In Vitro Studies of Bone Marrow in Refractory Anaemia

S ummary . Bone marrows of six patients with refractory anaemia have been studied for their capacity to form colonies in vitro on agar gel medium. They are not capable of forming colonies, and in this they behave as do bone marrows from patients with acute myeloblastic leukaemia in relapse.     

Bone Marrow Studies in Myelomatosis

The percentage of fat-cell areas in bone marrow particles from 22 patients with untreated myelomatosis was estimated. In only 1 patient was the mean fat cell area below 25 % of the bone marrow area measured. A negative correlation was found between the area of fat cells and plasma cells, indicating a displacement of the fat cell area by the plasma cells. 28 % of the patients had empty bone marrow deposits of iron. However, based on a normal iron saturation of S-transferrin and a normal sideroblast count in the bone marrow, the supply of iron to the erythropoiesis was considered sufficient. All patients but one had normoblastic bone marrows. Using a deoxyuridine suppression test in 10 patients, no biochemical defect could be demonstrated. To judge from the correlation coefficient a minor degree (9–14 %) of the variation in Hb values could be predicted from the cellularity in the bone marrow while a major degree (70 %) could be predicted from the renal glomerular filtration rate. The results do not support a displacement of blood-forming elements, iron deficiency, vitamin B₁₂ or folic acid deficiency to be of general significance in the pathogenesis of anaemia, but agrees with a causal relationship between anaemia and renal failure.     

Bone Marrow and Bone Mineral Scintigraphic Studies in Sickle Cell Disease

S ummary . Using discriminating radioisotopic techniques, a consistent pattern of abnormalities of both bone marrow and mineral was demonstrated in a series of patients with acute bone pain of sickle cell disease in which no roentgenographic evidence of infarction or infection was present. Bone scans utilizing reticulo-endothelial uptake of ^99mTc sulphur colloid showed discrete lesions of decreased radioisotopic localization in regions of acute pain suggesting decreased marrow blood flow. In these same areas, ⁸⁵Sr bone scans showed markedly increased uptake whereas ¹⁸F bone scans showed little or no alteration. The strontium findings are consistent with accelerated calcium turnover while the fluorine studies suggest that bone blood flow, in contrast to marrow blood flow, remains nearly normal. In one patient, ⁵²Fe positron scans confirmed an absence of effective erythropoiesis in those areas of involvement demonstrated by ^99mTc and ⁸⁵Sr. Marrow aspiration from one such area demonstrated sterile necrotic bone. These studies demonstrate that acute bone pain of sickle cell disease is associated with bone marrow death and increased bone mineral turnover.     

Progressive Cytopenia Developing during Treatment of Cryptococcosis in a Patient with HIV Infection and Bone Marrow Cryptococcal Infection

Cytopenia is a common complication in patients with human immunodeficiency virus (HIV) infection. Identifying the cause is demanding because of the wide range of possible diagnoses. We herein report an HIV-infected patient with disseminated cryptococcosis involving multiple organs including the blood, brain, lungs, and bone marrow, who developed progressive pancytopenia after initiation of anti-fungal treatment with liposomal amphotericin-B (L-AMB) and flucytosine (5FC). The pancytopenia persisted despite early 5FC discontinuation. A bone marrow biopsy revealed cryptococcal infiltration and the blood examination findings recovered quickly after resuming L-AMB. Thus, this HIV-infected patient''s pathological findings and clinical course suggested that the primary cause of the pancytopenia was bone marrow cryptococcosis.     

The Growth of Bone Marrow Cells in Liquid Culture

S ummary . Liquid suspension cultures of mouse bone marrow cells at high and low density were prepared in supplemented Eagle's medium containing 10% of a partially purified extract of mouse embryos and pregnant mouse uterus (PMU). In the low cell density cultures the number of cells decreased for 2 days; by 4 days the agar colony-forming cells (agar CFC) had risen ten-fold and the spleen colony-forming units (spleen CFU) had fallen to one tenth; between 4 and 8 days the total cell count showed a four-fold increase and the final cell number exceeded the number of the original culture. The cells produced were mainly macrophages. If PMU was not included in the culture the agar CFC disappeared after 4 days and there was no cell multiplication. In the high cell density cultures a similar pattern was observed; in the presence of PMU the agar CFC showed an increase in number, the spleen CFU decreased and an increase in cell number occurred between 4 and 8 days. However, the cells produced were predominantly granulocytes. In the absence of PMU from this cell culture, agar CFC were maintained for 6 days and the cell population remained predominantly granulocytic. These methods of growing cell enable cell recovery from the cultures to be made at any stage and provide an opportunity to study the kinetics and functional capacity of the cells produced.     

