The cell block preparation, which increases cellular yield by capturing any small tissue fragment... more The cell block preparation, which increases cellular yield by capturing any small tissue fragments in a fluid specimen, has been used to define the histologic pattern of the cells in question. This report describes a case in which processing a cell block on residual ThinPrep Pap Test material, using a thrombin-based technique, was useful in augmenting the diagnosis of a glandular lesion.
To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (N... more To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.
To compare a new spiral-shaped tissue-sampling brush with a standard cervical punch biopsy. Befor... more To compare a new spiral-shaped tissue-sampling brush with a standard cervical punch biopsy. Before large loop excision of the transformation zone, women with cervical intraepithelial neoplasia underwent a transepithelial brush biopsy of a portion of a colposcopically identified lesion, followed by a punch biopsy of the remaining portion. Brush biopsy samples were processed using liquid-based cytology and cell block techniques. Diagnoses were made using a consensus of three pathologists. Brush biopsy samples without basal cells were considered inadequate. The histological diagnosis was compared with the brush biopsy and punch biopsy samples. Patient-reported pain and physician-reported bleeding for punch and brush biopsies were compared. Fifty-two women were enrolled in the study; 47 successfully completed the study protocol. Eight brush biopsy specimens were inadequate. Thirty-nine women showed abnormal pathology (human papillomavirus/cervical intraepithelial neoplasia I or worse) on large loop excision of the transformation zone, and 32 women had high-grade (or worse) lesions. The punch biopsy correlated with high-grade disease in 53.1% of these women. The brush biopsy result correlated with high-grade disease in 79.3% of these women using a cell block technique and 76.7% using liquid cytology. There was significantly less pain (P <.001) and significantly less bleeding (P <.001) with the brush biopsy. When an adequate sample is collected, spiral brush biopsy is as good as a standard punch biopsy for detecting cervical pathology, with substantially less pain and bleeding. User training and guidelines for sampling are needed to assure that an adequate sample is collected.
In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-e... more In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-embedded lymphoma samples before performing FISH testing to detect the lymphoma-specific trans-location t(11;14) that defines mantle cell lymphoma. Well-characterized surgical pathology cases of mantle cell lymphoma were identified from pathology archives. Thin sections were cut from the paraffin-embedded tissue blocks. One section was stained using hematoxylin and eosin and an area composed exclusively of malignant cells was identified and marked on the slide. The corresponding area of the tissue block corresponding to this region underwent needle core biopsy, and the tissue was processed to isolate tumor cell nuclei and deposited onto a glass slide. The paired sample preparations underwent routine FISH testing for detection of the t(11;14)(q13;q32) chromosomal trans-location. DNA probe hybridization quality was compared between the tissue and isolated nuclei. Individual tumor cell nuclei were successfully extracted from each of the tissue blocks. The t(11;14) trans-location was detected by FISH in all of the samples diagnosed as mantle cell lymphoma. The hybridization signals found in the nuclei of extracted tumor cells were bright, planar, and easily identified. Detection of signal was superior to that on whole tissue samples, where signals often overlapped or were truncated. This technique produces intact nuclei for analysis, preserves the tissue block for additional studies, and allows sampling of a specific area of the tissue block. This approach may be particularly useful when the amount of diagnostic tissue is limited.
Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening ... more Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening of 10% of negative smears, to include cases selected randomly as well as smears from patients that may have a higher risk for developing cervical cancer based on clinical information. FDA approval of NeoPath's AutoPap 300 QC system (NeoPath, Inc., Redmond, WA) allows practical QC rescreening of all negatives. We tested the ability of AutoPap to help increase identification of detection errors compared to random 10%/high-risk selection. From March 1-August 30, 1997, we utilized AutoPap/high-risk status to select cases for manual rescreen, and compared the rate of identification of primary screening errors to that for the preceding year using 10% random selection/high-risk status. Of 35,027 smears accessioned, 31,240 (89.1%) were screened as negative and 7,965 were selected for manual rescreen. Of these, 353 were determined to be abnormal. Most abnormals identified by this protocol were classified as atypical squamous or glandular cells of undetermined significance (ASCUS or AGUS). However, 59 low-grade squamous intraepithelial lesions (LSIL) and 13 high-grade squamous intraepithelial lesions (HSIL), many with few abnormal cells, were also identified. These results represented an increase in pickup rate of false negative due to detection errors of 2.3-, 2.8- and 5.6-fold for atypical squamous or glandular cells of undetermined significance, LSIL, and HSIL, respectively, when accounting for the volume differences over the time period measured. Our findings strongly support the conclusions drawn from clinical trials of the AutoPap that false negatives due to detection error can be significantly reduced when using AutoPap as part of a routine quality control program.
