ELISA定量免疫组化技术在瘤源性骨软化病中的应用及非干预性危险因素的循证医学研究
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摘要
1、研究目的：瘤源性骨软化病（TIO）是一种罕见的获得性副肿瘤代谢性疾病，其临床特点为低磷血症与骨软化症，成纤维细胞生长因子23（FGF-23）在其发病中起重要作用，但临床上该肿瘤的定位往往极为困难。本研究的目的是系统性阐述TIO的临床特征、化验特征及病理特征，同时报道一种新型FGF-23的定量检测技术。
2、研究方法：检索纳入本中心诊治的14例TIO患者，从各方面详细阐述总结该病的特征。纳入对照组26例TIO肿瘤易误诊为的其它肿瘤，应用改进的ELISA定量免疫组化技术（通过将其操作转移至载玻片上而简化操作步骤）检测福尔马林固定-石蜡包埋的TIO标本及对照组标本中的FGF-23表达水平，并将本技术与文献所报道的RT-PCR技术作对比。
3、结果：总结该14例TIO患者的临床资料，其临床特点为长期的骨软化症病史、低磷血症与尿磷酸盐的丢失。TIO相关肿瘤可以位于全身各部位，大多可由常规检测技术或奥曲肽扫描定位，其病理类型大多为良性尿磷酸盐间质肿瘤混合结缔组织亚型（PMTMCT），手术完整肿瘤切除是该病的有效治疗方式。在肿瘤完整手术切除后，血磷在3.5±1.8天恢复正常，临床症状也将在数月内完全改善。通过ELISA定量免疫组化技术，13/14例TIO肿瘤检测出FGF-23的高表达（0.99±0.56），而对照组的26例非TIO肿瘤全部为FGF-23低表达（0.08±0.04），统计差异明显（p<0.001，95%CI0.48～1.14）。该检测技术明显优于文献所报道的石蜡标本中FGF-23的RT-PCR检测技术。
4、结论：TIO为一种罕见的经常漏诊、延迟诊断或误诊的疾病，临床医师与病理医师都应用分别重视TIO与PMTMCT的诊断。ELISA定量免疫组化技术可用于福尔马林固定-石蜡包埋的手术或活检标本中FGF-23的检测，该方法可行性好、重复性高，从而可为TIO提供新的临床诊断途径。
1、研究目的：髋部骨折术后患者死亡率高，文献报道了很多死亡风险预测的术前危险因子，但目前循证医学仍未系统性的鉴定总结这些危险因子。骨科下肢大手术后血栓栓塞（Venousthromboembolism，VTE）发病率高，各指南共同推荐该类患者必须术前评估有无VTE增高的风险。但是，各指南推荐的危险因素不尽相同，并且也并不是特异性针对于骨科下肢大手术患者。本系统评价与Meta分析的目的是系统性鉴定髋部骨折患者术后高死亡风险的术前危险因子，并鉴定骨科下肢大手术后发生VTE的术前危险因素。
2、研究方法：通过系统性检索Pubmed、Embase与Cochrane数据库系统性检索相关研究，仅纳入前瞻性研究与前瞻方式收集数据的回顾性研究。通过最佳证据分析模型同时融合了各纳入研究的实验设计质量与研究结果，进行系统评价与Meta分析。高循证等级危险因素的危险比仅由高质量等级的文献合并而来。
3、结果：髋部骨折术后死亡预测因子分析：共纳入75篇文献64,316例患者，患者总体死亡率1月为13.3%，3~6月为15.8%，1年为24.5%，2年为34.5%；通过最佳证据分析模型，共鉴定出12个高循证等级危险因子，包括高龄、男性、骨折前养老或医疗机构居住、术前行走能力差、术前日常生活能力差、高ASA分级、精神状态差、多种并存病、老年痴呆或认知障碍、糖尿病、恶性肿瘤与心血管疾病；另外鉴定了7个中等循证等级危险因素与12个有限循证等级危险因素，仅种族因素被鉴定为矛盾等级危险因素。VTE危险因素分析：共纳入47篇文献97,082例患者，总VTE发生率为25.8%，症状性VTE的发生率为1.7%，PE发生率为0.6%。通过最佳证据分析模型，我们鉴定了2个高循证等级危险因素，包括高龄与TKA手术（与THA相比）；鉴定了5个中等循证等级危险因素，包括V因子Leiden突变或APC（Activitieso fproteinC）抵抗，既往血栓病史，术前行走能力差，肥胖与女性患者；另外鉴定了12个有限循证等级危险因素与2个矛盾循证等级危险因素（心血管疾病与活化部分凝血活酶时间）。
4、结论：髋部骨折术后患者死亡率高，临床上应该特别注意以上12个高循证等级术前危险因子。VTE的预防应特别注意以上2个高循证等级与5个中循证等级的术前危险因素。由于本研究的局限性，这些危险因子还有待进一步的研究。
Part Ⅰ:Tumor-induced osteoaalacia and Quantitative ELISA-like immunohistocheaistry in the diagnosis of the disease
Objective:Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disorder and fibroblast growth factor23(FGF-23) plays a key role in its pathogenesis. This study was conducted to comprehensively describe the clinical, laboratory and pathological manifestations of the disease, and also to document a novel FGF-23detecting and quantifying procedure.
