S-adenosylhomocysteine vs. homocysteine as a cardiac marker
Elevated plasma total homocysteine (tHcy) has been identified as an independent risk factor for vascular disease. The underlying relationship between increased homocysteine levels and vascular disease, however, is somewhat controversial. Some have suggested that elevated plasma homocysteine levels are an effect rather than a cause of atherosclerotic disease. Others have argued that hyperhomocysteinemia is a proximate risk factor that provokes the acute event. In any case, the use of plasma tHcy levels as an indicator of vascular disease risk is widespread. There is considerable overlap in values for patients with vascular disease and control subjects such that marked elevations are necessary to revise an individual's risk of developing vascular disease. S-adenosylhomocysteine is a metabolic precursor of homocysteine produced in the body. It is formed by methyltransferase reactions involving S-adenosylmethionine (SAM) as the methyl donor. Some have suggested that the effect of homocysteine on vascular endothelial function may be due to the production of S-adenosylhomocysteine from homocysteine resulting from the action of the enzyme adenosylhomocysteinase, or SAH hydrolase. The authors became interested in measuring plasma SAH levels to determine whether it might be more informative than plasma tHcy for determining the risk of vascular disease. They measured plasma tHcy, SAH, SAM, creatinine, folate, and vitamin B₁₂ in 30 patients with proven cardiovascular disease and in 29 age- and sex-matched control subjects. Values for folate and vitamin B₁₂ did not differ significantly between the groups. The creatinine values were 110 µmol/L on average for patients and 97 µmol/L on average for controls. The homocysteine values were 12.8 µmol/L on average for patients and 11.0 µmol/L on average for controls. The SAH values were 40 nmol/L on average for patients and 27 nmol/L on average for controls. The P value for this difference was highly significant at 0.0021. The authors concluded that plasma SAH appears to be more sensitive than homocysteine as an indicator of the difference between patients with cardiovascular disease and control subjects. Total homocysteine and SAH levels were significantly correlated with plasma creatinine in patients.

Kerins DM, et al. Plasma S-adenosylhomocysteine is a more sensitive indicator of cardiovascular disease than plasma homocysteine. Am J Clin Nutr. 2001;74:723-729.

Reprints: C. Wagner, Dept. of Biochemistry and Medicine, Vanderbilt University School of Medicine, 620 Light Hall, Nashville, TN 37232-0146; conrad.wagner@vanderbilt.edu

Measuring LDL-cholesterol: calculation vs. homogeneous assay
In the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) guidelines, diagnostic and treatment efforts in cardiac risk assessment are focused on total cholesterol and low-density lipoprotein cholesterol. Therapy is intended to lower LDL-C values below a target value. These target values depend on a number of independent risk factors. Accurate and standardized measurements of LDL-C are therefore necessary to reliably classify patients. LDL-C is most commonly determined using the Friedewald formula, which estimates LDL-C from measurements of total cholesterol, triglycerides, and high-density lipoprotein cholesterol. This calculation, however, has limitations. Direct methods for measuring LDL-C also have limitations for general use. Recently, however, a new generation of homogeneous assay methods capable of full automation has been introduced. It uses specific reagents of various types to selectively expose and directly measure the cholesterol associated with LDL. The authors critically reviewed the literature on the new homogeneous assays. They concluded that the new assays seem to conform to NCEP requirements for LDL-C testing with regard to precision (coefficients of variation of less than four percent) and accuracy (bias of less than four percent) in specimens collected from nonfasting individuals. All five of the available homogeneous assays have been certified by the Cholesterol Reference Methods Laboratory Network. These assays appear to better classify individuals into NCEP cutpoints than the Friedewald calculation. Limited evaluations to date, however, raise questions about the reliability and specificity of the assays. Yet evidence supports using the homogeneous assays for LDL-C as a supplement to the Friedewald calculation in those cases where the calculation is known to be unreliable—for example, with triglycerides of more than 4,000 mg/L.

Nauck M, Warnick GR, Rifai N. Methods for measurement of LDL-cholesterol: a critical assessment of direct measurement by homogeneous assays versus calculation. Clin Chem. 2002;48:236-254.

