Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice

The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. The technique comprises two phases: (1) slides preparation and stages involved for the reaction; (2) interpretation and quantification of the obtained expression. Immunohistochemistry is an important tool for scientific research and also a complementary technique for the elucidation of differential diagnoses which are not determinable by conventional analysis with hematoxylin and eosin. In the last couple of decades there has been an exponential increase in publications on immunohistochemistry and immunocytochemistry techniques. This review covers the immunohistochemistry technique; its history, applications, importance, limitations, difficulties, problems and some aspects related to results interpretation and quantification. Future developments on the immunohistochemistry technique and its expression quantification should not be disseminated in two languages—that of the pathologist and another of clinician or surgeon. The scientific, diagnostic and prognostic applications of this methodology must be explored in a bid to benefit of patient. In order to achieve this goal a collaboration and pooling of knowledge from both of these valuable medical areas is vita

Superficial cervical plexus blockade improves pain control after thyroidectomy: A randomized controlled trial

OJECTIVES: The aim was to evaluate the ability of bilateral superficial cervical plexus blockade to control pain and to reduce the side effects of general anesthesia in patients submitted to thyroidectomy. METHODS: In this randomized controlled trial, we prospectively studied 100 consecutive patients who underwent total thyroidectomy. The simple random patient sample was divided into two groups: 50 patients received general anesthesia alone (group 1 [G1]), and 50 patients received general anesthesia with bilateral superficial cervical plexus blockade (group 2 [G2]). Statistical analyses were performed, and a 5% significance level was adopted. RESULTS: The mean arterial blood pressure and heart rate were 12% lower in G2 patients than in G1 patients 60 minutes after surgery (101 mmHg for G1 vs. 92.3 mmHg for G2; po0.001). G2 patients reported less pain than G1 patients, and opioid consumption was lower in G2 patients than in G1 patients, not upon postanesthesia care unit arrival, but at 30 minutes (2% vs. 34%; po0.001, respectively), 45 minutes (0% vs. 16%; p=0.006, respectively), and 4 hours postoperatively (6% vs. 20%; p=0.037, respectively). The incidence of nausea and vomiting was lower in G2 patients than in G1 patients from 45 minutes (0% vs. 16%; p=0.006, respectively) to 8 hours postoperatively (0% vs. 14%; p=0.012, respectively). CONCLUSIONS: The present study demonstrated that the combination of bilateral superficial cervical plexus blockade with general anesthesia for thyroidectomy is feasible, safe, and effective for achieving pain control and improving patient outcomes

Adjuvant treatment delay in breast cancer patients

Background: to evaluate if time between surgery and the first adjuvant treatment (chemotherapy, radiotherapy or hormone therapy) in patients with breast cancer is a risk factor for lower overall survival (OS). Method: data from a five-year retrospective cohort study of all women diagnosed with invasive breast cancer at an academic oncology service were collected and analyzed. Results: three hundred forty-eight consecutive women were included. Time between surgery and the first adjuvant treatment was a risk factor for shorter overall survival (HR=1.3, 95CI 1.06-1.71, p=0.015), along with negative estrogen receptor, the presence of lymphovascular invasion and greater tumor size. A delay longer than 4 months between surgery and the first adjuvant treatment was also associated with shorter overall survival (cumulative survival of 80.9% for delays ≤ 4 months vs. 72.6% for delays > 4 months; p=0.041, log rank test). Conclusion: each month of delay between surgery and the first adjuvant treatment in women with invasive breast cancer increases the risk of death in 1.3-fold, and this effect is independent of all other well-established risk factors. Based on these results, we recommend further public strategies to decrease this interval

Método digital assistido por computador de quantificação imunohistoquímica comparado à análise semiquantitativa

