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June 2002
Dipstick measurements of urine specific gravity
An easy and valuable way to determine a patients fluid and electrolyte status
is to determine the degree of urinary concentration. This measurement depends
on the presence of small and large particles per unit of urine volume. It
is related to the patient’s state of hydration and depends on the amount of
fluid reabsorbed along the renal tubal. The degree of concentration of solutes
in the urine can be determined by measuring its osmolality or specific gravity.
Measuring urine osmolality by freezing point depression is a central lab function
and is not performed at the bedside. Many physicians, therefore, rely on a
dipstick reagent strip or refractometer measurements of the U-SG, or both.
Controversy surrounds the comparative accuracy of dipstick measurements relative
to refractometry. The authors conducted a study to compare dipstick U-SG measurements
with other U-SG measurements on the same urine samples. They collected 135
fresh voided urine specimens from children visiting an outpatient renal clinic.
The specimens were immediately tested for U-SG without being preserved. They
were tested for urine pH and osmolality using a pH meter and an osmometer.
Then they were tested for U-SG using visual change of color of a dipstick,
automatic readout of a dipstick using the Clinitek-50 device, and refractometry.
Only the refractometer U-SG and urine osmolality had good linear correlations.
The dipsticks that were tested were unreliable for the bedside determination
of U-SG, even after correcting for urine pH, as recommended by the manufacturer.
Therefore, the correlations between the visual U-SG measurements and those
determined by refractometer and the Clinitek-50 comparison with osmolality
were all less than optimal, with a wide scatter of values. The authors concluded
that among bedside determinations, only refractometry gives reliable results
for U-SG, and they recommended that dipstick U-SG be abandoned.
De Buys Roessingh AS, Drukker A, Guignard J-P. Dipstick measurements of
urine specific gravity are unreliable. Arch Dis Child. 2001;85:155-157.
Reprints: Jean-Pierre Guignard, Renal Unit, Dept. of Pediatrics, University
Medical Center (CHUV), CH-1011 Lausanne, Switzerland; guignard@chuv.hospvd.ch
Testing genomic alterations in minimal residual cancer
cells in breast cancer
After the resection of a primary tumor, minimal residual cancer cells are
targets for chemotherapeutic agents. A number of attempts have been made to
develop methods for detecting or isolating these disseminated tumor cells
from peripheral blood or bone marrow. Most of these analytical approaches
involved the use of RT-PCR analyses to detect micrometastatic disseminated
cancer cells. In contrast to an analysis of RNA, an analysis depending on
determination of genomic imbalances in DNA requires a pure sample of cancer
cells. The authors, therefore, established a new density and size-dependent
methodology for isolating minimal residual cancer cells from blood. Their
method is based on observing that the extravasation of tumor cells often occurs
in clusters of malignant cells, and clotting in the blood appears by aggregation
of tumor cells and thrombocytes, leading to the formation of large particles.
In their approach, identifying and characterizing the purified disseminated
tumor cells is based on the analysis of genomic DNA. They measured genomic
imbalances of 15 different genes—13 tumor-suppressor genes and two oncogenes.
The authors investigated the prognostic value of performing this assay on
cells derived from 353 breast cancer patients. They used their technique to
purify disseminated tumor cells from the blood and isolate the nucleic acids.
Significant correlations were found between genomic alterations of the DCC
and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free
survival. In addition, worse prognosis of recurrent disease was significantly
associated with increasing numbers of these genomic imbalances. The genomic
imbalances were identified as an independent prognostic factor using logistic
regression and Cox multivariate analysis. The authors concluded that determining
which are disseminated tumor cells by genotyping oncogenes and tumor-suppressor
genes seems to be useful in following up carcinoma patients and offers additional
individualized prognostic and predictive information beyond that provided
by the tumor-nodes-metastases system.
Uciechowski P, Eder C, Böckmann B, et al. Prognostic value of genomic alterations
in minimal residual cancer cells purified from the blood of breast cancer
patients. Br J Cancer. 2000;83:1664-1673.
Reprints: H-J Grill, Institut für Molekulare NanoTechnologie, Berghäuser
Str. 295, 45659 Recklinghausen, Germany
A comparison of semen parameters in fertile and subfertile
populations
In in vitro fertilization and intra-uterine insemination programs, medical
professionals have found value in attempting to identify male partners in
couples at risk for a significantly lower chance of fertilization. Discussions
have centered on the criteria used for sperm morphology—that is, a five
percent threshold versus a 10 percent threshold. The authors addressed the
question of whether the threshold value might vary in different parts of the
world. They identified the semen parameters of a proven fertile population
and compared them with the parameters of a subfertile group in the same region.