Reprints: Matthias Nauck, University Hospital Freiburg, Dept. of Clinical Chemistry, Hugstetter Strasse 55, D-79106 Freiburg i.Br., Germany; manauck@med1.uk1.uni-freiburg.de

Hospital deaths potentially associated with nosocomial infection
Various studies of nosocomial infection have placed the incidence risk at between four and nine cases per 100 discharges. Nosocomial infection is a leading cause of death in the United States and Europe. It has been estimated that as many as one percent of all patients with a nosocomial infection die as a direct result of the infection and that nosocomial infections contribute to the death of 2.7 percent of patients admitted to hospitals. Few, if any, studies have used population attributable risk as a measure of the effect of nosocomial infections on the rate of intrahospital death. The authors conducted a study to determine the fraction of hospital deaths associated with nosocomial infection. They examined the association for all nosocomial infections and for different types of nosocomial infections. The authors used a matched case-control study design in an 800-bed tertiary care teaching hospital. All patients older than 14 years who were admitted to the hospital between Jan. 1, 1990 and Jan. 1, 1991 were eligible. The authors recorded the 524 consecutive deaths that occurred in the hospital, which constituted the case group. For each case, a control patient was matched on primary admitting diagnosis and admission date. The population attributable risk (PAR) was adjusted for age, gender, service, severity of illness, length of stay, and quality of the medical record. The proportion of hospital deaths that potentially could be attributed to nosocomial infection was estimated from this PAR. The PAR for all nosocomial infections was estimated to be 21.3 percent. The greatest proportion of deaths that were potentially associated with nosocomial infections was observed in patients with only a single infection and bacteremia or sepsis. The PAR for a single infection was 15 percent and for bacteremia or sepsis was 7.7 percent. The authors concluded that nosocomial infections are associated with a large proportion of intrahospital deaths.

Garcia-Martin M, Lardelli-Claret P, Jimenez-Moleon J, et al. Proportion of hospital deaths potentially attributable to nosocomial infection. Infect Control Hosp Epidemiol. 2001;22:708-714.

Reprints: Miguel Garcia-Martin, Departmento de Medicina Preventiva y Salud Publica, Facultad de Medicina, Universidad de Granada, 18071 Granada, Spain

Blood cell count changes in blood stored at room temperature
Blood specimens submitted for a complete blood cell count and differential leukocyte count may be delayed in transit. When such a delayed specimen arrives in the lab, staff must decide whether to accept it. To make such a decision, laboratorians need to be familiar with the changes known to occur in blood specimens during storage. Information regarding the suitability of specimens older than one day for various lab tests, including the CBC and automated differential, is fairly scarce in the recent literature. The authors attempted to delineate changes that occur in various parameters of the automated CBC and differential during storage of blood at room temperature for several days. They performed a CBC and automated white-cell differential count using a Coulter Gen.S on 40 K₃ EDTA-anticoagulated blood specimens once a day for three to seven days. The specimens were stored at room temperature during the study and results were tabulated in an Excel spreadsheet. The hemoglobin, red blood cell count, and mean corpuscular hemoglobin were stable for the duration of the study, which was seven days. The white blood cell count was stable for at least three days and up to seven days if the count was within or above the normal range. Platelet count was stable for four days and up to seven days if the count was within or above the normal range. The mean corpuscular volume, mean platelet volume, hematocrit, and RBC distribution width increased, and the mean corpuscular hemoglobin concentration decreased from day two onward. Among the differential parameters, the relative percentages and absolute numbers of neutrophils, lymphocytes, and eosinophils tended to increase, whereas those of monocytes trended downward over time. There was no appreciable change in basophils, although data were limited.

Gulati GL, Hyland LJ, Kocher W, et al. Changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature. Arch Pathol Lab Med. 2002;146:336-342.

