Diagnosis of renal amyloidosis using Congo red fluorescence

Early diagnosis and classification of amyloid deposition and differentiation from other glomerular fibrillar deposits relies on routine Congo red (CR) histochemistry. Congo red fluorescence (CRF) is an alternative method based on examination of the CR-stained section by ultraviolet (UV) light. The aim of this study is to investigate the usefulness of CRF, especially when applied to frozen kidney sections. Congo red fluorescence was applied to sections of frozen kidney biopsies prospectively and to paraffin sections retrospectively. The findings of CRF were compared to CR staining in bright light. Prospectively, 15 cases of amyloidosis were diagnosed on frozen sections and identical CR staining was found in all of the paraffin-stained sections. There were no false positives or negatives. Retrospectively, 146 renal biopsies previously stained with CR were re-evaluated with CRF. Eighty-seven CR positive cases were confirmed by CRF, and one new case was identified. Congo red fluorescence is simple to perform and more pronounced, therefore easier to evaluate than CR in bright light. Congo red, when combined with immunohistochemistry, is still visible under UV whereas CR is masked in bright light. Although not widely used, the CRF method for detecting amyloid is simple to use with a high specificity and sensitivity, and may be applied successfully to frozen sections.     

Highly sensitive diagnosis of amyloid and various amyloid syndromes using Congo red fluorescence

In order to find how best to diagnose amyloid deposits as early as possible, the sensitivity of three different methods that can be applied to the diagnosis of amyloid in tissue sections have been compared: the Congo red staining method (CR), the combination of CR and immunocytochemistry (CRIC) and Congo red fluorescence (CRF). Tissue blocks were available from 25 patients, including 11 with immunohistochemically distinct and 3 with chemically undefined amyloid diseases. The results revealed (a) that CRF is more sensitive than either CR or CRIC, as shown qualitatively and quantitatively, (b) that CRF can therefore be utilized to track down even minute amyloid deposits, which can be missed by the other two methods; (c) that the specificity of CRF and CRIC is secured on double-stained sections by the demonstration of green birefringence (GB) of the CRF-marked and IC-marked areas; (d) that CRF can be performed on the spot by just changing the light source; and (e) that CRF is not hampered by the congruent IC chromogen overlay, which ensures the specific classification of the amyloid deposits as applied to different amyloid classes. In conclusion, CRF was demonstrated to be the most sensitive method for direct diagnosis of amyloid in tissue sections. This method can, therefore, allow the earliest diagnosis and classification of amyloid, which is a good basis for an amyloid class-specific therapy while organ damage is still minimal. Received: 29 September 1999 / Accepted: 2 December 1999     

Prevalence of amyloid and positive Congo red frequency among routine surgical specimens

Amyloidosis is characterized by an extracellular tissue deposition of one of a family of biochemical proteins that are abnormally folded. The deposits are often subtle, and can be missed on routine hematoxylin and eosin (H&E)-stained slides. Current literature does not offer an expected prevalence rate of amyloid or frequency of Congo red positivity among routine surgical pathology specimens in a referral bias-free setting. The objective of this study was to determine these parameters at a large community hospital. The pathology database was searched for all surgical pathology and autopsy cases diagnosed with amyloidosis from 2001–2013. All cases were reviewed and clinical parameters were recorded. Based on H&E interpretation, Congo red was performed on 218 cases. Of these, 36% confirmed positive birefringence. The prevalence of amyloid among routinely submitted pathology specimens was calculated as 0.027% over the 13-year interval. Amyloid was detected in less than 1% of routine surgical specimens. When suspicious H&E findings prompted Congo red staining, amyloidosis was confirmed about a third of the time over the 13-year period. Establishing an optimal rate of Congo red utilization may provide a standard measurement needed to ensure high amyloid detection rates among pathologists at the community level.     

Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy

Amyloid fat pad aspiration specimens for cases with a clinical suspicion of amyloid typically are stained with Congo red and examined by brightfield microscopy. Congophilia with apple-green birefringence by polarization microscopy (PM) is considered diagnostic for amyloid. Examination of Congo red-stained slides by fluorescent microscopy (FM) is considered by some to be a more sensitive detection method. In this study, we assessed the utility of this technique in cytopathology archival slides from abdominal fat pad aspirations previously stained with Congo red dye. Seventy-eight cases of abdominal fat pad aspirations collected during the last 5 yr and stained with the Congo red procedure were obtained from archival files. Additionally, 20 adipose tissue material slides prepared from the surgical pathology specimens were examined as controls. One representative smear was examined in each case using FM equipped with rhodamine excitation/absorption (540/570 nm) filters. Relevant clinical information was obtained in all cases. Twelve cases (15.4%) of the 78 fat pad aspiration cases were reported originally as positive by Congo red stain using polarization and apple-green birefringence as diagnostic criteria. On review, four cases were deemed unsatisfactory. By FM examination 29 of the 74 (39.2%) cases were reclassified as positive for amyloid. The results were confirmed by immunohistochemical stain for amyloid P protein and electron microscopy. A number of similar distinct fluorescence and immunohistochemical patterns were recognized in the positive cases. Minimally weak fluorescence in the adipose tissue was observed in the control cases. The use of FM in Congo red-stained fat pad smears can improve the detection of amyloid in cytology preparations.     

Identification of elastic fibers and lamellae in porcine pericardium and aorta by confocal, fluorescence and polarized light microscopy

Pericardial connective tissue has been used to construct bioprostheses to repair various types of injuries, including aortic wall repairs. The arrangement and the distribution of elastic and collagen staple fibers are related directly to the biomechanical properties of the tissue and thus determine the choice tissue for the construction of bioprostheses. Although elastic fibers can be visualized using several histochemical methods, the specificity and mechanism of binding involved remain to be clarified. In this work, we compared the elastic net of the porcine pericardial matrix with that of the aortic wall using 1-anilino-8-naphthalene sulfonate (ANS) and dansyl chloride (DCl) as fluorescent probes and the permanganate-bisulfite-toluidine (PBT) method. Polarized light and fluorescence microscopy were simultaneously used to analyze the tissues. Some samples also were examined using confocal microscopy. Aorta and pericardium treated with ANS and DCl showed elastic fibers and lamellae with an intense blue fluorescence. When stained with the PBT method, the aortic elastic lamellae were clearly metachromatic and, under polarized light, they showed a greenish birefringence. DCl provided clearer fluorescent labelling of elastic fibers when examined using confocal microscopy.     

Evidence of the presence of amyloid substance in the blood of familial amyloidotic polyneuropathy patients with ATTR Val30Met mutation

Transthyretin (TTR) is a major amyloid fibril protein found in patients with familial amyloidotic polynuropathy (FAP) and senile systemic amyloidosis (SSA). Mainly synthesized in the live, TTR is transferred in the form of tetramer bound with thyroxine, retinol-binding protein (RBP) and lipoprotein in the blood. The aim of this study was to demonstrate the presence of amyloid substances in the blood by investigated the hemocoelom amyloid in different tissue sections from autopsies such as brain, kidney, heart and aorta arch tissue. Congo red staining was employed following by application of polarized light examination, to verify the presence of amyloid deposition in the tissues. Immunohistochemical staining was then performed to identify the specific type of amyloid deposition. Matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF/MS) was also used to analyze TTR mutation in FAP patients. All subjects were FAP ATTR Val30Met patients. In FAP patients, TTR amyloid deposition was found mainly in the tunica intima of the aortic arch. Interestingly, amyloid substance was found in the blood of FAP patient. Our results suggest that amyloid substance was present in the blood of FAP ATTR Val30Met patients.     

An ultrastructural study of nodular amyloidosis of the tongue

We have treated a patient with a nodular amyloidosis in the center of the dorsum of the tongue. Ultrastructural observations of human amyloidosis have been reported by many investigators, but there have been few reports of nodular amyloidosis of the tongue. Ultrastructural investigation showed a number of amyloid fibrils around and in the plasmocytoid cells, and severe pressure atrophy was found in many stromal cells, especially in endothelial cells.This study was presented at the 27th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Kurashiki, September 28–30, 1995.     

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis (“phantom amyloidosis”).     

Unsuspected systemic amyloidosis diagnosed by fine-needle aspiration of the salivary gland: case report

Amyloidosis of the head and neck region may represent a local amyloidoma or a manifestation of systemic disease. Involvement of major salivary glands by either primary or secondary forms of amyloidosis is very rare. We describe a case of systemic amyloidosis that initially presented as submandibular gland mass and was diagnosed by fine-needle aspiration (FNA). A 69-year-old male presented with submandibular mass. His past medical history was significant for left forearm melanoma that was excised 6 years ago and tricuspid valve endocarditis after valvular replacement 3 months prior to FNA of the submandibular gland. The patient had no symptoms or clinical and laboratory data suggestive of amyloidosis. FNA specimen showed salivary gland tissue and abundant amorphous material, which stained positive for amyloid with Congo red stain and showed typical birefringence when examined by polarized microscopy. Further workup of the patient revealed generalized amyloidosis with multiorgan involvement by the disease. This case demonstrates that FNA can be a useful technique in the diagnosis of unsuspected amyloidosis.     

