Bone marrow cryopreservation and clinical implications in autologous bone marrow transplantation

A simple, rapid and effective technique using the IBM (Cobe)-2991 cell processor for the concentration of buffy coat cells from large volume marrow has been well adopted (n = 16). Only about one-eighth of the original volume was obtained while retaining more than 90% of the total nucleated cells to be cryopreserved in polyolefine bags with TC-199 culture medium and final 10% dimethylsulfoxide (DMSO) (n = 9), processed by a computerized Nicool ST-20 (France) programmed freezer and stored in a vapor phase of liquid nitrogen at -196 degrees C. Stem cell assay by CFU-GM after thawing yielded a mean of 50.39 +/- 19.54% which has been satisfactory for clinical implementation. So far, three cases with hematological malignancies had been rescued by autologous cryopreserved marrow after supralethal doses of chemoradiotherapy. Two patients with acute nonlymphocytic leukemia transplanted in 1st remission as of Oct. 31 had been disease free for 178+ and 157+ days, respectively, after transplant which was taken at the corresponding age of 53 and 42 years. The other patient who was a victim of Hodgkin's disease, stage IV, and was transplanted in 3rd remission, expired on the 59th day because of the complication of idiopathic interstitial pneumonitis despite excellent granulocytopoietic reconstitution. The preliminary results are encouraging for further exploitation, especially for those who would otherwise be candidates for allogeneic bone marrow transplantation but are limited by age or lack of an HLA-identical sibling to serve as marrow donors.     

No narcosis for bone marrow harvest in autologous bone marrow transplantation

A prospective study with mild general analgesia and sedation together with local anesthesia during bone marrow harvest was performed. Thirty-one patients underwent 33 bone marrow collections. Pretreatment consisted of 100 mg meperidine i.m. and 20 mg diazepam i.m. 1 h before start of procedure. Eight patients got additional meperidine and diazepam during the procedure, all patients got lidocaine 1% locally. A mean volume of 1.321 was obtained with 42.5 punctures. Twenty-two patients had no complications, 4 vomited, 4 had easily correctable hypotension of short duration, one got oxygen for cyanosis of short duration. Acceptance was good in 23 patients, in 6 reasonably well, in two bad. Only one patient experienced pain problems, due to suction. Anxiety was no major problem due to good information before the procedure and mild sedation. This form of anesthesia for bone marrow collection is a safe procedure, it is generally well accepted by the patient and it can be performed on an out-patient basis.     

Bone marrow interferon]

The nucleated cells of the bone marrow of mouse, rat, guinea pig, chick, cattle and humans proved to be capable of producing interferon in vitro following induction with the Newcastle disease virus. The production of interferon by these cells was characterized by high stability. The bone marrow interferon was not inferior in its activity to the corresponding interferon prepared with the blood leukocytes or splenic cells.     

Cryopreservation of marrow, purging and autologous bone marrow transplantation in childhood

Since 1984 bone marrow from 42 children with acute lymphoblastic leukaemia, non Hodgkin's lymphoma and neuroblastoma was cryopreserved. In 5 cases (c-ALL, NHL and B type) the marrow was purged by using a cocktail of three monoclonal antibodies (VIL A1, VIB C5, VIB-E3). Up to now 13 children (ALL/10, neuroblastoma/3) were autografted (one of them after purging) after supralethal chemoradiotherapy. Except one child with early death all patients had engraftment: a level of 1.0.10(9)/l leukocytes was reached at days 10-33 (median, 19); platelet level over 60.10(9)/l at days 32-60 (median, 41). 2 children died on treatment related complications, one on infection after full haematological restitution, 2 patients alive with relapse, 8/13 alive in CCR and well.     

Iron in bone marrow

In the bone-marrow, non-haemoglobin iron can predominantly be found in the reticulum. Slight granules containing iron can also be observed in parts of erythroblasts by means of the Berlin blue reaction. These cells are called sideroblasts. In chemical respect, non-haemoglobin iron consists of ferritin soluble in water and haemosiderin insoluble in water. Erythroblasts will only take their iron from plasma transferrin. For the most part, this iron uptake is being regulated by erythropoietin adapting erythropoiesis to the oxygen requirements of the tissue. The iron contained in erythroblasts is predominantly utilized for haemoglobin synthesis in these cells. A slight part is being taken up by ferritin. The bone-marrow reticulum will phagocytise aged erythrocytes and store liberated iron as ferritin and haemosiderin. Part of the iron is being delivered again to plasma transferrin. With constant serum iron level the liberation of iron from the reticulo-endothelial tissue must correspond to the iron uptake by erythropoiesis. The absence of iron capable of being coloured in the bone-marrow reticulum is considered to be a reliable parameter of iron deficiency. It enables the diagnosis of iron deficiency anaemia to be made even in those patients with serum iron level and a total iron binding capacity lying within the normal range and no hypochromia of erythrocytes being present. It enables iron deficiency anaemia to be separated from sideropenic anaemia with reticulo-endothelial siderosis in differential-diagnostic manner. Even in patients with sideroblastic anaemia, iron colouring of bone-marrow smears is required for ensuring the diagnosis. Recently, a separation has also been made for idiopathic anaemia with abnormal sideroblasts. In these patients there is an increased risk for acute leukemia to develop.     

Elimination of T lymphocytes from the bone marrow in HLA-incompatible bone marrow transplantation

The method presented is very well suited to eliminate T-lymphocytes from great amounts of bone-marrow. The stem cells required to reconstitute the bone-marrow are enriched in this way. It can be completely performed in a closed system. Any contamination with germs is excluded. It can be reproduced well and learnt quickly. It takes 10 hours for two trained co-workers to process 1,500 ml of bone-marrow. The vitality of cells is very good (100%). Its suitability for transplantation has still to be checked.