Clinical Abstracts
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March 2004 Clinical pathology abstracts editors: Michael Bissell, MD, PdD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus, and Ronald Domen, MD, professor or pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pa.
Hemoglobin A1c is the result of irreversible post-translational attachment of glucose to the N-terminal amino acid (valine) of the Hb A0 β chain. It is widely used as an indicator of degree of glycemic control in routine monitoring of patients with diabetes mellitus and is accepted as the most suitable marker for long-term diabetes care. The methods for measuring Hb A1c are based on its physical or chemical properties and on antibodies specific for the glycated N-terminal amino acid of the β chain. These results may be affected by comorbid, analytic, and pharmacologic interference, such as mutant variants and chemical modifications of Hb—for example, carbamylation and acetylation—that cause erroneous results for Hb A1c . This may lead to inconsistent Hb A1c values for certain patients and indicates a need to search for Hb variants. The authors attempted to characterize a new Hb mutant found in a German patient with an exceptionally low Hb A1c value of 1.5 percent as measured by an automated ion-exchange HPLC analyzer (Tosoh HLC-723 GHb V, A1c 3.0). They analyzed hemolysates from the propositus and a healthy control by electrophoresis, cation-exchange HPLC, boronate affinity chromatography, and electrospray ionization-mass spectrometry. They amplified genomic DNA by polymerase chain reaction and performed the sequencing on an ABI 310 sequencer. They determined functional properties of Hb by oxygen equilibrium studies and CO recombination kinetics after flash photodissociation. Glycohemoglobin species were synthesized by incubating hemolysates with glucose. The authors detected a novel, electrophoretically silent β chain, b5(A2)Pro→Ala, or Hb Görwihl, by cation-exchange HPLC. It accounted for about 44 percent of the total Hb and had functional properties similar to those of normal Hb A plus a mild degree of heat instability. During incubation with glucose, glycation of the β chains (assessed by electrospray ionization-mass spectrometry) in the hemolysate of a healthy volunteer was twice as fast as in hemolysate from the index case. The authors concluded that the b5(A2)Pro→Ala substitution seems to affect neither the functional properties nor the heterotropic interactions of Hb, but it slows glycation of the N-terminal valine by an unknown mechanism. Bissé E, Schauber C, Zorn N, et al. Hemoglobin Görwihl [α2β25(A2)ProÆAla], an electrophoretically silent variant with impaired glycation. Clin Chem. 2003;49: 137–143. Reprints: Emmanuel Bissé, Dept. of Clinical Chemistry, University Hospital, Hugstetterstrasse 55, D-79106, Freiburg, Germany; bisse@med1.uk1.uni-freiburg.de Effects of microgravity on the immune system Longer space voyages have provided the need and the opportunity to study the effects of space flight on numerous physiological functions. Among these are the effects of space flight on the immune system, especially in animal models, and the newly emerging area of the interaction of the immune system with the neuroendocrine system during space flight. Space flight and models that created conditions similar to those that occur during space flight have been shown to affect a variety of immunological responses, primarily cell-mediated immune responses such as leukocyte proliferation, cytokine production, and leukocyte subset distribution. The mechanisms and biomedical consequences of these changes remain to be established. Among the many possible causes of space flight-induced alterations in immune responses are exposure to microgravity, stress, and radiation. The author reviewed known effects of space flight on the immune system and explored the possible role of stress in these changes. Previous studies were limited by small sample size, relatively small number of flights available for immunological studies, and experimental conditions. Although it is clear that immune responses change as a result of space flight, the possible effects of space flight on resistance to infection have not been established. Several studies in rats flown in space have indicated that prolonged space flight resulted in hypoplasia of lymphoid organs and alterations in mitogen-induced blastogenesis. These effects were transient and the rats’ health returned to normal after a postflight recovery period. Data obtained from human studies showed inhibition of mitogen-induced blastogenesis of lymphocytes obtained from astronauts and cosmonauts after the Apollo-Soyuz test project, Skylab, Space Shuttle, and various Soviet flights. In these cases, the lymphocytes were obtained from the crews after their return flight. Samples taken from the crews as late as seven days postflight indicated that the blastogenic response had not returned to normal. These defects in immune function may have been due to inhibition of macrophage function, but this has not been confirmed. Sonnenfeld G. The immune system in space and microgravity. Med Sci Sports Exerc. 