PURPOSE: To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis. METHODS: Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab®). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity. RESULTS: There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001). CONCLUSION: Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.OBJETIVO: Comparar a quantificação da imunomarcação através de um método digital assistido por computador à bem estabelecida análise semiquantitativa. MÉTODO: A marcação citoplasmática de galectina-3 foi obtida por reações imunohistoquímicas em 25 casos de carcinoma bem-diferenciado da glândula tireóide. Determinou-se o índice de expressão da análise semiquantitativa que associa a convencional fração de área de células marcadas com os escores de intensidade da imunoexpressão, com base na observação visual qualitativa. O método digital assistido por computador foi desenvolvido com o uso de um programa de análise de imagem (ImageLab®). Três parâmetros foram obtidos: (1) porcentagem de células marcadas; (2) intensidade de imunoexpressão digital e (3) índice de expressão digital. O método proposto resulta na análise numérica da intensidade de imunoexpressão. RESULTADOS: Houve importante correlação entre as intensidades de imunoexpressão obtidas pelos dois métodos (coeficiente de correlação de Pearson, r=0,71, p=0,0001). O mesmo foi observado entre os índices de expressão (coeficiente de correlação de Pearson, r=0,66, p=0,0001). DISCUSSÃO: Os resultados de intensidade de imunoexpressão obtidos com o emprego do método digital assistido por computador foram concordantes com os escores da análise semiquantitativa. Entretanto, os resultados alcançados com o emprego do método digital podem resolver a discordância entre diferentes observadores com relação a esta intensidade de imunomarcação. Além disso, o método proposto não categoriza os resultados em grupos, o que torna a análise imunohistoquímica numericamente mensurável individualmente, aumentando seu poder diagnóstico e, sobretudo, prognóstico

Vocal fold mobility alteration reversed after thyroidectomy

The involvement of the inferior or recurrent laryngeal nerve (RLN) in mobility derangement of the vocal folds occurs more frequently due to thyroid malignancy invasion. Although uncommon, the same derangement, which is caused by benign thyroid entities, is also described and reverts to normality after a thyroidectomy in up to 89% of cases. In these cases, the pathogenesis of the vocal cord mobility disturbance is attributed to the direct compression of the RLN by massive thyroid enlargement. The authors describe three cases of patients presenting unilateral vocal cord palsy, which, before surgery, was diagnosed by laryngoscopy concomitantly with large and compressive goiter. Vocal fold mobility became normal after the thyroidectomy in all three cases. Therefore, it is noteworthy that these alterations may present reversibility after appropriate surgical treatment. An early surgical approach is recommended to reduce the nerve injury as much as possible; to preserve the integrity of both RLNs since the nerve function will be restored in some patient

Pre-operative sera levels of CEA and CA19-9 and tissular distribution of tumor marker CA19-9 in colorectal carcinoma: correlation with morphological features of neoplasia

OBJETIVO: Comparar os níveis séricos de CA19-9 e CEA e a expressão tecidual do CA19-9 e relacioná-los com os aspectos morfológicos do carcinoma colorretal. MÉTODOS: Quarenta e cinco pacientes com carcinoma colorretal foram operados com coleta de CEA e CA19-9 séricos pré-operatórios. Valores séricos de CEA = 5,0ng/mL e de CA19-9 = 37UI/mL foram considerados aumentados. A avaliação da imunoexpressão do CA19-9 no tecido neoplásico foi realizada por meio de estudo imunoistoquímico com anticorpo monoclonal anti-CA19-9. A intensidade de expressão do CA19-9 no tecido neoplásico foi semiquantificada em leve(+/+++), moderada(++/+++), intensa(+++/+++) e ausente. RESULTADOS: Os valores do CA19-9 sérico foram progressivamente maiores conforme o aumento da expressão do CA19-9 no tecido neoplásico, porém sem significância (p=0,06). O aumento do nível sérico do CA19-9 foi acompanhado de elevação significante (p<0,001) do nível sérico do CEA. O nível sérico do CA19-9, a imunoexpressão tecidual do CA19-9 e o nível sérico do CEA não apresentaram associação significante com características morfológicas do carcinoma colorretal. CONCLUSÃO: As expressões sérica e tissular do CA19-9 demonstraram relação diretamente proporcional entre si, enquanto que os aspectos morfológicos da neoplasia não tiveram influência no CEA e CA19-9 séricos ou na imunoexpressão do CA19-9 tissular.OBJECTIVE: To compare sera levels of CEA and CA19-9 and tissular expression of the CA19-9 and to correlate these with morphological features of the colorectal carcinoma. METHODS: Forty five patients with colorectal carcinoma underwent surgical treatment following measurement of pre-operative levels of CA19-9 and CEA. Sera levels of CEA = 5.0ng/ml and CA19-9 = 37UI were deemed high values. Evaluation of CA19-9 immunoexpression in neoplastic tissue was carried through by means of immunohistochemical study with monoclonal antibody anti-CA19-9. The intensity of expression of CA19-9 in neoplastic areas was semi-quantified in each area of tumor differentiation into mild(+/+++), moderate(++/+++), intense(+++/+++) or absent. RESULTS: Sera CA19-9 values were progressively higher in the presence of elevated CA19-9 immunoexpression in colorectal carcinoma tissue, although not significant (p=0.06). Increased sera CA19-9 levels were found to be associated with significantly elevated (p<0.001) sera CEA levels. Levels of sera CA19-9, tissular immunoexpression of CA19-9 and sera levels of CEA presented no significant association with morphological features of the colorectal carcinoma. CONCLUSION: Sera and tissular levels of the CA19-9 marker exhibited, each other, a directly proportional relationship. The morphological features of the neoplasia had no influence on sera CEA or CA19-9 levels or tissular immunoexpression of CA19-9

Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1

Is surgical debridement necessary in the diabetic foot treated with photodynamic therapy?

