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February 2003

  • Infrared spectroscopy to simultaneously determine HDL and LDL cholesterol
  • Association between ANAs and coronary atherosclerosis
  • Prenatal diagnosis of sex chromosome aneuploidy
  • A guideline for general practitioners ordering blood tests
  • Preferences of youths regarding HIV rapid testing
  • Immunophenotyping adult acute leukemias
  • Comparing NAT testing and serology for hepatitis B in donor samples
  • Gene therapy to correct sickle cell disease in transgenic mouse models
  • Application of proteomics to α1-antitrypsin testing

    Infrared spectroscopy to simultaneously determine HDL and LDL cholesterol
    More accurate methods for quantitating low-density lipoprotein cholesterol are in demand. The authors proposed using an infrared spectroscopic method to simultaneously determine high-density lipoprotein and low-density lipoprotein cholesterol. Infrared absorption patterns are exquisitely sensitive to molecular structure and conformation. The authors previously demonstrated that a wide array of serum and urine analytes can be determined by infrared spectroscopy of films dried from the fluid of interest. For this study, the authors obtained 90 serum specimens. Duplicate 5-┬ÁL aliquots of the serum specimens were dried onto infrared-transparent barium fluoride substrates. Transmission infrared spectra were measured for these dry films. The authors also determined HDL and LDL cholesterol concentrations separately for each specimen using standard methods (the Friedewald formula for LDL cholesterol and an automated homogeneous HDL cholesterol assay). Infrared spectral features were quantitatively correlated with the clinical analytical results for 60 randomly chosen specimens using a partial least-squares regression analysis. For the 60 specimens used to train the infrared-based method, the standard error between the infrared-predicted values and the clinical laboratory assays was 0.22 mmol/L (r=0.98) for LDL cholesterol and 0.15 mmol/L (r=0.91) for HDL cholesterol. The corresponding standard errors for the test spectra were 0.34 mmol/L (r=0.96) for LDL-C and 0.26 mmol/L (r=0.82) for HDL-C. The standard deviation of duplicate measurements yielded a precision estimate of 0.11 mmol/L for LDL-C and 0.09 mmol/L for HDL-C. The authors concluded that infrared spectroscopy has the potential to become a clinical method of choice for quick and simultaneous determinations of HDL and LDL cholesterol.

    Liu KZ, Shaw RA, Man A, et al. Reagent-free, simultaneous determination of serum cholesterol in HDL and LDL by infrared spectroscopy. Clin Chem. 2002;48:499-506.

    Reprints: Kan-Zhi Liu, Institute for Biodiagnostics, National Research Council Canada, 435 Ellice Ave., Winnipeg, Manitoba, R3B 1Y6, Canada; kan-zhi.liu@nrc.ca

    Association between ANAs and coronary atherosclerosis
    The importance of inflammation in the pathogenesis of atherosclerosis is increasingly becoming understood. Inappropriate inflammatory responses may be associated with increased chronic development of atherosclerotic plaques and increased plaque rupture related to acute myocardial infarction. Secretion of proteases by macrophages recruited to the plaque may play a role in determining the likelihood of plaque rupture and its clinical sequelae. Humoral immunity may also play an important role during chronic atherosclerotic plaque development. Endothelial damage through the formation of autoimmune complexes was first proposed to be an important step in the development of atherosclerosis during the 1970s. More recently, evidence indicates a causative role for immunoglobulins in chronic vascular lipid lesion development in mice. In humans, however, specific autoantibodies against cytoskeletal proteins, cardiolipin, and oxidized or otherwise modified low-density lipoprotein have been associated with atherosclerosis. The literature does not contain studies of the prevalence of a systemic autoimmune reaction characterized by the presence of high titers of antinuclear antibodies (ANAs) in patients with advanced atherosclerosis. The authors analyzed serum from 40 patients with angiographically defined coronary artery disease resulting in stenosis in three major coronary arteries. They compared this with serum from 30 patients with no evidence of coronary artery disease on angiography. Both groups were tested for ANAs. None of the study subjects had been diagnosed with an autoimmune disorder. The ANAs were characterized by immunofluorescent detection of human antibodies bound to HEp-2000 cells. They were detected at a titer of at least 1:40 in 28 of the atherosclerotic subjects but only five of the controls (odds ratio, 11.67). Most of the ANA-positive atherosclerotic subjects had a pattern typical of antibodies directed against nucleolar antigens. Several common, extractable antigens were excluded, but the antigen involved has not been identified. The authors concluded that ANAs commonly associated with autoimmune diseases are substantially more prevalent among subjects with severe coronary atherosclerosis than those with normal coronary arteries. This preliminary association needs to be assessed further to determine whether ANAs are potentially useful as a biomarker.

