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.

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.

Folic acid binding protein in acute myeloid leukaemia.

Folic acid binding protein was estimated in the serum of 94 control subjects and a normal range was established. Raised levels were found in folate deficiency and chronic myeloid leukaemia. Considerably raised levels were found in untreated acute myeloid leukaemia, most often in cases with a marked monocytic element.

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.

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.

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.

An evaluation of commercial radioisotope methods for the determination of folate and vitamin B12.

Five commercial kits for the determination of folate and six kits for the determination of vitamin B12 were investigated. Their performance has been compared with microbiological methods for the two vitamins and with a non-commercial radioisotopic method for B12. The results show the importance of the determination of the reference range for an individual laboratory for each method. The precision of the kits varied appreciably, as did their performance using specimens from patients with different haematological disorders. In particular, certain kits failed to detect all patients with pernicious anaemia. The relative accuracy of the kits was assessed. Various factors which should be taken into account in the final selection of a satisfactory kit are discussed.

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.

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.

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.

Aplastic anaemia, acute myeloblastic leukaemia, and oxymetholone.

Four patients developed acute myeloblastic leukaemia after treatment of aplastic anaemia with oxymetholone. Two had previously shown an excellent clinical response to the drug and a third some improvement. Though no definite causal relationship between therapy and the onset of leukaemia has been established, the number of cases is greater than might have been expected by chance. The association of the two diseases is discussed.

Hypouricaemia and proximal renal tubular dysfunction in acute myeloid leukaemia.

Two patients with acute myeloid leukaemia developed hypouricaemia during the period of their illness. Renal clearance studies showed that the hypouricaemia was associated with an increased urate clearance, renal aminoaciduria, and an episodic increase in phosphate clearance. These findings together with an inadequate suppression of urinary urate exceretion after the administration of pyrazinamide suggest proximal tubular dysfunction affecting reabsorption of a wide variety of substances. Ten more patients with acute leukaemia were studied and the results indicate that this lesion develops in a large proportion of patients with acute myeloid leukaemia.

Combination therapy for myelomatosis.

Twenty-eight patients with multiple myeloma have been treated with a quadruple chemotherapeutic regimen consisting of 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU), cyclophosphamide, melphalan, and prednisolone. Nineteen new patients and nine who had escaped from previous single-agent therapy were included in the study. The results to date, on eight criteria of response, seem to be superior to those obtained from previous chemotherapeutic regimens. The study has been in progress for 18 months and only three patients have died. Only one who had not received previous therapy died, and she had complicating hyperparathyroidism, which almost certainly contributed to her death.

Hyponatraemia syndrome in acute myeloid leukaemia.

Hyponatraemia was observed in 11 out of 14 consecutive patients with acute myeloid leukaemia and its variants. Metabolic studies on these patients revealed an early increase in the urinary sodium excretion, negative free water clearance, and urine osmolality inappropriately higher than that of the serum. It is postulated that this syndrome is caused by a substance released from the primitive cells of the abnormal myeloid series.

Metabolic disorders in acute myeloid leukaemia.

Metabolic balance studies were carried out in 17 unselected patients with acute myeloid leukaemia. Widespread metabolic disturbances were observed. Serum Na fell below 135 mmol/1 in 14 patients (82%) and 11 patients (64%) developed hypokalaemia. An increased osmolal clearance caused by a release of electrolyte and blast cell waste (i.e. urea, urate, etc.) during chemotherapy appeared to be the principle cause of natriuresis and hyperkaluria. Seven patients had proteinuria before and eight others developed it during antileukemic therapy. Nine patients (53%) developed proximal renal tubular dysfunction with aminoaciduria, hyperphosphaturia and incomplete reabsorption of urate. No significant relation was found between this widespread glomerulo-tubular dysfunction and lysozymuria. We suggest that antileukaemic drugs release unidentified substances from blast cells which are toxic to the kidney. Metabolic alkalosis in six patients (35%) was probably related to volume depletion and hypokalaemia, while two patients developed acidaemia with the onset of renal failure. Hypocalcemia in seven patients (41%) had a multifactorial basis: hyperphosphaturia, septicaemia, malnutrition and cytotoxic drugs were among the probable causes.

Familial Hodgkin's disease.

A family with Hodgkin's disease is described. Each affected member of the family had mediastinal involvement and in each case the histology was that of nodular sclerosing Hodgkin's disease. The significance of these findings with reference to the aetiology of the disease is discussed.