CFU-mix are no better than CFU-GM in predicting hemopoietic reconstitutive capacity of peripheral blood stem cells collected in the very early remission phase of acute nonlymphoblastic leukemia.

High levels of circulating myeloid progenitor cells (CFU-GM) occur during the very early remission phase of acute nonlymphoblastic leukemia (ANLL). Autologous stem cell rescue using blood cells collected during this phase has shown that successful hemopoietic reconstitution can be achieved, but a higher CFU-GM dose appears to be required than when bone marrow cells are used. This suggests that during very early remission, the level of marrow repopulating pluripotent stem cells (PSC) in blood does not undergo the same amount of increase as does the CFU-GM. This study set out to determine whether the levels of the multilineage progenitor cell (CFU-Mix) would be better indicators of the PSC in these cells than the CFU-GM. Serial peripheral blood CFU-Mix and CFU-GM measurements were carried out in six ANLL patients during very early remission. The levels of peripheral blood CFU-Mix showed a mean 12-fold increase, as compared to a mean 20-fold increase in the CFU-GM. The timing of the increase in the CFU-Mix paralleled that of the CFU-GM. These findings suggest that the CFU-Mix is no better than the CFU-GM in predicting PSC levels during very early remission of ANLL, and is closer to the CFU-GM than to the PSC in ontogeny.

Bone marrow biopsy during induction chemotherapy for acute myeloid leukaemia identifies only 50% of patients with resistant disease.

A study was carried out to determine whether bone marrow biopsy performed on day 6 of induction therapy for acute myeloid leukaemia (AML) can identify those patients with resistant disease who would need an intensification of the first course of induction. Bone marrow biopsies were performed on day 6 of induction chemotherapy in 44 patients with AML treated with daunorubicin, cytosine arabinoside and thioguanine. Biopsies were assessed for blast count, trephine cellularity and leukaemic index. Discrimination between patients who went on to achieve remission and those with resistant disease was best achieved using the reduction in bone marrow cellularity from pretreatment marrow to day-6 marrow. However, this discriminator identified only 50% of the patients with resistant disease and included 13% of patients who achieved remission with the first course of chemotherapy. The other parameters of response were even less effective at discriminating between chemotherapy-resistant and chemotherapy-responsive disease.

Peripheral blood stem cells collected in very early remission produce rapid and sustained autologous haemopoietic reconstitution in acute non-lymphoblastic leukaemia.

Haemopoietic reconstitution was achieved in a patient with acute non-lymphoblastic leukaemia (ANLL) in relapse who was autografted with blood-derived stem cells collected during very early remission. The patient received a myeloid progenitor cell dose of 230 x 10(4) CFU-GM/kg body weight. Engraftment was evident in the bone marrow 7 days post-graft. Normal neutrophil and platelet counts were attained by day 14 and blood counts remained normal thereafter. An overshoot in peripheral blood haemopoietic progenitor levels occurred at the end of the second week, presumably the progeny of a family of early progenitor cells. The completeness of haemopoietic reconstitution is further illustrated by the satisfactory nucleated cell and myeloid progenitor cell yield when a bone marrow harvest was performed 4 1/2 months post-graft. Seven months post-graft, the patient remained in complete remission with normal blood counts and bone marrow cellularity, although haemopoietic progenitor levels were slightly reduced. The rapid recovery minimises aplasia-related risks and suggests that such autografting can be carried out safely in first remission. We propose that autografting using very early remission blood cells is a new therapeutic option for patients with acute ANLL.

The expression of mature myeloid cell differentiation markers in acute leukemia.

Cells from 82 patients with leukemia in acute phase (40 ANLL, 1 AUL, 36 ALL, 5 CGL in blast crisis) were studied for the expression of mature cell markers of the major nonlymphocytic cell lineages (monocytes, granulocytes, erythrocytes and platelets) using monoclonal antibodies. In addition, cells were examined for the presence of HLA-A, B, C antigens, Ia antigens and common ALL antigen, as well as Fc receptors capable of binding murine immunoglobulins. Approximately one-third of ANLL specimens lacked any of the mature-cell differentiation markers studied. These were always in the relatively undifferentiated morphological subgroups (M1 and M2). Some of the specimens in these groups also expressed little or no HLA-A, B, C and/or Ia antigen. Of the lineage-specific MAb, FMC32 and FMC34, which bind to monocytes, and monocytes plus granulocytes respectively, gave the most interesting results. Together with the anti-CALLA antibody J5, they contributed to the differential diagnosis of ANLL and ALL. In addition they detected phenotypic heterogeneity within the FAB types of ANLL, particularly the M1 and M2 groups. Binding of murine IgG2a and IgG3 antibodies, apparently via Fc receptors, was commonly observed with ANLL cells. This is a potentially serious source of "false positives" in studies using murine MAb with human leukemic cells.

