Experimental and computer modelling speciation studies of the effect of pH and phosphate on the precipitation of calcium and magnesium salts in urine.

BACKGROUND: pH and phosphate concentration are the major determinants of precipitation in urine of the salts of calcium and magnesium. This study aims to model the process of salt precipitation and establish whether the acidification of urine samples is necessary for the accurate measurement of calcium and magnesium in a clinical laboratory setting. METHODS: Urine samples were collected from 21 patients, aliquots were taken from each patient sample and the pH was adjusted to cover the range 2.0-10.0. The analytical and biological variation for each analyte was established and used to calculate percentage changes and critical differences. The critical difference was used to assess whether there was a significant difference between acidified and un-acidified samples. The JESS (Joint Expert Speciation System) thermodynamic computer-modelling program was used to predict the distribution of salt species formed with varying pH values and phosphate levels in simulated urine. RESULTS: The results showed that at a pH greater than 6.5, measured calcium, magnesium and phosphate significantly decreased as a result of precipitation (p<0.0001), although the critical difference was generally not exceeded. Computer modelling showed that both pH and phosphate concentration affected the distribution of salt species formed, as well as the precipitation patterns of calcium and magnesium phosphates. Overall, calcium phosphate precipitation tends to predominate at lower phosphate concentrations and at pH values below about 6.5, while both calcium and magnesium phosphate precipitation occur at higher phosphate concentrations and pH values greater than 6.5. CONCLUSIONS: For accurate analysis of these analytes in urine, the pH should be routinely measured and acidification should be undertaken prior to analysis if the pH is greater than 6.5. Based on the findings of this study, acidification or the lack of it does not result in a clinically significant change in calcium, magnesium and phosphate measured in urine. This study also predicted the likely salt species formed at varying urinary pH values and phosphate concentrations.

Can lithium-heparin plasma be used for protein electrophoresis and paraprotein identification?

INTRODUCTION: Lithium-heparin plasma is the most commonly used sample type in many hospitals, but it has been suggested that it is not suitable for protein electrophoresis due to the presence of fibrinogen, which can potentially mask a paraprotein band or be misconstrued as one. Here we aimed to demonstrate that lithium-heparin plasma samples could be used for protein electrophoresis and paraprotein typing without or with ethanol treatment to remove the fibrinogen. METHOD: A lithium-heparin sample from a patient with IgGlambda, IgGkappa, IgAlambda and IgAkappa myeloma, a non-specific polyclonal increase and a serum control were treated with ethanol prior to protein electrophoresis. Immunofixation electrophoresis was undertaken to investigate the effect of ethanol treatment on immunoglobulin and light chains. Nephelometry was undertaken to investigate whether ethanol treatment affected the quantification of IgG levels. Densitometric evaluation of proteins after electrophoresis was used to study whether ethanol treatment affected other serum proteins. An audit was also undertaken to ascertain the magnitude of the potential interference from the fibrinogen band in heparinized samples. RESULTS AND CONCLUSIONS: Ethanol treatment significantly but incompletely removed the fibrinogen in lithium-heparin plasma samples and did not affect the integrity of any of the proteins investigated. Even without ethanol treatment, lithium-heparin plasma can be used for protein electrophoresis and paraprotein identification as the instances of interference between fibrinogen and paraproteins was low (2.3%). In rare cases where there is uncertainty or ambiguity, immuno-fixation electrophoresis is recommended. This report has implications in terms of reducing costs and turn-around time as it prevents the need for requesting another serum sample from patients. This may be one step towards a universal sample for all tests.

Mechanism of interference by haemolysis in the cardiac troponin T immunoassay.

BACKGROUND: The cardiac troponins have been shown to be sensitive and specific biochemical markers of myocardial infarction and highly prognostic for future adverse events in patients with acute coronary syndromes. There have been reports suggesting that haemolysis causes a negative interference in the cardiac troponin T (cTnT) assay but the mechanism(s) involved remain unknown. Here we show the effects of haemolysis and haemoglobin per se on the cTnT assay. METHODS: The effect of haemolysis was studied by the addition of prepared haemolysate to serum samples with known and clinically relevant cTnT levels. The effect of haemoglobin was studied by the addition of haemoglobin of increasing concentrations and noting its effect on the level of cTnT measured. The effect of putative proteases was determined indirectly by incubating samples with spiked cTnT with various protease inhibitors and observing the changes in the measured cTnT levels. RESULTS: The results show that both haemolysis, which is the release of haemoglobin and corpuscular contents, and haemoglobin itself negatively interfere in the cTnT assay in a concentration-dependent manner, although the former had a greater magnitude of effect. On haemolysis, indirect evidence suggests that proteases are released which degrade the cTnT in serum, thus causing the decreased levels detected. Pepstatin A, a reversible inhibitor of aspartic proteinases, effectively inhibited the loss of cTnT in serum at 37 degrees C and pH 7.4 over a 48-h period. We found that at a haemoglobin level of 0.75 g/L, cTnT declined by more than 10% of the initial concentration, suggesting that falsely decreased levels due to haemolysis may significantly affect the clinical utility of the assay. CONCLUSIONS: Haemolysis, haemoglobin per se and possibly proteolysis play a role in the negative interference in cTnT assays. Measures to reduce this interference must be implemented.

Pneumatic tube system induced haemolysis: assessing sample type susceptibility to haemolysis.

BACKGROUND: The pneumatic tube system (PTS) has been implicated in inducing haemolysis. It is not known whether certain sample types are more susceptible to haemolysis than others. We assessed the level of haemolysis in commonly used sample types in the clinical biochemistry department when transported through the PTS. METHOD: Blood was collected in pairs for different sample types and sent to the laboratory via the pneumatic tube or delivered by a porter. Haemolysis indices were measured spectrophotometrically and compared for each pair of sample type. RESULTS: Our results suggest that plain serum samples are more susceptible to haemolysis than the other sample types when sent through our PTS (P <0.0001). Compared with serum with gel samples, plain serum samples are more prone to haemolysis (P <0.001). This suggests that gel may confer some protection against haemolysis. CONCLUSION: Different hospitals will have varying system configurations and use different sample types. We recommend that each hospital investigate their own system to assess whether haemolysis is a recurring problem in any of the sample types transported.