Usefulness of intraoperative rapid immunohistochemistry in the surgical treatment of brain tumors.

In the treatment of primary central nervous system lymphoma (PCNSL), intraoperative rapid pathological diagnosis can dramatically change the surgical strategy, and more accurate diagnostic methods are required. In April 2020, we adopted intraoperative rapid immunohistochemistry (IHC) in addition to conventional rapid intraoperative diagnosis based on morphological assessment, mainly for patients with PCNSL. Here, we investigate the usefulness and significance of intraoperative rapid IHC based on our initial experience. We performed intraoperative rapid IHC using antibodies for cluster of differentiation (CD)20, CD3, leukocyte common antigen (LCA) and glial fibrillary acidic protein (GFAP) using enzyme-labeled antibody methods in 25 patients, including PCNSL patients, from April 2020 to July 2022. We examined the utility of this approach in determining treatment strategies for brain tumors. Postoperative final pathological diagnoses from paraffin-embedded sections were as follows: diffuse large B-cell lymphoma, 16 cases; glioblastoma, six cases; pilocytic astrocytoma, one case; adenocarcinoma, one case; and inflammatory disorder, one case. The entire process took 32 min and staining for CD20, CD3, LCA, and GFAP was comparable to that using paraffin-embedded sections. In all cases, the results of intraoperative rapid IHC were consistent with final pathological diagnoses from paraffin-embedded sections. In addition, in two cases, the results of conventional intraoperative rapid pathological diagnosis based on morphological assessments using frozen sections were drastically changed by adding intraoperative rapid IHC. Intraoperative rapid IHC contributes to deciding appropriate treatment strategies and facilitating early initiation of chemotherapy for PCNSL. This may allow new therapeutic strategies not only for PCNSL but also for other brain tumors.

Rapid intraoperative Ki-67 immunohistochemistry for lung cancer using non-contact alternating current electric field mixing.

OBJECTIVES: Locoregional recurrence of non-small cell lung cancer (NSCLC) occurs even among patients with stage I disease, as a result of tumor proliferative activity. The aim of this study was to evaluate the clinical reliability of a new rapid immunohistochemistry (IHC) technique for assessing malignant potential through detection of tumoral Ki-67 expression. MATERIALS AND METHODS: The rapid IHC method uses non-contact alternating current (AC) mixing to achieve more rapid/stable staining within 20 min during surgery. First, to investigate the association between clinical outcomes and tumoral Ki-67 labeling with rapid IHC, 21 pairs of surgical patients treated between 2012 and 2020 for pStage IA1-3 NSCLC with/without recurrence were retrospectively reviewed. Second, 40 frozen section (FS) samples in patients with NSCLC for whom radical surgery was planned between April 2021 and February 2022 were deemed eligible for comparison of the clinical performance of conventional IHC and intraoperative rapid Ki-67 IHC with FS. RESULTS: Detection of tumoral Ki-67 expression using rapid IHC with formalin-fixed, paraffin-embedded (FFPE) blocks was significantly associated with clinical outcomes in R0 pStage IA NSCLC surgical patients, including overall and recurrence-free survival (P = 0.0043 and P < 0.0001, respectively). Levels of Ki-67 expression among resectable NSCLC patients detected using rapid IHC with FS significantly correlated with those detected using conventional FFPE-IHC (p < 0.001). An intraoperative cut-off of > 7.5 % tumor cell Ki-67 positivity accurately predicted pathological stage more advanced than IA3 [P = 0.0185, Odds ratio = 20.477, 95 % confidence interval (CI): 1.660-252.55]. CONCLUSION: Rapid Ki-67 IHC with AC mixing could potentially serve as a clinical tool for intraoperative determination of tumor malignancy status. The present study suggests that segmentectomy for early small NSCLCs is oncologically safe and a reasonable alternative to lobectomy, but only when there is adequate intraoperative selection for primary tumors with low-grade malignancy, which could be verified using intraoperative rapid Ki-67 IHC with FS.

Intraoperative diagnosis of lymph node metastasis during segmentectomy for non-small cell lung cancer by rapid immunohistochemistry using noncontact alternating current electric field mixing.