Changes in serum ferritin levels after intravenous iron.

The effect of intravenous iron dextran on serum ferritin levels was observed in two patients with iron deficiency anaemia. Serum ferritin levels rose sharply and reached peak levels seven to nine days after infusion when at least 90% of the infused iron had been removed from the plasma. A linear relation was shown for each patient between the logarithm of the serum ferritin levels and the logarithm of the calculated cellular non-haem iron levels.

The relationship of hookworm infection, anaemia and iron status in a Papua New Guinea highland population and the response to treatment with iron and mebendazole.

In 345 apparently healthy Papua New Guinea male subjects, predominantly highlanders, 89% of whom were infected with hookworm (Necator americanus), there was a statistically significant inverse correlation of hookworm egg count with haemoglobin and serum ferritin level, but no significant correlation with serum albumin, folate or B12 values. A sub-group of 128 was chosen for a six-month study on the effect of treatment with the anthelmintic mebendazole and/or parenteral iron on haemoglobin and serum ferritin levels. Mebendazole-treated subjects remained worm-free and the hookworm egg counts of the controls decreased during the study period. Parenteral iron treatment had the expected effect of raising haemoglobin to a normal level. There was a statistically significant improvement in haemoglobin level in all treated groups but not in the control. Serum ferritin levels decreased significantly in all groups, but more in the control than in the treated groups, although treatment groups were not significantly different. Although probable inadequate uptake of iron by the subjects and blood donation by some subjects was apparently more detrimental to iron status than hookworm infection, the results of this study support the view that hookworm infection in this country contributes to lowered haemoglobin levels and iron status.

Some haematological and physical characteristics of a group of Papua New Guinea highlanders.

Full blood counts and some serum biochemistry were carried out on 349 male and 22 female apparently healthy Papua New Guinea subjects, most of whom were highlanders. In males, RBC, Hb, PCV, MCH, MCHC, serum albumin, ferritin, iron, per cent transferrin saturation and folate means were significantly lower than the Australian normal means; WBC and MCV means were similar to Australian values; and eosinophil and reticulocyte counts and serum transferrin and vitamin B12 means were higher than Australian means. In females, WBC, RBC, Hb, PCV, MCV, MHC, MCHC and serum ferritin means were lower than Australian means; eosinophil counts, serum iron, per cent transferrin saturation, and folate means were similar to Australian means; and serum transferrin and B12 means were higher than the Australian means. Hepatomegaly and mild splenomegaly were common. beta-Thalassemia was present in three subjects two of whom were from Simbu Province.

Adult acute lymphoblastic leukaemia: a five-year experience.

The case histories of all patients for whom the diagnosis of acute lymphoblastic leukaemia (ALL) was made over a five-year period at the Royal Adelaide Hospital and Institute of Medical and Veterinary Science were studied. There were 34 patients with a median age of 47.5 years. Adequate follow-up was obtained on 33 patients, and their median survival was 5.4 months. The remission rate was 77% in the under-30 years age group, but only 15% in the over-30 years age group. The median duration of remission was 24 months while the median survival of remitters has not yet been reached. The median survival of non-remitters was two months with 100% being dead at 12-3 months. Of 20 patients without remission, seven died of haemorrhage and/or infection before an adequate trial of therapy had been completed, while nine patients had "adequate" intensive therapy but failed to achieve remission.

An abnormality of the bone marrow associated with vitamin E deficiency in sheep.

1. Sheep fed on a maintenance ration of wheaten-hay chaff or of wheaten-hay chaff-lucerne-hay chaff (I : I, W/W) became deficient or incipiently deficient in vitamin E. 2. Degenerative changes were observed in bone marrow and muscle, and liver function was imparied in some animals. These abnormalities were not influenced by the vitamin B12 status of the animals or by a shortage of cobalt in the rumen. 3. Plasma ascorbic acid levels may not have been optimum, and folic may not have been fully utilized by some sheep. 4. Liver function responded faily rapidly to alpha-tocopheryl acetate, but skeletal muscle had not returned to normal after 28 weeks of treatment. A variable trend towards normal cellularity was found in bone marrow following supplementation with alpha-tocopheryl acetate. 5. A secondary deficiency or, alternatively, inefficient excretion or metabolism of a toxic material, may occur in vitamin E deficiency as a result of degenerative changes in the absorptive or excretory areas of the intestinal tract and be responsible for the bone marrow abnormality.

