College of American Pathologists
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December 2003

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.

Sperm DNA denaturation and fertility
In conventional semen analysis, the semen parameters may help differentiate between infertile and fertile men, but none of these parameters-sperm concentration, motility, morphology-is a powerful discriminator because the overlap between the parameters of fertile and infertile men is significant. A role exists for identifying and applying new markers of male fertility potential that may better discriminate infertile from fertile men. These markers may also provide a better understanding of sperm function and dysfunction and predict outcome with natural or assisted reproduction. This would enable clinicians to select appropriate therapy for couples and could potentially help to better characterize a subgroup of infertile men previously diagnosed as having unexplained infertility. Sperm DNA integrity has been shown to correlate with male fertility potential in vivo and in vitro, and some evidence suggests that sperm DNA integrity may be a better discriminator of male fertility potential than conventional semen parameters. Couples in whom the husband's semen exhibits a high percentage of damaged spermatozoa with denatured DNA (greater than 30 percent) have very low potential for natural fertility, and poor sperm DNA integrity can negatively influence pregnancy rates after in vitro fertilization. The authors demonstrated that infertile men have significantly poorer sperm DNA denaturation, a marker of sperm DNA integrity, than do fertile men, with little overlap in the level of sperm DNA denaturation between infertile and fertile men. The authors examined 88 nonazoospermic, infertile men and 13 fertile men who underwent standard semen analysis and acridine orange sperm DNA integrity studies. Of the 88 infertile men, 13 had completely normal semen parameters, and the remaining 75 had at least one abnormal semen parameter. The mean (± SE) sperm DNA denaturation level was significantly lower in the population of infertile men with normal semen parameters than in those having abnormal parameters (11.1 versus 23.1 percent, respectively; P<0.001). Only one (eight percent) of the 13 men with normal semen parameters had elevated sperm DNA denaturation (greater than 30 percent verified on two separate analyses) compared with 13 (17 percent) of the 75 infertile men with abnormal semen parameters (P>0.05). None of the fertile controls had elevated sperm DNA denaturation. The authors concluded that sperm DNA denaturation correlates negatively with standard semen parameters and that an isolated abnormality of sperm DNA denaturation is uncommon in infertile men.

Zini A, Fischer MA, Sharir S, et al. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology. 2002; 60: 1069-1072.

Reprints: Dr. Armand Zini, Division of Urology, Dept. of Surgery, Mount Sinai Hospital, 600 University Ave., Ste. 1525, Toronto, Ontario M5G 1X5, Canada

Free thyroxine levels during early development
Very preterm infants lack the maternal thyroid hormone contribution that would normally be present at a developmental stage when maturation of the hypothalamo-pituitary-thyroid system and thyroid hormone metabolism hasn't been completed. This results in a period of transient low thyroxine during which bound and free plasma concentrations of T4 and T3 are low. This period lasts longer and is more severe in infants of lower gestational age. Three retrospective studies in large cohorts of preterm infants have studied the relationship between a single total T4 measurement in the first week of life or the lowest total T3 measurement during a 10-week period and developmental outcome at 18 months to nine years of age. The studies uniformly showed an increased risk of impaired developmental outcome in infants with the lowest thyroid hormone values. Free thyroxine (FT4) levels, not total or bound levels, are important for clinical effects. Part of the low total T4 and T3 levels that are found in preterm infants are caused by low concentrations of thyroid-binding globulin. Thus it is important to demonstrate associations between low FT4 levels and later development. The authors conducted a randomized trial of T4 supplementation in 200 infants who were younger than 30 weeks' gestation. T4 treatment was associated with better five-year outcome in infants younger than 29 weeks' gestation but with worse outcome in infants who were at 29 weeks' gestation, which could be related to low, respectively high free thyroxine levels. In the placebo group, low FT4 was associated with worse outcome on all domains in both time points. After correcting for confounding variables, mental and neurologic outcome remained significantly different at two years, as did motor outcome at five years. In the T4 group, high FT4 was not associated with worse outcome at two and five years. The authors concluded that in untreated infants, low FT4 values during the first four weeks of life in infants born at younger than 30 weeks' gestation are associated with worse neurodevelopmental outcome at two and five years. In the T4-treated infants, high FT4 was not associated with worse outcome. Factors other than high FT4 concentration presumably play a role in the worse outcome of the T4-treated group of 29 weeks' gestational age.

