College of American Pathologists
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  Clinical Abstracts





June 2011

Michael Bissell, MD, PhD, MPH

Cerebrospinal fluid biomarkers in normal and abnormal aging brains Cerebrospinal fluid biomarkers in normal and abnormal aging brains

The dominant pathologic findings in Alzheimer disease are Aβ-rich amyloid plaques, fibrillary tau deposits in neurofibrillary tangles, neurophil threads, neuronal dysfunction, and neurodegeneration. Accepted biomarker surrogates of the dominant pathologies in Alzheimer disease are Aβ1-42 and total-tau (t-tau) levels measured in cerebrospinal fluid (CSF) and atrophy seen on magnetic resonance imaging (MRI). Low CSF Aβ1-42 levels reflect deposition of Aβ in amyloid plaques. Elevated CSF t-tau levels reflect abnormal tau accumulation in neurofibrillary tangles and neurophil threads, as well as active axonal and neuronal damage. Atrophy on MRI is the macroscopic manifestation of microscopic neurodegenerative changes and reflects the cumulative loss of neurons, synapses, and dendritic arborization. Longitudinal biomarker measures of change over time provide additive diagnostic and prognostic information about the rate of change in disease-related pathology and can serve as outcome measures in therapeutic trials. MRI and CSF biomarkers have been studied extensively in a cross-sectional manner and, to a lesser extent, longitudinally in small cohorts or single centers. However, few reports have compared longitudinal change to CSF and MRI biomarkers in the same subjects examined serially in multicenter studies of large cohorts of cognitively normal individuals, subjects with amnestic-mild cognitive impairment (aMCI), and patients with Alzheimer disease. Therefore, the authors conducted a study to do the following: measure the annual change in CSF Aβ1-42, CSF t-tau, and ventricular volume on MRI by clinical group and compare the annual change in biomarkers between clinical groups; assess the correlation between annual change in CSF and MRI measures and annual change on continuous measures of cognitive and functional performance; evaluate the effect of APOE ε4 status on annual change in the biomarkers; and compare sample sizes needed in a hypothetical clinical trial. Comparisons were based on intergroup discrimination, correlation with concurrent cognitive/functional changes, relationships to APOE genotype, and sample sizes for clinical trials. The authors used data from the Alzheimer’s Disease Neuroimaging Initiative study consisting of cognitively normal, aMCI, and Alzheimer disease cohorts with baseline and 12-month followup CSF and MRI. They obtained the annual change in CSF (t-tau, Aβ1-42) and MRI (change in ventricular volume) in 312 subjects (92 cognitively normal, 149 aMCI, 71 Alz- heimer disease). The authors found no significant average annual change in either CSF biomarker in any clinical group, except t-tau in the cognitively normal group. Moreover, the annual change did not differ by clinical group in pairwise comparisons. In contrast, annual increase in ventricular volume increased in the following order: AD>aMCI>CN. Differences were significant between all clinical groups in pairwise comparisons. Ventricular volume increase correlated with concurrent worsening on cognitive/functional indices in aMCI and Alzheimer disease, whereas evidence of a similar correlation with change in CSF measures was unclear. The annual changes in MRI differed by APOE ε4 status overall and among aMCI, while annual changes in CSF biomarkers did not. Estimated sample sizes for clinical trials were notably less for MRI than for the CSF or clinical measures. The authors concluded that unlike the CSF biomarkers evaluated, changes in serial structural MRI correlate with concurrent change on general cognitive and functional indices in impaired subjects, track with clinical disease stage, and are influenced by APOE genotype.

Vemuri P, Wiste HJ, Weigand SD, et al. Serial MRI and CSF biomarkers in normal aging, MCI, and AD. Neurology. 2010;75:143–151.