The clinical value of the radioassay of serum folate.

Measurement of serum folate levels in humans using a radioassay has now been frequenly reported. However, very little data is available assessing the diagnostic value of this test in comparison with other laboratory and clinical information pertaining to folate status. A radioassay, using the folate binder in dried milk was used in parallel with the well established Lactobacillus casei microbiological assay. Three slightly different modifications of the radioassay were tested. At first, a partially purified milk binder was used, in the second the unpurified milk and, third, the unpurified milk with variation of the incubation periods during the assay, were utilized. A total of 159 sera were tested by one of the modifications of the radioassay as well as by the L. casei method. Duplicate estimations were done in all cases. Using the first method, the correlation between the radio- and microbiological assays was 0.89, with the second 0.52, and with the third 0.85. The final assay system gave the most reliable results when compared to the L. casei assay which was taken as the standard of reference. However, in a number of cases, normal levels by L. casei gave low levels by radioassay and vice versa. In 13 such instances, clinical and other laboratory data were assessed and it was concluded that the microbiological assay gave the more accurate indication of folate stores in humans. Some reasons for the discrepancies between the assay systems were discussed.

Diffuse histiocytic lymphoma: the 20-year experience of an Australian teaching hospital.

There is a paucity of information regarding the natural history and treatment outcome of diffuse histiocytic lymphoma (DHL) in Australia. Case records from 80 patients treated for DHL at the Royal Adelaide Hospital between 1965 and 1985 were reviewed to determine treatment outcome and prognostic information. Pathological review of biopsy specimens confirmed the correct diagnosis in 78 patients. The Ann Arbor staging criteria were unsatisfactory for prognostic purposes. We identified three prognostic groups: Localised disease (82% five-year survival), Advanced disease marrow negative (36% five-year survival), and Advanced disease marrow positive (11% five-year survival). An elevated plasma lactate dehydrogenase (LDH) and calcium (Ca++) predicted a poorer outcome; no patient with a LDH greater than 500 IU achieved longterm survival (p less than 0.001). Survival was identical for patients reclassified histologically as intermediate grade or high grade (Working Formulation). Localised disease was associated with a good prognosis (82% five-year survival) regardless of treatment modality. The outcome of patients with advanced disease has markedly improved over the last two decades, particularly with the introduction of combination chemotherapy containing doxorubicin in 1974 (p less than 0.005). Using these regimens, complete remission was achieved in 65% of patients, with a 39% five-year survival.

Urinary ferritin protein in subjects with prosthetic heart valves and other disorders.

Twenty-four-hour urine ferritin protein excretion (UFPE) was elevated in the great majority of patients with prosthetic cardiac valves. In this group, there was a positive correlation with the presence of haemosiderin in the urine and with another indirect measure of reduced red-cell survival, namely, serum lactic dehydrogenase. In a number of hospital patients with various disorders, UFPE was also increased. In this latter group, however, there was no constant accompanying haemosiderinuria or elevation of lactic dehydrogenase. The measurement of UFPE can be added to the list of screening tests for intravascular haemolysis, but analysis of the different isoferritins in urine is necessary to fully understand the mechanism of excretion in the different groups.

The effect of monocytes in the peripheral blood CFU-C assay system.

The effect of cellular interactions in the in vitro assay of myeloid progenitor cells in peripheral blood (PB CFU-C) was investigated. Ficoll-Paque-separated peripheral blood mononuclear cells (PB MNC) from 7 healthy subjects were cultured at cell concentrations from 10 to 0.625 X 10(5) MNC/plate in doubling dilutions. The number of colonies per 10(6) lymphocytes plated (corrected colony count, CC) was significantly higher when 2.5 X 10(5) or less PB MNC were cultured than when 5 or 10 X 10(5) cells were cultured. This decrease in CC when large numbers of cells were cultured was not present when the nonadherent cells only were cultured. The inhibition was reproduced when adherent cells were added back to the nonadherent cells. The inhibition appeared to be proportional to the number of monocytes present. A model depicting the role of monocytes in the PB CFU-C assay system is presented. The increased understanding of cellular interaction represents an important step towards the standardization of the PB CFU-C assay.

High levels of circulating haemopoietic stem cells in very early remission from acute non-lymphoblastic leukaemia and their collection and cryopreservation.

