportunity to study a 27 year-old male with acute leukaemia whose morphological, cytochemical, immunocytochemical (HLA-DR, CD-10, CD-19, CD-20, CD-21 all positive) and ultrastructural features were clearly in accordance with acute lymphoblastic leukaemia, except for 42% Sudan black B positivity of the bone marrow blast cells. The practical interest of this case comes from the necessity to take this rare ALL case into account.

Fibrinogen Sevilla, a congenital dysfibrinogenemia characterized by an abnormal monomer aggregation and a defective plasmin lysis.

A dysfibrinogenemia (fibrinogen Sevilla) was detected in a 64-yr-old woman with no previous history of hemorrhagic diathesis or thrombosis. Thrombin and reptilase times were prolonged. The aggregation of fibrin monomers showed a prolonged latency time with a defective slope although fibrinopeptide release and clot stabilization were found to be normal. Plasmin proteolysis was abnormal with a much slower plasmic degradation in patient's purified fibrinogen. By chromatofocussing the patient's fibrinogen showed an abnormality in pattern elution with a second peak eluting at a pH slightly more basic than the normal one (pH 5.5). Likewise, the isoelectrofocussing of purified non-reduced patient's fibrinogen in agarose gel showed an abnormal distribution in its focussed bands, especially in a group which focussed in a pI-interval between 5.20-5.85. By two-dimensional electrophoresis we did not find any abnormality in the fibrinogen-reduced chains. These results could indicate that the abnormal monomer aggregation, as well as the defective plasmin lysis, could be due to conformational aspects of fibrinogen rather than to structural defects.