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March 2012

Editors:
Deborah Sesok-Pizzini, MD, MBA
Michael Bissell, MD, PhD, MPH

Amikacin added to blood to reduce fall in platelet count Amikacin added to blood to reduce fall in platelet count

Pseudothrombocytopenia is associated with blood specimens collected in anticoagulants such as EDTA, sodium citrate, heparin, and sodium fluoride. Patient specimens with this spuriously low platelet count may have antibodies that cause agglutination and induce in vitro platelet clumping. Most of the antibodies described are IgG and react best at 37°C, but IgM and IgA antibodies have also been described. The antibodies involved in EDTA-induced agglutination are most likely reactive with GP IIb/IIIa on the platelet surface. Pseudothrombocytopenia may lead to misdiagnosis, unnecessary repeat blood specimen collection, delay in care, or unnecessary treatment, such as platelet transfusion. Although this condition is most often found in ill hospitalized patients, healthy patients are also reported to have EDTA-induced thrombocytopenia. The authors attempted to use a variety of reagents, including aminoglycosides (gentamicin and amikacin), vitamin B6, and aminophylline, to inhibit platelet clumping in a specimen from a healthy person known to have multianticoagulant-dependent thrombocytopenia. The blood samples were supplemented with each reagent separately and then mixed with K2-EDTA to observe changes in platelets at different times to identify the optimal reagent. The optimal reagent was then added to blood samples mixed with K2-EDTA, 109 mmol/L sodium citrate, NaF, or heparin lithium. Results showed that NaF affected the platelet count most significantly, with obvious platelet aggregation just 10 minutes after mixing. The authors showed that platelet counts remained unchanged for up to four hours after sampling with the amikacin-treated EDTA-anticoagulated blood. The other reagents did not prevent the platelet count from decreasing. Furthermore, EDTA blood samples showed a rapid dissociation of platelet aggregation without morphological changes or changes in other blood cell indices. This suggests that platelet aggregation was inhibited by amikacin and may be a useful method to avoid additional blood sampling and inaccurate platelet counts. The authors concluded that adding amikacin to EDTA-anticoagulated blood collection tubes may prevent multianticoagulant-dependent pseudothrombocytopenia and EDTA-induced pseudothrombo- cytopenia.

Zhou X, Wu X, Deng W, et al. Amikacin can be added to blood to reduce the fall in platelet count. Am J Clin Pathol. 2011;136:646–652.

Correspondence: Dr. X. Zhou, Dept. of Laboratory Medicine, Guangzhou First Municipal People’s Hospital, Affiliate of Guangzhou Medical College, 510180 Guangzhou, PR China

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Targeting cellular structures and cell-signaling pathways for influenza treatment Targeting cellular structures and cell-signaling pathways for influenza treatment

