College of American Pathologists
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  Clinical Abstracts





October 2008

Michael Bissell, MD, PhD, MPH

Serial cardiac biomarkers in chronic heart failure
Effect of hemoglobin on NT-proBNP levels
Platelet counts on automated hematology analyzers
Molecular RHD typing of blood donors
Overall performance of MRSA PCR assay in high-volume clinical use
Association of anti-CCP levels with rheumatoid arthritis haplotypes
Evaluation of a point-of-care D-dimer assay for excluding deep-vein thrombosis

Serial cardiac biomarkers in chronic heart failure Serial cardiac biomarkers in chronic heart failure

Despite advances in medical and device therapies, patients with chronic heart failure have an adverse prognosis over time. Progression often occurs even in the absence of overt clinical events. Because heart failure activates multiple neurohormones, such as the renin-angio­ten­sin-aldosterone, endothelin, sympathetic, and natriuretic peptide systems, there has been enthusiasm for the use of related biomarkers to define prognosis. B-type natriuretic peptides (BNP and N-terminal pro­BNP [NT-pro­BNP]) and, to a lesser extent, cardiac troponin T (cTnT) have been used for this purpose. Elevations of these biomarkers identify patients at risk. Most studies, however, have examined acutely decompensated hospitalized patients rather than stable ambulatory heart failure patients and have evaluated measurements only at a single point in time. Therefore, how much additional information would be available to potentially guide management if values were monitored more frequently and for a longer period of time has not been well defined. In addition, how this approach applies to more stable patients is unclear. Accordingly, the authors evaluated serial measurements of cardiac cTnT and BNP every three months during a two-year period in clinically stable hospital outpatients with New York Heart Association (NYHA) class III–IV heart failure to determine the optimal way to use these biomarkers to follow ambulatory patients. They hypothesized that serial measurements of these biomarkers would provide added information to predict events such as death, cardiac transplantation, and hospitalization. A co­hort of 190 New York Heart Association class III–IV heart failure patients was prospectively enrolled in the study from June 2001 to January 2004. Primary endpoints were death, cardiac transplantation, or hospitalization. At study enrollment, cTnT was <0.01 ng/mL in 87 (45.8 percent) patients, 0.01 to 0.03 ng/mL in 50 (26.3 percent) patients, and >0.03 ng/mL in 53 (27.9 percent) patients. An increase in cTnT above normal (<0.01 ng/mL) carried a 3.4-fold increased risk (P=.019) of adverse events. Further increases (approximately 20 percent) from an elevated level worsened overall risk (hazard ratio, 5.09; P<.001). BNP was elevated (>95th percentile for a normal population for age and gender) in 122 (64.2 percent) patients. An elevation of BNP from normal at any time during the study was associated with a poor outcome, but, once elevated, additional increases or decreases in BNP remained associated with the same level of risk (hazard ratio, 5.09; P<.001). Combined elevations of cTnT (>0.03 ng/mL) and BNP defined the highest risk group (hazard ratio, 8.58; P<.001). The authors concluded that elevations of cTnT or BNP from normal detected at any time during clinical followup in ambulatory patients with chronic heart failure are highly associated with an increased risk of adverse events. Further increases in cTnT contribute to additional risk. Combined elevations of cTnT and BNP generate the highest level of risk. The ability to monitor changes by serial measurements adds substantially to the assessment of risk in this patient population.

Miller WL, Hartman KA, Burritt MF, et al. Serial biomarker measurements in ambulatory patients with chronic heart failure. The importance of change over time. Circulation. 2007;116:249–257.

Correspondence: Dr. Wayne L. Miller, at
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Effect of hemoglobin on NT-proBNP levels Effect of hemoglobin on NT-proBNP levels

Diagnostic measurement of plasma B-type natriuretic peptide and its molecular precursor, proBNP, is increasing in hospitals and primary care centers. Both peptides are released from the heart in response to myocyte strain and neurohormonal activity. Age, gender, and renal function also affect the plasma concentrations of B-type natriuretic peptide and proBNP. Recent studies suggest that anemia is associated with increased proBNP concentrations in patients with heart failure, coronary artery disease, and stroke. These studies examined the impact of hemoglobin concentration in patients referred to an emergency department or for further cardiac examination. The findings suggested that anemia could be associated with BNP or proBNP concentrations, or both, in the general population. The high prevalence of anemia will overlap the diagnostic use of BNP and proBNP measurement, perhaps most frequently for elderly patients, in whom diffuse symptoms, such as discomfort, shortness of breath, and fatigue, can be signs of anemia and cardiac disease. The authors examined the relationship between hemoglobin and proBNP concentrations in the general population (n=5,892). They also assessed the association between proBNP concentrations and hemoglobin status in selected individuals (n=2,855) with healthy left ventricular systolic function and without a history of heart disease. In the fourth examination in the Copenhagen City Heart Study, the authors performed a nested case-control study of 6,238 individuals from a Danish general population. Of these, 3,497 randomly selected participants also underwent an echocardiographic examination. The population was stratified into groups depending on health and hemoglobin status. Correlations between hemoglobin and proBNP concentrations were examined by simple and multiple regression analyses, adjusted for variables known to influence the proBNP plasma concentration. The authors found that the mean pro­BNP concentration was increased 1.7-fold in the group with anemia versus the nonanemic group (mean [SD], 42 [45] pmol/L versus 25 [29] pmol/L; P<.0001; n=5,892). Multiple regression analysis confirmed an independent effect of hemoglobin on proBNP concentrations. In the selected subgroup without signs or symptoms of heart disease (n=2,855), lower hemoglobin concentrations, defined as P<120 g/L in women and P<130 g/L in men, were associated with increased circulating proBNP concentrations, but their contribution to the overall variation in proBNP concentrations was modest. The authors concluded that because moderate anemia is associated with a 1.7-fold increase in proBNP concentrations, hemoglobin concentrations should be considered in patients with nonspecific symptoms of heart disease and increased proBNP concentrations.

Nybo M, Benn M, Mogelvang R, et al. Impact of hemoglobin on plasma pro-B-type natriuretic peptide concentrations in the general population. Clin Chem. 2007; 53: 1921– 1927.

Correspondence: Jens Peter Goetze at
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Platelet counts on automated hematology analyzers Platelet counts on automated hematology analyzers

Hematology analyzers are designed to count cells in whole blood from patients. But in blood banking, hematology analyzers are also used to count cells in blood products derived from centrifuged whole blood. In most European blood banks, whole blood generally is separated into a red blood cell (RBC) unit, unit of plasma, and buffy coat. The buffy coat contains approximately 70 percent of the white blood cells (WBCs) and approximately 90 percent of the platelets. Buffy coats can be further processed into platelet concentrates (PCs). A final PC contains approximately 1,000 ¥ 109 plate­lets/L, compared to a mean of 300 ¥ 109 platelets/L in normal blood, and a very low number of RBCs (<5 ¥ 109/PC) and WBCs (<1 ¥ 106/PC). Platelet counting in PCs should be validated separately from platelet counting in whole blood because most analyzers are designed to count platelets in the presence of RBCs. Hematology analyzers count cells based on impedance, optical technology, immunologic technology, or a combination of these. Impedance is based on monitoring the voltage required to send a constant current through an aperture. The immunologic method is based on flow cytometry, in which platelets are labeled with a platelet-specific monoclonal antibody, such as CD41a (against platelet membrane glycoprotein IIb/IIIa), CD42b (against platelet membrane glycoprotein Ib-a), or CD61 (against platelet membrane glycoprotein IIIa). The authors conducted a study to test hematology analyzers that use different principles for platelet counting. They tested the analyzers for accuracy, precision, linearity, and carryover. They also studied the effect of EDTA on platelet count and dilution of PC samples using various analyzers. The Cell-Dyn 4000 CD61 technique was used as a reference in the study. Six hematology analyzers (AcT 8, Beckman Coulter; Advia 2120, Bayer; Cell-Dyn 4000, Abbott; Onyx, Beckman Coulter; K4500, Sysmex; and XT 2000i, Sysmex) were tested for platelet counting. PC samples with various platelet concentrations were made (0–1,700 ¥ 109/L) and measured 10 times. Carryover was determined five times. The authors found that PC samples (1,000 ¥ 109 platelets/L) in EDTA tubes showed significantly higher platelet counts than samples in “dry” tubes for all analyzers, except the Cell-Dyn 4000 with the impedance technique. Carryover was no more than 0.3 percent for all analyzers. The K4500 provided the most accurate results, whereas the Cell-Dyn 4000 with the impedance technique had low accuracy, with an overestimation of more than 20 percent. The authors concluded that most of the analyzers tested seemed to be suitable for counting platelets in PCs. All hematology analyzers should be validated for counting platelets in the absence of RBCs, as with PCs, in addition to being validated for platelet counting in whole blood.

Dijkstra-Tiekstra MJ, Kuipers W, Setroikromo AC, et al. Platelet counting in platelet concentrates with various automated hematology analyzers. Transfusion. 2007; 47: 1651–1657.

Correspondence: M. J. Dijkstra-Tiekstra at
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Molecular RHD typing of blood donors Molecular RHD typing of blood donors

More than 70,000 blood products are transfused every year in Upper Austria. Routine blood typing with standard serologic methods is performed for the ABO and Rhesus blood group systems and K2 for every donor. Approximately one percent of the European population exhibit aberrant RHD alleles. More than 50 weak D phenotypes have been described, respectively. At the molecular level, single-point mutations leading to missense mutations are located in the transmembranous or intracellular regions of the RhD protein. Partial and weak D alleles must be distinguished to identify donor characteristics and avoid the risk of transfusion-related antibodies in recipients. The authors developed and tested a real-time-based RHD typing scheme during an eight-month period. A total of 53,347 blood donors and patients were tested with standardized immunohematologic methods. A total of 201 DNA samples with weak D reactions underwent molecular characterization by weak D real-time PCR, exon screening real-time PCR, and nucleotide sequencing of RHD exons 1 through 10. A total of 2,427 samples with D– phenotype were tested for RHD markers. The authors found that molecular typing of 201 samples with weak D expression revealed 15 different known aberrant alleles and one new weak D type, dubbed weak D type 49. Approximately 60 percent of the alleles were determined to be weak D types 1 through 3 and detected by only one amplification run. Weak D type 1 represented the most frequent allele (n=72). Three samples with D– phenotype showed amplification of RHD-specific markers. Sequence-based typing of these samples revealed a DEL allele, RHD (IVS3+1G>A), in two samples and one weak D type 4.3. The authors concluded that this scheme for RHD genotyping of weak D red blood cell units was reliable for detecting aberrant alleles. Testing D– blood samples as a quality control measure seems to overcome the limitations of standard serology by detecting samples with weak D or DEL phenotype.

Polin H, Danzer M, Hofer K, et al. Effective molecular RHD typing strategy for blood donations. Transfusion. 2007;47:1350–1355.

Correspondence: Helene Polin at
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Overall performance of MRSA PCR assay in high-volume clinical use Overall performance of MRSA PCR assay in high-volume clinical use

Methicillin-resistant Staphylococcus aureus (MRSA) continues to increase in prevalence. Furthermore, the burden of MRSA on U.S. health care organizations is often underestimated due to widespread unrecognized, asymptomatic colonization. Culture-based detection of MRSA with traditional media requires 48 to 96 hours to produce results. A combination of molecular methods with culture decreases the time to results to about 24 to 40 hours. In contrast, the BD GeneOhm MRSA real-time PCR assay, formerly called the IDI-MRSA assay, rapidly identifies MRSA-colonized patients in as little as two hours. This real-time polymerase chain-reaction (PCR) method recently has been compared to plating of samples onto agar, and if any test, including PCR, was assumed to be a true positive, the sensitivity of culture was 62 percent for direct plating, increasing to 85 percent with broth enrichment, while the sensitivity of PCR was 95 percent. The authors conducted a study to validate the alternative lysis procedure they developed for their institution (to facilitate the processing of 100 to 150 nasal swabs a day by a single laboratory worker) for use with the BD GeneOhm MRSA real-time PCR assay and to evaluate the overall performance of the PCR test during high-volume clinical use. The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and using an in-house lysis method that was developed specifically to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay were 98 percent, 96 percent, 77 percent, and 99.7 percent with the kit lysate, and 98 percent, 95 percent, 75 percent, and 99.7 percent with the ACP lysate, respectively. The second evaluation was done after implementing all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed a negative predictive value of 99.9 percent and a positive predictive value of 73.5 percent (prevalence, six percent), consistent with the initial findings. The authors concluded that the BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.

Paule SM, Hacek DM, Kufner B, et al. Performance of the BD GeneOhm methicillin-resistant Staphylococcus aureus test before and during high-volume clinical use. J Clin Microbiol. 2007;45:2993–2998

Correspondence: Suzanne M. Paule at
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Association of anti-CCP levels with rheumatoid arthritis haplotypes Association of anti-CCP levels with rheumatoid arthritis haplotypes

Rheumatoid arthritis, a chronic systemic autoimmune disease characterized by destructive inflammation of the synovial joints, affects approximately one percent of the world population and is more common in women than in men. Rheumatoid arthritis (RA) is a multifactorial disease involving a variety of environmental and genetic components, with genetics determining 50 to 60 percent of the RA phenotype. Early diagnosis is critical to treatment. Rheumatoid factor has been used as a serologic marker for diagnosing RA, but it has relatively low specificity, as it can be detected in patients with other autoimmune diseases and in unaffected people. In contrast, antibodies against cyclic citrullinated peptides (anti-CCP) are good serologic markers for RA, with a specificity of more than 95 percent and a sensitivity of approximately 80 percent, comparable with that of rheumatoid factor. Members of the peptidyl arginine deiminase (PAD) gene family (PADI1 to PADI4 and PADI6), which generate the citrullinated peptides recognized by anti-CCP via post-translational modification of arginine residues to citrul­lines, appear to be good candidates for involvement in the path­o­genesis of RA. Indeed, several single-nucleotide polymorphisms (SNPs) and related haplotypes in PADI4 have been associated with RA. HLA-DRB1 shared epitope alleles have been found to be associated with increased anti-CCP levels in anti-CCP-positive RA patients, and anti-CCP has been associated with the development of RA. However, shared epitope alleles have not been found to contribute to the development of RA in patients with undifferentiated arthritis. The authors had previously reported that a haplotype in PADI4 consisting of minor alleles at three nonsynonymous SNPs was associated with RA susceptibility in Koreans. For this study, the authors recruited a new cohort of Korean patients with RA and measured their serum anti-CCP levels. The authors examined possible relationships between anti-CCP levels and PADI4 haplotype or shared epitope alleles, or both. They genotyped three nonsynonymous SNPs in PADI4 (padi4_89, padi4_90, and padi4_92) and shared epitope alleles and measured serum anti-CCP levels in 311 patients with nonerosive or erosive RA. They then analyzed statistically the relationships between anti-CCP levels and PADI4 haplotypes or shared epitope al­leles, or both. The authors found that among anti-CCP-positive patients with RA with a disease duration of no more than 34 months (P=.041), anti-CCP levels were significantly higher in patients carrying the PADI4 RA risk haplotype than in patients who did not have the risk haplotype. However, this was not found among patients with a longer disease duration or among those who had erosive RA versus nonerosive RA. In contrast, the levels were significantly higher in shared epitope carriers than in noncarriers among patients with RA with a disease duration of 141 months or more (P=.0037) and those who had erosive RA (P=.000098). But this was not found among patients who had a shorter disease duration or those who had nonerosive RA. The authors concluded that the PADI4 RA risk haplotype is associated with increased anti-CCP levels in RA patients with disease of short duration, and PADI4 may play a role in early RA. In contrast, shared epitope alleles are associated with increased anti-CCP levels in RA patients with very long-standing disease and in patients with erosive RA, suggesting that shared epitope alleles play a role in very late RA.

Cha S, Choi C-B, Han T-U, et al. Association of anti-cyclic citrullinated peptide antibody levels with PADI4 haplotypes in early rheumatoid arthritis and with shared epitope alleles in very late rheumatoid arthritis. Arthritis Rheum. 2007; 56: 1454– 1463.

Correspondence: Dr. Changwon Kang at
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Evaluation of a point-of-care D-dimer assay for excluding deep-vein thrombosis Evaluation of a point-of-care D-dimer assay for excluding deep-vein thrombosis

Compression ultrasonography and contrast venography are the most reliable methods for diagnosing deep-vein thrombosis, which has a lifetime cumulative incidence of two percent to five percent. Contract venography is the gold standard for the diagnosis, but it is not ideal because of its invasive nature and the risks associated with contrast media. D-dimer is a mixture of cross-linked fibrin degradation products, which is a marker of endogenous fibrinolysis, and thus it can be detected in patients with deep-vein thrombosis (DVT). Retrospective studies have shown a high negative predictive value of a D-dimer concentration for excluding DVT when taking into account a defined cut-off value for the assay used. Moreover, various studies have proven the safety of withholding anticoagulation in those patients with a negative D-dimer test result and a normal ultrasonography. Several D-dimer assays, includ­ing enzyme immu­no­assays, latex assays, and immunoturbidimetric assays, are available, but their clinical efficiency can differ markedly. Enzyme immunoassays, in comparison to latex assays, show a high sensitivity and negative predictive value in the presence of DVT. Some of these assays can be used as point-of-care tests for an emergency. The Vidas D-dimer assay is a well-established method for diagnosing or excluding DVT. And the newly developed PathFast D-dimer assay is a sensitive and quantitative method based on the principle of an enzyme immunoassay using a chemiluminescent substrate. Whole blood and plasma can be used as samples, and the reaction time is only five minutes. The authors conducted a study to evaluate the PathFast assay when used as a screening test to exclude DVT. Eighty-two consecutive patients (34 percent DVT, 66 percent non-DVT) with suspected DVT of a lower limb were tested with the D-dimer assay using a PathFast analyzer. The diagnostic value of the PathFast assay for DVT was evaluated with pre-test clinical probability, compression ultrasonography (CUS). Furthermore, each patient underwent contrast venography and computed tomography, if necessary. The sensitivity and specificity of the assay using 0.570 µg/mL FEU fibrinogen equivalent unit as a clinical cut-off value was found to be 100 percent and 63.2 percent, respectively, for the diagnosis of DVT, with a positive predictive value and negative predictive value of 66.7 percent and 100 percent, respectively. The correlation between the results of the PathFast D-dimer and Vidas D-dimer was acceptable (y= 1.134x+ 0.003; r=0.902). The test reproducibility was good (CV%, 4.0–5.0 percent for plasma and 7.1–7.5 percent for whole blood), and the total imprecision was very good (CV%, 3.6–5.7 percent). Whole blood and plasma can be used as samples in this assay (y= 1.013x –0.010; r=0.971 for heparinized specimens; y=1.068x+ 0.003; r=0.989 for citrated specimens). The authors concluded that because of its high sensitivity and negative predictive value, the PathFast D-dimer assay can be useful for rapidly ruling out DVT in patients admitted to the hospital with suspected thrombosis.

Fukuda T, Kasai H, Kusano T, et al. A rapid and quantitative D-dimer assay in whole blood and plasma on the point-of-care PATHFAST analyzer. Thromb Res. 2007; 120; 695– 701.

Correspondence: Teruko Fukuda at
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Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.