Immunocytochemical detection of ferritin in human bone marrow and peripheral blood cells using monoclonal antibodies specific for the H and L subunit

We have used the monoclonal antibodies 2A4 (specific for the H subunit of human ferritin) and LO3 (specific for the L subunit) for immunocytochemical detection of ferritin in bone marrow and peripheral blood cells from normal subjects and patients with various haematological disorders. Formalin-fixed slides were stained by the immunoalkaline phosphatase procedure (APAAP). In normal subjects, ferritin could be found only in bone marrow smears and appeared to be largely confined to erythroid precursors and reticuloendothelial cells. The more immature erythroid precursors contained higher concentrations of cellular ferritin. Although evaluation could be only semiquantitative, erythroblast ferritin appeared to be more reactive with the monoclonal 2A4 (15 +/- 7% positive erythroblasts) than with the monoclonal LO3 (6 +/- 5% positive erythroblasts), indicating that H-type ferritin was predominant, particularly in proerythroblasts and basophilic erythroblasts. By contrast, the ferritin present in reticuloendothelial cells appeared to be predominantly of L-type. Patients with iron deficiency showed low levels of positive erythroblast, whereas the reverse was true in patients with transfusional iron overload. Intense positivity for reticuloendothelial cell ferritin was found in patients with anaemia of chronic disease. In myelodysplastic syndromes and acute myeloid leukaemia (AML), ferritin positivity was generally very strong at any stage of erythroblast development, particularly with the monoclonal antibody 2A4. Perls-positive perinuclear granules of ring sideroblasts were not stained, confirming that mitochondrial iron deposition is not in the form of ferritin. In AML and myelodysplastic syndromes with excess of blasts, ferritin could be detected also in immature myeloid cells. These data indicate that: (a) in normal conditions ferritin is mainly expressed in red cell precursors and reticuloendothelial cells, and this is in keeping with the peculiar role of these cells in iron metabolism; (b) abnormal cell ferritin contents can be observed in both iron overload and malignancy.     

Effects of zileuton,a new 5-lipoxygenase inhibitor,in experimentally induced colitis in rats

Intravenous injection of platelet-activating factor (PAF) (0.36 mol/kg b.w.) in mice induced severe hemoconcentration, leucopenia, thrombocytopenia and finally the death of 85% of the tested animals. Combined inhibition of histamine and serotonin by promethazine and chlorpromazine, 6.24 and 3.12 mg/kg b.w. subcutaneously, protected the mice from PAF in part, reducing the death rate to 43%. These drugs did not protect the mice against the PAF-induced hemoconcentration, leucopenia and thrombocytopenia. Sulfinpyrazone (100 mg/kg b.w.) intravenously was the most effective both in protecting mice from PAF-induced death, reducing the death rate to 17%, and from thrombocytopenia, although hemoconcentration persisted. These results indicated that an important component of the PAF-induced systemic effects is mediated by reactions which can be inhibited by sulfinpyrazone. Furthermore, PAF-induced thrombocytopenia is not a direct PAF effect since it can be inhibited by sulfinpyrazone.     

Duchenne/becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner? in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.     

Early increase precedes a depletion of VIP and PGP-9.5 in the skin of insulin-dependent diabetics—correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18–46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n=6), with diabetes for 6 months to 3 years; group 2 (n=5), with the disease for 5–10 years; and group 3 (n=7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P <0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P <0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (>95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (<5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P=0·002), presence of clinical autonomic neuropathy (Fisher exact test P=0.04), and a reduced sural nerve conduction velocity (Fisher exact test P=0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.     

<Superscript>11</Superscript>C-Choline PET/CT for restaging prostate cancer. Results from 4,426 scans in a single-centre patient series

Purpose

To evaluate ¹¹C-choline PET/CT as a diagnostic tool for restaging prostate cancer (PCa), in a large, homogeneous and clinically relevant population of patients with biochemical recurrence (BCR) of PCa after primary therapy. The secondary aim was to assess the best timing for performing ¹¹C-choline PET/CT during BCR.

Methods

We retrospectively analysed 9,632 ¹¹C-choline PET/CT scans performed in our institution for restaging PCa from January 2007 to June 2015. The inclusion criteria were: (1) proven PCa radically treated with radical prostatectomy (RP) or with primary external beam radiotherapy (EBRT); (2) PSA serum values available; (3) proven BCR (PSA >0.2 ng/mL after RP or PSA >2 ng/mL above the nadir after primary EBRT with rising PSA levels). Finally, 3,203 patients with recurrent PCa matching all the inclusion criteria were retrospectively enrolled and 4,426 scans were analysed.