Influenza morbidity and mortality rates, as well as the occurrence of influenza pandemics and the emergence of new strains of influenza, support the need for effective antiviral therapy. Clinical antiviral therapy targets viral factors such as the M2 ion channel or neuraminidase. In recent years, reports of resistance to the four FDA-approved anti-influenza virus drugs have increased. In this review, the authors described newer approaches to influenza treatment, including targeting cellular structures and cell-signaling pathways. The author suggested that overcoming the problem of antiviral therapy resistance may involve changing the mechanism of therapy to include both attachment-disrupting agents and prevention of viral-dependent cellular signal transmission. The author highlighted some of the recent advances to target cellular factors for anti-influenza therapy. One approach is to disrupt virus-host cell interaction by blocking viral HA protein interaction with sialic acids on cell surfaces. Under clinical development is the use of DAS181, a fusion construct of sialidase linked to a human epithelium-anchoring domain, which cleaves the surface receptors recognized by HA on the respiratory epithelium. DAS181 is inhibitory for influenza A and B viruses and avian influenza. Another attachment disruption mechanism described was the use of cell-penetrating peptides, which likely interact with the HA protein to prevent cell attachment. Soluble receptor analogs using multimeric sialoglycoconjugates are also shown to inhibit adhesion and influenza virus replication in vitro and in a mouse model. Since cleavage of influenza’s HA protein is necessary for viral cell infection, the use of protease inhibitors in antiviral therapy is also being researched. The author described mechanisms of blocking intracellular events during viral replication using the viral polymerase complex and the interferon antagonist NS1 protein. Additional mechanisms addressed disrupting cellular signaling processes exploited by viruses for successful replication. Two signaling pathways show promise for use as antivirals: the Raf/MED/ERK mitogenic kinase cascade and the IKK/NF-kB module. Since viral replication requires activating this kinase cascade to induce RNP viral nuclear release, inhibitors of the cascade show promise. Several are under investigation. Pre-activated NF-kB cells showed more efficient influenza replication, so inhibiting this factor in cells was proposed to affect virus life cycle. A common NF-kB inhibitor, ASA (aspirin), was shown to block replication of influenza viruses in cell culture and in vivo, with no resistance strains found. The authors concluded that first proof-of-concept data are available for many of these newer inhibitors without toxic effects or induction of viral resistance.

Ludwig S. Disruption of virus-host cell interactions and cell signaling pathways as an anti-viral approach against influenza virus infections. Biol Chem. 2011;392:837–847.

Correspondence: Stephan Ludwig at ludwigs@uni-muenster.de

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Assessing resistance to Raf inhibition in melanoma by tumor genomic profiling Assessing resistance to Raf inhibition in melanoma by tumor genomic profiling

Expanding the use of personalized cancer medicine may involve investigating individual tumor genomic characterization to better guide treatment decisions. Genetic features of the tumor may prove to be just as important in the future as tissue origin when predicting the likelihood of response to anti-cancer drugs. For example, protein kinase inhibitors have shown efficacy when tumors have genetic alterations that dysregulate cellular signaling mechanisms. Newer kinase inhibitors targeting BRAF in melanoma are the focus of this study by investigators at the Dana-Farber Cancer Institute. The authors performed targeted massive parallel sequencing of 138 cancer genes in a tumor from a patient with melanoma who later developed resistance to PLX4032 after a dramatic response. Investigators identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the pretreated tumor tissue. The MEK1C121S mutation showed increased kinase activity and the ability to create a resistance to Raf and MEK inhibition in vitro. The authors found that tumors with MEK1C121S or similar mutations may be predicted to have combined MEK/Raf inhibition. Clinical trials that targeted mutated BRAF in melanoma have shown favorable results. In a recent phase-one trial of the Raf inhibitor PLX4032, 81 percent of patients with BRAFV600E melanoma experienced at least 30 percent tumor shrinkage. However, resistance to PLX4032 re-emerged. Mechanisms for developing resistance to Raf inhibition are still poorly understood. The results of this study will provide a framework for future patients with BRAF-mutant melanoma in clinical trials and help characterize mechanisms of acquired resistance to kinase inhibitors. In addition, knowledge of resistance-associated MEK1 mutations in BRAF-mutant melanoma may help guide salvage or combination therapies that intercept the MAP kinase pathway at the level of MEK or further downstream.

Wagle N, Emery C, Berger MF, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol. 2011;29:3085–3096.

Correspondence: Dr. Levi A. Garraway at levi_garraway@dfci.harvard.edu

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Prostate-specific antigen and genetic variables Prostate-specific antigen and genetic variables

