College of American Pathologists
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  Clinical Abstracts





December 2009

Michael Bissell, MD, PhD, MPH

Importance of the volume of a blood culture Importance of the volume of a blood culture

According to the classic literature, the volume of blood per culture is the most important variable in recovering microorganisms from patients with sepsis. The higher the blood volume cultured, the higher the rate of detection of bloodstream infections (BSIs). However, reports regarding the requirements for blood volume with today’s automated and continuous-monitoring blood culture systems are scarce. In a previous study, the authors noted that blood cultures inoculated with lower volumes had a higher yield of detection of BSIs at their institution. The authors conducted the current study to reassess the importance of blood volume in the yield of blood cultures after introducing automated systems with continuous agitation and to correlate the findings with clinical variables. During a six-month period, random samples of blood cultures were weighed to determine the volume of injected blood (weight/density). The study involved 298 patients with significant BSIs and 303 patients with sepsis and negative blood cultures. The mean volume of blood cultured in patients with a BSI (30.03±14.96 mL [mean ± standard devia-tion]) was lower than in patients without a BSI (32.98±15.22 mL; P=.017), and more episodes of bacteremia were detected with less than 20 mL (58.9 percent) than with more than 40 mL (40.2 percent) of blood cultured (P=.022). When patients were stratified according to the severity of their underlying condition, patients with BSI had higher APACHE II (Acute Physiology and Chronic Health Evaluation II) scores, which were related to lower sample volumes (P<.001). A multivariate analysis showed that in the group of patients with APACHE II scores of 18 or greater, higher volumes yielded higher rates of bacteremia (odds ratio, 1.04 per mL of blood; 95 percent confidence interval, 1.001–1.08). The authors concluded that the higher yield of blood cultures inoculated with lower volumes of blood reflects the conditions of the population cultured.

Bouza E, Sousa D, Rodriguez-Creixems M, et al. Is the volume of blood cultured still a significant factor in the diagnosis of bloodstream infections? J Clin Microbiol. 2007;45:2765–2769.

Correspondence: Emilio Bouza at

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Autoantibodies to RNA polymerase-III in scleroderma Autoantibodies to RNA polymerase-III in scleroderma

Autoantibodies to RNA polymerase (RNAP) were first reported in 1982, and in 1987 the specificity of this reaction was refined to show that these antibodies targeted RNAP-I. Since then, it has been shown that antibodies also target RNAP-III and are found in four to 25 percent of systemic sclerosis (SSc) sera. They are associated with the diffuse cutaneous form of SSc (dcSSc) and an increased risk of kidney and cardiac involvement. Although antibodies to RNAP-II and RNAP-I have been described in a variety of diseases, RNAP-I antibodies are rarely observed in conditions other than SSc. Until recently, the detection of anti-RNAP antibodies was performed by time-consuming immunoprecipitation (IP) assays. More recently, ELISA kits that employ a recombinant RNAP-III fragment have become available. They have been employed in studies to evaluate the clinical and serological measures associated with these autoantibodies. Because the assay using this purified antigen is highly specific for SSc, it is likely to find a useful clinical application. Since RNAP-I antibodies nearly always coexist with RNAP-III antibodies, it is thought that an anti-RNAP-III assay would serve as a useful surrogate for both autoantibodies. Although early studies suggested that RNAP autoantibodies might be correlated with a speckled nucleolar pattern of indirect immunofluorescent (IIF) staining, a recent study found that antibodies to RNAP-III were not consistently associated with this IIF pattern on conventional HEp-2 cell substrates. Sera with anti-RNAP-III more consistently had a nuclear speckled IIF pattern, a finding consistent with observations that monoclonal anti-RNAP-III antibodies stain nuclei, whereas antibodies to RNAP-I stain nucleoli. Therefore, while the clinical usefulness of detecting anti-RNAP-III has been demonstrated, it cannot be assumed that IIF on conventional HEp-2 cell substrates serves as a useful screen to detect these antibodies. The authors investigated the prevalence and serological and clinical associations of anti-RNAP-III measured by a new commercial ELISA in an unselected SSc cohort and various control populations. They also determined the sensitivity and specificity of the assay for the diagnosis of SSc. Sera from 242 patients with SSc were collected from 14 Canadian clinics. Control sera were collected from 288 blood donors, 42 patients with infectious disease, 30 with rheumatoid arthritis (RA), and 30 with systemic lupus erythematosus (SLE). Antibodies to RNAP-III were detected by an ELISA kit, and anti-bodies to other cellular antigens were identified by IIF on HEp-2 cell substrate, line immunoassay, immunoprecipitation of recombinant protein, and addressable laser bead immunoassay (ALBIA). The authors reported that anti-RNAP-III antibodies were detected in 47 of 242 (19.4 percent) SSc sera, no RA and SLE sera, one of 287 blood donor sera, and two of 42 infectious disease sera. Diffuse disease (59.5 percent) was more common than limited disease (36.1 percent) in the anti-RNAP-III-positive patients (P=.006). An association was found between the presence of anti-RNAP-III and kidney and joint/tendon involvement, but not with nucleolar IIF pattern, lung involvement, or other clinical indicators. A negative association was found between anti-RNAP-III antibodies and anticentromere by IIF (P=.00004) and anti-Scl-70 by ALBIA (P=.0005) and line immunoassay (P=.003), suggesting a virtually exclusive presence of these antibodies in SSc. The authors concluded that anti-RNAP-III autoantibodies were found in nearly 20 percent of SSc patients but in less than one percent of controls. Therefore, the antibody is a useful marker to help diagnose SSc. The antibody also has prognostic utility since it is associated with scleroderma renal crisis and the diffuse cutaneous form of SSc.

