The Histochemical Detection of Thallium

Thallium can be histochemically localized in formalin-fixed, paraffin-processed tissues by treating sections, after passing them to water through xylene and graded alcohols, in a 0.5% aqueous Bi(NO₃)₃ solution acidified with 1 drop of concentrated HNO₃ per 100 ml. Sections are then washed and placed for 5 min in 2% aqueous KI, again washed, routinely dehydrated, cleared and covered. If desired, they may be lightly counterstained prior to dehydration. The tissue sites of T1 will be demonstrated by the fine wine-red crystals of (T1I)₂-BiI₃ resulting from the reaction, and will appear black with the usual microscopic illumination.     

The Histochemical Detection of iron in Tissues

The fixation and staining of iron in tissues is discussed. Procedures for demonstrating iron in hemoglobin and nuclei are also briefly considered.

Lillie's formalin buffered at neutrality gave the optimal fixation for iron. The Prussian blue method was preferred to the Turnbull blue. Lison's procedure of the former, slightly modified, gave the most satisfactory results. When a procedure is required that employs non-iron-containing reagents, Macallum's or Mallory's hema-toxylin and Quincke's ammonium sulfide method are useful. The former, though not entirely specific, is preferable under controlled conditions when the quantities of iron are small. Hemoglobin iron in paraffin sections can be demonstrated by the usual procedures for iron after previous exposure of the section to peroxide, as recommended by Brown. The property of nuclei to adsorb iron from inorganic sources and from hemoglobin can readily be shown; caution is required in interpreting the iron detected in nuclei after Macallum's sulfuric acid hydrolysis.     

一种简便的从石蜡包埋组织中提取RNA的方法

目的:建立一种从石蜡包埋组织提取高质量RNA的简便、经济的方法。方法:运用TRIzol法和改进的AGPC(酸-异硫氰酸胍-苯酚-氯仿)法等2种提取方法,从50例石蜡包埋组织中提取RNA,用紫外分光光度仪测定RNA的产率和纯度;用管家基因β-肌动蛋白做RT-PCR,检测RNA的质量。结果:TRIzol法提取RNA的成功率为96%(48/50),AGPC法为94%(47/50),2种方法所得RNA的吸光度值及得率在统计学上均无明显差异。结论:AGPC法和TRIzol法的提取效率相似,且提取费用只有TRIzol法的一半左右,是一种简便、经济、适合大量提取石蜡包埋组织中的RNA的方法,可用于临床。     

Histochemical Tests for Polyphenols in Plant Tissues

A satisfactory histochemical test for polyphenols in fresh plant tissues is described. The test is based upon a colorimetric method for phenolics using a nitrous acid reaction. Certain catechol derivatives are characterized by the formation of an intense cherry-red in fresh sections of plant tissues with the reagents used. The red color is sufficiently intense to be readily observed microscopically at 100X. Results obtained with other tests also are described.     

Histochemical Demonstration of Acid Phosphatase in Hard Tissues

The action of the following decalcifying solutions for the demonstration of acid phosphatase has been studied: buffer solution acetic-acetate 0.05 M, pH 5; 2, 5, 10, 20, and 50% formic acid and 20% sodium citrate in equal parts (pH 2.6, 3, 3.8, 4.2, and 5); 0.5 M citric acid-NaOH, pH 4.2; Versene solution, 5%, pH 7. A comparative study of fixatives has been made also (neutral formalin, 10-20%; formalin-chloral hydrate (Fishman), acetone and 80% alcohol). The best results were obtained with fixation at 4°C in 10-20% neutral formalin or formalin-chloral hydrate, for a period of 24 hr, and decalcification with 20% sodium citrate, 5% formic acid, in equal parts, pH 4.2, which can act on both specimens or sections for a period up to 2 wk with very little loss of enzyme. It is not necessary to reactivate the enzyme after decalcification; frozen sections should be used and should be washed in distilled water before proceeding with the demonstration of the enzyme (Gomori's method or azo dye coupling). Other fixatives (acetone and alcohol) and paraffin embedding produce a greater loss in enzymes and very irregular results.     

