Automated in situ hybridization and immunohistochemistry for cytomegalovirus detection in paraffin-embedded tissue sections.

Cytomegalovirus (CMV) infections cause serious morbidity and mortality in immunocompromised individuals. CMV detection methods include serology, viral culture, polymerase chain reaction, and histologic examination [hematoxylin and eosin, immunohistochemistry (IHC), in situ hybridization (ISH)]. Until recently, ISH was performed manually. We compared automated ISH and automated IHC (Bond-max system, Leica Microsystems) in 72 cases (multiple organ systems) previously evaluated for CMV by conventional methods [hematoxylin and eosin, IHC (Dako Autostainer Plus), polymerase chain reaction, and/or viral culture]. By automated ISH, 27 cases were CMV-positive (25 positive, 2 equivocal on original diagnosis), 43 were negative (10 positive, 29 negative, 4 equivocal on original diagnosis), and 2 were equivocal (positive on original diagnosis). By automated IHC, 31 cases were CMV-positive (28 positive, 3 equivocal on original diagnosis), 39 were negative (7 positive, 29 negative, 3 equivocal on original diagnosis), and 2 were equivocal (positive on original diagnosis). Using original CMV diagnosis as gold standard, automated ISH and automated IHC had sensitivities of 67.6% and 75.7%, respectively (P value=0.25), and both had specificities of 100%. Combined automated ISH and IHC had sensitivity of 75.7% and specificity of 100%. Positive predictive values for automated ISH, automated IHC, and combination of ISH and IHC were 92.6%, 90.3%, and 90.3%, respectively. Negative predictive values were 67.4%, 74.4%, and 76.3%, respectively. We recommend either automated ISH or IHC for CMV detection in formalin-fixed, paraffin-embedded tissues. Although with moderate sensitivities, these methods have high specificities and positive predictive values, permit rapid turnover, require only set-up time, and result in strong stain intensity with minimal background.

Multidetector row CT angiography of living related renal donors: is there a need for venous phase imaging?

OBJECTIVE: To prospectively evaluate whether renal venous anatomy can be detected from arterial phase images of multidetector row CT (MDCT) of renal donors. MATERIAL AND METHODS: Institutional review board approved our study protocol with waiver of consent. Forty-eight consecutive renal donors (age range, 21-56 years; M:F, 20:28) referred for MDCT evaluation were included. Two sub-specialty radiologists performed an independent and separate evaluation of renal venous anatomy in arterial and venous phase images. Opacification of renal venous structures was scored on a five-point scale (1-not seen; 3-minimal opacification; 5-excellent opacification). Arterial and venous phase opacification scores were compared by Wilcoxon signed rank test. RESULTS: Both readers detected all renal venous anomalies in arterial as well as venous phase images. Each reader detected accessory right renal veins (n=14), retroaortic left renal vein (n=2), circumaortic left renal vein (n=1), and left renal hilar arteriovenous malformation (n=1) in arterial phase images. Retroaortic left renal venous branch was difficult to differentiate from lumbar vein (reader-1, n=1; reader-2, n=2) in both arterial and venous phase images. Sensitivity of detection of renal veins, left adrenal, gonadal and lumbar veins in arterial phase images was 100, 83-88, 100, and 85-90%, respectively. As expected, venous phase images showed significantly greater opacification of renal veins, left gonadal, adrenal and lumbar veins (p<.05). However, this did not substantially limit the evaluation of renal venous anatomy in arterial phase images. Both readers had substantial interobserver agreement (kappa coefficient, 0.7; p<0.05). CONCLUSIONS: Arterial phase MDCT images alone can be used to detect renal venous anomalies, and to identify small left renal venous branches namely, the left gonadal, adrenal and lumbar veins in renal donors. Venous phase MDCT acquisition is not necessary for evaluation of renal venous anatomy in renal donors.