Santiago M, Baron M, Hudson M, et al. Antibodies to RNA polymerase III in systemic sclerosis detected by ELISA. J Rheumatol. 2007;34:1528–1534.

Correspondence: Dr. M. J. Fritzler at

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Chronic lymphocytosis of functionally immature natural killer cells Chronic lymphocytosis of functionally immature natural killer cells

Natural killer cells are innate lymphocytes that can recognize and kill tumors and tumor cell lines without prior sensitization. They play an important role in early antipathogen host defense and are believed to participate in tumor surveillance. The development of NK cells in bone marrow is only beginning to be characterized. The authors previously described a transgenic mouse (referred to as an NK cell-deficient [NKD] mouse) with substantially decreased numbers of peripheral NK cells that were functionally immature. Because IL-15 plays a crucial role in NK cell development, as well as in peripheral NK cell homeostasis and proliferation, the authors investigated whether excess IL-15 could overcome the apparent developmental block in NKD mice and allow the NK cells to reach functional maturity and resume normal homeostasis from the bone marrow to the periphery. For their study, double-transgenic mice were generated by crossing the NKD mice with transgenic mice overex-pressing IL-15. The authors found that the double-transgenic mice had a dramatic accumulation of phenotypically immature NK cells in the bone marrow and subsequently in the blood, liver, and spleen. NK cells from these double-transgenic mice manifested functional deficits similar to those observed in NK cells from NKD mice, as assessed by decreased cytokine production and cytotoxicity. The authors concluded that rather than bypass the observed developmental defect in NKD mice, excess IL-15 drove a massive accumulation of phenotypically and functionally immature NK cells in the bone marrow and periphery. They propose that these double-transgenic mice will serve as a murine model of chronic NK cell lymphocytosis in human patients.

French AR, Kim S, Fehniger TA, et al. Chronic lymphocytosis of functionally immature natural killer cells. J Allergy Clin Immunol. 2007;120(4):924–931.

Correspondence: Dr. Anthony R. French at

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Proteomics of cystic fibrosis Proteomics of cystic fibrosis