The use of the Congo red-related dye DBACR to recognize the heavy chain-derived abnormality of myeloma immunoglobulins

Introduction The aim of this study was to differentiate heavy and light chain-derived instability of monoclonal myeloma immunoglobulins by complexation of matched supramolecular dyes. These are composed of several micellar pieces of self-assembled dye molecules which may penetrate the protein interior of the binding locus with polypeptide chains. These dyes were used to elicit, by precipitation, the postulated higher aggregation tendency of the heavy chain derived from its higher hydrophobicity. Materials and Methods Agarose gel electrophoresis was used to create conditions for dye complexation and to reveal the precipitation. Results Congo red derivatives with aromatic ring substitutes, BACR and DBACR, of increased penetrating capability were chosen to provoke the precipitation of abnormal immunoglobulins by displacing association-prone polypeptide chains from the protein interior. Conclusions The results of this study confirm the heavy chain-related propensity of some monoclonal immunoglobulins to aggregate and precipitate. The simplicity of the technique may improve clinical diagnosis and facilitate predictions of disease complications.     

Silver Nanoparticles Engineered Polystyrene‐Poly(N‐isopropylmethacrylamide‐acrylic acid) Core Shell Hybrid Polymer Microgels for Catalytic Reduction of Congo Red

Polystyrene‐poly(N‐isopropylmethacrylamide‐acrylic acid) [pSty‐p(NIpmam‐AC)] core shell microgels loaded with silver nanoparticles (NPs) as a recyclable catalyst for rapid reduction of Congo Red (CR) dye in aqueous medium is reported for the first time. Catalytic activity of Ag‐pSty‐p(NIpmam‐AC) core shell composite microgels is measured by adopting the reduction of CR as a benchmark reaction. Restriction of Ag NPs in single layered p(NIpmam‐AC) shell of Ag‐pSty‐p(NIpmam‐AC) core shell composite microgels enhances the diffusion of reactants to access the Ag NPs, surface during reaction with respect to previously reported core shell and homogenous composite microgels, and thus enhances the rate of CR reduction. The solid nonresponsive pSty core induces easy recyclability in core shell composite microgel catalysts. The effect of different reaction parameters such as amount of catalyst, concentration of NaBH₄ and CR on the reaction completion time for CR reduction, as well as on the value of apparent rate constant (k_app), is also studied by keeping all other parameters constant, separately. Reduction efficiency of Ag‐pSty‐p(NIpmam‐AC) catalyst is also measured under different reaction conditions for the reduction of CR. A slight decrease in the percentage activity of Ag‐pSty‐p(NIpmam‐AC) catalyst is observed for up to four cycles for CR reduction. The reported Ag‐pSty‐p(NIpmam‐AC) composite microgel catalysts can be used to degrade other pollutants.     

Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods

Purpose: The purpose of this study was to evaluate three methods for detection of biofilm formation in staphylococci. Methods: For detection of biofilm formation, 152 clinical isolates of Staphylococcus spp. were screened by tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) method. Results: Of the 152 Staphylococcus spp. 88(57.8%) displayed a biofilm-positive phenotype under the optimized conditions in the TCP method and strains were further classified as high 22 (14.47 %) and moderate 60 (39.4 %) while in 70 (46.0 %) isolates weak or no biofilm was detected. Though TM correlated well with the TCP test for 18 (11.8 %) strongly biofilm producing strains, weak producers were difficult to discriminate from biofilm negative isolates. Screening on CRA does not correlate well with either of the two methods for detecting biofilm formation in staphylococci. Conclusion: The TCP method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices.     

