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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.
A new hemoglobin variant
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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