Spurious testosterone laboratory results in a patient taking synthetic alkaline phosphatase (asfotase alfa).

OBJECTIVES: We report a case of discordant total and free testosterone values in a patient with hypogonadism and juvenile hypophosphatasia after he initiated treatment with asfotase alfa, recombinant tissue non-specific alkaline phosphatase. METHODS: Total testosterone was evaluated using immunoassay pre and post initiation of therapy with asfotase alfa, and free testosterone was evaluated using radioimmunoassay and LC-MS/MS while on asfotase alfa therapy. RESULTS: Total testosterone measured by immunoassay was normal prior to therapy with asfotase alfa, and was low post initiation of therapy. During the same time frame, free testosterone measured using RAI and total testosterone measured using LC-MS/MS were normal on asfotase alfa therapy. This suggests assay interference with the total testosterone immunoassay. CONCLUSION: When laboratory results are discordant or do not match the clinical impression, the possibility of assay interference should be considered. Alternative laboratory methods free of the interference should be selected to evaluate these patients. HUMAN GENES DISCUSSED IN THE PAPER: ALPL gene, Approved name: Alkaline phosphatase, liver/bone/kidney, Synonym: Tissue non-specific alkaline phosphatase (TNSAP).

Clinically distinct presentations of copper deficiency myeloneuropathy and cytopenias in a patient using excessive zinc-containing denture adhesive.

OBJECTIVES: While copper deficiency has long been known to cause cytopenias, copper deficiency myeloneuropathy is a more recently described entity. Here, we present the case of two clinically distinct presentations of acquired copper deficiency syndromes secondary to excessive use of zinc-containing denture adhesive over five years: myeloneuropathy and severe macrocytic anemia and neutropenia. METHODS: Extensive laboratory testing and histologic evaluation of the liver and bone marrow, were necessary to rule out other disease processes and establish the diagnosis of copper deficiency. RESULTS: The initial presentation consisted of a myelopathy involving the posterior columns. Serum and urine copper were significantly decreased, and serum zinc was elevated. On second presentation (five years later), multiple hematological abnormalities were detected. Serum copper was again decreased, while serum zinc was elevated. CONCLUSIONS: Zinc overload is a preventable cause of copper deficiency syndromes. This rare entity presented herein highlights the importance of patient, as well as provider, education.

Transiently Pink-Tinged Serum in a Patient With Multiple Myeloma and Anemia Undergoing Lenalidomide Treatment.

OBJECTIVES: While in vitro hemolysis is a preanalytical interferent, in vivo hemolysis is a pathologic process requiring investigation. We present a case of an anemic patient with multiple myeloma undergoing chemotherapy with lenalidomide who had multiple serum samples drawn before and after chemotherapy treatment. Some of these samples showed hemolysis. This triggered further investigations to differentiate the cause of the hemolysis. METHODS: Various laboratory tests and additional investigations were necessary to establish the root of the hemolytic process. RESULTS: Multiple laboratory tests and a rigorous review of the samples, time of collection, and laboratory results revealed that only samples collected shortly after lenalidomide administration showed hemolysis. This indicates that the chemotherapeutic agent itself was most likely the proximate cause of the in vivo hemolysis in a non-immune-mediated manner. CONCLUSIONS: Upon administration, chemotherapeutic agents, such as lenalidomide, can immediately induce transient hemolysis, which can be visualized as transiently pink-tinged serum samples.

Effects of temperature and light on the stability of bilirubin in plasma samples.