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R²: 0.99–1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.MS based proteomics has traditionally been used to investigate complex biological systems, such as cell lines, plasma, or fresh-frozen tissues (1, 2). In the last decade however, MS proteomics has extended to the analysis of formalin-fixed paraffin-embedded (FFPE)¹ tissues (3). Formalin fixation is the gold standard for sample storage in clinical pathology because it allows optimal preservation of the morphological features of the tissue and it is economically attractive (storage at room temperature over several years or decades) (4). Several studies have shown that although the individual peptides retrieved and identified from fresh-frozen and FFPE tissues may differ, the biological information obtained from both types of material in terms of number of proteins identified, cellular location and molecular function is very similar (5–10). A number of proteomics studies were reported, which used untargeted MS on FFPE tissues to compare diseased and healthy samples in the search for potential novel biomarkers (10). Nevertheless, these untargeted MS workflows do not allow performing accurate protein quantification on large numbers of samples. One option is to use targeted MS approaches, such as selected reaction monitoring (SRM), which are highly quantitative and reproducible over many samples (11, 12). Additionally, SRM assays allow a high level of multiplexing (several hundreds of peptides can be measured in parallel in a single analysis) (13). The lack of access to a sufficient number of high-quality samples annotated with comprehensive clinical data sets may be a limiting factor for preclinical exploratory phase biomarker studies (14). The possibility to use FFPE samples for MS-based proteomics, in particular for quantitative targeted approaches, would therefore open tremendous perspectives for performing large retrospective biomarker discovery and verification studies. Indeed, in addition to being widely available, most FFPE tissues are annotated with clinical data. Moreover, targeted MS workflows applied to FFPE samples are complementary to techniques requiring high-quality antibodies, such as immunohistochemistry (IHC) or reverse-phase protein arrays (RPPA). These techniques all rely on the measurement of the target protein, with SRM measuring one or ideally several peptides as surrogates of the protein (15, 16). In opposition to IHC and RPPA however, SRM does not rely on the presence of a specific antibody for analyte detection, thereby avoiding cross-reaction issues and making assay development relatively rapid and cost effective. Although SRM is less advanced for protein analysis than for small molecules quantification, the technique was demonstrated to be selective, reproducible, and highly quantitative over large dynamic ranges for proteins as well (17–19). However, although the equivalence of qualitative analyses performed on fresh-frozen and FFPE samples has been investigated and demonstrated, only a few studies evaluated quantitative targeted MS approaches in FFPE samples (20, 21). Targeted proteomics performed on FFPE tissues is still in its early days and known limitations of this technique include the loss of morphologic features of the tissue and an extensive sample preparation, causing a low sample throughput (20). Moreover, targeted proteomics quantifies peptides as surrogates of a protein, with the former not necessarily agreeing in absolute terms with the latter. This is true for bottom-up proteomics in general, but it is of particular importance for FFPE tissues.In this study, we critically assessed the validity of targeted MS applied to peptide quantification in FFPE tissues. We developed and evaluated an SRM assay for the quantification of six peptides of the human receptor tyrosine-protein kinase erbB-2 (HER2) and compared the obtained results with those of standard methods used in clinical pathology, namely IHC and fluorescence in situ hybridization (FISH). HER2 was chosen as a candidate protein because its overexpression is routinely assessed in breast tumors in order to determine susceptibility to anti-HER2 treatment (22). Depending on the laboratory, HER2 overexpression can be assessed by IHC or the amplification of the corresponding gene (ERBB2) can be quantified by FISH (23). Several laboratories use both techniques for decision-making, as they are complementary.In a first step, we developed an SRM assay for the quantification of HER2 peptides in archival clinical FFPE tumor tissues and assessed its analytical performance, including linearity, precision and lower limit of quantification (LLOQ). We then demonstrated the applicability of the method by quantifying HER2 peptides in a cohort of 40 FFPE tumor tissues expressing different levels of HER2 (selected based on ERBB2 gene amplification status). The samples originated from surgical resections performed on women with invasive mammary carcinomas. We thereby investigated several options to normalize the results in regard to sample size. Finally, in order to confirm the validity of SRM as a suitable method for protein quantification in FFPE tissues, we determined the agreement between data generated by SRM and data generated by IHC or FISH.     

Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.     

应用高分辨率熔解曲线分析石蜡标本中KRAS基因突变

目的:KRAS基因在结直肠癌患者突变率为30％～50％,并且KRAS基因状态与抗EGFR抗体疗效的密切相关.常用的直接测序法其检测灵敏度低、容易出现假阴性结果.采用高分辨率溶解曲线分析法检测结直肠癌中KRAS基因突变,探索其应用于临床检测的可行性.方法:本文收集20例结直肠癌患者石蜡包埋组织标本,应用高分辨率熔解曲线方法检测KRAS基因突变(2,3号外显子).将KRAS基因突变型DNA(p.G12D)和野生型DNA梯度混合稀释,突变型DNA浓度分别为40％、20％、10％、2％,进行高分辨率熔解曲线方法进行检测,并将扩增产物进行直接测序,比较两种方法的灵敏度.结果:在20例结直肠癌患者中检出5例2号外显子突变,p.G12D 2例,p.G12V 2例,p.G13D 1例,3号外显子未检出突变.HRM方法检测基因突变的检出限可达2％,直接测序法检测基因突变的检出限为20％.结论:HRM法比直接测序法灵敏度高,能精确检测出结直肠癌石蜡标本中低浓度的KRAS突变,有望应用于临床基因突变检测.     

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed,Paraffin-Embedded Archival Tissues

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH₃/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.     

Optimal Conditions for the Retrieval of CD4 and CD8 Antigens in Formalin-Fixed, Paraffin-Embedded Tissues

The effects of buffer, NaCl, EDTA, and urea on the retrieval of CD4 and CD8 antigens in formaldehyde-fixed, paraffin-embedded tissues with a microwave pressure-cooker were evaluated. The optimal retrieval conditions were found to be borate buffer at pH 8 containing 1 mM NaCl and 1 mM EDTA. Urea was found to be less effective.     

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed,Paraffin-Embedded Tissues from Colorectal Cancer Patients

Background

Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.

Methods

We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.

Results

Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.

Conclusion

The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.     

The Autoradiographic Detection of Ions in Plant Tissues1

The autoradiographic techniques for the examination of the subcellularlocalization of ions in plant cells have been assessed. It hasbeen shown that the use of a controlled-temperature freezing-sectioningtechnique, together with low-temperature, stripping-film autoradiography,may provide a more suitable method than is at present availablefor studies on ion uptake and movement in plant cells.     

石蜡包埋组织的DNA提取及其在免疫球蛋白基因重排分析中的应用

基因重排分析在淋巴瘤诊断中具有重要意义.文章应用改良DNA提取方法,从30例淋巴增生性病变石蜡包埋组织获得的DNA虽有不同程度的降解,但适于PCR扩增Ig重链基因重排分析;约1/3病例提出高分子量DNA,可用于DNA印迹杂交.因此,石蜡包埋组织同样可为某些疾患,如淋巴瘤疑难和罕见病例的回顾性分子病理学研究提供基因诊断的DNA来源.     

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.     

High Fidelity Copy Number Analysis of Formalin-Fixed and Paraffin-Embedded Tissues Using Affymetrix Cytoscan HD Chip

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples.