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Previous studies have shown that enzyme supplementation therapy with alglucerase for type 1 Gaucher's disease is effective at doses of 30-130 U/kg per month. Since both the clinical presentation and the response to therapy in Gaucher's disease are highly variable, individual dosing seems indicated. This notion, as well as the high costs of alglucerase and the unknown long-term side-effects, led us to investigate the efficacy of an individualised very low dose of alglucerase. Twenty-five adults with symptomatic type 1 Gaucher's disease (thirteen splenectomised) received alglucerase 1.15 U/kg three times a week (15 U/kg per month). Every 6 months, the dose was halved, maintained, or doubled, according to the response (based on haematological variables and liver and spleen volume). After 6 months of treatment, eighteen (72%) patients had a response (seventeen moderate, one good). After 12 months (in nineteen patients) and 18 months (in seven patients), all had sustained improvement. Severe splenomegaly resulted in slower haematological responses. Our results are similar to those obtained by others with higher-dose regimens and better than a low-dose regimen of 10U/kg every 2 weeks. We conclude that very low initial doses of alglucerase, when administered frequently, are effective and cost-saving in the treatment of type 1 Gaucher's disease.

The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization, the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody, washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%, which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords, there is an overall increase in sensitivity of three- to fourfold, which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated, and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity, the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated, normal, and even subnormal Ep levels in human serum and plasma.

Serum ESF titers were measured in 42 polycythemic patients using the fetal mouse liver cell bioassay. ESF titers in patients with secondary polycythemia differed significantly from those in patients with polycythemia vera (p less than 0.0001). Among the 21 patients with secondary polycythemia, 1 patient had an ESF titer less than 10 mU/ml (the lower limit of sensitivity) and 20 had ESF titers that ranged between 11 and 112 mU/ml, with a mean titer of 56 mU/ml. Among the 21 patients with polycythemia vera, 13 patients had ESF titers less than 10 mU/ml and 8 had ESF titers ranging between 12 and 55 mU/ml, with a mean titer of 26 mU/ml. The mean hemoglobin concentration in the 8 patients with ESF titers greater than 10 mU/ml was significantly below that in the 13 polycythemia vera patients with ESF titers less than 10 mU/ml (p less than 0.03). If ESF titers less than 10 mU/ml had been indicative of polycythemia vera and ESF titers greater than 10 mU/ml had been indicative of secondary polycythemia in patients with hemoglobin concentrations greater than 17.7 g/dl, but not indicative of either condition in patients with hemoglobin concentrations less than 17.7 g/dl, 71.5% of the polycythemic patients in this study would have been diagnosed correctly, 9.5% incorrectly, and in the 19% the diagnosis would have remained uncertain. It was concluded that measurement of serum ESF titers using this in vitro bioassay can be of clinical importance in differentiating between polycythemia vera and secondary polycythemia.

We studied the clinical, hematologic, cytogenetic and molecular biologic features in four patients with Philadelphia (Ph) negative chronic myeloid leukemia (CML). In all four cases the clinical and hematologic characteristics were indistinguishable from Ph positive CML. Cytogenetic analysis showed a normal karyotype in two patients and chromosomal translocations apparently not affecting chromosome 22 in the other two cases. Southern blot analysis using probes of the bcr region, demonstrated a bcr break-point in all four patients. In situ hybridization with bcr, c-abl, and c-sis probes showed unusual hybridization sites for 5'-bcr and c-abl indicating complex chromosomal rearrangements affecting three different chromosomes in the four patients investigated. Using polymerase chain reaction (PCR) followed by hybridization to oligonucleotide probes specific for the bcr-abl fusion region, the expression of a chimeric bcr-abl mRNA was detected. In these patients we demonstrated that (a) CML with a breakpoint in the bcr region without cytogenetically detectable Ph chromosome is characterized by the same genomic recombination of 5'-bcr and c-abl as CML with standard Ph translocation and (b) unusual localization of 5'-bcr and c-abl sequences caused by complex Ph translocation does not interfere with transcription of the bcr-abl fusion gene.