Correspondence: Dr. Clifford R. Jack Jr. at

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Drug screen cross-reactivity Drug screen cross-reactivity

Rapid urine screens for drugs of abuse are often used in pediatric emergency departments. An unexpected positive result often leads to further clinical assessment, social evaluation, and increased stress and inconvenience for the patient and family. The authors presented a previously unreported false-positive methadone result, its clinical consequences, and an in vitro laboratory evaluation of the device for cross-reactivity with diphenhydramine (DPH) and its metabolites. A pediatric emergency department patient with suspected DPH ingestion had a positive methadone result on the rapid urine drug screen One Step Multi-Drug, Multi-Line Screen Test Device (Acon Laboratories). The patient had no history of methadone exposure and was admitted while confirmatory testing was performed. Gas chromatography/mass spectroscopy testing of the patient’s urine failed to confirm the presence of methadone. The same One Step urine drug screen was tested at an independent laboratory for cross-reactivity between methadone and DPH, including DPH metabolites. Drug-free urine was fortified with DPH nordiphenhydramine or dinordiphenhydramine at 0, 10, 25, 50, and 100 µg/mL for each analyte. Then 100 µL of the solutions were added to each of the four wells on test cassettes. Urine was allowed to migrate according to manufacturer instructions. Each cassette was interpreted by two analysts to ensure consistency and accurate recording of data. The in vitro laboratory testing results showed cross-reactivity between methadone and DPH but not for nordiphenhydramine or dinordiphenhydramine. The authors concluded that rapid urine drug screens using immunoassays based on the principle of competitive binding may show false-positive methadone results for patients who have ingested DPH. Product information for urine drug screens may not include all cross-reacting agents and should be used with caution when interpreting drug screen results for pediatric emergency department patients.

Rogers SC, Pruitt CW, Crouch DJ, et al. Rapid urine drug screens: diphenhydramine and methadone cross-reactivity. Pediatr Emerg Care. 2010;26:665–666.

Correspondence: Dr. Steven C. Rogers at

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Strengthening lab systems in resource-limited settings Strengthening lab systems in resource-limited settings

Public health laboratories are a critical component of global communicable disease detection, prevention, and control. However, access to reliable laboratory testing remains limited in many resource-challenged countries. This can result in delayed diagnosis, misdiagnosis, and ineffective and inappropriate treatment and lead to increased morbidity and mortality. Many factors have been cited as contributing to limited laboratory access, including lack of laboratory supplies, lack of essential equipment, limited numbers of skilled personnel, lack of educators and training programs, inadequate logistical support, de-emphasis of laboratory testing, insufficient monitoring of test quality, decentralization of laboratory facilities, and lack of government standards for laboratory testing. In part owing to efforts to improve the diagnosis and treatment of people with HIV, many governments and their external partners have been focusing on enhancing access to diagnostic services by expanding laboratory capacity in developing countries. A 2008 international conference in Maputo, Mozambique, that was convened by the World Health Organization (WHO) addressed laboratory challenges. The conference focused on diagnostic tests that should be available at each level in a tiered system, standardization of laboratory equipment and supplies, and key considerations for equipment maintenance and service contracts. It also deemed laboratory strategic plans, human capacity, infrastructure, and management of quality systems as essential to laboratory capacity. Efforts to improve laboratory capacity in resource-limited settings have not been systematically examined or reported. Therefore, the authors reviewed published reports, interviewed major donor organizations, and conducted case studies of laboratory systems in Ethiopia, Kenya, and Thailand to assess how those nations and donor organizations in those countries have worked together to improve laboratory services. The authors found that while infrastructure and the provision of services have improved, further advancement is needed. Implementation of national laboratory plans is inconsistent; human resources are limited; and quality laboratory services rarely extend to laboratories in health clinics, district hospitals, and other similar settings. Coordination within, between, and among the governments and donor organizations is also frequently problematic. The authors concluded that laboratory standardization and quality control are improving but remain challenging, making accreditation a difficult goal. The governments of resource-limited countries and their external funding partners should coordinate their efforts around the country’s national plan for advancing laboratory capacity.

Olmsted SS, Moore M, Meili RC, et al. Strengthening laboratory systems in resource-limited settings. Am J Clin Pathol. 2010;134:374–380.