Lung disease in cystic fibrosis is characterized by recurrent, and then persistent, bacterial infection accompanied by neutrophil-dominated airway inflammation that begins in the first years of life. Cystic fibrosis (CF) endobronchial infection is accompanied by an excessive inflammatory response, which appears to be directly related to lack of the affected gene product, the CF transmembrane conductance regulator protein (CFTR). Clinical approaches to reducing pulmonary inflammation in CF are limited in scope and by concerns about toxicity. Therefore, a greater understanding of the mechanisms involved in CF lung inflammation is required, including identification of new targets for novel intervention strategies. Sampling and analysis of bronchoalveolar lavage fluid (BALF) has been used widely in studies of pulmonary diseases. The BALF proteome may contain as many as 1,500 different proteins. Many are serum derived, but some are also specific products of airway epithelial cells and, depending on the disease state, originate from resident and infiltrating leukocytes. Identification and analysis of BALF proteins pro-vides information about the activities and functions of these cells during disease. To further define airway inflammation in CF, the authors con-ducted a proteome-based screen of BALF collected from young children with and without CF who were experiencing endobronchial infection. They collected BALF samples from 45 children younger than five years and grouped them according to the presence of respiratory pathogens: 1 × 105 colony-forming units (CFU)/mL BALF or more (18 and 12 samples with and without CF, respectively) and 1 × 105 CFU/mL or less (23 and 15 samples). BALF proteins were analyzed with SELDI-TOF mass spectrometry (MS) and H4 ProteinChips. Proteins were identified and charac-terized using trypsin digestion, tandem MS, Fourier transform ion cyclotron resonance MS, immunoblotting, and ELISA. The authors found that the SELDI-TOF MS BALF profiles contained 53 unique, reliably detected proteins. Peak intensities of 24 proteins differed significantly between the CF and non-CF samples. They included the neutrophil proteins α-defensin 1 and 2, S100A8, S100A9, and S100A12, as well as novel forms of S100A8 and S100A12 with equivalent C-terminal deletions. Peak intensities of these neutrophil proteins and immunoreactive concentrations of selected examples were significantly higher in CF than non-CF samples. The authors concluded that small neutrophil-derived BALF proteins, including novel C-terminal truncated forms of S100A proteins, are easily detected with SELDI-TOF MS. Concentrations of these molecules are abnormally high in early CF lung disease. The data provide new insights into CF lung disease and identify novel proteins strongly associated with CF airway inflammation.

McMorran BJ, Patat SAO, Carlin JB, et al. Novel neutrophil-derived proteins in bronchoalveolar lavage fluid indicate an exaggerated inflammatory response in pediatric cystic fibrosis patients. Clin Chem. 2007;53:1782–1791.

Correspondence: Brendan J. McMorran at

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Prenatal diagnosis of thalassemia in Thailand Prenatal diagnosis of thalassemia in Thailand

Thalassemias are common genetic disorders in Southeast Asia. In Thailand, the frequency of α-thalassemia is 20 to 30 percent. It is three to nine percent for β-thalassemia, and 20 to 30 percent for hemoglobin (Hb) E. Interaction of Hb E with β-thalassemia leads to a serious clinical dis-ease known as Hb E-β-thalassemia, with phenotype ranging from mild anemia to severe β-thalassemia major. Studies have shown that most cases referred for prenatal diagnosis of severe thalassemia in Thailand and other Southeast Asian countries result from this genetic interaction. Routine prenatal diagnosis of the disease relies on analyzing fetal tissues obtained by invasive procedures, including chorionic villus sampling, amniocentesis, and cordocentesis. Another source of fetal specimen can be fetal DNA in maternal plasma, which has been used for noninvasive prenatal diagnosis of several paternally inherited disorders. The authors evaluated this noninvasive approach for prenatal diagnosis of Hb E-β-thalassemia in Thai pregnant women at risk of having severely thalassemic fetuses. To establish a simple noninvasive prenatal diagnosis of common β-thalassemia in Southeast Asia, the authors evaluated the possibility of identifying the three most common β-thalassemia genes—βE, β17A-T, and β41/42 (-CTCC)—by analyzing fetal DNA in maternal plasma using combined conventional polymerase chain reaction (PCR) and real-time quantitative PCR. Maternal plasma was obtained from the peripheral blood of Thai pregnant women during the first and second trimesters of gestation. DNA was prepared from 200 µL plasma using a QIAmp Blood Mini Kit. Identifications of βE, β17A-T, and β41/42 (-CTCC) in plasma DNA were carried out using semi-nested (βE) and nested (β17 and β41/42) real-time allele-specific PCR methodologies. The results were compared with those obtained on fetal tissue analysis using a routine invasive procedure. Twenty-six fetal βE mutations were identified by maternal plasma DNA analysis of 39 pregnant women investigated. The fetal β17 and β41/42 mutations were detected in six of 12 and four of nine maternal plasma specimens, respectively, in concordance with the results obtained by the routine invasive procedure. The noninvasive prenatal diagnostic methods developed may prove useful for detecting paternally inherited mutations and for excluding pregnancies at risk for this common genetic disorder in the region.

Tungwiwat W, Fucharoen G, Fucharoen S, et al. Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E-β-thalassemia. Transl Res. 2007;150(5):319–325.

