College of American Pathologists
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  Clinical Abstracts





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February 2005

Michael Bissell, MD, PhD, MPH, Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus
Ronald Domen, MD, Professor of Pathology, Medicine, and Humanities, Penn State University College of Medicine, Hershey, Pennsylvania

Nitric oxide production in multiple sclerosis patients
Epstein-Barr virus and cancer
Breakpoint region of isochromosome 17q
Blood typing using flow cytometry
Plasma cell-like T-helper cell
Differential gene-expression profiling in leukemia
Bcr-abl gene expression in essential thrombocythemia
Questionable efficacy of acute normovolemic hemodilution
Use of vein vs. capillary blood as samples

Nitric oxide production in multiple sclerosis patients

Nitric oxide has been implicated in the pathogenesis of immune-mediated inflammation of the central nervous system in multiple sclerosis. Reactive astrocytes and microglia in multiple sclerosis (MS) plaques express high levels of inducible nitric oxide synthase (iNOS). Nitric oxide is thought to play a role in demyelination, oligodendrocyte destruction, and the functional and structural injury of axons. It is an unstable molecule and rapidly converted in vivo into its metabolites, nitrate and nitrite. Levels of nitrate and nitrite can be used as an index of nitric oxide generation. Nitric oxide metabolite concentrations are increased in the cerebrospinal fluid of patients with MS and seem to correlate with disease activity. Increased levels of nitric oxide metabolites have also been measured in the serum of patients with MS. The authors conducted a study to investigate peripheral blood leukocyte (PBL) nitric oxide production in patients with a benign course of MS (BMS) lacking progression, secondary progressive MS (SPMS), and primary progressive MS (PPMS). They used PBL because mononuclear cells and neutrophils can produce nitric oxide. The authors isolated PBL from 25 patients with BMS, 33 with SPMS, 21 with PPMS, and 29 healthy individuals. Leukocyte supernatants were assayed for nitrite concentration, which is an index of nitric oxide generation, using the Griess reaction. Serum levels of tumor necrosis factor-alpha (TNF-a) and interleukin (IL)-12 were measured using an enzyme-linked immunosorbent assay. The authors found that, compared with healthy controls, nitrite concentrations were higher in patients with BMS (P<0.001), SPMS (P<0.001), and PPMS (P<0.05). No significant differences were found among the three clinical subgroups of MS. A correlation was found between nitrite concentration and serum levels of IL-12 (P=0.04) but not with TNF-a. The authors concluded that increased nitric oxide production by PBL in patients with MS is independent of disease course.

Ramsaransing GSM, Teelken A, Arutjunyan AV, et al. Peripheral blood leukocyte NO production in MS patients with a benign vs progressive course. Neurology. 2004;62:239-242.

Reprints: Dr. Jacques De Keyser, Dept. of Neurology, Academisch Ziekenhuis Groningen, Hanzeplein 1, 9713 GZ Groningen, Netherlands;

Epstein-Barr virus and cancer

Abstract Epstein-Barr virus was the first human virus to be directly implicated in carcinogenesis. It infects more than 90 percent of the world's population. Although most humans coexist with the virus without serious sequelae, a small proportion will develop tumors. Normal host populations can vary markedly in their susceptibility to Epstein-Barr virus (EBV)-related tumors, as demonstrated by geographical and immunological variations in the prevalence of these cancers. EBV has been implicated in the pathogenesis of Burkitt's lymphomas and leiomyosarcomas arising in immunocompromised persons. The presence of this virus also has been associated with epithelial malignancies arising in the gastric region and the breast, although some of these findings remain in dispute. The virus uses its viral proteins, the actions of which mimic several growth factors, transcription factors, and anti-apoptotic factors, to usurp control of the cellular pathways that regulate diverse homeostatic cellular functions. Recent advances in antiviral therapeutics, application of monoclonal antibodies, and generation of EBV-specific cytologic T lymphocytes are beginning to show promise in treating EBV-related disorders.

Thompson MP, Kurzrock R. Epstein-Barr virus and cancer. Clin Cancer Research. 2004;10:803-821.