Van Wassenaer AG, Briët JM, van Baar A, et al. Free thyroxine levels during the first weeks of life and neurodevelopmental outcome until the age of 5 years in very preterm infants. Pediatr. 2002;109: 534-539.

Reprints: Dr. Aleid G. van Wassenaer, Emma Children's Hospital AMC, Dept. of Neonatology, H3N, Box 22700, 1100 DE Amsterdam, Netherlands;

Quantitating Pneumocystis carinii by real-time PCR
Pneumocystis cariniicannot be readily grown in vitro, so the usual method for quantifying P.carinii is to enumerate organisms by microscopic examination. This method, however, is subject to uncertainty because the organism grows in clusters and because different morphological forms are present. Different stains also vary in their sensitivities of detection. Definitive diagnosis of P.carinii pneumonia often relies on identifying cysts, which frequently occur within clusters of trophozoites, but this is not an optimal method since the cyst-to-trophozoite ratio may change under different conditions and stages of PCP. The authors developed a rapid, quantitative touchdown PCR assay for detecting P.carinii f. sp. carinii, the subspecies of P.carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect fewer than five copies of a plasmid standard per tube. The assay was reproducibly quantitative (r=0.99) over six log values for standards containing five or more copies per tube. Applying it to a series of 10-fold dilutions of P.carinii organisms isolated from rat lung demonstrated reproducible quantitation over five log values (r=0.99). The authors concluded that the assay is rapid, sensitive, reproducible, quantitative, and applicable to in vivo and in vitro systems. The assay should prove useful for determining organism burden or growth assessment, such as in in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

Larsen HH, Kovacs JA, Stock F, et al. Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii. J Clin Microbiol. 2002;40:2989-2993.

Reprints: Hans Henrik Larsen, Copenhagen HIV Programme, Dept. of Infectious Diseases 144, Hvidovre University Hospital, Kettegaard Aile 3D, 2650 Hvidovre, Denmark;

Leukocyte calprotectin in rheumatoid arthritis
Calprotectin is a leukocyte-derived intracellular regulatory and extracellular immunomodulating protein that increases in plasma secondary to various tissue injuries and inflammation. The protein has been studied in rheumatic diseases and numerous other clinical conditions. Fecal calprotectin is increased in patients with inflammatory bowel disease and in those with colorectal cancer. Plasma calprotectin is increased in cystic fibrosis and other conditions with reduced pulmonary gas diffusion. Furthermore, the protein has been shown to promote neurite outgrowth. Studies of patients with rheumatoid arthritis have described increased concentrations in circulating leukocytes, synovial neutrophil leukocytes, and synovial fluid. The plasma level of the protein correlates with C-reactive protein and other disease variables that have been associated with an unfavorable outcome in rheumatoid arthritis and other autoimmune conditions. Calprotectin, however, also inhibits immunoglobulin production of B-cells in vitro. In a rat model, calprotectin was found to protect against the development of arthritis. Calprotectin may also inhibit metalloproteinases, which suggests that it has a protective role in arthritis or that levels rise in response to tissue injury. The authors undertook a study to determine if calprotectin is predictive for outcome in patients with rheumatoid arthritis. They prospectively followed 56 rheumatoid arthritis inpatients for five years. Clinical and laboratory data were collected to assess disease activity. Health assessment questionnaire and radiographic scores of hands and wrists, as described by Larsen, were used as outcome measures. Plasma calprotectin levels were determined with the enzyme-linked immunosorbent assay technique. Significant correlations were found cross-sectionally at followup between calprotectin concentration and other known parameters of disease activity and severity: C-reactive protein (r=0.67), investigator's global assessment of disease activity (r=0.57), Waaler titer (r=0.50), HAQ score (r=0.48), and number of swollen joints (r=0.48). Calprotectin at baseline was not identified as an independent predictor for HAQ or radiographic progression in the multivariate analysis. The authors concluded that calprotectin is a good measure of disease activity and joint inflammation in rheumatoid arthritis, but the level of calprotectin at baseline is not predictive for radiographic damage or functional impairment five years later.

Madland TM, Hordvik M, Haga HJ, et al. Leukocyte protein calprotectin and outcome in rheumatoid arthritis. Scand J Rheumatol. 2002; 31: 351-354.

