Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.     

胰腺癌组织VEGF和MVD表达与CT灌注成像的关系

血管内皮生长因子作为肿瘤血管生成的一种主要调控因子,与微血管密度密切相关．胰腺癌组织的血管内皮生长因子和微血管密度的测定对于判断患者的预后,指导临床治疗和判断疗效等具有重要作用．CT灌注成像可以通过无创伤检查反映胰腺癌的肿瘤血管构成, 在治疗前后提供有价值依据,对胰腺癌的诊治有重要意义．     

免疫磁珠分选骨髓衍生肝干细胞亚群c-Kit+lin-

目的:利用免疫磁性细胞分选系统分离纯化骨髓衍生肝干细胞亚群c-Kit lin-。方法:实验于2006-07/08在南方医科大学实验动物中心完成。6～8周龄的SPF级纯系BALB/C雄性小鼠10只,体质量18～20g。收集小鼠股骨骨髓细胞,利用免疫磁性细胞分选系统,通过两步法分选纯化c-Kit lin-:将获取的lin-细胞悬液8℃条件下1500r/min离心10min,弃上清,按80μL/107加入Buffer重悬细胞。按20μL/107加生物素抗体磁珠,混匀,4℃冰箱孵育15min,按1mL/107加入Buffer洗细胞1次,8℃条件下1500r/min离心10min,弃上清,按500μL/108加入Buffer重悬细胞。Buffer500μL润MS柱,悬液过柱后,Buffer500μL/次洗柱3次,柱子脱离磁场,加1mLBuffer,用配套柱塞推出柱中的c-Kit lin-细胞,收集到c-kit lin-细胞,细胞计数。取2.0×106个细胞分成10等份,流式细胞仪分析c-Kit lin-细胞纯度,计算回收率,评估纯化效率,苔盼兰染色检测纯化前后的细胞活力。计算活细胞的百分率。细胞纯度和细胞回收率的计算:细胞纯度=分离产物中的阳性细胞数/分离细胞的总细胞数×100%,细胞回收率=分离产物中的阳性细胞数/起始标本阳性细胞总数×100%。结果:10只小鼠均进入结果分析。利用免疫磁性细胞分选系统分选出的骨髓衍生肝干细胞亚群c-Kit lin-细胞纯度和回收率分别为(77.98±2.34)%,75.40%,纯化前后细胞活力不受影响。结论:免疫磁性细胞分选系统能有效分选骨髓衍生肝干细胞亚群c-Kit lin-,纯度和回收率高,且不影响细胞活力。     

Differentiation of soft tissue benign and malignant peripheral nerve sheath tumors with magnetic resonance imaging

PURPOSE: The objective of this study was to differentiate the magnetic resonance (MR) imaging appearance of benign peripheral nerve sheath tumors (PNSTs) from that of malignant PNSTs. MATERIALS AND METHODS: Twenty-six patients who underwent MR imaging and had a histologic diagnosis of benign (schwannoma, n=16; neurofibroma, n=1) or malignant (n=9) PNST were retrospectively reviewed. The size, location, shape, margin, and signal intensities of the tumors on precontrast and gadolinium-enhanced MR imaging were analyzed. In each patient, the presence or absence of split fat, target, and fascicular signs was determined. RESULTS: The mean size of the benign PNSTs (3.4 cm, S.D.=2.5 cm) was significantly smaller than that of the malignant tumors (8.2 cm, S.D.=3.1 cm) (P<.001). Seventeen (65.4%) of the 26 tumors were spindle shaped or ovoid (12 benign and 5 malignant tumors). Contiguity with specific nerves was identified in 15 (88.2%) of the 17 benign PNSTs but in none of the malignant tumors (P<.05). Well-defined margins were noted in all 17 benign PNSTs but in only 3 (33.3%) of the 9 malignant tumors (P<.001). Five (55.6%) of the 9 malignant PNSTs but none of the benign tumors showed signal intensity change in adjacent soft tissue (P<.05). There was no significant difference in signal intensity between the benign and malignant tumors on T(1)-weighted, T(2)-weighted, and contrast-enhanced MR images. The split fat and target signs were present more frequently in the benign PNSTs than in the malignant PNSTs (P<.05).Conclusions: Benign and malignant PNSTs are often spindle shaped. Recognition of contiguity with adjacent nerves, a well-defined margin, and the presence of the split fat sign may suggest benignity. Imaging features suggestive of malignancy can be a larger size and an infiltrative margin.     

ECM1 and TMPRSS4 are diagnostic markers of malignant thyroid neoplasms and improve the accuracy of fine needle aspiration biopsy

OBJECTIVE: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. SUMMARY AND BACKGROUND DATA: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. METHODS: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. RESULTS: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P < or = 0.031). CONCLUSIONS: ECM1 and TMPRSS4 are excellent diagnostic markers of malignant thyroid nodules and may be used to improve the diagnostic accuracy of FNA biopsy. ECM1 is also a marker of the extent of disease in differentiated thyroid cancers.     

Can localization studies be used to direct focused parathyroid operations?