To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burling... more To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burlington, North Carolina, U.S.A.) in determing the absence of endocervical cells in conventional Pap smear slides. A consecutive series of conventional Pap smears, designated by FocalPoint as requiring no further review (NFR) and as lacking endocervical cells, was manually screened to determine the true presence or absence of endocervical cells. These results were compared to those obtained by FocalPoint. From January 1, 2000, to December 31, 2001, FocalPoint indicated that 797 NFR slides did not contain endocervical cells. In contrast, manual screening revealed that 504/797 (63.2%) did contain endocervical cells. The reliability of a negative FocalPoint determination for endocervical cells is limited. Manual screening of NFR slides designated by the instrument as lacking endocervical cells appears to be necessary.
Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening ... more Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening of 10% of negative smears, to include cases selected randomly as well as smears from patients that may have a higher risk for developing cervical cancer based on clinical information. FDA approval of NeoPath's AutoPap 300 QC system (NeoPath, Inc., Redmond, WA) allows practical QC rescreening of all negatives. We tested the ability of AutoPap to help increase identification of detection errors compared to random 10%/high-risk selection. From March 1-August 30, 1997, we utilized AutoPap/high-risk status to select cases for manual rescreen, and compared the rate of identification of primary screening errors to that for the preceding year using 10% random selection/high-risk status. Of 35,027 smears accessioned, 31,240 (89.1%) were screened as negative and 7,965 were selected for manual rescreen. Of these, 353 were determined to be abnormal. Most abnormals identified by this protocol were classified as atypical squamous or glandular cells of undetermined significance (ASCUS or AGUS). However, 59 low-grade squamous intraepithelial lesions (LSIL) and 13 high-grade squamous intraepithelial lesions (HSIL), many with few abnormal cells, were also identified. These results represented an increase in pickup rate of false negative due to detection errors of 2.3-, 2.8- and 5.6-fold for atypical squamous or glandular cells of undetermined significance, LSIL, and HSIL, respectively, when accounting for the volume differences over the time period measured. Our findings strongly support the conclusions drawn from clinical trials of the AutoPap that false negatives due to detection error can be significantly reduced when using AutoPap as part of a routine quality control program.
Secondary chromosomal changes are known to develop in Philadelphia chromosome-negative (Ph-) cell... more Secondary chromosomal changes are known to develop in Philadelphia chromosome-negative (Ph-) cells of chronic myelogenous leukemia (CML) patients after treatment with imatinib mesylate, an ABL kinase inhibitor. We report here a novel case of a pericentric inversion of chromosome 16 as the sole cytogenetic abnormality in Ph- cells after treatment of Ph+ CML with imatinib.