Prostate-specific antigen is the primary biomarker for screening prostate cancer. However, it has limited specificity as a marker and it can be increased in multiple noncancerous conditions, such as prostatitis and benign prostatic hyperplasia, and after prostatic manipulation via means such as catheterization. Emerging evidence also indicates that PSA concentrations may be affected by genetic variables. Through genome-wide association studies, investigators have identified more than 30 single-nucleotide polymorphisms (SNPs) that are associated with prostate cancer susceptibility. A report by Eeles, et al., showed a strong association of SNPs on chromosomes 10 (rs10993994) and 19 (rs2735839) with prostate-specific antigen (PSA) concentration and prostate cancer risk. It is noteworthy that rs2735839 is located near the KLK33 (kallikrein-related peptidase 3) gene on chromosome 19, which encodes PSA, potentially underlying the relationship. In 2009, the author’s group explored the potential implications of these findings for prostate cancer screening. For 505 men from the Baltimore Longitudinal Study of Aging, they used mixed effects models to evaluate the relationships between genotype, PSA, and prostate cancer risk. In a model with age and date, they noted a 1.18-fold (18 percent) increased risk ratio for prostate cancer per unit increase in PSA. In an expanded model that also considered the SNP genotypes on chromosomes 10 (rs10993994) and 19 (rs2659056 and rs2735839) and the interaction between genotype and PSA, they found significant differences in prostate cancer risk by PSA that depended on genotype. The risk ratio for prostate cancer was 1.28 (95 percent confidence interval [CI], 1.20–1.38) per unit increase in PSA for carriers of a minor allele and 1.10 (95 percent CI, 1.06–1.15) for men without a minor allele. The authors concluded that genetic variants significantly altered the risk of prostate cancer per unit increase in PSA concentration and that additional study is warranted to evaluate a potential role for genetic markers in the interpretation of PSA for prostate cancer risk assessment. Subsequently, Gudmundsson, et al., presented a large study of the relationship of germline sequence variants with serum PSA concentration. An important limitation of that study was that the study population comprised Caucasian men of European ancestry, potentially limiting its generalizability to other ethnic groups with different genetic profiles. In addition, although alternative PSA thresholds were suggested in this study, prospective studies are necessary to determine whether adjusting PSA concentrations based on genotype would improve clinical outcomes. Validated prospectively, these combined findings suggest that genetic markers may help improve screening protocols.

Loeb S. Germline sequence variants and prostate-specific antigen interpretation. Clin Chem. 2011;57:662–663.

Correspondence: Stacy Loeb at stacyloeb@gmail.com

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Cerebrospinal fluid test for posterior cortical atrophy Cerebrospinal fluid test for posterior cortical atrophy

Posterior cortical atrophy is a rare syndrome characterized by predominant progressive higher order visuoperceptual deficits associated with occipito-parieto-temporal brain dysfunction. Visual symptoms include simultanagnosia, hemispatial neglect, optic ataxia, and visual agnosia. Neuroimaging usually shows atrophic or metabolic changes in posterior brain regions. Evolution generally leads to dementia. This syndrome is usually related to Alzheimer disease (AD) but can also result from corticobasal degeneration, Creutzfeldt-Jakob disease (CJD), dementia with Lewy bodies (DLB), or subcortical gliosis. Etiologic diagnosis remains uncertain until postmortem examination. Cerebrospinal fluid (CSF) biomarkers aid in vivo AD diagnosis. In AD, the biomarkers are characterized by a combination of increased total tau proteins (t-tau) and phosphorylated tau on amino acid 181 (p-tau181) and decreased 42 amino acid amyloid β peptide (Aβ42). These tau and Aβ profiles are related to elementary AD lesions—neurofibrillary tangles and amyloid plaques, respectively. Therefore, CSF biomarker examination appears relevant to the etiologic diagnosis of posterior cortical atrophy (PCA) since the prevalence of AD pathology is high in this condition. The authors conducted a study to describe the CSF biomarker profile in a prospective series of well-defined patients with PCA. The PCA CSF profile was compared to that of typical AD and other dementia groups. CSF determination in PCA was assessed by comparing clinically based and CSF-based classifications of etiologic diagnosis. This prospective observational study included 22 patients with PCA who underwent CSF biomarker analysis of t-tau, p-tau181, and Aβ42. At group level, the CSF profiles of patients with PCA were compared to those of patients with typical AD and patients with other forms of dementia. Individually, the clinical presentation of patients with PCA was correlated to their CSF profiles to assess the predictability of clinical features for diagnosing underlying AD pathology. At group level, the PCA biomarker profile was not different from that of the AD group, but it was very different from that of the group with other forms of dementia (P<0.001). More than 90 percent of patients with PCA had CSF profiles consistent with AD. All patients with PCA with isolated higher order visual deficit (n=8) or visual deficit associated with memory impairment (n=11) had CSF profiles consistent with AD. Only one of the three patients with PCA with asymmetric motor signs fulfilled biological CSF criteria for AD. The authors concluded that PCA syndrome is usually associated with CSF biomarkers suggestive of AD, as shown by previous neuropathologic studies. This does not apply in cases of motor signs suggesting associated corticobasal syndrome. CSF biomarkers help to discriminate AD from non-AD processes associated with this condition.