    Grainger DJ, Bethell HWL. High titres of serum antinuclear antibodies, mostly directed against nucleolar antigens, are associated with the presence of coronary atherosclerosis. Ann Rheum Dis. 2002;61: 110-114.

    Reprints: Dr. D.J. Grainger, Dept. of Medicine, Box 157, Addenbrooke’s Hospital, Hills Rd., Cambridge CB2 2QQ, United Kingdom; djg15@mole.bio.cam.ac.uk

    Prenatal diagnosis of sex chromosome aneuploidy
    Most autosomal abnormalities result in serious defects of an intellectual, neurological, and physical nature. Sex chromosome abnormalities, however, are far less damaging to the phenotype. Sex chromosome aneuploidy (SCA) detected in amniocentesis is often an unexpected result of a test performed for another purpose. For most of these cases, the prognosis is milder and less predictable than trisomy 21 so parents are faced with the difficult decision of whether to terminate the pregnancy. Studies from Europe and the United States report a declining trend in the termination rates for SCA, but the experience in Israel is different. From 1989 to 1998 in Israel, the authors diagnosed 60 SCA cases in 20,106 amniocenteses, and 48 of these pregnancies were terminated, a significantly higher proportion than has been reported in Europe and the United States. The difference between the authors’ experience and that of others may be related to differences in cultural norms and values. Thirty women were interviewed, 23 of whom terminated pregnancy. Content analyses of the interviews showed that the main reason behind the decision to terminate was associated with the parents’ fear of a nonspecific abnormality in the child and concerns about abnormal sexual development. Genetic counseling at the authors’ center is intended to be nondirective, but 56 percent of the women reported that the counseling was directive toward termination or that they felt the counselor’s attitude was protermination. Of the women, 93 percent reported having come to terms with their decision.

    Sagi M, Meiner V, Reshef N, et al. Prenatal diagnosis of sex chromosome aneuploidy: possible reasons for high rates of pregnancy termination. Prenat Diagn. 2001;21: 461-465.

    Reprints: M. Sagi, Dept. of Human Genetics, Hadassah Hebrew University Hospital, Jerusalem, 91120, Israel; msagi@hadassah.org.il

    A guideline for general practitioners ordering blood tests
    The Dutch College of General Practitioners, in the Netherlands, issues recommendations for blood test ordering as defined in its guidelines for the general practitioner. Guideline creation is a four-stage process ultimately leading to publication in the journal of the Dutch College of General Practitioners. Published guidelines are revised at regular intervals. The authors sought to determine to what degree general practitioners in the Netherlands comply with the recommendations for blood test ordering defined in the aforementioned guidelines. The authors performed an audit of guideline compliance during a 12-month period from March 1996 through February 1997. A guideline-based decision-support system for blood test ordering, called BloodLink, was integrated with the electronic patient records of 31 general practitioners in 23 practices, 16 of which were solo practices. The authors determined compliance by comparing the recommendations for test ordering with the tests ordered by the clinicians. Compliance was presented as a percentage of the order forms that followed the recommendations for test ordering. Of 12,668 orders generated, 9,091 (71 percent) used decision-support software rather than paper order forms. Twelve indications accounted for more than 80 percent of the 7,346 order forms that selected a testing indication in BloodLink. The most frequently used of these categories was "vague complaints" (2,209 order forms; 30.1 percent). Of the 7,346 order forms, 39 percent were compliant. The most frequent type of noncompliance was the addition of tests. Six of the 12 tests most frequently added to the order forms were supported by guideline revisions that occurred within three years after the intervention. The authors concluded that the main source of noncompliance with guidelines involves adding tests. They also noted that noncompliance with guidelines appears to be partly due to practitioners applying new medical insight before it is incorporated into a revision of that guideline.

    Van Wijk MAM, Van Der Lei J, Mosseveld M, et al. Compliance of general practitioners with a guideline-based decision support system for ordering blood tests. Clin Chem. 2002;48:55-60.

