Artificially raised creatinine concentrations due to analytical interference for samples contaminated with total parenteral nutrition fluid.

BACKGROUND: A sample received in the laboratory from a patient receiving total parenteral nutrition (TPN) indicated that the patient may have renal dysfunction, but the results were not considered to be reliable enough to report. Investigations using a reference method for measurement of creatinine confirmed positive interference in the creatinine assay and distribution of samples via an External Quality Assessment (EQA) Scheme showed that this positive interference was method dependent. METHODS: Residual TPN fluid (Nutriflex Lipid Special) left in the bag after the patient had completed the infusion was collected and added to a patient serum pool in increasing amounts and distributed to different laboratories for analysis of creatinine and glucose through an EQA Scheme. RESULTS: Positive interference in a number of different creatinine assays was identified as a result of a component in the TPN fluid. Positive interference from high concentrations of glucose has been demonstrated to be a cause for falsely high results in Jaffe creatinine assays. CONCLUSIONS: The concern would be that a sample contaminated with TPN fluid would have both abnormal electrolytes and creatinine concentrations and give the impression that the patient was in renal failure due to analytical interference in the creatinine assay and laboratory staff need to be aware of this problem.

Use of Dried Blood Spot Specimens to Monitor Patients with Inherited Metabolic Disorders.

Monitoring of patients with inherited metabolic disorders (IMDs) using dried blood spot (DBS) specimens has been routinely used since the inception of newborn screening (NBS) for phenylketonuria in the 1960s. The introduction of flow injection analysis tandem mass spectrometry (FIA-MS/MS) in the 1990s facilitated the expansion of NBS for IMDs. This has led to increased identification of patients who require biochemical monitoring. Monitoring of IMD patients using DBS specimens is widely favoured due to the convenience of collecting blood from a finger prick onto filter paper devices in the patient's home, which can then be mailed directly to the laboratory. Ideally, analytical methodologies with a short analysis time and high sample throughput are required to enable results to be communicated to patients in a timely manner, allowing prompt therapy adjustment. The development of ultra-performance liquid chromatography (UPLC-MS/MS), means that metabolic laboratories now have the capability to routinely analyse DBS specimens with superior specificity and sensitivity. This advancement in analytical technology has led to the development of numerous assays to detect analytes at low concentrations (pmol/L) in DBS specimens that can be used to monitor IMD patients. In this review, we discuss the pre-analytical, analytical and post-analytical variables that may affect the final test result obtained using DBS specimens used for monitoring of patients with an IMD.

Effect of blood volume on analytical bias in dried blood spots prepared for newborn screening external quality assurance.

Aim: Dried blood spots (DBS) are used for the analysis of more than 2000 biomarkers. We assessed a range of analyte concentrations and diameters of DBS. Materials & methods: DBS samples were created by the application of increasing volumes of whole blood prepared by the UK NEQAS Quality Assurance Laboratory. Samples were analyzed in four separate laboratories. Results: Volumes less than 25 µl (8 mm) and more than 75 µl (14 mm) created unsatisfactory analytical biases. Results obtained from peripheral subpunches tended to be higher than those from a central subpunch. Conclusion: DBS diameters formed from nonvolumetric application of blood to filter paper can be used to assess whether measurement bias will be within acceptable limits according to the analyte being quantified. DBS received for newborn screening in the UK with diameters less than 8 mm and those more than 14 mm should be rejected.

Newborn screening for sickle cell disorders using tandem mass spectrometry: three years' experience of using a protocol to detect only the disease states.