Hypothesis: Engrafted Human Bone Marrow and Blood Cells in Culture

Lymphoblasts appearing in immunosuppressed patients after bone marrow transfusion are compared to thosethat can be established in vitro as permanent lymphoid cell lines. It is suggested that Epstein-Barr virus (EBV)could be responsible for the recurrent "lymphoblastic leukemia" inthese patients and that the transplanted cells may be a clone of nonmalignant cells that has becomecapable of growing without normalrestraints. It is important that in futurepatients the transplanted cells be characterized as to morphology, chromosome constitution, relative clonabilityand transplantability, the presence ofEBV, T or B cell-like traits, and theirgrowth potential in immunosuppressedheterologous hosts. The antibody titerto EBV should be measured beforeand after leukocyte transfusion.

     

Ultrastructural Studies of the Bone Marrow in Sickle Cell Anaemia

Marrow aspirates from sickle cell patients were examined without prior deoxygenation and revealed the presence of a variable proportion (10-30%) of sickled red cells and reticulocytes. The main feature of sickled red cells was the presence of 17.6 nm fibres arranged in hexagonal order, and to a lesser extent in square or rectangular array, to form bundles of varying size and compactness which occupied the entire cytoplasm. The sickling pattern in reticulocytes was more variable. Some reticulocytes contained highly-ordered bundles of 17.6 nm fibres whose structure and distribution was identical to that in red cells from whiich they could be distinguished only by their content of organelles. Many reticulocytes exhibited less-organized fibre patterns ranging from localized aggregates to poorly-ordered regions of short fibres and filaments lacking apparent preferential orientation. In these cells, the bulk of the cytoplasm was not polymerized. Haemoglobin polymerization in reticulocytes led to entrapment of ribosomes in concentrated foci among the fibres. Frequently, iron-laden mitochondria were associated with sickled reticulocytes. The variation in pattern of sickling seen in reticulocytes is attributed to possible differences in concentration of Hb S. Correlative studies have shown that fibres were not induced by fixation procedures. Marked phagocytic degradation of sickled cells by macrophages was observed. The results are interpreted to indicate the possible intramedullary phagocytosis of red cells and reticulocytes, predisposed to sickling in the marrow. However, the data are also consistent with the removal of sickled elements from the circulating blood.     

Erythrokinetic Studies in Severe Bone Marrow Failure of Diverse Etiology

1. Erythropoiesis was studied in seven patients with refractory anemia.

2. In all seven patients the total red cell volume was low and the plasmavolume elevated.

3. The serum iron was elevated and iron binding protein saturated in sixof seven patients.

4. The T for clearance plasma Fe⁵⁹ was decreased in one, normal in two,and increased in four patients.

5. The red cell iron turnover was decreased in six of seven patients.

6. Radioiron accumulated in liver and spleen.

7. Red cell life span was difficult to measure but probably shortened.

8. Androgen therapy in two cases was ineffective, and was associated witha slight effect in one patient.

Submitted on April 18, 1961 Accepted on September 14, 1961     

大鼠血管内皮祖细胞培养及鉴定

目的探索骨髓来源的血管内皮祖细胞的分离培养方法,为缺血性疾病治疗找到新的移植细胞来源。方法采集SD雄性大鼠骨髓细胞,用梯度密度离心法分离单核个细胞,种植于提前包埋了纤维连接蛋白的培养皿中培养,将血管内皮生长因子(VEGF)、重组纤维细胞生长因子(bFGF)和重组上皮生长因子(EGF)作为诱导剂,将72h后贴壁细胞(A组)和非贴壁细胞(B组)分别培养,观察细胞形态的变化以及数量变化,取A组在第14天,应用免疫组化和免疫荧光技术鉴定内皮祖细胞系列标志。结果A组细胞经过体外诱导后大量扩增,在28d时仍有强大扩增的活力,呈铺路石样,而B组细胞短期内可以大量增殖,呈长索样,之后逐渐变少;在细胞迁移实验中,A组细胞可以相互融合形成血管结构,而B组细胞之间则不能融合,随着时间推移,逐渐凋亡;A组经过CD34免疫荧光、CD133、FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA-和内吞DIL-Ac-LDL。结论自体骨髓细胞通过贴壁换液后可以分离培养出内皮祖细胞,取贴壁细胞经过体外诱导后大量扩增,并通过表面标记物鉴定可以确认为内皮祖细胞,而非贴壁细胞不能大量增殖,故将贴壁分离方法来筛选内皮祖细胞,此方法简易,并能满足细胞移植的需要,因而,在移植治疗脑缺血引起的内皮损伤应该有较好的临床应用前景。     

Chromosome Studies on Bone Marrow from a Male Control Population

Summary Chromosome analyses of bone marrow cells prepared by the direct method were carried out on 32 male patients, who had no primary haematological disease. One patient was found to have a pericentric inversion of the Y chromosome, which was also present in a skin fibroblast culture and in a lymphocyte culture. Three cases were found to have a high percentage of bone marrow cells with a ₄₅,X chromosome constitution and one case had a ₄₅, X?Y - or G- cell line. The high degree of aneuploidy was not found on examination of cells from leucocyte cultures. This observation is discussed in relation with other similar findings in patients with haematological diseases. The remaining 27 patients had a normal male karyo-type and showed only small deviations from the modal chromosome number. The percentage of bone marrow cells with either chromatid aberrations or stable chromosome abnormalities was not significantly different from previous observations on lymphocyte cultures. However, the proportion of cells with unstable chromosome abnormalities was found to be substantially reduced in comparison with results from cultured lymphocytes examined in their first in vitro division.     

