A sensitive sandwich ELISA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra- and inter-assay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose–response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross-react with a variety of blood components and cytokines to produce false-positive results.
The serum TPO concentrations from 29 normal males and 21 females were 0.79 ± 0.35 and 0.70 ± 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukaemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET ( n  = 6, 2.80 ± 1.55 fmol/ml) were higher than the normal level. The patients with AA ( n  = 7, 18.53 ± 12.37 fmol/ml) and ALL ( n  = 5, 10.36 ± 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.     

Differences in the clinical characteristics of Pneumocystis jirovecii pneumonia in immunocompromized patients with and without HIV infection

Background and objective: The incidence of Pneumocystis jirovecii pneumonia (PCP) in patients with predisposing immunodeficiencies other than AIDS is growing. Knowing the different characteristics and outcomes of PCP according to HIV status would help physicians manage and treat patients with PCP. Methods: The medical charts of all patients with a proven first episode of PCP, diagnosed between 1997 and 2007 were retrospectively reviewed, and clinical and laboratory data abstracted. Results: Of the 35 patients with PCP, 18 were HIV‐positive and 17 were HIV‐negative with other immunosuppressive conditions. HIV‐negative patients were significantly older than HIV‐positive patients. The WCC (10 952 ± 5669 vs 9750 ± 3133/µL; P = 0.015), neutrophil counts (9631 ± 5421 vs 5680 ± 2628/µL; P = 0.01) and CD4+ lymphocyte counts (329 ± 502 vs 47 ± 50/µL; P < 0.001) were significantly higher in HIV‐negative patients. Six of the 17 HIV‐negative patients had a CD4+ lymphocyte count >300/µL. Serum IgG levels were lower in HIV‐negative patients (943 ± 379 vs 1635 ± 657 mg/dL; P = 0.017). Mortality was higher in HIV‐negative (52.9%) than in HIV‐positive patients (0%). On univariate analysis, risk factors for mortality from PCP were the presence of underlying pulmonary disease (odds ratio 4.000, 95% CI: 1.501–10.658) and HIV‐negative status (odds ratio 2.125, 95% CI: 1.283–3.518). Conclusions: The characteristics and outcomes of PCP differ significantly depending on HIV status. The existence of underlying pulmonary diseases may be associated with the prognosis of HIV‐negative patients with PCP.     

Influence of thrombopoietin on platelet activation in myeloproliferative disorders

Although thrombopoietin itself does not influence platelet aggregation, it enhances platelet activation in response to certain agonists. We evaluated the effects of thrombopoietin on platelet activation using platelet-rich plasma from 16 patients with myeloproliferative disorders (MPD group) and 16 healthy volunteers (control group). Preincubation with thrombopoietin significantly enhanced platelet aggregation stimulated by ADP, collagen, or epinephrine in the MPD group as well as the control group. However, aggregation induced by 3 μ M ADP or 16 μ M epinephrine showed significantly less augmentation by thrombopoietin in the MPD group than in the control group. Thrombopoietin significantly shortened the lag time between the addition of 3 μ M ADP or 16 μ M epinephrine and initiation of secondary aggregation and the lag time between addition of 2 μg/ml collagen and initiation of aggregation in both groups. When platelet-rich plasma was used without adjustment of the platelet count, thrombopoietin itself induced aggregation in two patients. Hypoaggregation after addition of 0.5 μg/ml collagen was observed in seven out of nine patients with normal thrombopoietin levels and only one of six patients with high levels ( P =0.04). Enhancement of 0.5 μg/ml collagen-induced aggregation by thrombopoietin was seen in five out of nine patients with normal thrombopoietin levels and none of the six patients with elevated levels ( P =0.04). These results indicate that platelet activation by certain agonists is enhanced by thrombopoietin in patients with these diseases as well as in normal controls and that the serum thrombopoietin level may regulate the function of circulating platelets in vivo .     

Hepatic angiomyolipoma pre-operatively diagnosed by imaging

An asymptomatic patient with pre-operatively diagnosed solitary angiomyolipoma is reported. The tumour was a well defined, fat density mass on computerized tomography (CT) and magnetic resonance imaging (MRI), and a hyperechoic nodule with a sharp margin on ultrasonography (US). The lesion was hypervascular on arteriography and CT during hepatic angiography. Chemical shift MRI confirmed the fat component within the lesion. Although a review of the literature shows this tumour to be rare and difficult to differentiate from a malignant lesion pre-operatively, the pre-operative diagnosis of angiomyolipoma is considered feasible when the characteristic findings as described here are present.