Background Tandem mass spectrometry (MS/MS) has recently become an alternative method for the newborn screening of sickle cell disorders (SCD), as it is able to detect haemoglobin (Hb) peptides following digestion of bloodspots with trypsin. Using the SpOtOn Diagnostics Reagent Kit, we previously developed a screening protocol to detect only the disease states of SCD, using action values based on the ratio between the variant Hb peptide to wild-type peptide abundances for the HbS, C, DPunjab, OArab, E and Lepore peptides. Methods Action values using the ratios between the wild type HbA (ßT1-3) peptides and the foetal Hb (γT2) peptide were developed to identify bloodspot samples from premature and transfused infants. An evaluation was undertaken to assess the transferability of the action values onto an additional MS/MS instrument. We report here our experience using this MS/MS protocol. Results During a three-year period, we screened 100,456 babies and identified 10 SCD cases (1 HbS/HPFH, 5 HbS/S and 4 HbS/C) and a case of HbE/ß-thalassaemia that was identified as a by-product. The Hb variant to wild-type peptide ratio action values were transferable to a second MS/MS instrument. Our protocol prevented the identification of an estimated 810 carrier infants. Gestational age-related action values for HbA to HbF peptide ratios were required to minimize the number of samples referred for second-line testing to exclude ß-thalassaemia. Conclusion MS/MS is a robust alternative screening technology for SCD; in addition, it also optimizes the use of equipment and expertise that currently exist in newborn screening laboratories.

Effect of Dried Blood Spot Quality on Newborn Screening Analyte Concentrations and Recommendations for Minimum Acceptance Criteria for Sample Analysis.

BACKGROUND: The analysis of dried blood spots has been used routinely for newborn screening since the early 1970s, and the number of disorders screened has expanded substantially in recent years. However, there is a lack of evidence regarding minimum blood spot quality acceptance criteria for sample analysis. METHODS: Blood pools were spiked with phenylalanine, tyrosine, leucine, methionine, octanoylcarnitine, decanoylcarnitine, isovalerylcarnitine, glutarylcarnitine, thyroid-stimulating hormone, and immunoreactive trypsinogen to concentrations at the analytical cutoffs used in UK screening protocols. We evaluated the effect of sample volume applied to the card (10, 20, 50, 75, and 100 µL), punch location (central vs peripheral), and sample quality (double layering, applying blood to both sides of the filter paper, multispotting, applying insufficient sample, and compressing the sample after application). RESULTS: Compression of blood spots produced significantly lower results (14%-44%) for all analytes measured (P < 0.001). Smaller blood spots produced significantly lower results (15%-24% for 10-µL vs 50-µL sample size) for all analytes at all concentrations measured (P < 0.001). Results obtained from peripheral punches were higher than those from a central punch, although this did not reach statistical significance for all analytes. Insufficient and multispotted samples demonstrated heterogeneous results. CONCLUSIONS: All blood spots containing ≤20 µL (blood spot diameter <8 mm), those in which blood has not fully penetrated the filter paper, and all samples with evidence of compression should be rejected, since there is a risk of producing false-negative results.

The use of trypsin to confirm the presence of macrocreatine kinase on isoenzyme electrophoresis.

BACKGROUND: Macrocreatine kinase (MCK) type 1 is a high molecular weight form of CK that is non-pathological. Identification of MCK is beneficial in preventing unnecessary investigations that may follow a persistently elevated CK of unknown origin. Currently, gel filtration chromatography can be used as a confirmatory technique, but it is laborious, time-consuming and expensive. The aim of this work, carried out as part of a larger investigation into the prevalence of MCK, was to determine whether trypsin can be used as an alternative to confirm the presence of MCK on isoenzyme electrophoresis. METHODS: Five samples found to have bands running in the MCK region on isoenzyme electrophoresis were treated with trypsin. Electrophoresis was carried out using the Helena Biosciences Sas-1 Plus system. These samples were also analysed by the confirmatory technique of gel filtration chromatography. RESULTS: Of the five samples treated with trypsin, three were found to be MCK-positive and two MCK-negative. These results correlated with those obtained by the reference method of gel filtration chromatography. CONCLUSIONS: There appears to be a potential use of trypsin in confirming the presence of MCK following isoenzyme electrophoresis. If these findings were verified, this would provide a less labour-intensive, less time-consuming and more cost-effective confirmatory technique. Further study is required, which needs to be expanded to include a larger number of patients, before this method can be adopted routinely.