Rapid intraoperative immunohistochemical evaluation of sentinel lymph nodes for metastatic breast carcinoma.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is an integral part of the surgical management of patients with breast cancer. Rapid immunohistochemistry (RIHC) has the potential to increase detection of metastatic carcinoma at the time of frozen section consultation. The authors assessed the accuracy and turnaround time of a newly developed RIHC method for pancytokeratin (RIHC-CK). METHODS: Sixty-six SLNs from 32 patients with breast carcinoma were examined for metastasis using the Zymed Sentinel Lymph Node Rapid IHC Kit. Intraoperative frozen sections (6 mum) of the SLNs were incubated with Zymed anti-pan-cytokeratin/HRP conjugate, diaminobenzidine (DAB), and stained with hematoxylin. Slides were ready within 8 minutes and were interpreted as positive or negative for metastatic carcinoma. Results were compared with previous intraoperative touch preparations, frozen sections, hematoxylin and eosin (Perm H&E), and AEl/3-immunostained permanent sections (Perm CK). RESULTS: Fourteen lymph nodes (19%) in 13 patients tested positive for metastatic carcinoma in Perm H&E, the gold standard. RIHC-CK had the highest sensitivity (92%) of the intraoperative tests, compared with touch preparations (64%) and frozen sections (80%). RIHC-CK showed 94% accuracy, compared with 96% (frozen section) and 93% (touch preparation). The RIHC technique took 8 minutes and was easy to perform and interpret. CONCLUSIONS: Zymed RIHC is a sensitive method for detecting breast cancer metastases in SLNs. The speed, accuracy, and ease of interpretation of the test allow for recognition of micrometastases (<2 mm) that might otherwise be undetectable by current methods of intraoperative evaluation. The prognostic significance and effect on surgical management of micrometastases in SLNs have yet to be determined.

Detection of simulated lesions on data-compressed digital mammograms.

PURPOSE: To evaluate retrospectively the effect of a wavelet-based compression method on the detection of simulated masses of various sizes and clustered microcalcifications on data-compressed digital mammograms. MATERIALS AND METHODS: The images used in this study were acquired with institutional review board approval and patient informed consent, both of which allowed subsequent image data analysis. Patient identification was removed from images, and the study complied with requirements of the Health Insurance Portability and Accountability Act. Masses 3, 6, and 8 mm in diameter were analytically simulated and added to clinical mammographic backgrounds. In addition, microcalcifications were extracted from a clinical mammogram and hybridized with simulated microcalcifications for use in this study. Image compression conditions of 1:1, 15:1, and 30:1 were investigated. Observer responses were recorded with a six-point rating scale, and receiver operating characteristic (ROC) analysis was performed. In addition, two well-established numeric observer models were used to study the effect of image compression under the same compression conditions as were used with human observers. Analysis of variance was performed after observer adjustment to compare the mean values for area under the ROC curve (A(z)) across the three compression levels for the masses and microcalcification clusters. RESULTS: The results of the study indicated no significant differences in the A(z) values for masses with the compression conditions investigated. For images of microcalcifications, there were significant differences in A(z) values between compression ratios of 1:1 and 30:1 (P = .0005) and of 15:1 and 30:1 (P = .004); the difference between compression ratios of 1:1 and 15:1 was nonsignificant (P = .053). The observer models and human observers exhibited similar trends in detection of the masses investigated in this study. CONCLUSION: Detection of simulated masses was not affected by the compression method with the conditions used in this study, while the detection of microcalcifications was significantly reduced with a compression ratio of more than 15:1.

Gd(ABE-DTTA)-enhanced cardiac MRI for the diagnosis of ischemic events in the heart.