The commercially available hemagglutination inhibition (HAI) assay kit for erythropoietin (ESF) was compared with the fetal mouse liver cell (FMLC) bioassay. No correlation was obtained ESF levels determined by both methods in a variety of pathologic sera. The HAI kit showed a great batch variability. Significant immunoreactivity was found in those fractions of a normal human serum and a human urinary ESF preparation that were not active in the FMLC bioassay. A very poor recovery of immunoreactivity was found when the international reference preparation for erythropoietin (second IRPE) was added to a normal human serum.

Erythropoietin (ESF) titers were determined in sera from patients with different types of anemia using the fetal mouse liver cell bioassay. An inverse relationship was found between hemoglobin concentration and ESF titer. However, ESF titers differed markedly between patients at comparable degrees of anemia. Several groups of patients were distinguished on the basis of the activity of their erythroid bone marrow. In each of these groups, a significant negative correlation was found between the hemoglobin concentration and the logarithm of the ESF titer. ESF titers in patients with pure red cell aplasia were fourfold higher than those in patients with iron-deficiency anemia and tenfold higher than those in patients with megaloblastic anemia and homozygous sickle cell anemia at comparable hemoglobin concentrations. Following the initiation of specific therapy in patients with pernicious anemia and patients wit iron-deficiency anemia, serum ESF titers were found to decrease prior to any substantial rise in hemoglobin concentrations. In the patients with pernicious anemia, the lowest ESF levels were found 1 day after administration of vitamin B12, whereas in the patients with iron-deficiency anemia, the lowest ESF levels were reached in the second week of oral iron therapy. ON the basis of these data it was concluded that serum ESF titers in anemic patients are not only inversely related to the hemoglobin concentration but also to the activity of the erythroid bone marrow.

A modification of the mouse fetal liver cell bioassay for erythropoietin (ESF) that allows quantitative detection of ESF in human serum is described. The modification consists of (1) correction for the effect of serum iron on 59Fe incorporation into heme and (2) the use of an "internal standard," i.e., a standard ESF preparation dissolved in the assayed test serum. As a result of this modification the statistical method of bioassay analysis had to be changed fundamentally. The mean serum concentration of ESF measured in 20 normal males was 48 microunit/ml, as compared to 29 microunit/ml in 18 females. The difference was significant at p = 0.017. The stimulatory activity of a human serum on 59Fe incorporation into heme could be neutralized by an anti-human ESF immune serum.

Investigations on the mouse fetal liver cell bioassay for erythropoietin (ESF) have revealed that iron present in test sera significantly dilutes the radiolabel (59Fe) and thus decreases 59Fe incorporation into heme. A method of correction for the influence of iron on the dose-response relationship of human sera is presented. Application of this method made it possible to assay human sera up to culture concentrations of 150 microliter/ml. It was shown that a corrected serum dose-response curve showed parallelism to the curve of an ESF standard preparation. This suggests similarity of the active principles and allows valid estimation of a potency ratio.

ESF titers were determined in 99 patients of various stages of chronic renal failure, by using the fetal mouse liver cell bioassay. Of these patients 45 were receiving conservative therapy and 54 on maintenance hemodialysis. ESF levels were significantly below normal in both groups of patients. A significant inverse relationship was found between hemoglobin concentration and ESF level in the predialysis patients with chronic glomerulonephritis. No correlation was found between both parameters in the predialysis patients with chronic nonglomerular renal disease. A significant positive correlation was found between hemoglobin concentration and ESF level in nephric dialysis patients who were transfusion independent. Transfusion-dependent nephric dialysis patients had lower hemoglobin concentrations and lower ESF levels before transfusion than did nephric dialysis patients who were transfusion independent. In nephric dialysis patients ESF levels fell sharply after blood transfusion, whereas in anephric dialysis patients such a physiologic ESF response was not found. It was concluded that despite the presence of an absolute ESF deficiency in all anemia uremic patients, this anemia cannot be explained by ESF deficiency alone. The increasing degree of anemia found in predialysis patients with deteriorating renal function appears to be primarily caused by factors other than ESF deficiency, the most likely being accumulation of uremic inhibitors of erythropoiesis. In dialysis patients in whom inhibitor levels are relatively homogeneous, the degree of anemia appears to be directly related to the residual capability of the kidney or the extrarenal sites to produce ESF.