BACKGROUND: There is considerable controversy today concerning the most appropriate surgical approach for patients with primary hyperparathyroidism. The conventional surgical operation involves a bilateral neck exploration through a collar incision with identification of all parathyroid tissue and removal of abnormal parathyroid glands while the patient is under general anesthesia. The success rate of this operation is about 95% or greater in the hands of an experienced endocrine surgeon. Preoperative localization techniques are generally considered to be unnecessary before initial parathyroid operations. The purpose of this investigation was (1) to evaluate the individual and combined accuracy of ultrasonography and technetium 99m sestamibi scans in localizing abnormal parathyroid glands and (2) to determine whether such scans could be used to direct a focused operation. METHODS: We retrospectively studied 338 patients with sporadic primary hyperparathyroidism who had preoperative neck localization studies, ultrasonography and/or technetium 99m sestamibi scans, and parathyroid exploration (238 patients or, reexploration, 60 patients) from January 1996 to April 2000 at the University of California San Francisco/Mount Zion Medical Center. The preoperative localization studies were recorded as true-positive, false-positive, and false-negative and compared with the surgical and pathologic findings and with the outcome of the operation. RESULTS: All of the abnormal parathyroid glands were correctly identified by ultrasonography in 184 of 303 patients (60.7%) and by technetium 99m sestamibi scanning in 183 of 237 patients (77.2%). The sensitivities of ultrasonography and sestamibi were 65% and 80%, respectively. Among the 202 patients who received both ultrasonography and sestamibi scans, a parathyroid tumor was identified at the same site in 105 (52%) of them. When both techniques identified a parathyroid tumor at the same site, the tests were correct in 101 of 105 patients and the sensitivity increased to 96%. CONCLUSIONS: When both the ultrasonography and sestamibi scans identified the same, solitary parathyroid tumor in patients with sporadic primary hyperparathyroidism, this was the only abnormal parathyroid gland in 96% of the patients. A focused parathyroidectomy could therefore be performed in such patients with an acceptable ( approximately 95%) success rate.     

Results of surgical treatment for hyperparathyroidism associated with renal disease

Recently, the role and timing of surgery for treating secondary and tertiary hyperparathyroidism (HPT) have been questioned. In order to delineate the indications for surgery in these patients, a retrospective analysis of 53 consecutive patients treated with parathyroidectomy was conducted. Subtotal thyroidectomy was done in 37 of 45 patients undergoing their initial operations for HPT. Eight additional patients were referred after failed operations. Of 33 patients with preoperative bone pain, 70% improved. Joint pain improved in 87% of 30 patients, pruritus improved in 81% of 27 patients, and preoperative malaise improved in 73% of 33 patients after parathyroidectomy. Abdominal pain and irritated eyes were unlikely to improve. The best predictors of a successful outcome were a markedly elevated preoperative immunoreactive parathyroid hormone (mid-region) level and an elevated alkaline phosphatase level. There were no perioperative deaths. One patient (1.6%) had a recurrent laryngeal nerve injury, and one patient required reoperation for a neck hematoma. No patient had permanent hypoparathyroidism, but transient hypocalcemia (less than 7 mg/dL) occurred in 22%. Postoperative hypocalcemia correlated with elevated preoperative alkaline phosphatase levels (r2 = 0.247).     

Total Thyroidectomy or Thyroid Lobectomy in Patients with Low-risk Differentiated Thyroid Cancer: Surgical Decision Analysis of a Controversy Using a Mathematical Model

There is a general consensus that total or near-total thyroidectomy is the optimal treatment for patients with high risk differentiated thyroid cancer (DTC), but the optimal extent of thyroidectomy in patients with low risk DTC continues to be controversial. To determine the optimal extent of thyroidectomy in patients with low risk DTC, we used decision analysis to compare the trade-offs of total thyroidectomy (TT) to thyroid lobectomy (TL). The decision analysis model included the probabilities of thyroidectomy complications, risk of DTC recurrence, and death from DTC. This information was obtained from the literature and from outcome data for patients with low risk DTC from our institution. In addition, the concept of utilities was used in the analysis. To determine the utility of each health outcome state (thyroidectomy complication, DTC recurrence, and DTC mortality for low risk patients) a survey was conducted. Overall, prospective patients viewed DTC recurrence as less desirable than thyroidectomy complication. The utilities assigned by the survey participants varied over a wide range, with 61.5% of the individuals viewing the occurrence of a thyroidectomy complication as better than DTC recurrence. At baseline utilities and probabilities, TT had a higher expected utility than TL. One-way sensitivity analysis varying the rates of (1) thyroidectomy complication, (2) DTC recurrence, and (3) DTC mortality over the possible range showed that complication from initial thyroidectomy was the most important factor that determined the preferred extent of thyroidectomy. TL was the preferred surgical approach only if a complication rate of > 33:1, TT/TL complication rate ratio, was assumed. When no differences in DTC recurrence between the two approaches was assumed in the model, TL had a higher expected utility using the baseline utilities of thyroidectomy complication and DTC mortality. The analysis indicates that TT in patients with low risk DTC is preferable to TL. However, TL is preferred if (1) no difference in the DTC recurrence rate between the two approaches is assumed, (2) a higher complication rate for TT is used (> 33 times higher), or (3) the utility ratio of thyroidectomy complication to DTC recurrence is < 0.8 TL. We believe this decision analysis model provides an objective approach that others can use to select the optimal extent of thyroidectomy based on patient preference of health outcome states, institution-specific outcome data for DTC recurrence or mortality, and the surgeon-specific complication rate.     

The brain‐derived neutrophic factor val66met polymorphism and sudden unexpected infant death

Aim: Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain‐derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls. Methods: The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real‐time PCR and fluorescence melting curve analysis. Results: There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively). Conclusion: The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G‐protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.