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.     

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.     

Frontiers in bone marrow transplantation

The early murine experiments and human studies that indicated the potential of marrow transplantation are reviewed. The results of marrow grafting for a variety of human diseases are summarized. Current directions of research are indicated.     

Gene-transfer into bone marrow cells

We have intended to improve gene-transfer technique into hematopoietic stem cells for somatic gene therapy. 1) We have developed a new packaging cell line, ampGPE for retroviral production. LTR-less gag, pol or env genes from Moloney murine leukemia virus were separately inserted into BMGNeo vector. Packaging cell lines containing 20-50 copies of these two kinds of plasmid were obtained. Retrovirus stock for gene-transfer have been produced at a high titer (10(5)-10(6) cfu/ml) and without replication-competent viruses by using ampGPE. 2) Retrovirus-transduced murine CFU-GM have been found to selectively proliferate (5-10 fold/week) in liquid capture with recombinant murine IL-3, human IL-6 and G418 to consequently obtain enough amount of, highly concentrated (70-100%), and gene-transferred murine CFU-GM for gene-delivery system.     

Antioxidants for bone marrow cryopreservation

Addition of antioxidants into the preservation solution improved the cryoprotection of human bone marrow cells. The viability was studied by the growth of GM-CFC in agar culture before and after storage at -196 degrees C. All used antioxidative drugs (selenomethionine, methionine, tocopherol, penicillin/Fe++) increased the tolerance of the stem cells to freezing and thawing and elevated the number of surviving GM-CFC up to the twofold in comparison with that of controls. More immature colony forming cells were especially protected.