BACKGROUND: Although lobectomy is considered the standard surgery for any non-small cell lung cancer (NSCLC), recent evidence indicates that for early NSCLCs segmentectomy may be equally effective. For segmentectomy to be oncologically safe, however, adequate intraoperative lymph node staging is essential. The aim of this study was to compare the results of a new rapid-IHC system to the HE analysis for intraoperative nodal diagnosis in lung cancer patients considered for segmentectomy. METHODS: This retrospective study analyzed the pathological reports from NSCLC resections over a six-year period between 2014 and 2020. Using a new device for rapid-IHC, we applied a high-voltage, low-frequency alternating current (AC) field, which mixes the antipancytokeratin antibody as the voltage is switched on/off. Rapid-IHC can provide a nodal diagnosis within 20 minutes. RESULTS: Frozen sections from 106 resected lymph nodes from 70 patients were intraoperatively evaluated for metastasis. Of those, five nodes were deemed positive based on both HE staining and rapid-IHC. In addition, rapid-IHC alone detected isolated tumor cells in one hilar lymph node. Three cStage IA patients with nodal metastasis detected with HE staining and rapid-IHC received complete lobectomies. Five-year relapse-free survival and overall survival among patients receiving segmentectomy with rapid-IHC were 88.77% and 88.79%, respectively. CONCLUSIONS: Rapid-IHC driven by AC mixing is simple, highly accurate, and preserves nodal tissue for subsequent tests. This system can be used effectively for intraoperative nodal diagnosis. Rapid immunohistochemistry based on alternating-current field mixing (completed within 20 minutes) is simple and highly accurate. This system will assist clinicians when making intraoperative diagnoses of lymph node metastasis and deciding upon the appropriate surgical procedure in segmentectomy for lung cancer. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Rapid immunohistochemistry driven by alternating-current field mixing (completed within 20 minutes intraoperatively) is simple, highly accurate, and preserves lymph node tissue for subsequent pathological examination, including molecular assessments. WHAT THIS STUDY ADDS: Segmentectomy for lung cancer is oncologically safe, but only when there is adequate intraoperative node staging. Rapid immunohistochemistry will assist clinicians when making intraoperative nodal diagnoses.

Detection of rabies virus antigen by the indirect rapid immunohistochemistry test in equines and comparisons with other diagnostic techniques.

Laboratory diagnosis of rabies in equines is essential for distinguishing the disease from other sources of encephalitis. Diagnosis by conventional techniques such as a direct fluorescent antibody test (dFAT) or viral isolation in mice or cell culture can be difficult, and the application of molecular biological methods may be necessary. We performed an indirect rapid immunohistochemistry test (iRIT) for the detection of the rabies virus (RABV) antigen in the central nervous system (CNS) of equines and compared the results with those of other diagnostic techniques. We reviewed result records from the Rabies Diagnosis Laboratory at Instituto Pasteur, São Paulo, Brazil, of 174 samples of equine CNS from July 2014 to June 2016, which were investigated by dFAT, rabies tissue culture infection test (RTCIT), mouse inoculation test (MIT) and reverse transcription-polymerase chain reaction (RT-PCR) followed by genetic sequencing. These samples, 29 presented divergent results among techniques and were selected for the performed in the iRIT. The detected positivity rate was 4/29 (14%) by dFAT, 5/28 (18%) by RTCIT, 10/29 (35%) by MIT and 26/27 (96%) by RT-PCR. We analysed 29 samples through imprints of the cortex, hippocampus, cerebellum and brainstem in slides fixed in 10% buffered formaldehyde. Eighteen samples were identified as positive (62%) by iRIT assay, representing a greater number of positive cases than that detected by dFAT, MIT and RTCIT but not by RT-PCR. Among the brain regions, the brainstem presented the highest positivity (78%), followed by the hippocampus (69%), cerebellum (67%) and cortex (67%). Our results provide evidence that iRIT can contribute to a rapid diagnosis of rabies in equines and that complementary tests should be used to improve diagnostic accuracy in this species.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test.

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.

Immuno-Laser-Capture Microdissection for the Isolation of Enriched Glial Populations from Frozen Post-Mortem Human Brain.

Laser-capture microdissection (LCM) has revolutionized the isolation of defined regions and specific cell populations from human tissue. This approach used in combination with immunohistochemistry (immuno-LCM) has become a valuable method for isolating individual cell-types from a complex heterogeneous population. Here, we describe the detailed methodology required for the isolation of enriched populations of GFAP+ astrocytes, OSP+ oligodendrocytes, and CD68+ microglia from frozen post-mortem human central nervous system tissue using immuno-LCM.