Facial analysis technology for the detection of Down syndrome in the Democratic Republic of the Congo

Down syndrome is one of the most common chromosomal anomalies affecting the world's population, with an estimated frequency of 1 in 700 live births. Despite its relatively high prevalence, diagnostic rates based on clinical features have remained under 70% for most of the developed world and even lower in countries with limited resources. While genetic and cytogenetic confirmation greatly increases the diagnostic rate, such resources are often non-existent in many low- and middle-income countries, particularly in Sub-Saharan Africa. To address the needs of countries with limited resources, the implementation of mobile, user-friendly and affordable technologies that aid in diagnosis would greatly increase the odds of success for a child born with a genetic condition. Given that the Democratic Republic of the Congo is estimated to have one of the highest rates of birth defects in the world, our team sought to determine if smartphone-based facial analysis technology could accurately detect Down syndrome in individuals of Congolese descent. Prior to technology training, we confirmed the presence of trisomy 21 using low-cost genomic applications that do not need advanced expertise to utilize and are available in many low-resourced countries. Our software technology trained on 132 Congolese subjects had a significantly improved performance (91.67% accuracy, 95.45% sensitivity, 87.88% specificity) when compared to previous technology trained on individuals who are not of Congolese origin (p < 5%). In addition, we provide the list of most discriminative facial features of Down syndrome and their ranges in the Congolese population. Collectively, our technology provides low-cost and accurate diagnosis of Down syndrome in the local population.     

荧光原位杂交技术在胎儿染色体异常产前诊断中的应用

目的探讨荧光原位杂交(FISH)技术在检测胎儿染色体异常的临床应用。方法选用13、21、18、X、Y特异性探针对1128例孕16～22周有产前诊断指征的妊娠妇女羊水间期细胞进行分析,并与同时进行的羊水细胞培养核型分析结果进行对照。结果被检1128例羊水间期细胞FISH检测均成功,其中检出正常核型1081例,数目异常核型20例,与常规细胞染色体核型分析结果一致,另外27例结构异常FISH技术未能检出。结论 FISH技术检测胎儿染色体数目异常具有快速、简便、准确性高、特异性强等优点,有较大的临床应用价值,并对产前细胞遗传学诊断有重要意义。     

Cell-based fluorescence assay for evaluation of new-drugs potential for phospholipidosis in an early stage of drug development

To evaluate new-drugs potential for phospholipidosis (PL), we developed a cell-based fluorescence assay using a fluorescent-labeled phospholipid analogue (NBD-PE). CHL/IU cells derived from newborn hamster lung were exposed to positive reference compounds (amiodarone, imipramine, chloroquine, propranolol, chlorpromazine and amantadine) in the presence of NBD-PE, and the level of PL, as indicated by accumulation of fluorescent inclusions in the cytoplasm, was evaluated using fluorescence microscopy and fluorometry. All positive reference compounds induced accumulation of fluorescent inclusions in a concentration-dependent manner with an increase in fluorescence intensity. Fluorescence microscopically, the positive dose of test compound was determined as the concentration with a grade equivalent to or above that of 3.13 μM of amiodarone. Based on this criterion, 8 of 20 test compounds including PL-positive or -negative compounds were judged positive that were concurrent with the pathological results from rat toxicity studies. Furthermore, a positive criterion for fluorometry was decided as equivalent to or above 25% of maximum intensity induced by 1.56–25.0 μM amiodarone. In comparison of fluorometry methods with fluorescence microscopy method, 19 of 20 compounds were judged same. From these findings, we concluded that the assay developed in this study is a rapid and reliable method to predict new-drugs potential for PL at an early stage of drug development.     

On the physical properties of red cell ghost membranes in the affective disorders and psychoses A fluorescence polarization study

Membrane order was measured in the erythrocyte ghost membranes of DSM-III schizophreniform disorder (SF), DSM-III schizophrenic (SCZ) and DSM-III manic (bipolar) (M) patients and a group of age- and sex-matched controls. Fluorescence polarization with the probe 1,6-diphenyl1-1,3,5-hexatriene was used to determine the steady-state fluorescence anisotropy (r_s). The SF group showed a significant increase in r_s (Δr_s = 0.037) from the control group. Although the means were not significantly different, 3 of 8 Ms and 5 of 8 SCZs also had r_s values > the highest control value. Thermotropic behavior of the membranes was evaluated over the range of 40 to 20°C. No difference among groups in membrane enthalpy was detected. Thus, the differences in r_s appear to be associated with differences in entropy. Phosphatidylcholine (PC) levels, which were known to be abnormal in these patients, were compared with the r_s values. A significant (P < 0.001, R= -0.63) linear correlation between r_s and membrane PC levels was observed. Overall these data further support the view that unusual membrane biophysical factors may occur with high frequency in the psychoses and affective disorders.     

双重荧光PCR检测HIV前病毒DNA及其在婴幼儿HIV感染早期诊断中的应用

目的建立双重荧光PCR检测HIV前病毒DNA的方法,并应用于婴幼儿HIV感染的早期诊断。方法采用TaqMan技术,组建针对人类核糖核酸酶P（ RNase P）和HIV的长末端重复序列（ LTR）基因的双重荧光PCR体系;采用TA克隆技术构建pTG19-T重组质粒作为模板进行该方法灵敏度的评价;采用11例已知健康人的血样和98例已知HIV感染者血样进行该方法的特异性验证;收集2011年1月至2012年9月浙江省各地妇幼保健医疗机构上送的96份婴幼儿样本,用新方法进行HIV的早期诊断,并将检测结果与罗氏HIV DNA定性检测试剂盒作比较。结果双重荧光PCR新方法能特异性检测HIV前病毒DNA,特异性为100％,检测灵敏度为100拷贝/反应;新方法和罗氏HIV DNA定性检测试剂盒检测96份婴幼儿样本,结果完全一致（符合率为100％）。结论建立的双重荧光PCR方法经济便捷、特异性好、灵敏度高、结果准确、易于推广,有望用于婴幼儿HIV早期诊断,并为HIV前病毒DNA的检测提供了一个通用技术平台。     

Knowledge and practices of seeking informed consent for medical examinations and procedures by health workers in the Democratic Republic of Congo

BackgroundInformed consent (IC) is linked to the ethical principle of respecting patient autonomy, respect for human rights and ethical practice, while in many countries it is a standard procedure. Anecdotally, it should be noted that in the Democratic Republic of Congo (DRC) in many instances ICs are not obtained systematically. To date, no research appears to have been conducted on this matter. This study aimed to assess the knowledge and practice of obtaining IC from patients among health care providers (HCP) in the DRC.MethodsThis was a cross-sectional study, with a convenient sampling of 422 participants. Data from the questions were collected on an imported Microsoft Excel spreadsheet for review at INSTAT.TM The authors set IC''s accurate knowledge and practice at 80% or higher. The Fisher Exact test was used to compare categorical association results, and a p-value < 0.05 was considered statistically significant.ResultsResults showed that giving information in detail to patients on their medical condition was associated with formal training on medical ethics and IC (p: 0.0028; OR: 1.894; CI: 1.246 to 2.881), which was also associated with answering the patient''s questions in detail (p: 0.0035; OR: 1.852; CI: 1.236 to 2.774). About 127(30.09 %) of participants scored 80% or higher. Extracurricular training was associated with withholding information from patients, up to 27 times more than other factors (p< 0.0001; OR: 27.042; CI: 13.628 to 53.657). when it comes to get IC, HCP with many years of practice scored better than others, in one of the question the odd ratio was closer to 7 (p< 0.0001; OR: 6.713; CI: 4.352 to 10.356). Only 47(11.14%) of the participants scored 80% or more of the questions about practice of IC.ConclusionFor a variety of reasons, knowledge and practice of IC among HCPs was very low. A common programme for the country as part of formal training might lead to an improvement. In addition, patients'' education on IC should be displayed in waiting areas at all medical centres.     

单核苷酸多态性微阵列和荧光原位杂交技术在Pallister-Killian综合征的产前诊断中的应用CSCD

目的探讨Pallister-Killian综合征（Pallister-Killian syndrome,PKS）的临床特征及遗传学特点,分析Affymetrix CytoScan 750K SNP Array和荧光原位杂志（fluorescence in situ hybridization, FISH）技术在产前诊断中的应用价值。方法对1例36岁孕34＋2周高龄孕妇脐血标本进行G显带染色体核型分析,芯片技术鉴定异常片段来源,运用12pter/12qter探针FISH确认。结果胎儿脐血G显带染色体核型46,XY[77]/47,XY,＋mar[23],出生后新生儿外周血芯片分析结果为arr[hg19] 12p13.33p11.1（173 786～34 835 641）×4,显示12号染色体12p13.33p11.1区段存在34.6 Mb的2次重复;FISH检测显示39%细胞有12号染色体短臂四体。结论通过结合临床特征,综合应用染色体G显带核型分析、FISH技术和芯片技术能准确确认染色体异常片段来源,有效诊断PKS患者。