Development and testing of a new disposable sterile device for labelling white blood cells.

AIM: White blood cell (WBC) labelling requires isolation of cells from patient's blood under sterile conditions using sterile materials, buffers and disposables under good manufacturing practice (GMP) conditions. Till now, this limited the use of white blood cell scintigraphy (WBC-S) only to well equipped laboratories with trained personnel. We invented, developed and tested a disposable, sterile, closed device for blood manipulation, WBC purification and radionuclide labelling without exposing patient's blood and the operator to contamination risks. This device prototype and a final industrialized device (Leukokit®) were tested for WBC labelling and compared to standard procedure. Leukokit® was also tested in an international multi-centre study for easiness of WBC purification and labelling. METHODS: On the device prototype we tested in parallel, with blood samples from 7 volunteers, the labelling procedure compared to the standard procedure of the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol with respect to cell recovery, labelling efficiency (LE), cell viability (Trypan Blue test) and sterility (haemoculture). On the final Leukokit® we tested the biocompatibility of all components, and again the LE, erythro-sedimentation rate, cell viability, sterility and apyrogenicity. ACD-A, HES and PBS provided by Leukokit® were also compared to Heparin, Dextran and autologous plasma, respectively. In 4 samples, we tested the chemotactic activity of purified WBC against 1 mg/ml of lipopolysaccharide (LPS) and chemotaxis of 99mTc-HMPAO-labelled WBC (925 MBq) was compared to that of unlabelled cells. For the multi-centre study, 70 labellings were performed with the Leukokit® by 9 expert operators and 3 beginners from five centers using blood from both patients and volunteers. Finally, Media-Fill tests were performed by 3 operators on two different days (11 procedures) by replacing blood and kit reagents with bacterial culture media (Tryptic Soy Broth) and testing sterility of aliquots of the medium at the end of procedure. RESULTS: Tests performed with the prototype showed no significant differences with the standard procedure but a faster and safer approach. Tests performed with the final Leukokit® confirmed full biocompatibility, sterility and apyrogenicity of all reagents and plastic ware. Average WBC recovery with Leukokit® was comparable to that of the ISORBE protocol (117x106±24x106 vs. 132x106±29x106 cells, P=not significant). No differences in red blood cells and platelet content were observed. LE was 82% ± 3% for Leukokit® and 65±5% for control (P=0.0003) being PBS vs autologous plasma the main reason of such difference. Cell viability was always >99.9% in both conditions. Chemotactic tests showed no differences between all Leukokit® samples and controls. Haemocultures and Media-Fill tests were always sterile. The procedure was well accepted by expert operators and beginners, with a very fast learning curve (confidence after 2±2 labellings). CONCLUSION: The invented device offers high level of protection to operators and patients. The derived Leukokit® is safe and easy to use, and gives a high LE of WBC without affecting cell viability and function. Being a registered closed, sterile medical device, it may allow easier and faster WBC labelling that is not limited to only well equipped laboratories. Also simultaneously labelling of multiple patients is possible.

The dilemma of diagnosing fever of unknown origin: large arteries vasculitis revealed by 18F-FDG PET/CT imaging. A case report.

Fever of unknown origin (FUO) is an uncommon disease, and its underlying etiology may include a number causes, i.e., infections, malignancies, autoimmune conditions. Diagnosis is often a difficult task, and usually physician spend time and money in order to define the etiology of FUO. We report a case of patient who presented with FUO and headache, and positron emission tomography (PET) with 2-deoxy-2-[fluorine-18] fluoro-D-glucose (18F-FDG) allowed to reveal the presence of a large vessel vasculitis. 18F-FDG PET may represent an useful tool in patients with FUO, since it can early depict an hypermetabolic activity due to inflammation and so help to achieve a final diagnosis in some cases of FUO.

[18FDG-PET and large vessel vasculitis: preliminary data on 25 patients]. / 18FDG-PET e vasculiti dei grandi vasi: studio preliminare su 25 pazienti.

OBJECTIVE: To evaluate the predictive value of clinical and biochemical features when compared to 18FDG-PET in the diagnostic work-up of large vessel vasculitis (LVV). METHODS: Twenty-five patients underwent 18FDG-PET for the clinical suspect of LVV. All of them presented history of systemic symptoms lasting >or=6 months and laboratoristic evidence of persistently high markers of inflammation. The patients were stratified according with: i) clinical manifestations, defined as presence of one or more ACR criteria for the classification of LVV; ii) laboratory investigations: Erythrocyte Sedimentation Rate (ESR) higher or lower than 50 mm/h, C-Reactive Protein (CRP) higher or lower than 2 mg/dl; iii) prednisone dose in the 4 weeks preceding PET examination. RESULTS: The total number of positive PET was higher in the group without clinical ACR criteria and in the group with inflammation markers under the established cut-off. The number of scans consistent with LVV was higher in the groups presenting one or more clinical criteria for LVV but in those with very high ESR and CRP. In all the cases differences between groups were not statistically significative. A clear cut negative correlation between steroid dose and number of scans suggestive for LVV has been observed. CONCLUSIONS: Diagnosis of LVV remains challenging, especially in patients presenting with a constellation of non-specific symptoms and laboratory findings. In this study, both clinical and biochemical features show low correlation with a vasculitic pattern of FDG uptake. In our experience 18FDG-PET represents an useful diagnostic tool in early stages of LVV and a powerful instrument to follow the treatment responses.

