New enzyme immunoassay for detecting total, type I, and type II intrinsic factor antibodies.

A method for the detection of total, type I, and type II intrinsic factor antibodies was devised. The technique comprises a two-site solid phase enzyme linked immunosorbent assay (ELISA), with human intrinsic factor conjugated with horseradish peroxidase as label and attached to polystyrene tubes as solid phase. One conjugation provides sufficient material to assay more than 10,000 patient samples. The label proved stable during the course of this evaluation and was still in use more than 12 months after preparation. When applied to 45 serum samples from cases of pernicious anaemia, intrinsic factor antibodies were shown in 30 (67%). Simplicity, high capacity, low cost and label stability, combined with relatively high clinical sensitivity make the method suitable for cost effective screening of large numbers of samples. Simple modifications to the basic assay reagents permitted type I and type II intrinsic factor antibodies to be differentiated.

Inhibition of normal human granulopoiesis in vitro by amiloride: evidence for the involvement of facilitated sodium influx in the induction of differentiation.

The formation of colonies by normal human bone marrow granulopoietic progenitor cells in vitro in soft agar was inhibited in a simple, dose-dependent manner by amiloride (50% inhibitory concentration 26.4 +/- 3.4 microM, n = 9). Such inhibition was reversible and is evidence for the involvement of amiloride-sensitive sodium influx in granulopoietic cell proliferation. Colony-forming cells were capable of some proliferation at concentrations of amiloride 3-fold in excess of that required to inhibit full colony formation. A two-stage model is invoked to explain this observation: an early amiloride-insensitive stage and a late amiloride-sensitive stage which includes terminal differentiation. We conclude that the cellular mechanism by which differentiation is induced includes activation of the amiloride-sensitive sodium influx channel, which would indicate a new approach to the therapeutic induction of differentiation in vivo.

Systemic infection with Aureobasidium pullulans in a leukaemic patient.

Aureobasidium pullulans, a conidial fungus widely distributed in the environment, was repeatedly isolated from the blood of a 28-year-old man with acute myeloid leukaemia. Amphotericin B failed to eliminate the organism.

The assay of factor VIII: C in heparinized plasma: a polybrene neutralization method.

Factor VIII coagulant activity (VIII:C) has been shown by several investigators to exhibit increased stability in vitro when physiological levels of plasma ionized calcium are maintained by anticoagulation with heparin rather than citrate. An increase in initial activity of VIII:C in heparin over that of VIII:C in citrate has been reported but this has not been confirmed. In order to assay VIII:C in heparinized plasma, the heparin anticoagulant effect must be excluded without interfering with the validity of the assay. A one-stage clotting assay for VIII:C has been developed where heparin is neutralized by Polybrene, a synthetic polymerized quaternary ammonium salt. VIII:C may be accurately measured by this method which satisfies the requirements for a valid assay of parallelism and linearity.

Assessment of the role of an immunofluorescent terminal deoxynucleotidyl transferase kit.

The laboratory use of a commercial kit for the immunofluorescent measurement of terminal deoxynucleotidyl transferase (BRL Terminal Deoxynucleotidyl Transferase Immunofluorescent Assay System) has been investigated. The precision of the assay system with stored patient material and its performance with regard to ease of microscopic examination were assessed. The intensity of the fluorescence obtained was found to vary. However, the precision of the assay system on fixed slides stored at -30 degrees C was satisfactory; the method simple, and results obtainable in a relatively short space of time.

Tubular function in multiple myeloma.

In 42 myeloma patients our results confirm the association of light chain proteinuria and renal damage, but suggest that while the amount of light chain excreted is an important factor, only some light chains are nephrotoxic. The excretion of the proximal tubular cell lysosomal enzyme N acetyl B D glucosaminidase was a sensitive index of tubular injury, while the presence of low molecular weight proteinuria (Retinol Binding Protein and Lysozyme) was shown to indicate tubular dysfunction in a kidney sufficiently damaged to produce an impaired GFR. Isolated defects of distal tubular function (acid load response and concentrating ability) were rare. Such changes were seen mainly as part of global renal impairment and were usually associated with such specific pathophysiological conditions as plasma hyperviscosity or tubular crystal deposition. Hypercalcemia had a specific effect on the concentrating ability independent of any impairment of renal acidification.

Disseminated zygomycosis associated with erythroleukaemia: confirmation by lectin stains.

Zygomycosis is not often diagnosed in the United Kingdom, and so the possible importance of the findings in a patient with disseminated zygomycosis who had been treated with chemotherapy for erythroleukaemia was not appreciated until histological examination of specimens obtained at necropsy provided a presumptive diagnosis. No attempt had therefore been made to identify the organism by culture, and lectin binding methods were used to try to compensate for this. The characteristics of the hyphae on staining with lectins were similar to those previously shown in Rhizopus oryzae and were unlike those of a wide range of other hyphal fungi. Although definite speciation of the fungus was not achieved, these findings confirm that this was a case of zygomycosis and would seem to represent the first such reported confirmation in the absence of culture.

Pulmonary infiltrates and adult acute leukaemia: empirical treatment and survival related to the extent of pulmonary radiological disease.