Methods:14cases of diagnosed TIO or cases with typical clinical manifestations of TIO were retrieved from our institution.26non-TIO tumors which TIO tumors always misdiagnosed of were also included. We simplified the quantitative ELISA-like immunohistochemistry procedure on the slide and FGF-23level was measured by the procedure using formalin-fixed and paraffin-embedded tissues. The results were also compared with a previously reported FGF-23RT-PCR assay.
Results:Summarized by the14included TIO cases, the clinical manifestations of TIO were a long-standing history of osteomalacia, hypophosphatemia and urinary phosphate wasting. The associated tumors were mostly benign phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT) that could be located anywhere of the body and most of which could be localized by conventional examinations and octreotide scanning. After complete tumor removal, serum phosphate would return to normal level within3.5±1.8days and clinical symptoms would be resolved within several months. By the quantitative ELISA-like immunohistochemistry,13/14cases were identified and quantified of high FGF-23level (0.99±0.56, compared with the26non-TIO tumors of0.08±0.04, p<0.001,95%CI0.48-1.14). The results were also confirmed and even had superiority over the FGF-23RT-PCR assay using paraffin-embedded tissues.
Conclusion:Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of the disease of TIO and PMTMCT, respectively. The quantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect and quantify the high FGF-23level using formalin-fixed and paraffin-embedded biopsies or specimens.
Part II:Evidence-based medicine analysis of non-interventional risk factors
Objective:Hip fractures are always associated with a high postoperative mortality, the preoperative predictors for mortality have neither been well identified nor well summarized. Major lower extremity surgeries are associated with a high postoperative VTE (venous thromboembolism) incidence. It is all recommended that these patients should be assessed preoperatively for the increased VTE risks. However, the risk factors have not been well identified. The recommended risk factors differred from each other among these guidelines and they were also not specificly recommended for major lower extremity surgery patients either. This systematic review and meta-analysis was performed to identify the preoperative non-interventional predictors for mortality in hip fracture patients, and was also performed to identify the preoperative non-interventional predictors of VTE for THA, TKA and hip fracture patients.
Methods:Non-interventional studies were searched in Pubmed, Embase, Cochrane central database (All to February26th,2011). Only prospective studies and retrospective studies with prospective collected data were included. Qualities of included studies were assessed by a standardized scale previous reported for observational studies. The effects of individual studies were combined with the study quality score using a previous reported model of best-evidence synthesis. The Hazard Ratios of strong evidence predictors were combined only by high quality studies in which risk factors were identified.
Results:Preoperative risk factor analysis of mortality after hip fracture surgery:75studies involving64,316patients were included, the overall inpatient or one month mortality was13.3%,3-6months was15.8%,1year24.5%and2years34.5%; there were strong evidence for12mortality predictors, including advanced age, male gender, nursing home or facility residence, poor preoperative walking capacity, poor activities of daily living, higher ASA grading, poor mental state, multiple comorbidities, dementia or cognitive impairment, diabetes, cancer and cardiac disease; we also identified7moderate evidence and12limited evidence mortality predictors, and only the race was identified as the conflicting evidence predictor. VTE risk factor analysis for the major lower extremity surgery patients:of the47included studies involving97,082patients, the incidence of total VTE was25.8%, symptomatic DVT was1.7%and PE0.6%; by best-evidence, we had identified2strong evidence predictors including advanced age and TKA (versus THA) surgery, as well as5moderate evidence predictors including factor V Leiden mutation or APC resistance, history of previous thromboembolism,, impaired walking capacity, obesity and female gender; we also identified other12limited evidence predictors and2conflicting evidence predictors (including cardiovascular diseases and activated partial thromboplastin time).
Conclusion:While there is no conclusive evidence of these preoperative predictors for mortality following hip fractures and preoperative predictors of VTE for patients undergoing THA, TKA and hip fracture surgery, special attention should be paid to the above strong and moderate evidence predictors. Further researches are still needed to evaluate these risk factors.
2、研究方法：检索纳入本中心诊治的14例TIO患者，从各方面详细阐述总结该病的特征。纳入对照组26例TIO肿瘤易误诊为的其它肿瘤，应用改进的ELISA定量免疫组化技术（通过将其操作转移至载玻片上而简化操作步骤）检测福尔马林固定-石蜡包埋的TIO标本及对照组标本中的FGF-23表达水平，并将本技术与文献所报道的RT-PCR技术作对比。
3、结果：总结该14例TIO患者的临床资料，其临床特点为长期的骨软化症病史、低磷血症与尿磷酸盐的丢失。TIO相关肿瘤可以位于全身各部位，大多可由常规检测技术或奥曲肽扫描定位，其病理类型大多为良性尿磷酸盐间质肿瘤混合结缔组织亚型（PMTMCT），手术完整肿瘤切除是该病的有效治疗方式。在肿瘤完整手术切除后，血磷在3.5±1.8天恢复正常，临床症状也将在数月内完全改善。通过ELISA定量免疫组化技术，13/14例TIO肿瘤检测出FGF-23的高表达（0.99±0.56），而对照组的26例非TIO肿瘤全部为FGF-23低表达（0.08±0.04），统计差异明显（p<0.001，95%CI0.48～1.14）。该检测技术明显优于文献所报道的石蜡标本中FGF-23的RT-PCR检测技术。
4、结论：TIO为一种罕见的经常漏诊、延迟诊断或误诊的疾病，临床医师与病理医师都应用分别重视TIO与PMTMCT的诊断。ELISA定量免疫组化技术可用于福尔马林固定-石蜡包埋的手术或活检标本中FGF-23的检测，该方法可行性好、重复性高，从而可为TIO提供新的临床诊断途径。
1、研究目的：髋部骨折术后患者死亡率高，文献报道了很多死亡风险预测的术前危险因子，但目前循证医学仍未系统性的鉴定总结这些危险因子。骨科下肢大手术后血栓栓塞（Venousthromboembolism，VTE）发病率高，各指南共同推荐该类患者必须术前评估有无VTE增高的风险。但是，各指南推荐的危险因素不尽相同，并且也并不是特异性针对于骨科下肢大手术患者。本系统评价与Meta分析的目的是系统性鉴定髋部骨折患者术后高死亡风险的术前危险因子，并鉴定骨科下肢大手术后发生VTE的术前危险因素。
2、研究方法：通过系统性检索Pubmed、Embase与Cochrane数据库系统性检索相关研究，仅纳入前瞻性研究与前瞻方式收集数据的回顾性研究。通过最佳证据分析模型同时融合了各纳入研究的实验设计质量与研究结果，进行系统评价与Meta分析。高循证等级危险因素的危险比仅由高质量等级的文献合并而来。
3、结果：髋部骨折术后死亡预测因子分析：共纳入75篇文献64,316例患者，患者总体死亡率1月为13.3%，3~6月为15.8%，1年为24.5%，2年为34.5%；通过最佳证据分析模型，共鉴定出12个高循证等级危险因子，包括高龄、男性、骨折前养老或医疗机构居住、术前行走能力差、术前日常生活能力差、高ASA分级、精神状态差、多种并存病、老年痴呆或认知障碍、糖尿病、恶性肿瘤与心血管疾病；另外鉴定了7个中等循证等级危险因素与12个有限循证等级危险因素，仅种族因素被鉴定为矛盾等级危险因素。VTE危险因素分析：共纳入47篇文献97,082例患者，总VTE发生率为25.8%，症状性VTE的发生率为1.7%，PE发生率为0.6%。通过最佳证据分析模型，我们鉴定了2个高循证等级危险因素，包括高龄与TKA手术（与THA相比）；鉴定了5个中等循证等级危险因素，包括V因子Leiden突变或APC（Activitieso fproteinC）抵抗，既往血栓病史，术前行走能力差，肥胖与女性患者；另外鉴定了12个有限循证等级危险因素与2个矛盾循证等级危险因素（心血管疾病与活化部分凝血活酶时间）。
4、结论：髋部骨折术后患者死亡率高，临床上应该特别注意以上12个高循证等级术前危险因子。VTE的预防应特别注意以上2个高循证等级与5个中循证等级的术前危险因素。由于本研究的局限性，这些危险因子还有待进一步的研究。
Part Ⅰ:Tumor-induced osteoaalacia and Quantitative ELISA-like immunohistocheaistry in the diagnosis of the disease
Objective:Tumor-induced osteomalacia (TIO) is a rare acquired paraneoplastic disorder and fibroblast growth factor23(FGF-23) plays a key role in its pathogenesis. This study was conducted to comprehensively describe the clinical, laboratory and pathological manifestations of the disease, and also to document a novel FGF-23detecting and quantifying procedure.
