The role of calculated non-caeruloplasmin-bound copper in Wilson's disease.

Background US and European guidelines suggest the use of calculated non-caeruloplasmin-bound copper (free copper index) for the diagnosis and management of Wilson's Disease. However, there is concern that the required analytical measurements of caeruloplasmin and copper may not be sufficiently robust at the concentrations usually found. Methods Aliquots of six plasma specimens were sent to laboratories participating in the UK National External Quality Assessment Scheme for copper and caeruloplasmin. The variability of these two reported measurements and the calculated non-caeruloplasmin-bound copper concentrations were compared. The variability of caeruloplasmin reference ranges quoted by laboratories was also investigated. Results No laboratories use the required enzymatic methods in the calculation of non-caeruloplasmin-bound copper. The interlaboratory variations in caeruloplasmin concentrations and calculated non-caeruloplasmin-bound copper concentrations were very considerable so making clinical interpretation unreliable. Wide differences in the caeruloplasmin reference ranges used were also found. Conclusions Such variations of the calculated non-caeruloplasmin-bound copper concentrations and the predominant use of immunological methods for measuring caeruloplasmin preclude a clinical role for this calculated value in the investigation of Wilson's disease.

The importance of commutability of reference materials used as calibrators: the example of ceruloplasmin.

BACKGROUND: Different methods for ceruloplasmin tend to give different results in external quality assessment schemes. During the production of the certified reference material ERM-DA470k/IFCC discrepant measurement results were also found for ceruloplasmin measured with different methods, and consequently the protein could not be certified in the material. METHODS: We performed a commutability study with 30 serum samples and the reference materials ERM-DA470, ERM-DA470k/IFCC, and ERM-DA472/IFCC, using 6 different methods. Data were analyzed according to the CLSI Guideline C53-A to assess whether the reference materials had the same behavior as the serum samples with respect to measurement results obtained with combinations of the methods used. RESULTS: Measurement results from different methods showed a good linear correlation for the serum samples. ERM-DA470 showed marked noncommutability for certain combinations of methods. ERM-DA470k/IFCC and ERM-DA472/IFCC were commutable for more combinations of methods. The lack of commutability of ERM-DA470 for certain combinations of methods correlates with results from the UK National External Quality Assessment Service showing discrepancies between results from these methods. For serum stored in the presence of sodium azide the results from different methods are essentially equivalent. CONCLUSIONS: Ceruloplasmin in ERM-DA470 is a fully documented example of a situation in which, due to lack of commutability, the use of a common material for calibration did not lead to harmonization .

The UKNEQAS scheme for cerebrospinal fluid haem pigments: a paradigm for service improvement.

We describe the programme of an established External Quality Assurance (EQA) provider and a Specialist Advisory Group (SAG) to develop a successful EQA scheme for cerebrospinal fluid (CSF) haem pigments as an example of a professionally led, unfunded initiative with the real potential to benefit patients. Within three years, we had assured sample stability, stoichiometry, and published best practice guidelines, enabling both analytical results and interpretation to be assessed and reported with an educative summary of the desired responses. Misclassification scoring of analysis and interpretation was introduced. Following audit, guidelines were modified and republished. The outcomes were as follows: Participant numbers increased from 63 at inception to 150 10 years later; The percentage of participants using visual inspection, a poor practice indicator, decreased from 27% to less than 1%; In all, 94-100% of participants consistently detected minor increases in bilirubin over the last four years of the scheme; More than 93% of participants were able to interpret analytical results linked to straightforward clinical scenarios; Misclassification scoring demonstrated that more complex scenarios repeatedly posed problems and is the next challenge to address. Scheme success is attributed to the experience of the operator and the formation of a voluntary expert advisory group, with both concerned to advance science and patient safety and thus contribute unpaid time and effort in order to succeed. In times of fiscal constraint, such resource may not be so readily available, yet is a vital part of continuous quality improvement for the benefit of patients.

A monoclonal protein identified by an anomalous lipaemia index.

We describe a patient being investigated for anaemia where the lipaemia index on a Beckman Coulter DxC800 analyser was markedly elevated and out of keeping with the visual appearance of the serum. Subsequent investigation revealed a monoclonal IgM kappa immunoglobulin with type I cryoglobulin behaviour. The patient was then diagnosed with a non-Hodgkin B-cell lymphoma. We later identified a second patient with a similar anomalous index with an IgM lambda paraprotein, and a known marginal zone splenic lymphoma but were unable to confirm cryoglobulin behaviour prior to treatment. A review of 50 consecutive IgM paraproteins revealed no other anomalous lipaemia indices. We postulate that it is the properties of the paraprotein that determine its cryoglobulin behaviour that also render it susceptible to precipitation in the index diluent, not the fact of it being an IgM paraprotein per se. This appears to be the first reported case of a paraprotein identified following an anomalous lipaemia index.

CSF spectrophotometry for bilirubin--why and how?