Reprints: Tor Magne Madland, Dept. of Rheumatology, Haukeland University Hospital, NO-5021 Bergen, Norway;

Genetics of Pseudomonas aeruginosa
Pseudomonas aeruginosa is generally believed to be acquired environmentally, and person-to-person spread is thought to occur only rarely. However, recent reports of the transmission of virulent multidrug-resistant mucoid strains of P.aeruginosa have raised concerns about the effectiveness of standard infection control practices for this organism. P.aeruginosa in cystic fibrosis patients is associated with a unique in vivo microbial adaptation process that involves transformation of the nonmucoid phenotype to the mucoid phenotype. Mucoidy inhibits phagocytosis and penetration by antibiotics. The authors investigated 50 P.aeruginosa isolates from the sputa of 18 patients treated at the CF Centre, Royal Prince Alfred Hospital, Sydney, Australia, between 2000 and 2001. The isolates were examined by pulsed-field gel electrophoresis to determine inter- and intrapatient variabilities, and direct sequence analysis of PCR products derived from the mucA gene was carried out to identify changes potentially leading to mucoidy. Associations between phenotype, genotype, and mucA mutations were sought. The isolates were analyzed by restriction fragment length polymorphism analysis by pulsed-field gel electrophoresis. Genetic similarity ranged from 76 to 100 percent on phylogenetic analysis of the macrorestriction patterns. Fifteen isolates from seven patients were infected with clonal or subclonal strains. Sequence analysis of PCR products derived from the mucA gene showed 20 mutations, with the number of mutations in individual isolates ranging from one to four. Potentially functional changes were found in 22 (44 percent) isolates. Eight changes led to premature stop codons, providing support for the presence of mucA mutations as one pathway to mucoidy. The findings highlight the need for further investigation of the transmissibility of P.aeruginosa in cystic fibrosis patients.

Anthony M, Rose B, Pegler MB, et al. Analysis of Pseudomonas aeruginosa isolates from the sputa of Australian adult cystic fibrosis patients. J Clin Microbiol. 2002; 40: 2772-2778.

Reprints: Colin Harbour, Dept. of Infectious Diseases, Blackburn Bldg., D06, University of Sydney, Sydney, NSW, 2006, Australia;

Fluoroimmuno assay for determining rheumatoid factor
Rheumatoid factor as a marker for rheumatoid arthritis shows significant correlations with C-reactive protein and swollen joint counts but not with the erythrocyte sedimentation rate. A clear association has been shown between rheumatoid factor at baseline and later development of erosions, suggesting that RF predicts radiological outcome. In these studies, RFs were measured by qualitative or semiquantitative methods, such as Waaler-Rose and latex fixation tests, or by more quantitative immunoassays. However, these immunoassays, such as enzyme-linked immunosorbent assay and nephelometry, still apply several dilutions. Time-resolved fluoroimmunoassay has recently become available as a sensitive method for determining IgA and IgM RF. This method measures RF as a continuous variable with a wide range of measurement in a single dilution. The authors conducted a study in which they examined three questions: Can RFs be considered parameters for disease activity in rheumatoid arthritis? What is the relation between RF and radiological damage due to rheumatoid arthritis in groups of patients? Is there an individually determined relation between RF and radiological damage in single patients? The RF levels in 155 patients were strongly associated with disease activity parameters, especially erythrocyte sedimentation rate and C-reactive protein, and with each other. All disease activity parameters, at baseline as well as time-integrated parameters, were associated with radiographic damage. Moreover, in individual patients, a linear relationship between time-integrated disease activity parameters and progression of radiological damage was seen. The authors concluded that RFs measured as continuous variables can be considered disease activity parameters in patients with rheumatoid arthritis. And in patients, there is a constant relation between disease activity and radiological damage.

Knijff-Dutmer E, Drossaers-Bakker W, Verhoeven A, et al. Rheumatoid factor measured by fluoroimmunoassay: a responsive measure of rheumatoid arthritis disease activity that is associated with joint damage. Ann Rheum Dis. 2002;61:603-607.

Reprints: Dr. E. Knijff-Dutmer, Rheumatology Twente, Dept. for Rheumatology, Medisch Spectrum Twente, P.O. Box 50.000, 7500 KA Enschede, Netherlands;