The authors studied 69 fertile male patients and compared them with 93 patients
recruited at an infertility clinic in Ankara, Turkey. They matched a subsample
of the patients according to age. The group was composed of 61 fertile males
and 62 infertile males. Receiver operator characteristics curves were computed
for the subsample of the population. On the progressive motility parameter,
the threshold value was 42 percent, which was the best parameter with sperm
morphology to distinguish between the fertile and infertile groups. A sperm
morphology threshold of 12 percent normal forms had a sensitivity of 69 percent
and a specificity of 67 percent. If the positive and negative predictive value
was used to screen the general population to identify the subfertile group,
a concentration of 9 x 106/mL or lower, 30 percent motility, 14
percent progressive motility, and a five percent normal morphology threshold
was indicated. The negative predictive value of these parameters was 90 percent
in most cases. Sensitivity of the semen parameters at the reported thresholds
was poor and indicated a large overlap in the distributions of these variables
in the fertile and infertile groups. The most significant finding in this
study was the progressive motility with a threshold level of 14 percent to
distinguish between fertile and subfertile populations. The cut-off value
of sperm morphology at five percent in vivo was consistent with previous publications
in assisted reproduction programs for sperm morphology.
Gunalp S, Onculoglu C, Gurgan T, et al. A study of semen parameters with
emphasis on sperm morphology in a fertile population: an attempt to develop
clinical thresholds. Hum Reproduction. 2001;16:110-114.
Reprints: Thinus F. Kruger, Reproductive Biology Unit, Dept. of Obstetrics
and Gynaecology, University of Stellenbosch and Tygerberg Hospital, P.O. Box
19063, Tygerberg 7505, South Africa; tfk@gerga.sun.ac.za
Using PCR to differentiate mycobacterial species
Of the more than 70 species comprising the genus Mycobacterium, some
are pathogenic or potentially pathogenic to humans and animals and others
are saprobes. It is the slower growing mycobacteria, such as Mycobacterium
tuberculosis, M. avium complex, and M. kansasii, that are
primarily responsible for human infections. Reports of infections due to mycobacteria
other than M. tuberculosis complex have been increasing. Rapidly differentiating
causative mycobacteria, is, therefore, important because of their clinical
importance in terms of strategies for treatments and their epidemiological
implications. Polymerase chain-reaction restriction analysis is the preferred
PCR-mediated method for rapidly detecting and identifying mycobacterial species.
It is a simple and cost-effective method that does not involve radioisotopes.
It has been applied to several genes, such as the 16S ribosomal DNA, hsp65
and dnaJ, for rapidly differentiating closely related clinical isolates.
The authors previously reported that comparative sequence analysis of the
RNA polymerase gene (rpoB) DNA encompassing the region related to rifampin
resistance (Rifr) in M. tuberculosis is useful for identifying
mycobacterial species. In this study, the authors demonstrated that the same
region can be used for the differential identification of mycobacteria in
sputa and culture isolates. PCR-restriction analysis (PRA) of amplified rpoB
DNA can differentiate the two mycobacteria groups—rapidly growing and
slowly growing—and can distinguish M. tuberculosis from MOTT.
Most of the human pathogens of the MOTT group can be differentiated using
this technique. In all, the authors undertook the differential identification
of 49 mycobacterial species. They used DNA that had been used previously to
identify mycobacterial species by comparative sequence analysis. On the basis
of rpoB DNA sequences, the authors chose to digest the specimens with
four restriction enzymes—HaeIII, HindII, MvaI, and AccII.
These generated distinctive PCR amplification-restriction analysis patterns
that allowed the clinical isolates of mycobacteria to be distinguished. The
rapidly and slowly growing mycobacteria were distinctly differentiated by
HaeIII digestion of the amplified rpoB DNA. The M. tuberculosis
complex was distinguished from other bacteria by HindII digestion.
Six subspecies of M. kansasii (subspecies I to VI) as well as the closely
related M. gastri, and other closely related species, were distinguished
by simultaneous digestion of MvaI and AccII. In all, 240 strains
of clinical isolates could be identified according to the rpoB PRA
scheme. The authors also showed that it was possible to identify MTB directly
from sputa and BAL specimens. They concluded that PRA of rpoB DNA is
a simple and feasible method for differentiating cultured isolates and for
rapidly detecting and identifying pathogenic mycobacteria in clinical specimens.
Kim B-J, Lee K-H, Park B-N, et al. Differentiation of mycobacterial species
by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase
gene (rpoB). J Clin Microbiol. 2001;39:2102-2109.
Reprints: Yoon-Hoh Kook, Dept. of Microbiology, Seoul National University
College of Medicine, 28 Yongondong, Chongno-gu, Seoul 110-799, Korea; yhkook@plaza.snu.ac.kr
Serum homocysteine and subsequent preeclampsia
Hyperhomocysteinemia is widely regarded as an independent risk factor for
vascular disease. The pathogenetic effects of elevated homocysteine include
endothelial cell dysfunction, increased platelet aggregation, and thrombus
formation. Elevated plasma homocysteine levels in pregnant women with preeclampsia
have been noted previously but whether elevated homocysteine precedes the
development of preeclampsia is questionable. The authors addressed this question
in a study. They selected subjects from a population-based cohort of 1,049
nulliparous women from whom serum had been collected at 16 weeks’ gestation
and banked for Down syndrome screening. Thirty-four of the women became pre-eclamptic,
and serum samples from these women and from 68 women who remained normotensive
were analyzed for homocysteine level using high-pressure liquid chromatography
and fluorescence detection. The mean serum homocysteine in the pre-eclamptic
subjects was 6.99 µmol/L (95 percent confidence interval=6.42-7.55 µmol/L)
and the mean value for the normotensive subjects was 6.91 µmol/L (95 percent
confidence interval=6.45-7.34 µmol/L). These values were not significantly
different from one another, leading the authors to conclude that hyperhomocysteinemia
does not predate preeclampsia.
Hietala R, Turpeinen U, Laatikainen T. Serum homocysteine at 16 weeks and
subsequent preeclampsia. Obstet Gynecol. 2001;97:527-529.
Reprints: Dr. Timo Laatikainen, Dept. of Obstetrics and Gynecology, Helsinki
University Central Hospital, Sofianlehdonkatu 5 A, Helsinki, FIN-00610, Finland;
timo.laatikainen@hus.fi
Ability of cord blood vs. adult blood to produce cytokines
Umbilical cord blood transplantations show a reduced incidence of graft-versus-host
disease compared to allogeneic bone marrow transplantation. GVHD involves
activating donor-derived T cells recognizing alloantigens presented by host-
or donor-derived antigen-presenting cells. The clinical manifestations of
GVHD result in large part from cytokine dysregulation. Several groups have
compared the ability of cord blood and adult blood cells to produce cytokines.
The authors’ previous work showed that after recombinant IFN-γ stimulation,
cord blood monocytes released lower levels of IL-1ß and TNF-α
than did adult blood monocytes. These cytokines play a critical role in the
pathogenesis of GVHD. The authors undertook the current study to determine
intracellular production levels of cytokines to investigate whether the reduced
capacity of cord blood monocytes to secrete IL-1ß and TNF-α could
be related to impaired functional activity and a particular phenotypic profile
of cord blood monocytes. The authors evaluated the detection of intracellular
IL-1ß and TNF-α produced by monocytes in response to tuberculin
purified protein derivative. They did so to investigate whether the reduced
capacity of the cord blood monocytes in secreting cytokines could be related
to impaired functional activity or a particular phenotype, or both. The percentage
of CD64+ monocytes producing intracellular IL-1ß and TNF-α was
significantly lower in cord blood, and the phenotypic profile of cord blood
monocytes producing these cytokines was different from that of adult blood
monocytes. The phenotypic profile of the cord blood monocytes was CD64+CD14+,
whereas the adult blood monocytes were a combination of CD64+CD14+, CD64+CD33+,
and CD64+CD45RO+. The authors suggested that the lower capacity of cord blood
monocyte populations to produce IL-1ß and TNF-α might be due to
a functional immaturity of the cells as reflected by the different phenotypic
profile of CB monocytes.
Brichard B, Varis I, Latinne D, et al. Intracellular cytokine profile of
cord and adult blood monocytes. Bone Marrow Transplant. 2001;27:1081-1086.