BACKGROUND: Although it is known that bilirubin is photo-sensitive, detailed effects of both temperature and artificial light exposure on bilirubin stability in plasma have not been well investigated. We determined the impact of temperature and artificial light on bilirubin stability in plasma. METHODS: Plasma total and direct bilirubin were analyzed using a diazo method. The aliquots of 38 samples were stored at 3°C and 22°C with light protection for 2, 4, 8, and 24h respectively before analysis. The aliquots of 20 samples with normal bilirubin and additional 20 with elevated bilirubin were exposed to artificial light for 2, 4, 8, 24, and 48 h at 22°C, and total and direct bilirubin were measured. The differences between the baselines and subsequent measurements were analyzed with analysis of variance. RESULTS: The baseline total bilirubin was 9.6±8.1 mg/dl (mean±SD) and the concentrations were 9.6±8.2, 9.0±7.4, 9.0±7.5, and 8.8±7.5 mg/dl at 3°C and 9.5±8.1, 9.0±7.4, 9.6±8.1, and 9.5±8.0 mg/dl at 22°C after 2, 4, 8, and 24 h (p>0.05, n=38). The baseline direct bilirubin was 1.3±1.2 mg/dl and the concentrations after 2, 4, 8, and 24 h were 1.4±1.2, 1.4±1.2, 1.5±1.2, and 1.3±1.1 mg/dl at 3°C and 1.4±1.1, 1.3±1.1, 1.3±1.1, and 1.3±1.0 mg/dl at 22°C (p>0.05, n=19). In samples with elevated bilirubin exposed to light at 22°C, the baseline total and direct bilirubin concentrations were 10.2±1.7 mg/dl and 5.0±1.9 mg/dl, respectively. After 2, 4, 8, 24, and 48 h, total bilirubin concentrations were 10.1±1.8, 10.0±1.8, 10.0±1.8, 9.3±2.0 (p>0.05, n=20), and 8.4±2.3 (p<0.01, n=20) mg/dl and direct bilirubin concentrations were 4.9±1.8, 4.9±1.9, 4.8±1.8, 4.2±1.6 (p>0.05, n=20), and 3.5±1.5 (p<0.01, n=20) mg/dl. For samples with normal bilirubin levels under the same conditions, the average baseline total and direct bilirubin concentrations were 0.7±0.1 mg/dl and below the lower limit of quantification (LLOQ), respectively. After 2, 4, 8, 24, and 48 h, the average total bilirubin concentrations were 0.7±0.1, 0.6±0.1, 0.6±0.1 (p>0.05, n=20), 0.5±0.1, and 0.4±0.1 mg/dl (p<0.01, n=20) and direct bilirubin concentrations were still below LLOQ. CONCLUSIONS: Bilirubin in plasma is stable in refrigerator or at room temperature without light exposure for at least 24 h. In normal laboratory environment, a delay of up to 8 h in the measurement of bilirubin left unprotected from light at room temperature does not significantly affect the results. Under these conditions, the changes in bilirubin concentration are not clinically significant until 24 h (direct bilirubin) and after 48 h (total bilirubin).

FGF-16 is a target for adrenergic stimulation through NF-kappaB activation in postnatal cardiac cells and adult mouse heart.

AIMS: The fibroblast growth factor (FGF) family plays an important role in cardiac growth and development. However, only FGF-16 RNA levels are reported to increase during the perinatal period and to be expressed preferentially in the myocardium, suggesting control at the transcriptional level and a role for FGF-16 in the postnatal heart. Beyond the identification of two TATA-like elements (TATA1 and TATA2) in the mouse FGF-16 promoter region and the preferential cardiac activity of TATA2, there is no report of Fgf-16 gene regulation. Assessment of promoter sequences, however, reveals putative nuclear factor-kappaB (NF-kappaB) elements, suggesting that Fgf-16 is regulated via NF-kappaB activation and thereby implicated in a number of cardiac events. Thus, the Fgf-16 gene was investigated as a target for NF-kappaB activation in cardiac cells. METHODS AND RESULTS: Assessments of Fgf-16 promoter activity were made using truncated and transfected hybrid genes with NF-kappaB inhibitors and/or beta-adrenergic stimulation via isoproterenol (IsP) treatment (a known NF-kappaB activator) in culture, and on endogenous mouse and human Fgf-16 genes in situ. The mouse Fgf-16 promoter region was stimulated in response to IsP treatment, but this response was lost with NF-kappaB inhibitor pretreatment. Deletion analysis revealed IsP responsiveness linked to sequences between TATA2 and TATA1 and, more specifically, a NF-kappaB element upstream and adjacent to TATA1 that associates with NF-kappaB p50/p65 subunits in chromatin. Finally, TATA1 and the proximal NF-kappaB element are conserved in the human genome and responsive to IsP. CONCLUSION: The mouse and human Fgf-16 gene is a target for NF-kappaB activation in the postnatal heart.

A myocyte enhancer factor 2 (MEF2) site located in a hypersensitive region of the FGF16 gene locus is required for preferential promoter activity in neonatal cardiac myocytes.

Fibroblast growth factor 16 (FGF16) is preferentially expressed in the heart after birth, suggesting its regulation is associated with tissue-specific chromatin remodeling and DNA-protein interactions. Here we have mapped the transcription initiation site of murine FGF16 to approximately 1.1 kilobases (kb) upstream of the translation start codon (ATG). Hybrid reporter genes directed by about 4.7 kb of upstream FGF16 DNA were expressed specifically in transfected neonatal rat cardiac myocytes, as well as in the heart of transgenic mice. A DNaseI hypersensitive site was mapped to a region about 1.2 kb upstream of the transcription initiation site in heart but not kidney tissue, and a nuclease protection assay gave evidence of a cardiac-specific protein-DNA interaction in this region. Deletion analysis indicated that a hybrid gene with 1205 bp but not 1054 bp of upstream DNA directed FGF16 promoter activity in transfected neonatal rat cardiac myocytes. We identified a putative myocyte enhancer factor 2 (MEF2)-binding site at nucleotides -1159/-1148, confirmed by electrophoretic mobility shift assay and MEF2 antibody binding. Mutation of the MEF2 site resulted in a blunting of FGF16 promoter activity in transfected neonatal rat cardiac myocytes. These data suggest that chromatin remodeling and MEF2 binding in the FGF16 promoter contribute to expression in the postnatal heart.