Seguin J, Formaglio M, Perret-Liaudet A, et al. CSF biomarkers in posterior cortical atrophy. Neurology. 2011;76:1782–1788.

Correspondence: Dr. Pierre Krolak-Salmon at pierre.krolak-salmon@chu-lyon.fr

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Viral serology in workup of suspected myocarditis Viral serology in workup of suspected myocarditis

Myocarditis is an inflammatory heart disease classified by clinical, immunohistochemical, and clinicopathological criteria and is usually caused by infectious agents. In Europe and North America, viral infections are the most common causes of myocarditis. The clinical picture of patients with myocarditis is highly variable, ranging from asymptomatic courses to severe illness requiring intensive care. The diagnosis of myocarditis is based on assessing endomyocardial biopsies (EMBs) histopathologically and immunohistologically. Molecular pathological analyses, such as polymerase chain reaction and in situ hybridization, allow detection and quantification of the viral genome in the heart. Immunohistological signs of inflammation in the myocardium have recently been reported to predict poor outcome. EMB might be underused due to lack of accessibility, clinical experience, or skill. Nevertheless, immediate diagnosis and treatment may be needed to prevent the development of inflammatory dilated cardiomyopathy (DCMi), an important cause of heart failure that appears to be triggered by viral myocarditis in approximately 35 percent to 50 percent of cases. Serological analyses to diagnose viral infection in patients with suspected myocarditis are still used frequently in clinical practice, although the assays are costly and data are lacking when compared with state-of-the-art evaluation of EMB findings. The authors conducted a prospective study to determine the diagnostic value of virus serology in comparison with analyses of EMB including viral genome detection in patients with clinically suspected myocarditis. Virus serology and a state-of-the-art evaluation of EMB were performed in 124 patients (age, 40±15 years) with suspected myocarditis. Endomyocardial biopsy was studied for inflammation with histological and immunohistological criteria. The viral genome was detected in the myocardium by polymerase chain reaction. Acute viral infection with enterovirus, adenovirus, parvovirus B19, cytomegalovirus, human herpesvirus, and Epstein-Barr virus was diagnosed by IgM or IgA in the initial sample or IgG seroconversion in the followup sample. Immunohistological signs of inflammation were present in 54 patients. The viral genome was detected in the myocardium of 58 patients (47 percent). In 20 patients (16 percent), acute viral infection was diagnosed by serology. Only in five of 124 patients (four percent) was there serological evidence of an infection with the same virus that was detected by EMB. Sensitivity and specificity of virus serology were nine percent and 77 percent, respectively. The positive and negative predictive values were 25 percent and 49 percent, respectively. The lack of correlation between serology and EMB remained also for patients with biopsy-proven myocarditis and patients with time from initial symptoms to EMB procedure of one month or less. The authors concluded that for patients with suspected myocarditis, virus serology is not relevant to the diagnosis of myocardial infection. Endomyocardial biopsy remains the gold standard for the diagnosis of viral myocarditis.

Mahfoud F, Gartner B, Kindermann M, et al. Virus serology in patients with suspected myocarditis: utility or futility? Eur Heart J. 2011;32:897–903.

Correspondence: Felix Mahfoud at felix.mahfoud@uks.eu

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Prenatal diagnosis of esophageal atresia Prenatal diagnosis of esophageal atresia

Esophageal atresia and tracheoesophageal fistula are major congenital malformations affecting one in 3,500 live births. They are characterized by disruption of the continuity of the esophagus with or without a persistent communication with the trachea. Five subtypes (type I to type V) have been defined based on the location of the atresia and the type of connection between the trachea and esophagus. Type III (esophageal atresia [EA] with distal tracheoesophageal fistula) is the most frequent variety, representing 86 percent of cases. Associated anomalies occur in 50 percent of patients and include vertebral, anal, cardiovascular, renal, and limb anomalies (occurring together in the Vertebral, Anal atresia, Cardiac defect, TracheoEsophageal fistula, Renal, Limb association [VACTERL]) or chromosomal anomalies. The etiology of EA is unknown but is thought to be multifactorial, involving genetic and environmental factors. Prenatal diagnosis of EA may improve the outcome of affected neonates by allowing optimization of prenatal and postnatal care. The prenatal sonographic detection of EA relies on finding a small or nonvisualizable fetal stomach bubble associated with polyhydramnios. However, because of the large number of etiologies for polyhydramnios or nonvisualization of the fetal stomach bubble, or both, as well as the subjective nature of a small fetal stomach, EA prenatal diagnosis remains difficult. It is characterized by a high false-positive rate and a poor detection rate. Several studies have documented that the sensitivity of sonography ranges from 8.9 percent to 42 percent. Direct visualization of the fluid-filled, blind-ending esophagus during fetal swallowing or upper neck “pouch sign” was described as early as 23 weeks’ gestation. However, it may be difficult to visualize the pouch because of technical difficulties or time limitations. Polyhydramnios can be observed in a large number of etiologies, such as maternal diabetes, digestive tract atresia, chloride diarrhea, Pierre Robin syndrome, and Bartter syndrome. In addition to fetal karyotyping, amniotic fluid digestive enzyme assay could be of interest for polyhydramnios etiology. The authors conducted a study to evaluate the potential value of amniotic fluid digestive enzyme assay for EA prenatal diagnosis when suggested at routine ultrasound scan by indirect signs. The authors compared amniotic fluid biochemical markers in 44 EA cases with 88 polyhydramnios and 88 nonpolyhydramnios controls. Total proteins, alpha-fetoprotein (AFP), and digestive enzyme activities were assayed, including gammaglutamyl transpeptidase (GGTP). The authors defined an EA index (AFP multiplied by GGTP). A significant difference (P<0.0001) was observed for total protein, AFP, GGTP, and EA index between the EA group and each of the two control groups. No statistical difference was observed for any marker between the two most frequent EA subgroups (type I and type III) or between the two control groups. Using a cutoff of three for the EA index, 98 percent sensitivity and 100 percent specificity were observed for amniotic fluid prenatal diagnosis of EA, regardless of anatomical type. The authors concluded that a large prospective series is required to confirm these results.

Czerkiewicz I, Dreux S, Beckmezian A, et al. Biochemical amniotic fluid pattern for prenatal diagnosis of esophageal atresia. Pediatr Res. 2011;70:199–202.

Correspondence: Francoise Muller at fran coise.muller@rdb.aphp.fr

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Clinical pathology abstracts editors: Deborah Sesok-Pizzini, MD, MBA, associate professor, Department of Clinical Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, and medical director, Blood Bank and Transfusion Medicine, Children's Hospital of Philadelphia; Brooke Allen, MT(ASCP), lead technologist, Clinical Virology Laboratory, Children's Hospital of Philadelphia; Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.