Methods:14cases of diagnosed TIO or cases with typical clinical manifestations of TIO were retrieved from our institution.26non-TIO tumors which TIO tumors always misdiagnosed of were also included. We simplified the quantitative ELISA-like immunohistochemistry procedure on the slide and FGF-23level was measured by the procedure using formalin-fixed and paraffin-embedded tissues. The results were also compared with a previously reported FGF-23RT-PCR assay.
Results:Summarized by the14included TIO cases, the clinical manifestations of TIO were a long-standing history of osteomalacia, hypophosphatemia and urinary phosphate wasting. The associated tumors were mostly benign phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT) that could be located anywhere of the body and most of which could be localized by conventional examinations and octreotide scanning. After complete tumor removal, serum phosphate would return to normal level within3.5±1.8days and clinical symptoms would be resolved within several months. By the quantitative ELISA-like immunohistochemistry,13/14cases were identified and quantified of high FGF-23level (0.99±0.56, compared with the26non-TIO tumors of0.08±0.04, p<0.001,95%CI0.48-1.14). The results were also confirmed and even had superiority over the FGF-23RT-PCR assay using paraffin-embedded tissues.
Conclusion:Since TIO was often delay-diagnosed or misdiagnosed, clinicians and pathologists should be aware of the disease of TIO and PMTMCT, respectively. The quantitative ELISA-like immunohistochemistry was a feasible and reproducible procedure to detect and quantify the high FGF-23level using formalin-fixed and paraffin-embedded biopsies or specimens.
Part II:Evidence-based medicine analysis of non-interventional risk factors
Objective:Hip fractures are always associated with a high postoperative mortality, the preoperative predictors for mortality have neither been well identified nor well summarized. Major lower extremity surgeries are associated with a high postoperative VTE (venous thromboembolism) incidence. It is all recommended that these patients should be assessed preoperatively for the increased VTE risks. However, the risk factors have not been well identified. The recommended risk factors differred from each other among these guidelines and they were also not specificly recommended for major lower extremity surgery patients either. This systematic review and meta-analysis was performed to identify the preoperative non-interventional predictors for mortality in hip fracture patients, and was also performed to identify the preoperative non-interventional predictors of VTE for THA, TKA and hip fracture patients.
Methods:Non-interventional studies were searched in Pubmed, Embase, Cochrane central database (All to February26th,2011). Only prospective studies and retrospective studies with prospective collected data were included. Qualities of included studies were assessed by a standardized scale previous reported for observational studies. The effects of individual studies were combined with the study quality score using a previous reported model of best-evidence synthesis. The Hazard Ratios of strong evidence predictors were combined only by high quality studies in which risk factors were identified.
Results:Preoperative risk factor analysis of mortality after hip fracture surgery:75studies involving64,316patients were included, the overall inpatient or one month mortality was13.3%,3-6months was15.8%,1year24.5%and2years34.5%; there were strong evidence for12mortality predictors, including advanced age, male gender, nursing home or facility residence, poor preoperative walking capacity, poor activities of daily living, higher ASA grading, poor mental state, multiple comorbidities, dementia or cognitive impairment, diabetes, cancer and cardiac disease; we also identified7moderate evidence and12limited evidence mortality predictors, and only the race was identified as the conflicting evidence predictor. VTE risk factor analysis for the major lower extremity surgery patients:of the47included studies involving97,082patients, the incidence of total VTE was25.8%, symptomatic DVT was1.7%and PE0.6%; by best-evidence, we had identified2strong evidence predictors including advanced age and TKA (versus THA) surgery, as well as5moderate evidence predictors including factor V Leiden mutation or APC resistance, history of previous thromboembolism,, impaired walking capacity, obesity and female gender; we also identified other12limited evidence predictors and2conflicting evidence predictors (including cardiovascular diseases and activated partial thromboplastin time).
Conclusion:While there is no conclusive evidence of these preoperative predictors for mortality following hip fractures and preoperative predictors of VTE for patients undergoing THA, TKA and hip fracture surgery, special attention should be paid to the above strong and moderate evidence predictors. Further researches are still needed to evaluate these risk factors.
引文
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