Chromosomal rearrangements resulting in an interstitial inverted duplication with concomitant ter... more Chromosomal rearrangements resulting in an interstitial inverted duplication with concomitant terminal deletion were first described for the short arm of chromosome 8 in 1976. Since then, this type of alteration has been identified and characterised for most chromosome arms. Three mechanisms are commonly proposed to explain the origin of this type of rearrangement. All three mechanisms involve formation of a dicentric chromosome that then breaks in a subsequent meiotic division to produce a monocentric duplicated and deleted chromosome. However, the events leading to the formation of the dicentric chromosome differ between the mechanisms. In one mechanism, either parent carries a paracentric inversion. This results in formation of a loop during meiotic pairing with a recombination event occurring in the loop. In the second mechanism, inverted low copy repeats in the same chromosome arm allow partial folding of one homologue onto itself with a recombination event between the inverted repeats. The third mechanism involves a pre-meiotic double-strand break with subsequent fusion, or U-type exchange, between the sister chromatids. The first two mechanisms require a single copy region to exist between the duplicated and deleted regions on the derivative chromosome, and therefore high resolution analysis of the rearrangement can be used to distinguish between these mechanisms. Using G-banded chromosome analysis, fluorescence in situ hybridisation (FISH) and array comparative genomic hybridisation (CGH), we describe 17 new cases of inverted duplication with terminal deletion of 2q, 4p, 5p, 6q, 8p, 9p, 10q, 13q, 15q, 18p, 18q, and 22q. These new cases, combined with previously described cases, demonstrate that U-type exchange is the most frequent mechanism for this rearrangement and can be observed on most, or perhaps all, chromosome arms.
When a chromosome abnormality is identified in a child with a developmental delay and/or multiple... more When a chromosome abnormality is identified in a child with a developmental delay and/or multiple congenital anomalies and the chromosome rearrangement appears balanced, follow-up studies often examine both parents for this rearrangement. If either clinically unaffected parent has a chromosome abnormality with a banding pattern identical to the affected child's study, then it is assumed that the chromosome rearrangement is balanced and directly inherited from the normal carrier parent. It is therefore unlikely that the chromosome rearrangement is responsible for the child's clinical presentation. We present two unrelated cases in which an identical and apparently balanced abnormal chromosome banding pattern was identified in both an affected child and an unaffected parent of that child. Despite the identical banding patterns, molecular characterization through genomic microarray and fluorescence in situ hybridization showed the parent to be balanced whereas the affected child was significantly unbalanced. These two cases emphasize the utility of genomic microarray for further characterization of apparently balanced inherited chromosome rearrangements and caution against the assumption that identical banding patterns between a child and parent represent identical genomic rearrangements.
The cell block preparation, which increases cellular yield by capturing any small tissue fragment... more The cell block preparation, which increases cellular yield by capturing any small tissue fragments in a fluid specimen, has been used to define the histologic pattern of the cells in question. This report describes a case in which processing a cell block on residual ThinPrep Pap Test material, using a thrombin-based technique, was useful in augmenting the diagnosis of a glandular lesion.
To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (N... more To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.
To compare a new spiral-shaped tissue-sampling brush with a standard cervical punch biopsy. Befor... more To compare a new spiral-shaped tissue-sampling brush with a standard cervical punch biopsy. Before large loop excision of the transformation zone, women with cervical intraepithelial neoplasia underwent a transepithelial brush biopsy of a portion of a colposcopically identified lesion, followed by a punch biopsy of the remaining portion. Brush biopsy samples were processed using liquid-based cytology and cell block techniques. Diagnoses were made using a consensus of three pathologists. Brush biopsy samples without basal cells were considered inadequate. The histological diagnosis was compared with the brush biopsy and punch biopsy samples. Patient-reported pain and physician-reported bleeding for punch and brush biopsies were compared. Fifty-two women were enrolled in the study; 47 successfully completed the study protocol. Eight brush biopsy specimens were inadequate. Thirty-nine women showed abnormal pathology (human papillomavirus/cervical intraepithelial neoplasia I or worse) on large loop excision of the transformation zone, and 32 women had high-grade (or worse) lesions. The punch biopsy correlated with high-grade disease in 53.1% of these women. The brush biopsy result correlated with high-grade disease in 79.3% of these women using a cell block technique and 76.7% using liquid cytology. There was significantly less pain (P <.001) and significantly less bleeding (P <.001) with the brush biopsy. When an adequate sample is collected, spiral brush biopsy is as good as a standard punch biopsy for detecting cervical pathology, with substantially less pain and bleeding. User training and guidelines for sampling are needed to assure that an adequate sample is collected.