[Transient regional osteoporosis]. / Le osteoporosi transitorie regionali.

Transient osteoporosis of the hip and regional migratory osteoporosis are uncommon and probably underdiagnosed bone diseases characterized by pain and functional limitation mainly affecting weight-bearing joints of the lower limbs. These conditions are usually self-limiting and symptoms tend to abate within a few months without sequelae. Routine laboratory investigations are unremarkable. Middle aged men and women during the last months of pregnancy or in the immediate post-partum period are principally affected. Osteopenia with preservation of articular space and transitory edema of the bone marrow provided by magnetic resonance imaging are common to these two conditions, so they are also known by the term regional transitory osteoporosis. The appearance of bone marrow edema is not specific to regional transitory osteoporosis but can be observed in several diseases, i.e. trauma, reflex sympathetic dystrophy, avascular osteonecrosis, infections, tumors from which it must be differentiated. The etiology of this condition is unknown. Pathogenesis is still debated in particular the relationship with reflex sympathetic dystrophy, with which regional transitory osteoporosis is often identified. The purpose of the present review is to remark on the relationship between transient osteoporosis of the hip and regional migratory osteoporosis with particular attention to the bone marrow edema pattern and relative differential diagnosis.

Methods of measuring gallbladder motor functions--the need for standardization: scintigraphy.

Emptying the gallbladder is part of the complex process of food digestion. The gallbladder interacts with other gastrointestinal organs and its movements are coordinated and modified by functions of the stomach, intestine and pancreas. Many factors can modify gallbladder motility, for example, sex and age of the subject, their body mass, the kinds of food ingested and stimulus used. The assumption that the gallbladder progressively empties during meals and refills during fasting is incorrect. Using a combination of ultrasonography and cholescintigraphy, it is possible to measure absolute and net gallbladder emptying. In this way we demonstrated that the gallbladder begins to refill immediately after emptying begins, and the difference between net and absolute emptying of the gallbladder indicates the refilling of bile and provides a measure of bile turnover rate, an accurate index to assess gallbladder motility.

An original approach in the diagnosis of early breast cancer: use of the same radiopharmaceutical for both non-palpable lesions and sentinel node localisation.

A modern approach to the surgical treatment of early breast carcinoma requires intraoperative localisation of non-palpable lesions and assessment of the lymph node status. Localisation of breast lesions can be achieved by intratumoural injection of a small amount of radiotracer and intraoperative use of a gamma probe (i.e. radioguided occult lesion localisation, or ROLL). Assessment of the lymph node status is possible by means of the sentinel node approach. To date, two different radiopharmaceuticals have been used for localisation of tumour and sentinel node. We now propose the use of a single nanocolloidal tracer (Nanocoll, with a particle size of less than 80 nm) which is labelled with technetium-99m for simultaneous performance of ROLL and sentinel node identification. The aim of this study was to evaluate the feasibility of this approach, which should be easier and more practical than the dual-tracer injection method. We have employed this new technique in 73 patients with non-palpable, cytologically diagnosed breast cancer and non-palpable axillary lymph nodes. In all patients the radiocolloid, in a total volume of 0.3-0.4 cc, was injected under sonographic or stereotactic guidance. Half of the dose was injected intratumourally and half superficially, but very close to the tumour. Because of the slow lymphatic flow in the breast, Nanocoll must be injected some time before surgery in order to enable adequate migration to the axilla. We injected colloid in the afternoon before surgery (16-23 h before the start of the operation, with an average interval of 18 h). An average dose of 130 MBq (range 110-150) was injected in order to have about 10 MBq of radioactivity when surgery commenced. Lymphoscintigraphy was performed after 15-19 h, with an average interval of 17 h. The procedure was always successful in permitting the localisation of occult breast lesions. Lesions were always localised at the first attempt, and were always contained within the surgical margins. Histological examination revealed all 73 resected lesions to be malignant: there were 64 cases of infiltrating carcinoma and nine of intraductal carcinoma. All breast lesions were therefore confirmed to be early breast cancer. We achieved sentinel node localisation in 71 out of 73, either at scintigraphy or with the intraoperative probe; in two patients, radiopharmaceutical migration was absent. Lymphoscintigraphy showed only axillary drainage in 52 cases, only internal mammary chain (IMC) drainage in nine cases, and combined axillary and IMC drainage in eight cases. In two cases, lymphoscintigraphy suggested the sentinel node was located inside the same breast (intramammary lymph node). All the visualised sentinel nodes were biopsied except for four that were localised in the IMC. Histological examination of the nodes showed metastases in 20 cases: in 15 cases there were micrometastases, and in five, macrometastases. In conclusion, this study has demonstrated the feasibility of the proposed procedure. Simultaneous performance of ROLL and sentinel node localisation using a single tracer represents a useful and practicable choice in the management of early breast cancer.