Circulating myeloid progenitor cells (PB CFU-GM) were measured in the peripheral blood of 13 patients with acute non-lymphoblastic leukaemia (ANLL) as they entered first remission. The mean of the recorded peak levels was 2796 X 10(3) CFU-GM/l, representing a 25-fold increase above the mean value in normal subjects. These elevated levels of PB CFU-GM occurred regularly during the very early remission phase when platelet counts rose rapidly. Five of the patients had PB mononuclear cells collected by continuous-flow leukapheresis during this early recovery phase. CFU-GM were assayed as a measure of the number of haemopoietic stem cells in each collection. The cells were concentrated and then cryopreserved in liquid nitrogen. Leukapheresis was also performed on five normal subjects for comparison. Low numbers of CFU-GM were harvested from normal subjects, mean 0.33 +/- 0.06 X 10(4) CFU-GM/kg body weight for each leukapheresis. In ANLL patients entering remission, however, very large numbers of CFU-GM were regularly harvested. A mean of 11 +/- 2 X 10(4) CFU-GM/kg body weight were cryopreserved after each leukapheresis, representing 5 times the number of CFU-GM considered necessary for successful autologous haemopoietic reconstitution. Haemopoietic stem cell viability was assessed after varying periods of cryopreservation. There was no significant stem cell loss after up to 24 months storage. Thus, it is possible to collect and cryopreserve large numbers of CFU-GM and by inference pluripotent haemopoietic stem cells from the peripheral blood of patients with ANLL during very early remission. The possible biological and therapeutic implications are discussed.

Circulating autologous stem cells collected in very early remission from acute non-lymphoblastic leukaemia produce prompt but incomplete haemopoietic reconstitution after high dose melphalan or supralethal chemoradiotherapy.

Haemopoietic reconstitution (HR) using autologous peripheral blood stem cells (PBSC) was attempted after intensive chemotherapy or chemoradiotherapy in two patients with relapsed acute non-lymphoblastic leukaemia (ANLL). The PBSC were collected by leukapheresis very early in first remission and cryopreserved in liquid nitrogen. Both patients demonstrated early evidence of trilineage engraftment. The first patient received melphalan 200 mg/m2 followed by rescue with 1.3 X 10(8) mononuclear cells/kg body weight containing 29 X 10(4) granulocyte-macrophage progenitor cells (CFU-GM)/kg, and HR was evident by Day 14. The second patient was treated with supralethal chemoradiotherapy followed by rescue with 3.0 X 10(8) mononuclear cells/kg containing 23 X 10(4) CFU-GM/kg. He demonstrated early engraftment with near normal peripheral blood counts by Day 16. There was a subsequent fall in both bone marrow cellularity and peripheral blood counts to a level of low but persistent activity. There was a further phase of haematological recovery from 8 weeks following transplantation with an increase in peripheral blood counts and bone marrow cellularity until final relapse at 13 weeks. This study demonstrates that circulating stem cells have haemopoietic reconstitutive capacity, previously only shown with buffy coat cells from chronic granulocytic leukaemia. The minimum number of PBSC required for satisfactory engraftment remains unknown, although it seems probable that the ratio of pluripotent stem cells to committed progenitor cells is lower in very early remission peripheral blood than in either allogeneic normal bone marrow or autologous bone marrow collected later in stable remission. The question of leukaemic contamination of the PBSC remains to be answered.

Successful peripheral blood stem-cell autograft with a near-critical dose of myeloid progenitor cells in acute non-lymphoblastic leukaemia in relapse.

Rapid, complete and sustained haemopoietic reconstitution was achieved in a 69-year-old man with acute non-lymphoblastic leukaemia in relapse who received an autograft of peripheral blood cells that were collected during very early remission. The patient received 1.7 X 10(8) nucleated cells/kg bodyweight containing 63 X 10(4) myeloid progenitor cells (CFU-GM)/kg bodyweight. Trilineage engraftment was evident in the bone marrow seven days after the graft. Normal neutrophil and platelet counts were attained by day 17, on which day the patient was discharged from hospital. He remained in complete remission three months after the graft with normal blood counts and bone-marrow cellularity. The rapid and sustained haemopoietic activity in this patient, in conjunction with our previous experience of four other patients who received autografts with peripheral blood stem cells, supports the concept we have proposed that a minimum CFU-GM dose of 50 X 10(4)/kg bodyweight produces complete and sustained engraftment. The rapid recovery minimizes aplasia-related risks and suggests that such autografting can be carried out safely in first remission even in older patients. This technique should be considered as a new therapeutic option for patients with acute non-lymphoblastic leukaemia.