Reprints: Razelle Kurzrock, Dept. of Bioimmunotherapy, Box 422, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030;

Breakpoint region of isochromosome 17q

Isochromosome 17q, or i(17q), is the most common isochromosome characterizing human neoplasia and plays an important pathogenetic role in tumor development and progression. The high frequency of i(17q) formation in leukemia and solid tumors compared with other isochromosomes may potentially be explained by the presence of unique DNA sequence characteristics in the breakpoint region of i(17q) that favor genetic rearrangement, in combination with a selective advantage conferred by dysregulated genes in the vicinity of the breakpoint or, perhaps more likely, conferred by gene dosage imbalances resulting from the loss of 17p and gain of 17q material. However, limited information is available about the mechanism by which this chromosomal abnormality is formed and the resulting molecular genetic consequences. In this study, the authors used fluorescence in situ hybridization (FISH) analysis with a large set of BAC and PAC clones to further delineate the breakpoints of i(17q) in hematologic malignancies. Sequencing of individual BAC, PAC, and fosmid clones covering the breakpoint region and subsequent sequence comparison of individual clones allowed them to generate a physical map of an ~240-kb genomic interval, including the i(17q) breakpoint cluster region. The delineated region contained two unique low-copy repeats organized in a complex manner, with the capability to form a long DNA palindromic structure. Mapping of all breakpoints to within this unique genome architecture suggests its important role in the genesis of i(17q). The authors previously identified a breakpoint cluster region for i(17q) formation in 17p11.2 and hypothesized that genome architectural features could be responsible for this clustering. To test this hypothesis, they precisely mapped the i(17q) breakpoints in 11 patients with different hematologic malignancies and determined the genomic structure of the involved region. Their results revealed a complex genomic architecture in the i(17q) breakpoint cluster region characterized by large (~38-kb to 49-kb), palindromic, low-copy repeats, strongly suggesting that somatic rearrangements are not random events but rather reflect susceptibilities due to genomic structure.

Barbouti A, Stankiewicz P, Nusbaum C, et al. The breakpoint region of the most common isochromosome, i(17q), in human neoplasia is characterized by a complex genomic architecture with large, palindromic, low-copy repeats. Am J Hum Genetics. 2004;74:1-10.

Reprints: Dr. Thoas Fioretos, Dept. of Clinical Genetics, Lund University Hospital, Lund, SE-22185, Sweden;

Blood typing using flow cytometry

Testing before transfusion includes immunohematology assays to determine the ABO group and D type of blood donors and recipients, as well as tests to identify unexpected red blood cell alloantibodies and to confirm cross-match compatibility. These assays historically have been performed by visually scoring antibody-mediated RBC agglutination in test tubes following centrifugation. Column agglutination technology (CAT) (ID-micro typing system, Ortho-Clinical Diagnostics) and solid-phase RBC adherence assays (Capture-R, Immucor Inc.) have been developed to automate immunohematology testing. The authors recently reported on a fluorescence cytometry method for immunohematology testing. This fluorescence cytometry assay used a standard flow cytometer and demonstrated accuracy comparable to CAT and tube testing. In the process of developing an automated platform, a modified procedure was developed that used low-speed centrifugation of 96-well filter plates for RBC staining. And two small-footprint benchtop capillary cytom eters (PCA and PCA-96, Guava Technologies Inc.) were used to acquire data. Authentic clinical samples from hospitalized patients were tested for ABO group and D antigen (n=749) as well as for RBC alloantibodies (n=428). Challenging samples with mixed-field reactions and weak antibodies were included. Results were compared to those obtained by CAT, and discrepancies were resolved by standard tube methods. Detailed investigation of fluorescence cytometry sensitivity and reproducibility were also performed. The modified fluorescence cytometry method using the PCA determined the correct ABO group and D type for 98.7 percent of 520 samples, compared with 98.8 percent for CAT (P>0.05). No-type-determined (NTD) rates were 1.2 percent for both methods. In testing for unexpected alloantibodies, fluorescence cytometry determined the correct result for 98.6 percent of 215 samples, compared with 96.3 percent for CAT (P>0.05). When samples were automatically acquired in the 96-well plate format with the PCA-96, 98.7 percent of 229 samples had correct ABO group and D type determined by fluorescence cytometry, compared with 97.4 percent for CAT (P>0.05). NTD rates were 0.9 percent and 2.6 percent, respectively. Antibody screens were accurate for 99.1 percent of 213 samples with the PCA-96, compared with 99.5 percent for CAT (P>0.05). Further investigations demonstrated that fluorescence cytometry with the PCA-96 was better than CAT at detecting weak anti-A (P<0.0001) and alloantibodies.

