Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

BACKGROUND: Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. STUDY DESIGN AND METHODS: The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. RESULTS: Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. CONCLUSION: Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Relationship between B-cell epitope structural properties and the immunogenicity of blood group antigens: Outlier properties of the Kell K1 antigen.

BACKGROUND: The immunogenicities of polypeptide blood group antigens vary, despite most being created by single amino acid (AA) substitutions. To study the basis of these differences, we employed an immunoinformatics approach to determine whether AA substitution sites of blood group antigens have structural features typical of B-cell epitopes and whether the extent of B-cell epitope properties is positively related to immunogenicity. STUDY DESIGN AND METHODS: Fifteen structural property prediction programs were used to determine the likelihood of ß-turns, surface accessibility, flexibility, hydrophilicity, particular AA composition and AA pairs, and other B-cell epitope properties at AA substitution sites of polypeptide blood group antigens. RESULTS: AA substitution sites of Lua , Jka , E, c, M, Fya , C, and S were each located in regions with at least two structural features typical of B-cell epitopes. The substitution site of K, the most immunogenic non-ABO/D antigen, scored the lowest for most B-cell epitope properties and was the only one not predicted to be part of a linear B-cell epitope. The most immunogenic antigens studied (K, Jka , Lua , E) had B-cell epitope structural properties determined by the fewest programs; the least immunogenic antigens (e.g., Fya , S, C, c) had B-cell epitope properties according to the most programs. DISCUSSION: Counter to prediction, the immunogenicity of polypeptide blood group antigens was not positively related to B-cell epitope structural features present at their AA-substitution sites. Instead, it tended to be negatively related. The AA-substitution site of the most immunogenic non-ABO/D antigen, K, had the least B-cell epitope features.

The utility and limitations of B- and T-cell gene rearrangement studies in evaluating lymphoproliferative disorders.

A hallmark of lymphoid malignancies is the presence of a monoclonal lymphocyte population. Monoclonality of B- and T-cell populations can be established through immunoglobulin (IG) or T-cell receptor (TCR) gene rearrangement analysis, respectively. The biological rationale of IG and TCR gene rearrangement analysis is that due to the extensive combinatorial repertoire made possible by V(D)J recombination in lymphocytes, it is unlikely that any substantive lymphocyte population would share the same IG or TCR gene rearrangement pattern unless there is an underlying neoplastic or reactive origin. Modern IG and TCR gene rearrangement analysis is typically performed by polymerase chain reaction (PCR) using commercially available primer sets followed by gel capillary electrophoresis. This process is highly sensitive in the detection of nearly all lymphoid malignancies. Several pitfalls and limitations, both biological and technical, apply to IG/TCR gene rearrangement analysis, but these can be minimised with high quality controls, performance of assays in duplicate, and adherence to strict criteria for interpreting and reporting results. Next generation sequencing (NGS) will likely replace PCR based methods of IG/TCR gene rearrangement analysis but is not yet widespread due to the absence of standardised protocols and multicentre validation.

Relationship of epitope glycosylation and other properties of blood group proteins to the immunogenicity of blood group antigens.

BACKGROUND: The intrinsic properties of polypeptide blood group antigens that determine their relative immunogenicities are unknown. Because size, composition, charge, dose, and epitope glycosylation affect the immunogenicity of other polypeptides, we examined whether similar properties were related to the immunogenicity of blood group antigens. STUDY DESIGN AND METHODS: Amino acid (AA) sequences of antithetical blood group antigens were searched for N- and O-glycosylation sites. Regression analysis was carried out to determine whether blood group protein properties, including total and ectodomain size, red blood cell (RBC) antigen site density, number of mismatched AAs between an antigen and its closest homolog, and differences in mass, charge, and hydrophobicity of the mismatched AAs, were related to immunogenicity. RESULTS: The immunogenicities of non-RhD polypeptide antigens were directly related to the total and ectodomain sizes of their carrier proteins. A negative power relationship existed between RBC antigen site density and immunogenicity, such that the most immunogenic antigens had the lowest site density. The strong immunogenicity of RhD was related to the number of AA mismatches between RhD and RhCE, to their cumulative hydrophobicity and electrostatic mismatch scores, and the cumulative AA mass difference. No N- or O-glycosylation differences were predicted for antithetical or homologous antigens, other than a previously known N-glycosylation difference between K and k. CONCLUSION: Epitope glycosylation appeared not to be a determinant of immunogenicity for blood group antigens, except possibly for K. The immunogenicity of blood group antigens was positively related to total and ectodomain sizes of blood group proteins and negatively related to antigen site density. Such findings should be considered hypothesis generating for future, more definitive studies.