Exploratory development of rapid immunohistochemical staining technique in prostate biopsy tissue frozen section diagnosis / 中国基层医药

in 30minutes.Antibody expression located accurately.Background stained clearly and no interfering signal.The back-ground was better than conventional immunohistochemistry of frozen remaining tissue.The positive expression rate of p40,34βE12,p504s with rapid immunohistochemistry in prostatic hyperplasia was 97.4%(77 /79),93.7%(74 /79),0%(0 /79),and in prostatic carcinoma was 0%(0 /39),0%(0 /39),97.4%(38 /39).The positive expression rate of p40,34βE12,p504s with conventional immunohistochemistry in prostatic hyperplasia was 96.2%(76 /79), 93.7%(74 /79),2.5%(2 /79),and in prostatic carcinoma was 0%(0 /39),0%(0 /39),92.3%(36 /39).The difference of expression between prostatic hyperplasia and prostatic carcinoma with rapid immunohistochemical detec-tion p40 group:χ2 =109.402,P =0.000,34βE12 group:χ2 =97.971,P =0.000,p504s group:χ2 =113.537,P =0.000;The difference of expression between prostatic hyperplasia and prostatic carcinoma with conventional immuno-histochemical detection p40 group:χ2 =105.410,P =0.000,34βE12 group:χ2 =97.971,P =0.000,p504s group:χ2 =96.388,P =0.000;The expression of prostatic hyperplasia with between rapid immunohistochemical detection and conventional immunohistochemical detection 34βE12 group was identical,p40 group:χ2 =0.207,P =0.649, p504s group:χ2 =2.026,P =0.155;The expression of conventional immunohistochemical detection with between rap-id immunohistochemical detection and conventional immunohisto -chemical detection p40 group and 34βE12 group were identical,p504s group:χ2 =1.054,P =0.305.The expression of three markers between prostatic hyperplasia and prostatic carcinoma had statistical significance(P 0.05).Conclusion MaxVision rapid immunohisto -chemical staining technique has the advantages of rapid,accurate,timely.It could be used to rapid diagnosis of prostate biopsy tissue.The combined detection of p40,34βE12,p504s has very high practi-cal value in the differential diagnosis of benign and malignant lesions of the prostate.

Intraoperative Examination of Sentinel Lymph Nodes Using Rapid Cytokeratin Immunohistochemical Staining in Breast Cancer Patients / 한국유방암학회지

PURPOSE: Sentinel lymph node (SLN) biopsy has become a new standard procedure in the treatment of patients with early breast cancer. Furthermore, many institutions have begun offering the sentinel lymph node biopsy without simultaneous axillary dissection as a possible standard procedure when the SLN was free from tumors. For appropriate intraoperative decision making on the presence of cancer cells in axillary lymph nodes, a fast and accurate method to assess the SLN is required. The authors performed a prospective investigation of the relative merits of rapid cytokeratin immunohistochemical (IHC) staining of the SLN removed during the operations of breast cancer patients. METHODS: Between December 2002 and August 2003, 38 patients with T1and T2 breast cancer were enrolled after undergoing successful sentinel lymph node biopsy. A total of 60 sentinel lymph nodes (mean number, 1.58) were biopsied and first examined by hematoxylin-eosin (H&E) staining. All the tumor free sentinel lymph nodes by H&E stained section were immunostained for cytokeratin using a rapid immunohistochemical assay (Cytokeratin (PAN), 1:50, Newcastle, UK) during the operation. Finally, the sentinel lymph nodes were submitted for paraffin embedding and serial section after surgery. Both H&E stained and cytokeratin immunostained sections were also performed. RESULTS: This technique has a turnaround time of less than 20 minutes during the operation. Rapid IHC staining revealed 4 positive sentinel lymph nodes that were negative for metastasis by H&E staining. Among these false negative 4 cases, two cases had problems with the frozen section of H&E staining and the other 2 cases had problems due to micrometastasis. This study showed a sensitivity of 88.89%, a specificity of 100%, an accuracy of 98.33%, and a negative predictive value of 98.08%. The false-negative case (1 of 52), which was negative on H&E staining and rapid IHC staining during the operation, was disclosed as positive only after a serial permanent section examination with IHC stain. CONCLUSION: The introperative examination of sentinel lymph nodes is a highly accurate and effective way of predicting the axillary lymph node status of patients with breast cancers. This may be a promising technique in deciding whether to spare axillary lymph node dissection for the patient in the operating room.