Roback JD, Barclay S, Hillyer CD. Improved method for fluorescence cytometric immunohematology testing. Transfusion. 2004;44:187-196.

Reprints: Dr. J.D. Roback, Emory University, WMB 2307, 1639 Pierce Drive, Atlanta, GA 30322;

Plasma cell-like T-helper cells

T and B cells are the two actors of the immune response that interact with antigens and their derived peptides. After such activation, T- and B-cell lineages are characterized by a differentiation process. B cells internalize specific antigens via their surface immunoglobulin receptors and process the antigens into peptides, which are presented to T cells in the context of the B-cell class II major histocompatibility complex (MHC). Terminal differentiation of B cells results in the production of soluble immunoglobulins by plasma cells, which have lost their surface immunoglobulins. T cells are also characterized by the expression of antigen-specific T-cell receptor (TCR) associated with the CD3 complex. As opposed to B cells, the TCR is not secreted as a soluble form after TCR activation. However, T cells can secrete cytokines in response to antigen stimulation. The nature of secreted cytokines led to the classification of T-helper cells into Th1 proinflammatory cytokine-producing cells and Th2 anti-inflammatory cytokine-producing cells. A Th1/Th2 imbalance with a local predominance of Th1 cytokines can influence disease mechanisms such as rheumatoid arthritis. The authors observed the plasma cell appearance of some cytokine-producing T cells in rheumatoid arthritis synovium sections. To further clarify this subset, they focused on its morphology and phenotype in in vitro and in vivo situations. This morphology was observed in rheumatoid arthritis, dermatomyositis, and activated lymph node sections. In these situations, Th1-cytokine production was associated with a similar phenotype as in plasma cells producing immunoglobulins. The authors clarified the morphology and phenotype of interleukin (IL)-17 and interferon (IFN)-g-producing cells in in vitro and in vivo situations. Oligoclonal activation of normal peripheral blood mononuclear cells with the super-antigen Staphylococcus aureus enterotoxin B and polyclonal activation with phorbol myristate acetate/phytohemagglutinin were used as in vitro models. This study was extended to various in vivo situations, such as rheumatoid arthritis, dermatomyositis, and normal activated lymph nodes. The phenotype of IL-17- and IFN-g-producing cells was evaluated by immunohistochemistry using CD3 and CD4 T-cell markers and CD20, CD38, k, and a light chain B-cell lineage markers. The expression of two chemokine receptors, CCR6 and CCR7, involved with their associated ligands CCL20 and CCL19/CCL21 in the migration of T lymphocytes was evaluated in tissue sections. After polyclonal and oligoclonal activation, IL-17+ and IFN-g+ cells acquired a plasma cell-like morphology associated with a high secretory activity, reduced expression of CD3, and no change in CD4 expression. In rheumatoid arthritis, dermatomyositis, and activated lymph nodes, IL-17- and IFN-g-producing cells had the same morphology. These Th1 cytokine-producing cells were CD4+-, CD3-, and B-cell lineage marker negative. In in vitro and in vivo situations, expression of CCR6 or CCR7 was not associated with a particular subset. The authors concluded that activated T-helper CD4+ T cells, by their release of cytokines, seem to have functional similarities with plasma cells secreting immunoglobulins.

Page G, Sattler A, Kersten S, et al. Plasma cell-like morphology of Th1-cytokine-producing cells associated with the loss of CD3 expression. Am J Pathol. 2004;164:409-417.