Structural and functional impacts of amino acid substitutions that create blood group antigens: implications for immunogenicity.

BACKGROUND: The immunogenicities of polypeptide blood group antigens vary widely. One possible determinant of immunogenicity is antigenic foreignness. The goal was to employ alternative ways of assessing foreignness and determine whether foreignness was related to immunogenicity. STUDY DESIGN AND METHODS: Foreignness was assessed as the extent of protein functional disruption caused by the exofacial amino acid (AA) substitutions that create blood group antigens, using AA substitution prediction algorithms such as Meta-SNP and according to whether those substitutions were radical or conservative. RESULTS: AA substitutions that create the most immunogenic antigens had the highest Meta-SNP scores, predictive of greater protein structure and function changes. Four of the 11 exofacial AAs that distinguish the most immunogenic antigen, RhD, from RhCE, and substitutions creating four of the five next most immunogenic antigens had the highest Meta-SNP scores (0.293-0.649). Excluding the outlier Jka , the mean Meta-SNP score of the four most immunogenic non-RhD antigens (K, Lua , E, c) was 3.7-fold higher than the mean of the four least immunogenic (M, Fya , C, S), 0.459 versus 0.123 (p = 0.0026). Regression analysis revealed a relationship between immunogenicity and Meta-SNP score (R2 = 0.953). Actual protein functional disruption was predicted for the AA substitution creating the E antigen. An AA cluster at Positions 350, 353, and 354 of RhD was unique, containing radical substitutions according to two classification schemes and relatively high Meta-SNP scores (0.351-0.432). CONCLUSION: The immunogenicity of blood group antigens was related to the functional disruption caused by the AA substitutions that create the antigens, as measured by Meta-SNP score.

Comparison of assay systems for warfarin-related CYP2C9 and VKORC1 genotyping.

BACKGROUND: A variety of commercial genotyping assays is available to detect variants in the CYP2C9 and VKORC1 genes. The assay results are used in genotype-based warfarin dosing algorithms. We compared the performance of four such assay systems: Verigene, eSensor, Invader, and Luminex. METHODS: Result concordance and no call rates were determined on patient specimens tested on all four instruments. Turnaround times (TAT), hands-on time (HOT), pipetting steps and cost were obtained for runs of 1, 8 and 24 samples. RESULTS: The four assays were 100% concordant for the common CYP2C9 and VKORC1 alleles (n=100). Verigene had the shortest TAT and HOT for 1 and 8 samples. Verigene had the fewest pipetting steps for all sample sizes, while Invader had the most. Luminex had the longest TAT and highest cost for all sample run sizes. Verigene had the lowest cost for 1 and 8 samples and Invader the lowest for 24 samples. The no call rates for Verigene, Luminex, eSensor, and Invader were 10%, 4%, 1% and 0%, respectively. CONCLUSIONS: All assays gave comparable results for common variants. Each system offered unique advantages and disadvantages, whose relative importance depends on the needs of the adopting clinical laboratory.

Histopathologic and molecular aspects of CD56+ natural killer/ T-cell lymphoma of the testis.

Primary nasal-type natural killer/T-cell lymphoma of the testis is a rare malignancy. Although dissemination to the testis from other sites occurs somewhat more frequently than a primary presentation, even secondary testicular involvement is uncommon. In this article, the authors report on the comprehensive histopathologic, immunohistochemical, and molecular analysis of a case of primary testicular nasal-type natural killer/T-cell lymphoma, and review the features of 16 previously reported patients. The investigation carried out in this study indicates that the testicular nasal-type natural killer/T-cell lymphomas occur at a younger age than their B-cell counterparts, express cytoplasmic CD3 and surface CD56, and consistently show an infection by Epstein-Barr virus. These tumors have variable expression of T-cell antigens other than cytoplasmic CD3 and may show monoclonal rearrangement of T-cell receptor genes. Testicular natural killer/T-cell lymphomas of nasal type invariably follow an aggressive clinical course.

Curriculum content and evaluation of resident competency in clinical pathology (laboratory medicine): a proposal.

Ten years have passed since the Graylyn Conference Report on Laboratory Medicine Clinical Pathology training was issued. Over that period, the Accreditation Council for Graduate Medical Education substantially revised the requirements for training programs; the American Board of Pathology amended both the requirements and the periods needed for certification; and the discipline itself, along with the broader discipline of pathology, evolved significantly. Recently, a curriculum proposal in anatomical pathology was published as a potential template to be used by training programs to help meet these new and evolving needs. Toward the same end, the Academy of Clinical Laboratory Physicians and Scientists has now developed a template for a curriculum in clinical pathology (laboratory medicine), taking into account newly designated and revised areas of residency core competency, the alterations in training requirements promulgated by the Accreditation Council for Graduate Medical Education and American Board of Pathology, and the rapidly developing nature of the discipline itself. The proposed clinical pathology curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by pathology residency training programs.

Curriculum content and evaluation of resident competency in clinical pathology (laboratory medicine): a proposal.

Ten years have passed since the Graylyn Conference Report on Laboratory Medicine/Clinical Pathology training was issued. During that period, the Accreditation Council for Graduate Medical Education (ACGME) substantially revised the requirements for training programs, the American Board of Pathology (ABP) amended the requirements and the time needed for certification, and the discipline itself along with the broader discipline of pathology, evolved significantly. Recently, a curriculum proposal in anatomic pathology was published as a potential template to be used by training programs to help meet these new and evolving needs. Toward the same end, the Academy of Clinical Laboratory Physicians and Scientists has developed a template for a curriculum in clinical pathology (laboratory medicine), taking into account newly designated and revised areas of residency core competency, the alterations in training requirements promulgated by the ACGME and ABP, and the rapidly developing nature of the discipline itself The proposed clinical pathology curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by pathology residency training programs.

Curriculum content and evaluation of resident competency in clinical pathology (laboratory medicine): a proposal.

Ten years have passed since the Graylyn Conference Report on Laboratory Medicine/Clinical Pathology training was issued. Over that time period, the Accreditation Council for Graduate Medical Education (ACGME) substantially revised the requirements for training programs, the American Board of Pathology (ABP) amended both the requirements and the time periods needed for certification, and the discipline itself, along with the broader discipline of pathology, evolved significantly. Recently, a curriculum proposal in anatomic pathology was published as a potential template to be used by training programs to help meet these new and evolving needs. Toward the same end, the Academy of Clinical Laboratory Physicians and Scientists has now developed a template for a curriculum in clinical pathology (laboratory medicine), taking into account newly designated and revised areas of residency core competency, the alterations in training requirements promulgated by the ACGME and ABP, and the rapidly developing nature of the discipline itself. The proposed clinical pathology curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by pathology residency training programs.

Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma.

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.

Flow cytometric DNA ploidy analysis of peripheral blood from patients with sezary syndrome: detection of aneuploid neoplastic T cells in the blood is associated with large cell transformation in tissue.

We reviewed and screened 219 cutaneous T-cell lymphoma (CTCL) cases for Sezary syndrome (SS); 63 met the criteria for SS. Of these, 17 (27%) demonstrated circulating aneuploid cells and 46 (73%) showed only euploid cells in blood samples. Of 17 aneuploid cases, DNA ploidy study was essential for initial blood-based diagnosis of SS in 4 (24%) and important in monitoring minimal residual disease after treatment in 9 (53%) in which neoplastic T cells showed otherwise unremarkable or nonspecific flow cytometric immunophenotypic findings. Tissue biopsy slides (predominantly skin and lymph node) at the time of DNA ploidy studies were available for 47 of 63 cases. Of 14 cases with circulating aneuploid cells, 11 (79%) showed large cell transformation (LCT; 6 [43%]) or markedly increased large cells (ILC; 5 [36%]) in tissue, whereas only 10 (30%) of 33 euploid cases showed LCT (4 [12%]) or ILC (6 [18%]) (P < .01). There was no significant difference in blood tumor burden, immunophenotype, or proliferation index between euploid and aneuploid groups or histologic high- and low-grade groups. DNA ploidy study by flow cytometry is important for blood-based diagnosis of SS and detection of minimal residual disease in aneuploid SS after treatment. Detection of aneuploid neoplastic T cells in peripheral blood samples of patients with CTCL is associated with LCT in skin, lymph node, or other tissues.