Reprints: Dr. Gene L. Gulati, 307 Pavilion Bldg., Thomas Jefferson University Hospital, 125 S. 11th St., Philadelphia, PA 19107; gene.gulati@mail.tju.edu

HIV drug susceptibility testing
Human HIV-1 infections treated with antiretroviral drugs that are selected on the basis of drug-resistance testing have been associated with improved virologic responses. A number of phenotypic and genotypic assays are being used to monitor drug resistance in HIV. The genotypic assays detect resistance-related mutations and provide indirect evidence of drug resistance, whereas the phenotypic assays measure the ability of the virus to replicate in the presence of a drug and are therefore a direct measurement. The IC₅₀, or inhibitory dose for 50 percent of virus replication for a drug-susceptible reference virus, is the baseline used to measure reductions in drug susceptibility. The conventional phenotypic assays require isolating HIV-1 from blood mononuclear cells or plasma. This adds labor time and cost. Newer phenotypic assays circumvent this requirement for viral isolation. The authors evaluated the correlation between the results of drug-resistance testing assays for two commercial phenotypic assays—the Antivirogram and PhenoSense HIV assays. They analyzed HIV-1 isolates from 50 plasma specimens for phenotypic susceptibility to reverse transcriptase (RT) inhibitors and protease (PR) inhibitors using the Antivirogram and PhenoSense assays. Twenty of the specimens came from drug-naïve persons who had seroconverted and 30 were from patients who were sources of occupational exposures to HIV-1. Sixteen of the specimens in this latter group were from drug-experienced patients. The results of the phenotypic assays were characterized as sensitive or reduced susceptibility on the basis of cutoff values established by the manufacturers. Data for 12 to 15 drugs were available by both assays for 38 specimens and represented 529 pairs of results. The two data sets have a 91.5 percent concordance by phenotypic category. The 45 discordant results were distributed randomly among 26 specimens and included 28 results which were within a two-fold difference of the assay cutoff values. None of the discordant results were associated with primary resistance mutations that predicted high-level resistance—greater than 20 fold. The discordant results were distributed equally among specimens from the drug-experienced and drug-naïve subjects. The results were slightly higher for protease inhibitors than for nonnucleoside or nucleoside reverse transcriptase inhibitors. The authors concluded that the two assays correlate well, despite the use of different testing strategies.

Qari SH, Respess R, Weinstock H, et al. Comparative analysis of two commercial phenotypic assays for drug susceptibility testing of human immunodeficiency virus Type 1. J Clin Microbiol. 2002;40:31-35.

Reprints: HIV and Retrovirology Branch, CDC, MS G-19, 1600 Clifton Rd., Atlanta, GA 30333; wmh2@cdc.gov

A DNA-based assay for detecting prostate cancer in urine sediment
Promotor hypermethylation of the glutathione-S-transferase P1 (GSTP1) is an epigenetic DNA alteration that has been identified as the most common DNA alteration in prostate cancer. This alteration is absent in nonmalignant tissue. GSTP1 inactivation is being increasingly implicated as an early event in prostatic carcinogenesis. Hypermethylation of GSTP1 promoter can be detected by methylation-specific polymerase chain reaction. This method is capable of identifying hypermethylated alleles from tumor DNA within 10⁴- to 10⁵-fold excess amounts of unmethylated alleles from normal DNA. This situation may be encountered in analyzing urine samples for shedded prostate cancer cells in larger amounts of urothelial, prostatic, and other benign cells. The authors investigated the diagnostic potential of methylation-specific PCR as a DNA-based approach for molecular detection of prostate cancer in urine sediment. They used bisulfite treatment followed by methylation-specific PCR to detect GSTP1 promotor hypermethylation in DNA isolated from urine sediments obtained after prostatic massage of men with and without prostate cancer. GSTP1 promoter hypermethylation was demonstrated in the sediments of one of 45 patients diagnosed with benign prostatic hyperplasia, two of seven patients with prostatic intraepithelial neoplasia, 15 of 22 patients with early intracapsular cancer, and 14 of 18 patients with locally advanced or systemic prostate cancer. This resulted in a calculated specificity of 98 percent and a calculated overall sensitivity of 73 percent for detecting prostate cancer. The authors concluded that urinalysis for GSTP1 promoter hypermethylation may be a sensitive and specific marker for molecular detection of prostate cancer, including early stage cancer.