Correspondence: Dr. S. S. Olmsted, 4750 5th Ave., Suite 600, Pittsburgh, PA 15213

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Assessing recombinant human thyrotropin potency Assessing recombinant human thyrotropin potency

Recombinant human thyrotropin has been used extensively to diagnose recurrent differentiated thyroid cancer. The recommended dose is 0.9 mg intramuscular on two successive days. Serum thyroglobulin is measured as a tumor marker three days after the second injection. Recombinant human thyrotropin (rhTSH) is also used for ablation of remnants of thyroid tissue after surgery for thyroid cancer. In addition, several reports have detailed the use of a single dose of 0.03 to 0.45 mg rhTSH, which is administered to increase the thyroid uptake of a therapeutic dose of radioiodine-131 (131I) in patients with nodular goiter to reduce goiter size and compressive symptoms. The authors have used 0.1 mg rhTSH to increase thyroid uptake of a therapeutic dose of 131I in patients with subclinical hyperthyroidism and relatively low 24-hour radioiodine uptake. The increased uptake of the therapeutic dose of radioiodine induced by rhTSH elevates the effect of the therapeutic dose. This allows physicians to reduce the amount of 131I that is administered. Reducing the dose may decrease side effects, such as damage to salivary glands and lacrimal ducts. Thyrogen is prepared in ampules containing 1.1 mg rhTSH plus phosphate-buffered salt. It is diluted for injection in 1.2 mL sterile water so that 1 mL contains 0.9 mg. To determine whether the biologic activity of rhTSH can be preserved after dilution, the authors designed experiments to assess the biologic activity of rhTSH under different durations and temperatures of storage. The biologic activity was tested in vitro by measurement of 125I-iodide uptake in FRTL-5 rat thyroid cells. RhTSH was diluted in one percent bovine serum albumin in phosphate-buffered saline to a concentration of 0.9 mg/mL and further diluted to 0.1 mg/mL. Aliquots of 0.5 mL were stored at room temperature, 4°C, –11°C, and –60°C for various lengths of time. RhTSH aliquots were also subjected to incubation for one hour at 50°C and to 10 cycles of freezing in dry ice alternating with thawing at 37°C. Bioassays were performed with FRTL-5 cells. RhTSH was added to the media at a final concentration of 5 ng/mL or 20 ng/mL, and the cells were then incubated for 48 hours. Potency was assessed by measuring 125I-iodide uptake in comparison to cells treated with perchlorate to block iodide uptake. Samples underwent immunoassay at day 185 of storage. The authors found that samples stored at 4°C, –11°C,–60°C, and room temperature retained activity after storage periods of up to 204 days. Samples subjected to 10 freeze-thaw cycles or heated to 50°C for one hour retained full biologic activity. The samples subject to immunoassay at day 185 showed no difference in immunoactivity in relation to storage condition. The authors concluded that rhTSH kept at 4°C, –11°C,–60°C, and room temperature maintains good biologic potency for more than six months of storage when tested in vitro, indicating that the biologic activity is very stable. However, altered sialylation during storage could alter the half-life of rhTSH. Nevertheless, the data provide reassurance that cold storage for a few months does not result in significant loss of biologic activity.

Lin R, Hogen V, Cannon S, et al. Stability of recombinant human thyrotropin potency based on bioassay in FRTL-5 cells. Thyroid. 2010;20:1139–1143.

Correspondence: Jerome M. Hershman at

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False-negative congenital hypothyroid screens False-negative congenital hypothyroid screens