Sentinel node study in early breast cancer.

Since October 1997 60 patients with early breast cancer (T <3 cm) were studied. All patients underwent lymphoscintigraphy with two types of colloid: the first (17 pts) with a particle size <1,000 nm; the second (43 pts) with a particle size <80 nm. The standard procedure consists of injection, on the day before surgery, of 70 MBq of the smaller nanocolloid in 0.4 cc saline divided over four sites, around the lesion or subdermally around the surgical scar. We utilize a low-energy, high-resolution LFOV camera for scintigraphy and a probe specific for the sentinel node during surgery. In 56/60 patients (93.3%) lymphoscintigraphy showed the sentinel node (SN). In two cases the SN was not detected presumably because of lymphatic interruption by an old surgical scar; in the other two cases the sites of injection were too close to the SN, thus masking it. In five cases (9%) the SN was not visualized with the surgical probe but in two of these drainage to the internal mammary chain was observed. The apparently lower sensitivity of intraoperative localization was due to the extra-axillary lymphatic drainage or to the vicinity of the SN to the primary lesion. The SN proved to be metastatic in 12 cases. No false-negative SNs were found. In five cases (10%) the radiolabeled lymph node was the only node containing tumor cells (micrometastases): this result depends on the combined use of hematoxylin-eosin and rapid cytokeratin staining. The application of blue dye was useful for easier identification of the SN but did not allow detection of more SNs. Our preliminary results are extremely encouraging. Considering that at the early stages of breast cancer the likelihood of lymph node metastases is low (20% in our series) and no false negative were reported in this study, we conclude that with SN biopsy axillary lymph node dissection can be avoided, making surgery less aggressive but maintaining accuracy.

Massive bleeding in an adult patient suffering from Meckel's diverticulum.

The case of a 22-year-old male who bled from a Meckel's diverticulum is described. The diagnosis was achieved after 99mTechnetium pertechnetate scintigraphy. With the administration of somatostatin very clear images were obtained. The histological examination confirmed the presence of ectopic gastric mucosa. The literature, over the last 10 years, has been reviewed to identify factors associated with bleeding in adults. Ectopic gastric mucosa is the most important factor predicting bleeding. The diagnostic approach to bleeding Meckel's diverticulum and the improvement in the quality of 99mTechnetium pertechnetate scintigraphy, following administration of somatostatin, is discussed.

Postprandial refilling and turnover: specific gallbladder motor function defects in patients with gallstone recurrence.

BACKGROUND AND AIMS: Impaired gallbladder motor functions are important in the pathogenesis of primary cholesterol gallstones, and possibly in the pathogenesis of recurrent gallstones. By using ultrasonography and cholescintigraphy simultaneously, we recently defined new parameters of gallbladder motor function (postprandial refilling and turnover in addition to emptying), which were markedly impaired in gallstone patients. The aim of this study was to assess the value of these new parameters in distinguishing patients with from those without gallstone recurrence. METHODS: We studied 11 patients with gallstone recurrence, 11 without gallstone recurrence (at least 40 months after complete dissolution by oral bile acids) and 11 healthy controls. Simultaneous measurements of gallbladder volume (ultrasound) and gallbladder counts (gamma-camera scintigraphy) were carried out in the fasting state and at 10 min intervals following meal ingestion, for a period of 90 min. Gallbladder refilling, turnover of bile and turnover index were calculated, as well as gallbladder emptying by both cholescintigraphy and ultrasound. RESULTS: Patients with gallstone recurrence had reductions in gallbladder emptying, postprandial refilling and gallbladder bile turnover. They also had a significant reduction in the turnover index (1.7 +/- 1.4) compared to controls (3.5 +/- 0.3, P < 0.01) and to patients without gallstone recurrence (3.1 +/- 1.5, P < 0.05). Patients without gallstone recurrence had only a small reduction in emptying and no reduction in postprandial refilling or turnover compared to controls. CONCLUSIONS: We conclude that impairment of gallbladder emptying persists in all patients after gallstone dissolution, albeit to a more pronounced extent in patients with recurrence; but that impairment of postprandial refilling and turnover are specific defects in patients with recurrence.