Bone Marrow Necrosis

ABSTRACT Bone marrow necrosis (BMN) is a rare finding in specimens from living patients. It is most commonly found in patients with neoplastic disorders, severe infections and sickle cell disease. We present a patient with Hodgkin's disease who developed extensive BMN 11 months before death. A concise review of the literature is also presented.     

Ways of Expressing Results of Human Bone Marrow Progenitor Cell Culture

SUMMARY In human bone marrow culture studies the number of granulocyte progenitor cells (CFU_c) is expressed as the numbcr of colonies per 10⁵ incubated, nucleated cells. However, this provides little information on cell proliferation kinetics of granulopoiesis. The ratio of progenitors (CFU_c) to end cells (metamyelocytcs or granuloc)-tcs) is related to the effectiveness of granulopoiesis and this allows an estimate to be made of an amplification factor within the maturing and proliferating compartments. We measured an index equal to the number of CFU_c per 10⁵ nietamyelocytes incubated in both normal individuals and in patients. The index falls when a large number of granulocytes is produced by each progenitor cell. This was observed in many patients with a neutropenia. An estimation of the total number of mctamyelocytes is based on the ratio of the number of metamyelocytes to the number of blood granulocytes. This allows an estimate of the total number of CFU_cs, which is of clinical interest.     

Paroxysmal Nocturnal Haemoglobinuria and Refractory Marrow Failure Treated by Marrow Transplantation

S ummary . A patient with pancytopenia and paroxysmal nocturnal haemoglobinuria (PNH) following exposure to insecticide spray developed complete marrow failure after inhalation of vapours containing benzol. There was no sign of spontaneous recovery after more than 6 mth of conventional and supportive therapy. The patient was treated with the immunosuppressive agent cyclophosphamide, 50 mg/kg on each of four days, followed in 36 hr by transplantation of marrow from a sibling compatible at the major human histocompatibility locus (HL-A). Intermittent methotrexate therapy was given for 102 days after grafting to prevent graft-versushost disease. The patient showed prompt haemopoietic engraftment indicated by restoration of marrow cellularity and a rise in peripheral blood cell counts beginning on day 11 after the graft. The patient is alive and well with normal haemopoietic function and continued absence of PNH more than 1 yr and 4 mth after transplantation.     

Therapy in Cytopenia

Abstract. Intravenous immunoglobulin (IVIG) may be considered first-line maintenance therapy for idiopathic thrombocytopenic purpura (ITP) because it has been proven to be the least toxic. In a study of 25 children with acute ITP, treatment with IVIG maintained platelet counts above 40,000/mm³ in all of the children. After 1 year, none of these patients required further therapy. In another study group of 25 pediatric patients with chronic ITP, treatment with IVIG circumvented splenectomy in 60% of the cases. The therapeutic regimens for adults and children are described, as is a strategy to overcome IVIG resistance. Experiences with IVIG in hemolytic anemia and neutropenia are discussed. The mechanism of action is explored in some detail, specifically as it relates to reticuloendothelial system (RES) Fc receptor blockade and suppression of antiplatelet antibody synthesis.     

Studies of the Reticuloendothelial Mass and Sequestering Function of Rat Bone Marrow

Quantitative studies were made in rats of the total reticuloendothelial cellcontent of bone marrow and of the ability of marrow to sequester antibody-treated red cells, immature red cells and carbon particles. Although normalmarrow played a relatively minor role in clearing cells and particles from theblood stream, its clearing activity increased markedly when the marrow wasdepleted of its blood-forming cells. The greatly enhanced ability of hypoplasticmarrow to trap immature red cells suggests that this may be a homeostaticmechanism for repopulating the depleted marrow.

Carbon blockade resulted in decreased liver and spleen uptake of sensitizedred cells, but enhanced the marrow uptake. This suggests that marrowsequestration of cells, in contrast to that in the liver and spleen sequestration,is not affected by phagocytic activity.

In their functions and reactions the RE cells of the marrow usually actedin parallel with those of the liver and reciprocally with those of the spleen.

Submitted on July 17, 1964 Accepted on December 20, 1964