Correspondence: Dr. Supan Fucharoen at

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S-nitrosohemoglobin deficiency in banked blood S-nitrosohemoglobin deficiency in banked blood

Administration of red blood cells has become the main therapeutic intervention to treat acute and chronic anemia, with an estimated 14 million units of blood transfused annually in the United States. However, there is growing recognition that blood storage has a negative effect on RBC oxygen delivery, and emerging evidence suggests that allogenic RBC infusion may harm some recipients. RBC-derived nitric oxide (NO) bioactivity may play an important role in the respiratory cycle, and impairment of this activity may contribute to the pathophysiology of ischemic conditions. Based on these findings, the authors reasoned that the inability of banked blood to improve oxygen delivery might relate to a deficiency in S-nitrosohemoglobin (SNO-Hb). Specifically, they hypothesized that the impaired vasoregulation associated with administration of banked blood results, at least partly, from losses in RBC-derived NO bioactivity. To test their theory, the authors screened samples of expired blood (more than 42 days of storage). They found the blood to be markedly depleted in SNO-Hb and deficient in hypoxic vasodilatory activity. Motivated by these results, the authors conducted another study to quantitate the effect of storage duration on SNO-Hb content of human packed RBCs prepared and stored under standard American Blood Bank collection conditions and to assess the physiological consequences of a potential decline in SNO-Hb concentration. They also determined whether SNO-Hb levels and NO bioactivity of banked blood can be revived ex vivo. The authors reported that SNO-Hb concentrations declined rapidly after storage of fresh venous blood and that hypoxic vasodilation by banked RBCs correlated strongly with the amounts of SNO-Hb (r2=0.90; P<.0005). Renitrosylation of banked blood during storage increased the SNO-Hb content and restored its vasodilatory activity. In addition, canine coronary blood flow was greater during infusion of renitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs, and this difference in coronary flow was accentuated by hypoxemia (P<.001). The findings indicate that NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia, and they suggest that SNO-Hb repletion may improve transfusion efficacy.

Reynolds JD, Ahearn GS, Angelo M, et al. S-nitrosohemoglobin deficiency: a mechanism for loss of physiological activity in banked blood. Proc Natl Acad Sci USA. 2007;104:17058–17062.

Correspondence: Jonathan S. Stamler at

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Chromosome territory reorganization in a disease with altered DNA methylation Chromosome territory reorganization in a disease with altered DNA methylation

Understanding the orchestration of gene expression requires knowledge of how chromatin is spatially organized in the cell nucleus. The position of a gene with respect to its chromosome territory (CT) and the chromatin condensation of its genomic region, in particular, have been linked to gene activation and repression. Although individual genes can be transcribed from inside CTs, some regions that have constitutively high gene expression or are subject to coordinate gene activation during development or differentiation or in response to physiological stimuli locate at the edge or outside of their CT. This level of nuclear reorganization is often accompanied by a visible level of chromatin decondensation. The precise function, if any, of repositioning toward the outside of CTs remains unclear, but it may allow for genes to access a nuclear environment enriched in the components of the transcription or mRNA-processing machinery, or both, and so enhance the efficiency of transcription. The interplay between this level of higher-order chromatin organization and epigenetic mechanisms that act at primary levels of chromatin structure, such as DNA methylation, has not been explored extensively. DNMT3B mutations in immunodeficiency centromeric instability facial anomalies (ICF) syndrome result in loss of DNA methylation at particular sites, including at CpG islands on the inactive X chromosome (Xi). This allows the specific effects of DNA methylation on CTs to be examined. Using fluorescence in situ hybridization, the authors revealed a dif-ferential organization of the human pseudoautosomal region (PAR)2 between CTs of the X and Y chromosomes in healthy males and the active X (Xa) and Xi in females. There is a more condensed chromatin structure on Xi compared with Xa in this region. PAR2 genes are relocalized toward the outside of the Y and Xi CTs in ICF. The authors showed that on the Xi, this can extend to genes distant from the site of DNA hypo-methylation. This reorganization is not simply a reflection of the transcriptional activation of the relocalized genes. The authors concluded that DNA hypo-methylation at restricted sites in the genome can lead to more extensive changes in nuclear organization distant from the original site of epigenetic change.

Matarazzo MR, Boyle S, D’Esposito M, et al. Chromosome territory reorganization in a human disease with altered DNA methylation. Proc Natl Acad Sci USA. 2007;104(42):16546–16551.

Correspondence: Wendy A. Bickmore at

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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.