PURPOSE: To demonstrate that contrast-enhanced MRI (ceMRI) with the aid of Gd(ABE-DTTA) is able to detect ischemic events in the heart in a canine ischemia/reperfusion (30/40 minutes) model. MATERIALS AND METHODS: ECG-gated, T1-weighted MR image sets (four to five slices each) with three-minute time resolution were collected in transiently LAD-occluded dogs. Following the acquisition of control image sets, ischemia was started by occluding the LAD. Either Gd(ABE-DTTA) (N = 6) or Gd(DTPA) (N = 6) was injected, and imaging was continued for 30 minutes of ischemia and 40 minutes of reperfusion. The contrast agent (CA)-induced MRI signal intensity enhancement (SIE) and contrast were monitored. Microspheres measured myocardial perfusion (MP) to verify areas of ischemia and reperfusion. RESULTS: SIEs of 86% +/- 3% and 97% +/- 3% in nonischemic, and 25% +/- 5% and 29% +/- 8% in ischemic regions were found within three minutes of onset of ischemia with Gd(ABE-DTTA) and Gd(DTPA), respectively. For the rest of the 30 minutes of ischemia, with Gd(ABE-DTTA) SIE of 60% +/- 3% and 25% +/- 5% persisted in the nonischemic and ischemic regions, respectively. With Gd(DTPA), however, SIE in the nonischemic areas decreased rapidly after the first three minutes of ischemia, while SIE in the ischemic areas increased, abolishing contrast. Thus, there was a persistent contrast with Gd(ABE-DTTA) and a short-lived contrast with Gd(DTPA) during ischemia. Furthermore, with Gd(ABE-DTTA) some contrast was still visible in the early reperfusion period. CONCLUSION: Gd(ABE-DTTA) in an ischemia/reperfusion model induces a persistent MRI contrast between regions of normal and ischemic myocardium, and verifies reperfusion. Therefore, it can be used to detect myocardial ischemic events.

A perceptual evaluation of JPEG 2000 image compression for digital mammography: contrast-detail characteristics.

In this investigation the effect of JPEG 2000 compression on the contrast-detail (CD) characteristics of digital mammography images was studied using an alternative forced choice (AFC) technique. Images of a contrast-detail phantom, acquired using a clinical full-field digital mammography system, were compressed using a commercially available software product (JPEG 2000). Data compression was achieved at ratios of 1:1, 10:1, 20:1, and 30:1 and the images were reviewed by seven observers on a high-resolution display. Psychophysical detection characteristics were first computed by fitting perception data using a maximum-likelihood technique from which CD curves were derived at 50%, 62.5%, and 75% threshold levels. Statistical analysis indicated no significant difference in the perception of mean disk thickness up to 20:1 compression except for disk diameter of 1 mm. All other compression combinations exhibited significant degradation in CD characteristics.

Rapid immunohistochemistry of sentinel lymph nodes for metastatic melanoma.

Sentinel lymph node (SLN) biopsy is performed on patients with malignant melanoma (MM) to assess the need for selective complete lymphadenectomy. Melanoma metastasis to regional lymph nodes is an important prognostic indicator in patients with MM. This study assesses the sensitivity and specificity of rapid immunohistochemistry (RIHC) in intraoperative delineation of melanoma metastasis to SLN. RIHC for S-100 protein, HMB45, and a melanoma marker cocktail (melan A, HMB45, and tyrosinase) was performed on 71 SLNs obtained from 28 patients with MM. Frozen sections (6 micro thick) on plus slides were fixed for 2 to 3 minutes in cold acetone and then stored at -70 degrees C. The EnVision kit (Dako, Carpinteria, CA) for rapid immunohistochemistry (RIHC) on frozen tissue sections was used, and the staining technique took 19 minutes. Together with preparation of the frozen sections and fixation in acetone, immunostained slides were available in approximately 25 minutes. Of the 71 SNLs examined, 7 showed melanoma metastasis in permanent sections. RIHC of frozen sections detected metastatic melanoma in 6 SLNs, with a sensitivity of 86% for HMB45 and 71% for S-100 protein and the melanoma cocktail and a specificity of 97% for HMB45 and 100% for S-100 and the melanoma cocktail. We conclude that RIHC for HMB45, S-100 protein, and the melanoma cocktail may help detect melanoma metastasis in SLN intraoperatively, leading to total lymph node dissection and obviating the need for 2 surgical procedures. Section folds and background stain can make interpretation difficult. Intraoperative time constraints require a more rapid technique. A recent consensus group has discouraged frozen-section examination of SLN.