Reprints: Dr. Pierre Miossec, Clinical Immunology Unit, Departments of Immunology & Rheumatology, Hôpital Edouard Herriot, 69437 Lyon Cedex 03, France;

Differential gene-expression profiling in leukemia

T-cell large granular lymphocyte leukemia runs a chronic clinical course without need for therapy. However, some cases progress to an aggressive disease after the indolent clinical stage. The mechanism that transforms such cases into a high-grade malignancy has not been well studied. The authors have established two leukemia cell lines, MOTN-1 and PLT-2, derived from the same clone of CD56+ T-cell large granular lymphocyte (T-LGL) leukemia in chronic and aggressive phases, respectively. The paired availability of such cell lines is valuable in the biologic and genetic investigation of T-LGL leukemia. The authors used a microarray containing 406 cDNAs to elucidate alterations of gene expression between the two cell lines. They found a number of genes that were expressed differentially: 13 genes with increased expression and three genes with reduced expression in PLT-2 cells as compared to MOTN-1 cells. Increased expression of the dek, rac, Op18, CD6, CD58, CD106, Id2, ATF4, IRF5, ELL2, and D6 genes, and reduced expression of the GzmA and GzmK genes, were confirmed by real-time quantitative reverse transcription-polymerase chain reaction, the results of which paralleled the microarray data. These upregulated genes encode oncoproteins, cell surface antigens, including molecules related to T-cell proliferation, transcription factors, and chemokine receptor. The two downregulated genes encode granzymes that play an important role in induction of cell death. These findings suggest that there is differential gene expression in different clinical phases of T-LGL leukemia, and these differentially expressed genes would be potential targets for additional studies to identify the genes involved in the transformation process of T-LGL leukemia.

Daibata M, Matsuo Y, Machida H, et al. Differential gene-expression profiling in the leukemia cell lines derived from indolent and aggressive phases of CD56+ T-cell large granular lymphocyte leukemia. Int J Cancer. 2003;108:845-851.

Reprints: Dept. of Hematology and Respiratory Medicine, Kochi Medical School, Kochi 783-8505, Japan;

Bcr-abl gene expression in essential thrombocythemia

Chronic myeloproliferative disease is characterized by panmyelosis, splenomegaly, and a predisposition to venous/arterial thrombosis, myelofibrosis, and acute leukemia. Subtypes of chronic myeloproliferative disease (MPD) include chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis. The clinical and morphological features of these diseases frequently overlap. Significant variations based on region, practice type, specialty, and clinical experience are evident in the diagnostic approach to MPD patients. The major bcr-abl fusion gene is seen as a major marker of chronic myeloid leukemia (CML). However, whether the bcr-abl transcript can be detected in patients with ET is still a matter of controversy. The authors detected the messenger RNA expression of the bcr-abl gene using reverse transcription-polymerase chain reaction in peripheral-blood leukocytes (PBLs) from 63 patients with myeloproliferative disorders, including CML, ET, and PV, and in 51 healthy volunteers. The bcr-abl transcript was detected in four of the 30 ET patients (13.3 percent), 17 of the 17 CML patients (100 percent), none of the 16 PV patients, and one of the 51 normal subjects (1.9 percent). Compared with the normal controls, ET patients have a greater tendency to express the bcr-abl transcript in PBLs (P=.06, Fisher's exact test). Further semiquantitative analysis showed that the intensity of bcr-abl transcript expression in the four ET patients and a normal individual was 103 to 104 times less than in the CML patients. The authors concluded that the bcr-abl transcript can be detected in the PBLs of Philadelphia chromosome-negative ET patients but that the level of expression is markedly less than in CML patients. The clinical significance of this finding merits further investigation.

Hsu HC, Tan LY, Au LC, et al. Detection of bcr-abl gene expression at a low level in blood cells of some patients with essential thrombocythemia. J Lab Clin Med. 2004;143:125-129.