Screening programs for congenital hypothyroidism have virtually eradicated mental retardation and impaired somatic growth caused by thyroid hormone deficiency. This has been achieved through early diagnosis and treatment with L-thyroxine. Therefore, the screening programs are cost-effective, a fact that is taken into consideration by governmental health officials. Although the screening programs for congenital hypothyroidism (CH) were established more than 30 years ago, the optimal screening strategy, and particularly the thyroid-stimulating hormone (TSH) threshold, have not been defined satisfactorily. This was evidenced in a survey that outlined the status of neonatal screening in Europe in 2004. Screening TSH cut-off values ranging from 5 to 25 mU/L were applied in 178 screening centers using a variety of methods for TSH measurement and showed differences in the prevalence of CH and recall rates. Numerous studies reported missed CH cases during the operation of neonatal screening programs. The causes of CH in these missed cases were either defects in T4 synthesis or thyroid gland malformations. Newborn screening for CH was initiated in Greece as a pilot study in 1979 and was gradually generalized, covering the total newborn population by 1990. A blood spot TSH value of 30 mU/L was initially used as a cutoff point. It was lowered to 20 mU/L in the 1990s, a value recommended by the Working Group of the European Society of Pediatric Endocrinology. Based on this criterion, an overall prevalence of CH of 1:3,300 screened newborns was found, a figure comparable with that in other European countries and the United States. During the screening operation, however, it became evident that some CH cases had escaped diagnosis because they presented with spot TSH values below the cutoff point applied by that time (false-negative results). These patients were identified by their pediatricians, who suspected CH on the basis of clinical symptoms or signs of CH, or both, or because they ordered thyroid function tests due to familial CH. This prompted the authors to initiate a prospective study to determine the additional number of newborns found to have CH if the TSH cutoff point was lowered to 10 mU/L. The study included 311,390 screened newborns. The children with CH were followed up for a period of three years. Twenty-eight percent of infants diagnosed with CH had neonatal TSH values between 10 and 20 mU/L (56 of 200). Forty of 47 infants who were re-evaluated later (85.1 percent) suffered permanent CH. A thyroid scintiscan or echogram, or both, revealed that eight of 40 children (20 percent) had a structural defect, and the remaining children (32 of 40; 80 percent) had a functional defect of the thyroid gland without anatomical abnormality. Fourteen of 32 cases were familial. Eighteen of the 47 infants who were re-evaluated were born prematurely (38.3 percent), and 15 of those 18 had permanent CH (83.3 percent). Lowering the TSH cutoff point from 20 to 10 mU/L resulted in a 10-fold increase of recall rate. The authors concluded that a significant number of cases with permanent CH are missed when a TSH threshold of 20 mU/L is applied. Almost 40 percent of the missed CH cases were premature. A mild increase of TSH at screening is not a predictor of transient CH. The increase in recall rate constitutes a serious drawback and should be balanced against the possible consequences of thyroid dysfunction at this important developmental stage.

Mengreli C, Kanaka-Gantenbein C, Girginoudis P, et al. Screening for congenital hypothyroidism: the significance of threshold limit in false-negative results. J Clin Endocrinol Metab. 2010;95:4283–4290.

Correspondence: Dr. Chryssanthi Mengreli at

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Determination of fetal Rh  by cell-free DNA analysis Determination of fetal Rh by cell-free DNA analysis

The distribution of Rh-negative (D–) phenotype varies according to race. The D– blood group is found in 15 percent of whites and three percent to five percent of black Africans; it is rare in Asians. The mechanisms responsible for determining the D– phenotype also vary according to race. Most white D– individuals have a deletion of the Rh blood group, D antigen (RHD) segment on the short arm of chromosome 1. Black D– subjects have variations in their gene sequence that lead to nonexpression of the Rh D-positive (D+) genotype hybrid RHD gene or pseudogene RHD with a discrepancy between genotype and phenotype. Therefore, ethnicity may affect the antenatal diagnosis of fetal RHD genotype when noninvasive methods are used. The Brazilian population is a blend of many races, with many people of African descent. According to an official census performed in 2000, 38.5 percent of the Brazilian population self-reported as being mixed (of black and white ancestry) and 6.2 percent as black. Several investigators have described their experience with fetal RHD genotyping in maternal blood, but none with this kind of racial distribution, using the real-time polymerase chain-reaction (PCR) technique. Therefore, the authors conducted a study to present their experience in determining fetal RHD status from cell-free fetal DNA in maternal blood using real-time PCR in a population with an intense miscegenation of races. They performed a prospective study from January 2006 to December 2007 in which they analyzed fetal RHD genotype in the plasma of 102 D– pregnant women by real-time PCR, targeting exons 7 and 10 of the RHD gene. The authors compared genotype results with cord blood phenotype obtained after delivery or before the first intrauterine transfusion. Most of the participants (75.5 percent) were less than 28 weeks of pregnancy and 87.5 percent had at least one relative of black ancestry. By combining amplification of two exons, the accuracy of genotyping was 98 percent; sensitivity was 100 percent; and specificity was 92 percent. The positive likelihood ratio was 12.5, and the negative likelihood ratio was zero. The two false-positive cases were confirmed to be pseudogene RHD by real-time PCR. No differences were found between the patients with a positive or negative Coombs test (P=0.479). The authors concluded that determination of fetal RHD status in maternal peripheral blood was highly sensitive in this racially mixed population and was not influenced by anti-erythrocyte antibodies.

Chinen PA, Nardozza LMM, Martinhago CD, et al. Noninvasive determination of fetal Rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population. Am J Perinatol. 2010;27:759–762.

Correspondence: Dr. E. A. Junior at

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.