Background: Diabetic patients are susceptible to developing foot ulcerswith serious complications such as osteomyelitis and amputations. Treatment approaches are still empirical and the benefit of usual procedures such as surgical debridement has not been properly evaluated. Photodynamic Therapy (PDT) is a non-invasive and highly efficient method for the treatment of the diabetic foot, being able to eradicate the infection and to stimulate healing, decreasing considerably the amputation risk. In the day-to-day practice of our service, we have been faced with the question whether debridement is necessary before PDT. In here, we designed a study to answer that question. Methods: Patients were divided in two groups: In one of the groups (n = 17), debridement was performed before PDT and in the other (n = 40) only PDT treatment was performed. PDT sessions were performed once a week in all patients until healing was achieved, as indicated by visual inspection as well as by radiographic and laboratory exams. At the start of the study, the two groups had no statistical differences concerning their clinical features: average age, gender, insulin use, diabetes mellitus onset time and previous amputations. Results: PDT was effective in the treatment of 100% of the patients showing no relapses after one year of follow up. The group submitted to PDT without previous debridement had a statistically significant (p = 0.036, Mann-Whitney) shorter cure time (29 days, similar to 27%). Conclusion: Our data indicates that debridement is not necessary in the treatment of diabetic foot in patients that have enough peripheral arterial perfusion. In addition, we reproduced previous studies confirming that PDT is an efficient, safe, simple and affordable treatment method for the diabetic foot.Fundacao de Amparo a Pesquisa do Estado de Sao PauloCNPqHosp Anchieta, Fac Med ABC, Sao Bernardo Do Campo, BrazilFac Med ABC, Dept Bioquim, Santo Andre, BrazilUniv Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Sao Paulo, BrazilFAPESP: 12/50680-5FAPESP: 13/07937-8Web of Scienc

Expression of ck-19, galectin-3 and hbme-1 in the differentiation of thyroid lesions: systematic review and diagnostic meta-analysis

Background: To distinguish between malignant and benign lesions of the thyroid gland histological demonstration is often required since the fine-needle aspiration biopsy method applied pre-operatively has some limitations. in an attempt to improve diagnostic accuracy, markers using immunocytochemistry and immunohistochemistry techniques have been studied, mainly cytokeratin-19 (CK-19), galectin-3 (Gal-3) and Hector Battifora mesothelial-1 (HBME-1). However, current results remain controversial. the aim of the present article was to establish the diagnostic accuracy of CK-19, Gal-3 and HBME-1 markers, as well as their associations, in the differentiation of malignant and benign thyroid lesions.Methods: A systematic review of published articles on MEDLINE and the Cochrane Library was performed. After establishing inclusion and exclusion criteria, 66 articles were selected. the technique of meta-analysis of diagnostic accuracy was employed and global values of sensitivity, specificity, area under the summary ROC curve, and diagnostic odds ratio (dOR) were calculated.Results: for the immunohistochemistry technique, the positivity of CK-19 for the diagnosis of malignant thyroid lesions demonstrated global sensitivity of 81% and specificity of 73%; for Gal-3, sensitivity of 82% and specificity of 81%; and for HBME-1, sensitivity of 77% and specificity of 83%. the association of the three markers determined sensitivity of 85%, specificity of 97%, and diagnostic odds ratio of 95.1. Similar results were also found for the immunocytochemistry assay.Conclusion: This meta-analysis demonstrated that the three immunomarkers studied are accurate in pre- and postoperative diagnosis of benign and malignant thyroid lesions. Nevertheless, the search for other molecular markers must continue in order to enhance this diagnostic accuracy since the results found still show a persistency of false-negative and false-positive tests.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fac Med ABC, Dept Biochem, Santo Andre, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFac Med ABC, Dept Head & Neck Surg, Santo Andre, BrazilFac Med ABC, Med Sch Students, Santo Andre, BrazilFac Med ABC, Dept Hematol & Oncol, Santo Andre, BrazilAlbert Einstein Jewish Hosp, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc