College of American Pathologists
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  Clinical Abstracts





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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;

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;

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;

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;

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