    Reprints: Marc A.M. Van Wijk, Institute of Medical Informatics, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands; wijk@mi.fgg.eur.nl

    Preferences of youths regarding HIV rapid testing
    The Centers for Disease Control and Prevention estimates that 50 percent of people in the United States newly infected with human immunodeficiency virus are younger than 25 years. Nonetheless, the youth of America are underdiagnosed for HIV infection and are reluctant to seek HIV counseling and testing services. Improving early detection of HIV infection in youths remains a high priority for the public health care system. The authors undertook a study to determine youth preferences for FDA-approved and investigational HIV antibody collection and testing methods before and after the subjects learned of test result response times. Health educators explained and demonstrated six HIV antibody collection and testing strategies (three saliva, one urine, and two fingerstick methods), and the participants, who were aged 12 to 24 years, completed a confidential survey about their test method preference and tried the different testing methods. The participants could rerank their test method preference after learning about each test’s result response time. The study was conducted in health education sessions in clinical and community settings. The most highly preferred test method was an oral collection device with rapid saliva test. Preference for this method and the rapid response test methods via fingerstick improved significantly after subjects learned of the rapid result response time, while the other methods were given significantly lower preference rankings after subjects learned of the longer result response times. The shifts in preference rankings were not related to demographic variables. The authors’ research supports the use of noninvasive and rapid HIV testing methods with rapid response times for adolescents to assist in the early identification of HIV status, while offering HIV-prevention opportunities and immediate linkage to care.

    Peralta L, Constantine N, Deeds BG, et al. Evaluation of youth preferences for rapid and innovative human immunodeficiency virus antibody tests. Arch Pediatr Adolesc Med. 2001;155:838-843.

    Reprints: Dr. Ligia Peralta, University of Maryland School of Medicine, Dept. of Pediatrics, Division of Adolescent Medicine, 655 W. Lombard St., Suite 311, Baltimore, MD 21201; lperalta@peds.umaryland.edu

    Immunophenotyping adult acute leukemias
    The availability of well-characterized monoclonal antibodies to molecules associated with the major lineages of hematopoietic cell differentiation permits delineation of the cellular origin of leukemic cells in more than 99 percent of acute leukemias. Scoring systems based on the lineage specificity of particular antigens have been developed to clearly distinguish acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) and to define biphenotypic acute leukemia (BAL). The authors reported on the immunophenotypes of 325 consecutive acute leukemias, classified according to the aforementioned scoring systems. They compared the results with morphologic and molecular genetic characteristics. The bone marrow cells were immunophenotyped using a panel of monoclonal antibodies proposed by the European Group for the Immunological Characterization of Leukemias (EGIL). Of these cells, 97.2 percent could be easily assigned to myeloid or lymphoid lineage (254 acute myeloid leukemias, 48 B-cell lineage acute lymphoblastic leukemias, and 14 T-cell lineage ALLs), 1.8 percent as biphenotypic, and less than one percent as undifferentiated. Immunologic subtyping of ALLs revealed an association between early precursor phenotypes and coexpression of myeloid antigens. This was particularly true for CD15/ CD65s coexpression and pre-pre-B cell-specific phenotypes and genotypes. The common ALL phenotype was associated with BCR-ABL translocation. Among the AMLs, CD2 coexpression was restricted to French-American-British subtypes M3 variant and M4Eo and related molecular aberrations. The most valuable markers for differentiating between myeloperoxidase-negative AML subtypes M0 and ALLs were CD13, CD33, and CD117, typical of M0, and intracytoplasmic CD79a, intracytoplasmic CD3, CD10, and CD2, all of which are typical of B cell- or T cell-lineage ALL. The results confirm the practicability of the EGIL proposal for immunologic classification of acute leukemias. For myeloperoxidase-negative AMLs, the authors suggest a scoring system based on the markers most valuable for distinguishing between AML-M0 and ALLs.

    Thalhammer-Scherrer R, Mitterbauer G, Simonitsch I, et al. The immunophenotype of 325 adult acute leukemias. Am J Clin Pathol. 2002;117:380-389.