In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-e... more In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-embedded lymphoma samples before performing FISH testing to detect the lymphoma-specific trans-location t(11;14) that defines mantle cell lymphoma. Well-characterized surgical pathology cases of mantle cell lymphoma were identified from pathology archives. Thin sections were cut from the paraffin-embedded tissue blocks. One section was stained using hematoxylin and eosin and an area composed exclusively of malignant cells was identified and marked on the slide. The corresponding area of the tissue block corresponding to this region underwent needle core biopsy, and the tissue was processed to isolate tumor cell nuclei and deposited onto a glass slide. The paired sample preparations underwent routine FISH testing for detection of the t(11;14)(q13;q32) chromosomal trans-location. DNA probe hybridization quality was compared between the tissue and isolated nuclei. Individual tumor cell nuclei were successfully extracted from each of the tissue blocks. The t(11;14) trans-location was detected by FISH in all of the samples diagnosed as mantle cell lymphoma. The hybridization signals found in the nuclei of extracted tumor cells were bright, planar, and easily identified. Detection of signal was superior to that on whole tissue samples, where signals often overlapped or were truncated. This technique produces intact nuclei for analysis, preserves the tissue block for additional studies, and allows sampling of a specific area of the tissue block. This approach may be particularly useful when the amount of diagnostic tissue is limited.
Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening ... more Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening of 10% of negative smears, to include cases selected randomly as well as smears from patients that may have a higher risk for developing cervical cancer based on clinical information. FDA approval of NeoPath's AutoPap 300 QC system (NeoPath, Inc., Redmond, WA) allows practical QC rescreening of all negatives. We tested the ability of AutoPap to help increase identification of detection errors compared to random 10%/high-risk selection. From March 1-August 30, 1997, we utilized AutoPap/high-risk status to select cases for manual rescreen, and compared the rate of identification of primary screening errors to that for the preceding year using 10% random selection/high-risk status. Of 35,027 smears accessioned, 31,240 (89.1%) were screened as negative and 7,965 were selected for manual rescreen. Of these, 353 were determined to be abnormal. Most abnormals identified by this protocol were classified as atypical squamous or glandular cells of undetermined significance (ASCUS or AGUS). However, 59 low-grade squamous intraepithelial lesions (LSIL) and 13 high-grade squamous intraepithelial lesions (HSIL), many with few abnormal cells, were also identified. These results represented an increase in pickup rate of false negative due to detection errors of 2.3-, 2.8- and 5.6-fold for atypical squamous or glandular cells of undetermined significance, LSIL, and HSIL, respectively, when accounting for the volume differences over the time period measured. Our findings strongly support the conclusions drawn from clinical trials of the AutoPap that false negatives due to detection error can be significantly reduced when using AutoPap as part of a routine quality control program.
To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burling... more To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burlington, North Carolina, U.S.A.) in determing the absence of endocervical cells in conventional Pap smear slides. A consecutive series of conventional Pap smears, designated by FocalPoint as requiring no further review (NFR) and as lacking endocervical cells, was manually screened to determine the true presence or absence of endocervical cells. These results were compared to those obtained by FocalPoint. From January 1, 2000, to December 31, 2001, FocalPoint indicated that 797 NFR slides did not contain endocervical cells. In contrast, manual screening revealed that 504/797 (63.2%) did contain endocervical cells. The reliability of a negative FocalPoint determination for endocervical cells is limited. Manual screening of NFR slides designated by the instrument as lacking endocervical cells appears to be necessary.
Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening ... more Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening of 10% of negative smears, to include cases selected randomly as well as smears from patients that may have a higher risk for developing cervical cancer based on clinical information. FDA approval of NeoPath's AutoPap 300 QC system (NeoPath, Inc., Redmond, WA) allows practical QC rescreening of all negatives. We tested the ability of AutoPap to help increase identification of detection errors compared to random 10%/high-risk selection. From March 1-August 30, 1997, we utilized AutoPap/high-risk status to select cases for manual rescreen, and compared the rate of identification of primary screening errors to that for the preceding year using 10% random selection/high-risk status. Of 35,027 smears accessioned, 31,240 (89.1%) were screened as negative and 7,965 were selected for manual rescreen. Of these, 353 were determined to be abnormal. Most abnormals identified by this protocol were classified as atypical squamous or glandular cells of undetermined significance (ASCUS or AGUS). However, 59 low-grade squamous intraepithelial lesions (LSIL) and 13 high-grade squamous intraepithelial lesions (HSIL), many with few abnormal cells, were also identified. These results represented an increase in pickup rate of false negative due to detection errors of 2.3-, 2.8- and 5.6-fold for atypical squamous or glandular cells of undetermined significance, LSIL, and HSIL, respectively, when accounting for the volume differences over the time period measured. Our findings strongly support the conclusions drawn from clinical trials of the AutoPap that false negatives due to detection error can be significantly reduced when using AutoPap as part of a routine quality control program.
Secondary chromosomal changes are known to develop in Philadelphia chromosome-negative (Ph-) cell... more Secondary chromosomal changes are known to develop in Philadelphia chromosome-negative (Ph-) cells of chronic myelogenous leukemia (CML) patients after treatment with imatinib mesylate, an ABL kinase inhibitor. We report here a novel case of a pericentric inversion of chromosome 16 as the sole cytogenetic abnormality in Ph- cells after treatment of Ph+ CML with imatinib.
Chromosomal rearrangements resulting in an interstitial inverted duplication with concomitant ter... more Chromosomal rearrangements resulting in an interstitial inverted duplication with concomitant terminal deletion were first described for the short arm of chromosome 8 in 1976. Since then, this type of alteration has been identified and characterised for most chromosome arms. Three mechanisms are commonly proposed to explain the origin of this type of rearrangement. All three mechanisms involve formation of a dicentric chromosome that then breaks in a subsequent meiotic division to produce a monocentric duplicated and deleted chromosome. However, the events leading to the formation of the dicentric chromosome differ between the mechanisms. In one mechanism, either parent carries a paracentric inversion. This results in formation of a loop during meiotic pairing with a recombination event occurring in the loop. In the second mechanism, inverted low copy repeats in the same chromosome arm allow partial folding of one homologue onto itself with a recombination event between the inverted repeats. The third mechanism involves a pre-meiotic double-strand break with subsequent fusion, or U-type exchange, between the sister chromatids. The first two mechanisms require a single copy region to exist between the duplicated and deleted regions on the derivative chromosome, and therefore high resolution analysis of the rearrangement can be used to distinguish between these mechanisms. Using G-banded chromosome analysis, fluorescence in situ hybridisation (FISH) and array comparative genomic hybridisation (CGH), we describe 17 new cases of inverted duplication with terminal deletion of 2q, 4p, 5p, 6q, 8p, 9p, 10q, 13q, 15q, 18p, 18q, and 22q. These new cases, combined with previously described cases, demonstrate that U-type exchange is the most frequent mechanism for this rearrangement and can be observed on most, or perhaps all, chromosome arms.
When a chromosome abnormality is identified in a child with a developmental delay and/or multiple... more When a chromosome abnormality is identified in a child with a developmental delay and/or multiple congenital anomalies and the chromosome rearrangement appears balanced, follow-up studies often examine both parents for this rearrangement. If either clinically unaffected parent has a chromosome abnormality with a banding pattern identical to the affected child's study, then it is assumed that the chromosome rearrangement is balanced and directly inherited from the normal carrier parent. It is therefore unlikely that the chromosome rearrangement is responsible for the child's clinical presentation. We present two unrelated cases in which an identical and apparently balanced abnormal chromosome banding pattern was identified in both an affected child and an unaffected parent of that child. Despite the identical banding patterns, molecular characterization through genomic microarray and fluorescence in situ hybridization showed the parent to be balanced whereas the affected child was significantly unbalanced. These two cases emphasize the utility of genomic microarray for further characterization of apparently balanced inherited chromosome rearrangements and caution against the assumption that identical banding patterns between a child and parent represent identical genomic rearrangements.
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