Rapid immunohistochemistry for cytokeratin in the intraoperative evaluation of sentinel lymph nodes for metastatic breast carcinoma.

The sensitivity and specificity of detecting metastatic breast carcinoma in sentinel lymph nodes using a rapid immunohistochemistry technique was determined and compared with methods currently used at the authors' institution. At the time of intraoperative consultation, after routine diagnostic touch preparations and frozen sections were prepared, 6-microm frozen sections of 72 sentinel lymph nodes from 32 patients with breast carcinoma were placed on plus slides, fixed in cold acetone for 2 or 3 minutes, and stored at -70 degrees C. These sections were immunostained with a prediluted broad-spectrum anticytokeratin monoclonal antibody coupled to an inert polymer with horseradish peroxidase (DAKO EPOS). Slides were ready for interpretation within 16 minutes and were scored as positive, negative, or equivocal for metastatic carcinoma. Results were compared with those of the intraoperative touch preparations and frozen sections and with paraffin-embedded, hematoxylin and eosin-stained, and AE1/AE3 immunostained permanent sections. Fourteen (19%) sentinel lymph nodes were positive for metastatic carcinoma in 13 patients. All methods tested were 100% specific. The rapid immunohistochemistry method was the least sensitive (57% sensitivity) of all methods used to detect metastasis. Routine diagnostic touch preparations, frozen sections, and permanent sections had sensitivities of 69%, 86%, and 100% respectively. In conclusion, this rapid immunohistochemistry method would not be helpful in intraoperative assessment of sentinel lymph nodes in breast cancer patients due to its low sensitivity.

Immunohistochemical evaluation of sentinel lymph nodes in breast carcinoma patients.

Sentinel lymph node sampling has become an alternative to axillary lymph node dissection to provide prognostic and treatment information in breast cancer patients. The role of immunohistochemistry has yet to be established. A total of 241 sentinel lymph nodes (in 270 slides) from 91 patients with invasive carcinoma (73 ductal, 9 lobular, 8 mixed lobular/ductal, 1 NOS) were studied for presence of macrometastases (> 0.2 cm), identified in hematoxylin and eosin sections, and occult metastases (micrometastases [< or = 0.2 cm], clusters of cells, isolated carcinoma cells), identified only by immunohistochemistry. Intraoperative touch preparations, frozen sections, seven hematoxylin and eosin levels (L1-L7), and two AE1-3 cytokeratin immunohistochemistries (L1, L4-5) of the entire bisected or trisected sentinel lymph node were examined. Thirty-one (34%) patients had 50 positive sentinel lymph nodes. Twenty-six (33%) sentinel lymph nodes had metastatic carcinoma (11 macrometastases, 11 micrometastases, 3 clusters of cells, 1 isolated carcinoma cells) by touch preparations, frozen sections, and one hematoxylin and eosin (L1). Thirty-eight (43%) were positive by AE1-3 immunohistochemistry (L1) (11 macrometastases, 8 micrometastases, 13 clusters of cells, 6 isolated carcinoma cells), significantly more than by touch preparations, frozen sections, hematoxylin and eosin L1, or hematoxylin and eosin L2-7. Cytokeratin immunostain on L4-5 demonstrated 31 (34%) positive sentinel lymph nodes, a similar frequency to cytokeratin immunostain on L1. Size of sentinel lymph node metastasis did not correlate with size, histologic grade, or type of primary breast carcinoma. AE1-3 (L1) immunohistochemistry is highly sensitive in delineating sentinel lymph node metastasis, especially clusters of cells and isolated carcinoma cells. The prognostic significance of clusters of cells and isolated carcinoma cells and the value of AE1-3 immunohistochemistry on frozen sections need to be determined.