    Reprints: Dr. Ilse Schwarzinger, Dept. of Laboratory Medicine, University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria

    Comparing NAT testing and serology for hepatitis B in donor samples
    Copy numbers of hepatitis B virus in pooled donor specimens used for nucleic acid testing tend to be small during the window period. Chemoluminescence immunoassay for hepatitis B surface antigen (HBsAg) is effective at detecting HBV from donors in the window period. The authors conducted a study to determine the pool size at which NAT shows better sensitivity than chemoluminescence immunoassay for HBsAg serology. They tested HBV seroconversion panels for HBsAg by chemoluminescence immunoassay and for HBV DNA by nested PCR. PCR was carried out at various dilutions. HBV-positive samples detected serologically from the simultaneous screening of 540,161 routine whole-blood donations using the chemoluminescence immunoassay and agglutination assays were also characterized for additional HBV infection. In nine of the 10 HBV seroconversion panels, PCR had better sensitivity than chemoluminescence assay at dilutions of 1:25 or lower. Of the 65 chemoluminescence-only confirmed-positive donor samples (agglutination assay-negative), eight represented early infection, two of which were PCR-positive at a 1:50 dilution but negative at a 1:100 dilution. Two of 47 samples from probable late-stage HBV that were positive on chemoluminescence assay only were PCR positive with 0.1-mL sample volume and the S-region primer. The remaining 45 samples required a 1.0-mL sample input and C-region primer for increased PCR positivity. The remaining 10 chemoluminescence-only confirmed-positive donor samples were from HBV vaccine recipients. None of the 12 chemoluminescence and surface antigen serologic-negative donor samples that were strongly anti-HBc reactive could be detected by PCR at any dilution; all 12 were PCR positive when undiluted, but four required a 1.0-mL input volume for PCR positivity. PCR at dilutions of 1:25 or lower (equivalent to a pool of 25 or fewer members) had greater sensitivity than chemoluminescence HBsAg serology. In contrast, samples from late-stage HBV infection were detected by PCR only with undiluted samples, regardless of their chemoluminescence surface antigen reactivity. So, although NAT using minipools of 25 donations or less may be effective for detecting early stage HBV infection, it may not be effective for detecting persistent HBV.

    Sato S, Ohhashi W, Ihara H, et al. Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion. 2001;41:1107-1113.

    Reprints: Dr. Shinichiro Sato, Hokkaido Red Cross Blood Center, Yamanote 2-2, Nishi-ku, Sapporo, 063-0002, Japan; satolab@hokkaido.bc.jrc.or.jp

    Gene therapy to correct sickle cell disease in transgenic mouse models
    Sickle cell disease is one of the most prevalent autosomal recessive disorders worldwide and was the first genetic disorder for which a causative mutation was identified at the molecular level: the substitution of valine for glutamic acid in human βA-globin codon. The authors designed a βA-globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector that was optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Expression for up to 10 months was achieved without preselection in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52 percent of total hemoglobin and 99 percent of circulating red blood cells. In two sickle-cell mouse models, inhibition of RBC dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect. The authors concluded that before gene therapy for sickle cell disease can be proposed to humans on the basis of their findings, it is desirable to achieve large-scale lentiviral production devoid of replication-competent retrovirus and bone marrow reconstitution with transduced stem cells in the absence of toxic myeloablation regimens.

    Pawliuk R, Westerman KA, Fabry ME, et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science. 2001;294:2368-2371.

    Reprints: Philippe Leboulch, Harvard-MIT, Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139; pleboulch@ mit.edu

    Application of proteomics to α1-antitrypsin testing
    Proteomic technology supports the investigation of genetic metabolic diseases at the level of protein expression. The authors examined two applications of proteomics technology—the detection of mutant and polymorphic amino acid sequences and the detection of post-translational modifications. They chose plasma a1-antitrypsin as a target protein because plasma is readily available and because there are diseases that result from changes in the amino acid sequence and the post-translational modifications of the protein. The authors used high-resolution, two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass or charge, or both. They performed in-gel digestion with proteases and N-glycanases to confirm the identity of the separate proteins. They then analyzed the peptides that were released and the glycans that were released by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. They achieved complete characterization of the polypeptide sequences and glycosylation of α1-antitrypsin isoforms in plasma from controls and from patients with three different known α1-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. The authors concluded that proteomic technology is a powerful and sensitive means of detecting changes in amino acid sequence and abnormal post-translational modifications of specific proteins in a complex biologic matrix, such as serum.

    Mills K, Mills PB, Clayton PT, et al. Identification of a1-antitrypsin variants in plasma with the use of proteomic technology. Clin Chem. 2001;47:2012-2022.

    Reprints: Bryan G. Winchester, Biochemistry Endocrinology and Metabolism Unit, Institute of Child Health at Great Ormond Street Hospital, University College London, 30 Guilford St., London WC1 N 1EH, United Kingdom; b.winchester@ich.ucl.ac.uk

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