Application of proteomics to α1-antitrypsin
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-µL 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; email@example.com
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; firstname.lastname@example.org
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; email@example.com
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; firstname.lastname@example.org
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; email@example.com
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
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;
Gene therapy to
correct sickle cell disease in transgenic
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
Pawliuk R, Westerman KA, Fabry ME, et al. Correction
of sickle cell disease in transgenic mouse models by gene therapy.
Reprints: Philippe Leboulch, Harvard-MIT, Division
of Health Sciences and Technology, Massachusetts Institute of Technology,
Cambridge, MA 02139; pleboulch@
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; firstname.lastname@example.org