Identification of Bordetella pertussis in a critically ill human immunodeficiency virus-infected patient by direct genotypical analysis of Gram-stained material and discrimination from B. holmesii by using a unique recA gene restriction enzyme site.

Bordetella pertussis was diagnosed in a human immunodeficiency virus-infected patient by a newly developed method in which bacterial DNA is amplified directly from sputum Gram-stained slides. The validation of the method is described along with an additional new PCR-based assay that can distinguish between B. pertussis and Bordetella holmesii.

Systemic Tropheryma whippleii infection associated with monoclonal B-cell proliferation: a Helicobacter pylori-type pathogenesis?

We report a case of Whipple disease in a 55-year-old woman who presented with arthralgia, weight loss, and lymphadenopathy. Tropheryma whippleii bacilli were identified in the mesenteric lymph nodes by diastase-resistant periodic acid-Schiff stain and confirmed by electron microscopy. Retrospectively, previous biopsy specimens from the duodenum and right axillary lymph node of this patient, which were initially considered to demonstrate reactive changes, also showed features consistent with involvement by Whipple disease. At the time of presentation, a large kappa-restricted monoclonal B-cell population with the phenotype CD20+CD19+CD5-CD10- was identified in the patient's peripheral blood, lymph nodes, and bone marrow by flow cytometry study. The monoclonality of the mesenteric lymph node B cells was confirmed by immunohistochemical stain for kappa chain after antigen retrieval and also by polymerase chain reaction with the primer set targeting FR2-V(H). Routine cytogenetic study failed to reveal any chromosomal abnormalities, and polymerase chain reaction for Bcl-2 major and minor breakpoint cluster of t(14:18) was not detected. The monoclonal B cells have persisted in blood for the entire follow-up period (10 months). The possibility of reactive monoclonal B-cell proliferation versus Whipple disease-related B-cell lymphoma is discussed.

Real-time quantitative RT-PCR of cyclin D1 mRNA in mantle cell lymphoma: comparison with FISH and immunohistochemistry.

Presence of the balanced translocation t(11;14)(q13;q32) and the consequent overexpression of cyclin D1 found in mantle cell lymphoma (MCL) has been shown to be of important diagnostic value. Although many molecular and immunohistochemical approaches have been applied to analyze cyclin D1 status, correlative studies to compare different methods for the diagnosis of MCL are lacking. In this study, we examined 39 archived paraffin specimens from patients diagnosed with a variety of lymphoproliferative diseases including nine cases meeting morphologic and immunophenotypic criteria for MCL by: (1) real-time quantitative RT-PCR to evaluate cyclin D1 mRNA expression; (2) dual fluorescence in situ hybridization (FISH) to evaluate the t(11;14) translocation in interphase nuclei; and (3) tissue array immunohistochemistry to evaluate the cyclin D1 protein level. Among the nine cases of possible MCL, seven cases showed overexpression of cyclin D1 mRNA (cyclin D1 positive MCL) and two cases showed no cyclin D1 mRNA increase (cyclin D1 negative "MCL-like"). In six of seven cyclin D1 positive cases, the t(11;14) translocation was demonstrated by FISH analysis; in one case FISH was unsuccessful. Six of the seven cyclin D1 mRNA overexpressing cases showed increased cyclin D1 protein on tissue array immunohistochemistry; one was technically suboptimal. Among the two cyclin D1 negative MCL-like cases, FISH confirmed the absence of the t(11;14) translocation in both cases. All other lymphoproliferative diseases studied were found to have low or no cyclin D1 mRNA expression and were easily distinguishable from the cyclin D1 overexpressing MCLs by all three techniques. In addition, to confirming the need to assess cyclin D1 status, as well as, morphology and immunophenotyping to establish the diagnosis of MCL, this study demonstrates good correlation and comparability between measure of cyclin D1 mRNA, the 11;14 translocation and cyclin D1 protein.