Reprints: Dr. Hui-Chi Hsu, Division of Hematology, Dept. of Medicine, Taipei Veterans General Hospital, Shih-pai, Taipei, Taiwan 11217;

Questionable efficacy of acute normovolemic hemodilution

Acute normovolemic hemodilution is withdrawal of autologous whole blood with concurrent infusion of replacement fluids to maintain normovolemia. Acute normo vo lemic hemodilution (ANH) typically is performed in the operating room just prior to surgery. If needed, the autologous blood collected by ANH is then transfused during surgery. ANH was established as a possible alternative to allogeneic blood transfusion and preoperative autologous blood collection. Because the efficacy and safety of ANH is uncertain, the authors performed a meta-analysis of published reports. A total of 1,114 articles were identified, but 1,072 papers were excluded for various reasons. Forty-two studies published between 1972 and 2002 were identified for further analysis. While the volume of allogeneic blood transfused in the ANH groups was less than that transfused in the comparison groups (by one to two units), the results were heterogeneous, and recent studies were less likely to show a benefit with ANH. The aggregate data also suggested some hemostatic benefit with the use of ANH compared to the use of no blood conservation method, but further study is warranted. There was inconsistent reporting of adverse events related to ANH, and no study noted any transfusion reactions. The efficacy of ANH to avoid allogeneic transfusion was not proven in this analysis. The authors concluded that the efficacy of ANH is likely to be small, its safety remains unproven, and widespread adoption of ANH should not be encouraged.

Segal JB, Blasco-Colmenares E, Norris EJ, et al. Preoperative acute normovolemic hemodilution: a meta-analysis. Transfusion. 2004;44:632-644.

Correspondence: Dr. Jodi B. Segal, 720 Rutland Ave., Ross #1025, Baltimore, MD 21205;

Use of vein vs. capillary blood as samples

The accurate and precise measurement of glucose is influenced by a number of factors, including origin of the sample (arterial, capillary, or venous), sample preparation, analytical method, and whether the glucose is measured in plasma, whole blood, or serum. The World Health Organization (WHO) and American Diabetes Association (ADA) have published statements on glucose levels in blood for the diagnosis of diabetes and lesser degrees of glucose intolerance. However, the ADA has only published values for venous plasma glucose, while the WHO provides equivalence estimates of diagnostic values for venous plasma, venous whole blood, and capillary whole blood. It is commonly reported that venous samples give slightly lower values than capillary samples and that these differences generally can be ignored in the fasting state. But after a meal or glucose load, capillary samples are about eight percent higher than venous samples, and plasma samples are 14 percent to 16 percent higher than whole blood. The different diagnostic levels used by the WHO attempt to incorporate these differences. However, the few studies that have systematically compared glucose results in different blood samples raise doubt about published equivalence values. The authors conducted a study to examine the relationship between glucose measurements in capillary and venous samples at different time points—random, fasting, and two hours after oral glucose. Simultaneous measurements of venous plasma and capillary blood glucose were performed on random samples in 609 people, fasting samples in 685 people, and two hours after oral glucose samples in 463 people. Separate capillary and venous samples were collected each time. A variance component model was used to construct conversion algorithms between venous and capillary results. The relationship between venous and capillary glucose values varied, with venous plasma being higher than capillary blood for random and fasting samples but lower for sampling two hours after oral glucose. Discrepancies were observed between measured capillary blood values and the published WHO capillary blood equivalence values for venous plasma values for all except a fasting venous value of 7.0 mmol/L. For example, for a fasting venous plasma glucose of 6.1 mmol/L, the WHO equivalent value was 5.6 mmol/L, while the meaured value was 5.2 mmol/L; and for a two-hour venous plasma glucose of 11.1 mmol/L, the WHO value was 11.1 mmol/L, while the measured result was 11.7 mmol/L. The authors suggested that these results highlight the difficulty in equating glucose levels from one sampling and measuring procedure to another and raise uncertainties about published equivalence values, which could lead to misclassifications in glucose tolerance status.

Colaguiri S, Sanbaek A, Carstensent B, et al. Comparability of venous and capillary glucose measurements in blood. Diabet Med. 2003;20:953-956.

Reprints: Stephen Colaguiri, Dept. of Endocrinology and Diabetes, Prince of Wales Hospital, High St., Randwick 2031 New South Wales, Australia;