Postprandial gallbladder motor function: refilling and turnover of bile in health and in cholelithiasis.

BACKGROUND & AIMS: Impaired gallbladder emptying is implicated in gallstone disease. Ultrasonography and scintigraphy have shown conflicting results because the former is influenced by postprandial refilling, whereas the latter is not influenced by refilling. The aim of this study was to measure postprandial refilling and turnover of bile by combining the two techniques. METHODS: Simultaneous scintigraphy and ultrasonography were used in 14 patients with gallstones and 11 healthy controls. Measurements were performed while the patients were fasting and at 10-minute intervals after a standard meal for 90 minutes, and the measurements were used to calculate postprandial refilling, turnover of bile (in milliliters), and turnover index. RESULTS: Ultrasonography and scintigraphy provided different gallbladder emptying patterns. Compared with controls, patients with gallstones had impaired emptying by both scintigraphy (P < 0.0001) and ultrasonography (P < 0.01). Postprandial refilling and turnover were both reduced between 60 and 90 minutes (P < 0.05), and the turnover index was markedly reduced (1.8 vs. 3.5; P < 0.001). CONCLUSIONS: Simultaneous scintigraphy and ultrasonography provide a new model of gallbladder motor function showing that refilling begins immediately postprandially. In healthy controls, the gallbladder postprandially handles up to six times its basal volume within a period of 90 minutes, but this turnover of bile is markedly reduced in cholelithiasis causing a reduced washout effect of the gallbladder contents, including cholesterol crystals.

Immunoscintigraphy in malignant melanoma: a five-year clinical experience.

Immunoscintigraphy (IS) with 99Tcm-labelled anti-melanoma monoclonal antibody F(ab')2 fragments was performed in 135 melanoma patients, 64 males and 71 females, aged 19-82 years (mean 52.3 years) between December 1987 and December 1991. The first group of IS was performed in 50 patients before surgery to assess optimal management: seven true positive and one true negative were obtained in ocular and visceral melanomas, while in cutaneous MM sensitivity, specificity and accuracy in assessing lymph node involvement were, respectively, 61.5, 93.3 and 83.7%. The second group of 128 IS is relative to 85 patients in follow-up: excluding 13 cases with known metastatic disease and 12 inconclusive tests, sensitivity, specificity and accuracy were, respectively, 83.3, 98.8 and 96.1%. Immunoscintigraphy is free of side effects even after repeated administrations and is a useful adjunct to standard diagnostic techniques as a basis for treatment decisions.

Bone marrow imaging in plasma cell dyscrasias: review of 130 cases.

Skeletal radiography, bone and bone marrow scintigraphy have been performed in 130 patients with plasma cell dyscrasias (119 multiple myeloma, 9 MGUS and 2 Waldenström disease). Our results confirm: 1) that radiography is much more sensitive than scintigraphy in the identification of the lesions typical of myeloma, but in the first stage bone scintigraphy and especially bone marrow scintigraphy are more sensitive than x-ray for the detection of regions affected by focal lesions; 2) that bone scintigraphy is of particular value in detecting some abnormalities in specific sites not fully visualized by x-ray; 3) that bone marrow scintigraphy is a valuable diagnostic tool in the early stage of myeloma, especially for evaluating the progression of the disease, because it is able to demonstrate not only focal lesions, but also bone marrow expansion. We believe that bone marrow scintigraphy may be a useful technique in the early diagnosis and follow-up of multiple myeloma, particularly in the detection of unusual forms (i.e., "smouldering" myeloma), but it remains only an "additional" technique for bone imaging.

Pitfalls in scintigraphic detection of neuroendocrine tumours.

We report 4 cases of abnormal results using iodine-123 metaiodobenzylguanidine (123I-mIBG) or technetium-99m (V) dimercaptosuccinic acid (99mTc(V)-DMSA) scintigraphy in the diagnosis and follow-up of presumed neuroendocrine tumours. The present series consisted of 2 false-positive cases (1 adenomatous polyp of the caecum with mIBG and 1 follicular adenoma of the thyroid with DMSA) and 2 cases of anomalous uptake of (V)-DMSA in a non-neuroendocrine tissue.

Increased uptake of radio labelled white blood cells into abdomen of uremics on peritoneal dialysis.

Peritonitis and exit-site tunnel infection are frequent causes of CAPD drop out. We studied 9 patients, 8 treated by CAPD and 1 by IPD. These patients underwent sonographic and scintigraphic study of the abdomen. All scintigraphic examinations showed a visceral uptake. In two cases, sub-clinical bowel inflammation, demonstrated by scintigraphic study, preceded a gram negative peritonitis. The scintigraphic study with radiolabelled white blood cells may be useful in identifying chronic aseptic inflammations and some bowel and exit-site conditions which are possible risk factors in some cases of peritonitis.