Results

Overall, 52.8 % of the ¹¹C-choline PET/CT scans (2,337/4,426) and 54.8 % of the patients (1,755/3,203) were positive. In 29.4 % of the scans, at least one distant finding was observed. The mean and median PSA values were, respectively, 4.9 and 2.1 ng/mL at the time of the scan (range 0.2 – 50 ng/mL). In our series, 995 scans were performed in patients with PSA levels between 1 and 2 ng/mL. In this subpopulation the positivity rate in the 995 scans was 44.7 %, with an incidence of distant findings of 19.2 % and an incidence of oligometastatic disease (one to three lesions) of 37.7 %. The absolute PSA value at the time of the scan and ongoing androgen deprivation therapy were associated with an increased probability of a positive ¹¹C-choline PET/CT scan (p?<?0.0001). In the ROC analysis, a PSA value of 1.16 ng/mL was the optimal cut-off value. In patients with a PSA value <1.16 ng/mL, 26.8 % of 1,426 ¹¹C-choline PET/CT scans were positive, with oligometastatic disease in 84.7 % of positive scans.

Conclusion

In a large cohort of patients, the feasibility of ¹¹C-choline PET/CT for detecting the sites of metastatic disease in PCa patients with BCR was confirmed. The PSA level was the main predictor of a positive scan with 1.16 ng/mL as the optimal cut-off value. In the majority of positive scans oligometastatic disease, potentially treatable with salvage therapies, was observed.

     

Determinants of left ventricular mechanical dyssynchrony in patients submitted to myocardial perfusion imaging: A cardiac CZT study

Background

An interaction between coronary anatomy, myocardial perfusion, and left ventricular (LV) functional parameters in the development of mechanical LV dyssynchrony (LVD) has been suggested. This study examined the correlates of LVD in a large sample size of patients with known or suspected coronary artery disease (CAD) using cadmium-zinc-telluride camera.

Methods

Six-hundred and fifty-seven consecutive patients who underwent myocardial perfusion imaging (MPI) and coronary angiography were included. Coronary stenosis >70% was considered significant. LV perfusion and functional parameters were computed from MPI images. The presence of significant LVD was evaluated by phase standard deviation and histogram bandwidth.

Results

415/657 (63%) patients had significant CAD. LVD was present in 247 (38%) patients and was associated with the presence of a higher CAD burden (P < .001), more impaired measures of LV perfusion (P < .001), contractile function (P < .001), and larger LV volumes (P < .001). By multivariate analysis, the LV end-systolic volume index (P < .001) and ischemic burden (P < .001) were the strongest predictors of LVD independent of CAD extent and LV systolic dysfunction.

Conclusions

LVD is frequent in patients undergoing MPI for suspected or known CAD. Its presence is independent of CAD burden and LV systolic dysfunction, but is dependent on the presence of myocardial perfusion abnormalities and LV end-systolic volume.

     

I-123 MIBG scintigraphy and 68Ga-DOTANOC PET/CT negative but F-18 DOPA PET/CT positive pheochromocytoma: a case report

We observed a 34-year-old man who was incidentally found to have an adrenal mass during surgical follow-up for perforated ulcer. The patient was subjected to I-123 MIBG scintigraphy, 68Ga-DOTANOC PET/CT, and F-18 DOPA PET/CT. Only F-18 DOPA PET/CT showed evidence of an avid adrenal mass. A CT-guided biopsy was performed and it was suggestive for pheochromocytoma. He underwent surgery and a pheochromocytoma, about 40 mm in diameter, was detected. Traditionally, I-123 MIBG scintigraphy has been used in detecting chromaffin cell tumors, but more recently it had been demonstrated that a certain part of pheochromocytoma could be false-negative on scintigraphy.     

Choline PET/CT for prostate cancer: Main clinical applications

Several studies investigated the potential roles of imaging modalities in prostate cancer patients for the evaluation of intra-prostatic disease, stage and restage. However no precise guidelines exist about the use of imaging modalities, in particular about the role of PET/CT hybrid imaging. Considering the results of the literature and our experience, we tried to summarize the main applications of choline positron emission tomography (PET) in prostate cancer patients. The use of choline PET/CT for initial diagnosis and staging is not recommended as a first-line method. Instead the main and important application of choline PET/CT is represented by the restaging of the disease in case of biochemical relapse for the detection of lymph node and distant recurrence. In particular choline PET/CT could play a crucial role as first diagnostic procedure in prostate cancer patients who show a fast growing Prostate Specific Antigen (PSA) kinetics.