Goessl C, Müller M, Heicappell R, et al. DNA-based detection of prostate cancer in urine after prostatic massage. Urology. 2001;58:335-338.

Reprints: Dr. Carsten Goessl, Dept. of Urology, Benjamin Franklin Medical School, Free University of Berlin, Hindenburgdamm 30, Berlin, 1220, Germany

Use of pneumatic tube systems with blood gas specimens
Pneumatic tube systems are used widely in hospitals to rapidly deliver specimens to the clinic lab. The use of such systems is predicated on the notion that there are no significant effects on analytes. In response to a recent report of perturbations in pO₂ values subsequent to air contamination, the authors decided to study this issue in their hospital. They performed a three-stage study. In the first stage, consecutive samples were drawn in duplicate from patients during a two-week period. One of the samples was analyzed immediately in the intensive care unit using point-of-care methodology. The second sample was capped and sealed in a gas-tight plastic envelope and hand-delivered to the lab for analysis. In the second phase of the study, consecutive samples were drawn in duplicate from patients during a two-week period. One of these samples was analyzed immediately in the ICU and the second was capped and sealed in a gas-tight plastic envelope and sent to the lab via the pneumatic tube system. In the third phase, consecutive samples were drawn in triplicate from patients during a two-week period. One of the samples was analyzed on the floor and the remaining two samples were capped and sealed in separate gas-tight plastic envelopes. One of the latter was sent to the lab via the pneumatic tube system using the conventional canister and the second was sealed inside a pressure-tight container and sent to the lab via the pneumatic tube system inside the conventional canister. The authors found that the specimens that were sent via the pneumatic tube system tended to have significant alterations in pO₂ values, which was not seen with the other modes of transport. There was no effect on pCO₂ and pH values. Use of the pressure-sealed container abolished the negative effect on pO₂values that was otherwise observed. The authors recommended that samples for blood gas analyses be transported in a pneumatic tube system using a pressure-sealed container to avoid artefacts in the pO₂.

Collinson PO, et al. Changes in blood gas samples produced by a pneumatic tube system. J Clin Pathol. 2002;55:105-107.

Reprints: Dr. P.O. Collinson, Dept. of Clinical Biochemistry, 2nd floor - Jenner Wing, St. George's Hospital, Blackshaw Rd., London, SW17 OQT, United Kingdom; poctrop@poctrop.demon.co.uk

Maternal AFP and fetal karyotyping
More than 25,000 amniocenteses per year are estimated to be performed in the United States for the indication of an elevated maternal serum a-fetoprotein (AFP). It is common practice to send the amniotic fluid specimen obtained at the time of the procedure for not only AFP amniotic fluid levels but also for fetal karyotyping. Some studies have suggested, however, that obtaining a fetal karyotype routinely may not be necessary because of the relatively low incidence of chromosomal abnormalities in this subset of patients. The authors studied whether it is necessary to obtain a fetal karyotype on all patients undergoing amniocentesis for an elevated maternal serum AFP. They collected data retrospectively on patients under age 35 who were undergoing amniocentesis for elevated serum AFP. This included 537 patients with maternal serum AFP values greater than 2.5 multiples of the median. Of 509 patients in the group with normal amniotic fluid AFP levels, 505 had a normal karyotype (specificity, 94.9 percent; negative predictive value, 99.2 percent). Of the 28 patients in the group with an elevated amniotic fluid AFP, one had an abnormal karyotype (sensitivity, 20 percent; positive predictive value, 3.6 percent). The authors concluded that fetal chromosomal analysis of amniotic fluid amniocytes need not be performed routinely for patients who have a normal level of amniotic fluid AFP. The fetal karyotype is, however, recommended for those patients with an elevated amniotic fluid AFP.

Gonzalez D, Barrett T, Apuzzio J. Is routine fetal karyotyping necessary for patients undergoing amniocentesis for elevated maternal serum a-fetoprotein? J Matern Fetal Med. 2001;10:376-379.

Reprints: J. Apuzzio, Dept. of Obstetrics, Gynecology & Women’s Health, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714; apuzzijj@umdnj.edu