In a retrospective review of 141 consecutive adult patients with acute leukaemia there were 33 local and eight diffuse parenchymal pulmonary abnormalities noted on the first abnormal chest radiograph. Empirical treatment was given without any invasive diagnostic procedures. Twenty of the 41 patients (49%) with radiographic abnormalities died--10 of 31 patients (32%) in the "local" group (excluding two who were not actively treated) and all eight patients in the "diffuse" group. One patient in the local group and six in the diffuse group died within 48 hours of the first abnormal chest radiograph. The results in the local group suggest that failure to respond to the initial treatment prescribed would allow time to perform an invasive procedure in most patients that may lead to a change in management. In the diffuse group, however, an early invasive procedure would be required if the result were to have a chance to influence the outcome.

Urinary N-acetyl-beta-D-glucosaminidase as an indicator of tubular damage in multiple myeloma.

In 34 patients with multiple myeloma we have studied urinary N-acetyl-beta-D-glucosaminidase (NAG) as an index of tubular injury and related it to urinary light chain excretion and serum creatinine. Our results confirm the association of light chain proteinuria with both tubular damage and global renal impairment, but show that not all light chains are nephrotoxic. Tubular damage occurs independently of global renal impairment. Clinically, urinary NAG appears to be a useful index of early tubular damage in multiple myeloma.

The importance of urinary immunoglobulin light chain isoelectric point (pI) in nephrotoxicity in multiple myeloma.

There is clinical and experimental evidence to imply that urinary immunoglobulin light chains are nephrotoxic yet some patients pass large amounts for years with little renal damage. Animal experiments suggest that a light chain is more nephrotoxic if it has a high isoelectric point (i.e. is cationic in relation to urine pH). We studied 23 patients with multiple myeloma, measuring light chain excretion rate, light chain isoelectric point (pI) and creatinine clearance. Light chain excretion rate was independent of light chain isoelectric point and did not correlate with creatinine clearance. Light chain isoelectric point correlated with creatinine clearance, suggesting that this characteristic may be an important determinant of the nephrotoxic effect of these immunoglobulin constituents.

Should patients with renal failure associated with myeloma be dialysed?

In a study of renal function in multiple myeloma seven patients presented with renal failure and three developed it 16-106 months after diagnosis. All were dialysed. Infection with dehydration was a precipitating factor in all seven cases of acute or acute on chronic renal failure. Of these, two patients recovered normal renal function and one other was left with permanent renal impairment but no longer required dialysis. Results from the seven patients with acute renal failure and for the three with more chronic features support the practice of dialysis for all patients who present with renal failure. Dialysis is not indicated for those patients with progressive myelomatous disease. The study showed no evidence that chemotherapy permitted recovery from established renal failure. The prognosis in this elderly group is heavily dependent on the presence of cardiovascular or other degenerative disease.

The role of the cellular sodium pump in human granulopoiesis in vitro.

The specific sodium pump antagonist ouabain was used to study the effect of sodium pump inhibition on granulopoietic colony formation by normal human bone marrow cells cultured in soft agar for 7 d. Suppression of colony formation was dose-dependent and occurred at a low and reproducible concentration. The use of an extended range of concentrations (1 X 10(-19) M to 1 X 10(-4) M) revealed no additional effect. Inhibition occurred when colony formation was stimulated by either normal human white blood cells in a soft agar underlayer, or the inclusion of pre-formed colony-stimulating activity. Sensitivity to inhibition was similar under either set of conditions, the 50% inhibitory concentrations being 2.70 +/- 0.43 (SE) X 10(-8) M and 2.83 +/- 0.21 (SE) X 10(-8) M respectively. This is interpreted as showing an effect primarily on the colony-forming cells rather than on the cellular production of colony-stimulating activity. Inhibition of colony formation by ouabain was opposed by the addition of extra potassium to the culture medium, confirming that the inhibition was mediated via a perturbation of monovalent cation exchange. Inhibition by 2 X 10(-7) M ouabain appeared to be reversible by simple washing after exposure for up to 24 h, suggesting that the inhibitory effect of ouabain is not primarily cytotoxic.

Comparative studies of a new commercial kit for the estimation of vitamin B12 in serum.

A commercial kit method (Technia Diagnostics) for the estimation of serum vitamin B12 claiming certain practical advantages was examined. Analytical and clinical performance were compared with a non-commercial radioisotope B12 method, previously compared to other commercial radioisotope B12 methods. The kit's analytical performance in our hands was satisfactory, although the within-batch precision and recovery of added cyanocobalamin were disappointing. Clinical performance was comparable with the non-commercial B12 method. Establishment of suitable reference ranges as a prerequisite to diagnostic use is apparent.

Long survival in acute myelogenous leukaemia: an international collaborative study.

A group of 82 adult patients with acute myelogenous leukaemia had survived in continuous first remission for more than three years was studied. These long-surviving patients were being treated at 12 referral centres in Europe and the USA, and they were compared with other patients with acute myelogenous leukaemia from 10 of these centres. There was no clear difference in the amount of induction chemotherapy or the time taken to achieve remission. Immunotherapy was not found to improve chances of long-term survival. The 82 patients were also compared with a group of 115 patients who had no appreciable difference in the number of blood or marrow myeloblasts between these two groups at presentation, but the long survivors had significantly higher initial platelet counts and were slightly younger. The long survivors also tended to have a lower total white cell count at presentation and lower granulocyte counts; there was no obvious explanation for these differences. Eight of the 82 patients relapsed from three to four years after remission and two (of 69 patients) after four to five year. Thereafter relapse was rare, and it seems likely that some of the 40 patients who have survived for five years or more are cured.