Reprints: Dr. B. Brichard, Dept. of Pediatric Hematology, Cliniques Universitaires
Saint Luc & Laboratory of Immunohematology, University of Louvain Medical
School, Av Hippocrate, 10, 1200 Brussels, Belgium
Repeatability of protected specimen brush technique for
diagnosing pneumonia
No diagnostic criteria for nosocomial pneumonia are totally accurate but protected
specimen brush (PSB) and bronchoalveolar lavage (BAL) are the main diagnostic
tools. The authors assessed the repeatability of two pairs of PSB procedures
performed successively in the same lung area. They also analyzed PSB in nonventilated
patients and PSB specimens that were processed at the bedside or in the laboratory.
They collected bacteriological data on 39 cases of suspected nosocomial pneumonia
in nonventilated patients. They divided the four PSBs into two pairs; one
pair of brushes was prepared at the bedside and then sent to the laboratory
and the other pair was immediately sent to the laboratory for complete processing.
If a 103 colony forming units/mL threshold was used, 49 percent
of 156 PSBs were positive. Of the PSB brushes that were immediately sent to
the laboratory, 89.7 percent were concordant, while those done at the bedside
were only 76.9 percent concordant. The repeatability of the measurements,
as expressed by a K-value of the cultures of PSB, therefore was higher for
those sent immediately to the laboratory than for those done at the bedside
(K-values, 0.795 versus 0.537, respectively). Of the 156 PSB specimen isolates,
58.3 percent resulted in isolation of bacterial species. The cultures, in
14 cases, disclosed more than one microorganism and a concentration of more
than 103 cfu/mL. The most frequently isolated organisms were Pseudomonas
species, Enterobacteriaceae, Streptococcus species, and Staphylococcus
species. The polymicrobial cultures were more frequent if the patient had
a tracheostomy. Bacteriological discrepancies leading to a potentially troublesome
choice in antibiotherapy were observed in 31.8 percent of the patients whose
brushes were sent immediately to the laboratory and 56.5 percent of the patients
whose PSB was performed at the bedside. The authors concluded that there is
a low degree of repeatability of the PSB outside of ICUs which seem dependent
on sampling processing. The distribution of pathogens found in cases of suspected
nosocomial pneumonia in nonventilated patients appears to be similar to that
obtained in ventilator-associated pneumonia.
Herer B, Fuhrman C, Demontrond D, et al. Diagnosis of nosocomial pneumonia
in medical ward: repeatability of the protected specimen brush. Eur Respir
J. 2001;18:157-163.
Reprints: B. Herer, Centre Médical de Forcilles, F-77170 Férolles-Attilly,
France
Tools for managing suspected deep venous thrombosis in
outpatients
Clinical assessment can stratify a patient’s probability of having a deep
venous thrombosis, but it is insufficient to establish or exclude the diagnosis
on its own. D-dimer measurements are usually elevated in patients with venous
thromboembolism, so a normal D-dimer result can help exclude this diagnosis.
The authors analyzed results of previous studies and hypothesized that a combination
of a low pretest probability of deep vein thrombosis and a negative D-dimer
test result would exclude the diagnosis. They tested this hypothesis by performing
a prospective cohort study in which additional diagnostic testing and anticoagulant
therapy were withheld in consecutive outpatients presenting with a first episode
of deep venous thrombosis. These outpatients had a low pretest probability
and a negative result on a whole blood D-dimer test. The authors studied 445
outpatients with a suspected first episode of deep venous thrombosis who were
treated in three tertiary care hospitals in Canada. The patients were categorized
as having low, moderate, or high pretest probability of thrombosis and underwent
whole blood D-dimer testing. Patients with low pretest probability and a negative
result on the D-dimer test had no further diagnostic testing and received
no anticoagulant therapy. Additional diagnostic testing was done on the remaining
patients. The followup measurements were the reporting of venous thromboembolic
events during the three-month period following the work-up period. Forty percent
of the patients had a low pretest probability and a negative D-dimer result.
One of these patients had deep venous thrombosis during the followup period.
This resulted in a negative predictive value of 99.4 percent. The authors
concluded that the combination of a low pretest probability of deep venous
thrombosis and a negative result on a whole blood D-dimer test is effective
for ruling out deep venous thrombosis in a large portion of symptomatic outpatients.
Kearon C, Ginsberg JS, Douketis J, et al. Management of suspected deep venous
thrombosis in outpatients by using clinical assessment and D-dimer testing.
Ann Intern Med. 2001;135:108-111.
Reprints: Dr. Clive Kearon, McMaster University Clinic, Rm. 401, Henderson
General Hospital, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada
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