2002;34:2021–2027. Reprints: Dr. Gerald Sonnenfeld, Dept. of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Drive, SW, Atlanta, GA 30310-1495; sonneng@msm.edu The VIA LVM blood gas and chemistry monitoring system Technologic innovations in the development and fabrication of biosensors and microprocessors have led to the development and use of small, highly accurate point-of-care devices. These devices include analyzers that require permanent removal of blood specimens from patients and monitors that do not. Because monitors are designed to operate as closed systems with insignificant blood loss, the need for erythrocyte transfusions to neonates and the risk of nosocomial infections may be reduced. These advantages have led to the development and introduction of continuous and near-continuous patient-attached POC monitors. The authors prospectively determined the mean difference from laboratory methods of an in-line, ex vivo monitor, the VIA LVM blood gas and chemistry monitoring system (VIA LVM Monitor, Metracor Technologies Inc.), in 100 critically ill neonates and infants at seven children’s hospitals. They also examined monitor stability with continuous use. In vivo patient test results from laboratory benchtop analyzers were compared with those from the VIA LVM monitor on paired samples. In a separate in vitro comparison, benchtop analyzer and monitor test results were compared on whole-blood split samples. A total of 1,414 concurrent, paired-sample measurements were obtained. The mean differences (standard deviation of differences) from laboratory methods and r values for the combined data for the VIA LVM monitor from the seven sites were 0.001 (0.026) and 0.97 for pH, 0.7 (3.6) mmHg and 0.94 for Pco2, 4.2 (9.6) mmHg and 0.98 for Po2, 0.0 (2.9) mmol/L and 0.87 for sodium, 0.1 (0.2) mmol/L and 0.96 for potassium, and 0.3 percent (2.9 percent) and 0.90 for hematocrit. Performance results were similar among the study sites with increasing time of monitor use as well as between in vivo paired-sample and in vitro split-sample test results. The authors concluded that the VIA LVM monitor can be used to assess critically ill neonates and infants. Billman GF, Hughes AB, Dudell GG, et al. Clinical performance of an in-line ex vivo point-of-care monitor: a multicenter study. Clin Chem. 2002;48(11):2030–2043. Reprints: John Widness, University of Iowa Hospitals & Clinics, 200 Hawkins Drive, W222-1 GH, Iowa City, IA 52242-1083; john-widness@uiowa.edu Potential significance of aminopeptidase N/CD13 as a tumor marker Aminopeptidase N (APN; EC 3.4.11.2), also known as CD13, is a ZnZ+-dependent ectopeptidase that preferentially degrades proteins with a NH2-terminal neutral amino acid. CD13 is expressed by myeloid progenitors and monocytes, epithelial cells of the intestine and kidney, synaptic membranes in the central nervous system, fibroblasts, endothelial cells, and tumor cells. Known substrates of CD13 are small peptides such as enkephalins, as well as cytokines such as interleukin 1β. In addition, collagen type-IV, a protein present in the basement membrane of the extracellular matrix, has been suggested to be a substrate of CD13, and degradation of collagen type-IV by CD13 may contribute to the ability of tumor cells to invade the tissue and form metastases. Evidence supporting a role for CD13 in tumor angiogenesis recently was found. A previous study demonstrated the upregulation of CD13 expression in angiogenic vessels in MDA-MB-435 tumors in mice. In addition, angiogenic growth factors such as vascular endothelial growth factors (VEGF) and bFGF, but also tumor necrosis factor-α and hypoxia, appeared to induce CD13 expression of endothelial cells. These results suggest that in addition to matrix metalloproteinase (MMP) and the plasminogen proteolytic cascade, CD13 may facilitate the degradation of the extracellular matrix. The formation of PE and ascites is a common problem in patients with advanced cancer. The development of malignant effusions is usually a negative prognostic symptom and is associated with poor quality of life. The authors hypothesized that a soluble form of CD13 (sCD13) may be secreted from tumor cells and tumor-associated endothelial cells. If so, the level of sCD13 activity may be increased in the effusions and, in particular, intratumoral fluid of cancer patients. The authors measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition and studied its relationship with other major (anti-)angiogenic factors. They also studied the relationship of sCD13 activity in plasma with tumor load in a separate group of 41 patients. The sCD13 activity was highest in plasma from cancer patients at 71.9 (fmol/mL/s hydrolyzed substrate) versus 42.4 for healthy subjects. In intratumoral fluid, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. In comparing sCD13 level with tumor load and VEGF, endostatin, MMP-2, MMP-9, urokinase-type plasminogen activator, and plasmin, the authors found a significant correlation of plasma sCD13 activity with tumor load (r=0.68; P=0.01), suggesting that plasma sCD13 at least in part originates from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in intratumoral fluid compared with other effusions. In this fluid, a correlation was found between sCD13 and VEGF (r=0.67; P=0.03). No correlation was found between sCD13 and the other protease activities. Van Hensbergen Y, Broxterman HJ, Hanemaaijer R, et al. Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions and intratumoral fluid. Clin Cancer Res. 2002;8:3747–3754. Reprints: Henk Broxterman, VU Medical Center, Dept. of Medical Oncology, Br 232, De Boelelaan 1117, 1081 HV Amsterdam, Netherlands; h.broxterman@vumc.nl HCV viral load in HIV-positive patients Historically, many hemophilia patients transfused multiple times were coinfected with hepatitis C virus and HIV, and mortality remained high in this group even after the introduction of highly active antiretroviral therapy, or HAART. The presence of HIV increases HCV RNA load and greatly accelerates the progression of liver disease, whereas the presence of HCV increases the risk of death or an AIDS-defining illness as well as impairing CD4+ T cell recovery during HAART. The Royal Free Hospital Haemophilia cohort is one of the most extensively studied cohorts of male patients with hemophilia in the world. Within this cohort, six of 183 non-HIV-infected, HCV-positive patients died of liver-related disease, compared with 20 of 122 HIV-coinfected individuals (P=.0002). Liver disease is now also the primary cause of death in this, as well as other, HIV and HCV coinfected cohorts. HIV-1 RNA load is a useful marker of HIV disease progression, and its prognostic value is well documented. However, the prognostic value of HCV RNA load for progression to AIDS, death from liver disease, or all-cause mortality, remains unclear. No study has evaluated the prognostic value of the isolated HCV RNA loads that are measured in HCV-positive individuals for HIV progression and all-cause mortality. HCV RNA loads tend to be greater in HCV/HIV coinfected individuals than in those infected with HCV alone. HCV RNA load has been found in some studies to be associated with HIV-1 disease progression, response to antiretroviral therapy, and histological liver damage. There may also be an inverse correlation between CD4 cell counts and HCV RNA load, as well as HCV genotype-related effects. In the current study, the authors assessed the prognostic value of a single HCV RNA load measurement taken approximately four years after HIV seroconversion in a cohort of 96 HIV and HCV coinfected individuals attending the Royal Free Hospital Haemophilia Centre and Haemostasis Unit in London. HCV RNA loads are measured sporadically in HCV-positive individuals. However, the prognostic value of these isolated measurements for predicting progression to AIDS and all-cause mortality in coinfected individuals remains unclear. In this study, the prognostic value of a single HCV RNA load measurement taken early after HIV seroconversion was investigated in a cohort of 96 male patients with inherited bleeding disorders. Dates of HIV seroconversion had been estimated for all patients, and at least four HCV RNA load measurements per patient were done retrospectively after HIV seroconversion. HCV RNA load stabilized at four years after HIV seroconversion, and this point was used for analysis. There was a significant correlation between increased age and early HCV RNA load (r=0.25; P=0.1). Adjusting for HIV RNA levels, CD4 cell counts, and the age effect, HCV RNA load greater than 5.90 log10 copies/mL was predictive of progression to AIDS and all-cause mortality over a period of at least 15 years. Herrero-Martinez E, Sabin CA, Evans JG, et al. The prognostic value of a single hepatitis C virus RNA load measurement taken early after human immunodeficiency virus seroconversion. J Infect Dis. 2002;186:470– 476. Reprints: Vincent Emery, Royal Free and University College Medical School, Dept. of Virology, Royal Free Campus, Rowland Hill St., London NW3 2PF, United Kingdom; v.emery@rfc.ucl.ac.uk |
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