In patients presenting with a possible subarachnoid haemorrhage (SAH), a negative CT scan of the head does not exclude SAH and further investigations are therefore required. Cerebral angiography identifies aneurysms but does not inform on whether they have ruptured and is resource intensive. Examination of the CSF for blood cannot distinguish between an in-vivo bleed and a traumatic lumbar puncture. Visual inspection of the CSF supernatant fluid for xanthochromia is insensitive and should not be used on any account. The most appropriate investigation is spectrophotometry of the CSF for the haemoglobin breakdown products, oxyhaemoglobin and bilirubin. Guidelines for the performance of spectrophotometry and interpretation have been produced, modified and are reviewed here. From 5 years' data involving 2302 scans, 92% did not support the occurrence of SAH, 4% indicated the need for angiography to identify a possible aneurysm, while 4% were equivocal due to the presence of oxyhaemoglobin in sufficient concentrations to interfere with the ability to identify bilirubin reliably.

Revised national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage.

It is crucially important to detect subarachnoid haemorrhage (SAH) in all patients in whom it has occurred to select patients for angiography and preventative surgery. A computerized tomography (CT) scan is positive in up to 98% of patients with SAH presenting within 12 h, but is positive in only 50% of those presenting within one week. Cerebrospinal fluid (CSF) bilirubin spectrophotometry can be used to determine the need for angiography in those few CT-negative patients in whom clinical suspicion of SAH remains high; it may remain positive up to two weeks after the event. A lumbar puncture (LP) should only be performed >12 h after the onset of presenting symptoms. Whenever possible collect sequential specimens. Always ensure that the least blood-stained CSF sample taken (usually the last) is sent for bilirubin analysis. Protect the CSF from light and avoid vacuum tube transport systems, if possible. Always use spectrophotometry in preference to visual inspection. All CSF specimens are precious and should always be analysed unless insufficient sample is received. Centrifuge the specimen at >2000 rpm for 5 min as soon as possible after receipt in the laboratory. Store the supernatant at 4 degrees C in the dark until analysis. An increase in CSF bilirubin is the key finding, which supports the occurrence of SAH but is not specific for this. In most positive cases, bilirubin will occur with oxyhaemoglobin.

What is the role of cerebrospinal fluid ferritin in the diagnosis of subarachnoid haemorrhage in computed tomography-negative patients?

BACKGROUND: Spectrophotometry of cerebrospinal fluid (CSF) for bilirubin is the recommended method for investigation in suspected cases of subarachnoid haemorrhage (SAH), when a computed tomography (CT) of the head is negative for blood. There is a potential need for a simpler alternative. Measurement of CSF ferritin might fulfil this need. METHOD: We have measured ferritin in the CSF from 252 patients with suspected SAH who were negative on a CT of the head for blood, recruited on a consecutive intention to recruit basis from four centres. CSF spectrophotometry was performed on all samples. A positive outcome was taken as an aneurysm found on angiography that was treated or a discharge diagnosis of non-aneurysmal SAH. RESULTS: A final diagnosis of aneurysmal SAH was made in six patients, an arteriovenous malformation in one and non-aneurysmal SAH in nine. Receiver operating characteristic (ROC) analysis showed that at 6.4 microg/L, sensitivity, specificity, positive and negative predictive values were 1.0, 0.48, 0.12 and 1.0, respectively. At 12 microg/L, these values were 0.81, 0.91, 0.38 and 0.98, respectively. CONCLUSIONS: At an appropriate negative predictive value (1.0) for a rule-out test, ferritin has too low a specificity to function as a stand-alone test and we cannot recommend it as an initial screen to be followed by spectrophotometry.

Can serum free light chains replace urine electrophoresis in the detection of monoclonal gammopathies?

BACKGROUND: We investigated how sensitive serum free light chain (FLC) analysis was for the detection of Bence-Jones protein (BJP) and whether a serum kappa/lambda ratio could replace urine electrophoresis as part of the investigation algorithm for monoclonal gammopathy. METHODS: Serum kappa and lambda FLC analysis was performed on 932 consecutive patients investigated for monoclonal gammopathy, along with serum electrophoresis, serum immunofixation where appropriate, and in 483 individuals urine immunofixation. A reference interval for the kappa/lambda ratio was derived from all patients who had normal serum and urine electrophoresis and no B-cell dyscrasia (n = 312). RESULTS: The 100% reference interval was 0.21-1.44 (median 0.50), lower than that previously published. From the 483 patients who provided urines, BJP was detected in 34, with an abnormal kappa/lambda ratio in 26 of these. Using the reference interval, sensitivity, specificity, negative and positive predictive values of an abnormal kappa/lambda ratio for the presence of BJP were 0.76, 0.96, 0.98 and 0.59 respectively. Seven patients had an abnormal kappa/lambda ratio with no paraprotein. CONCLUSION: All kappa/lambda results should be interpreted against a reference interval appropriate for the analyser in use and the population under study. Some urines will be positive for BJP when the serum kappa/lambda ratio is normal, but there is little difference in outcome whether urine for BJP or serum FLC analysis is included in the screening strategy for monoclonal gammopathies. On balance, we believe that it is not yet time to dispense with urine BJP in the investigation of patients with suspected monoclonal gammopathy.

National audit of cerebrospinal fluid testing.

BACKGROUND: UK National External Quality Assessment Service (NEQAS) Specialist Advisory Group for EQA of CSF Proteins and Biochemistry was interested in current practice for the biochemical investigation of cerebrospinal fluid (CSF) in the UK. METHODS: A questionnaire was sent to laboratories via regional audit committees and the results collated. RESULTS: Most laboratories were analysing CSF in a satisfactory manner. There was some variation in the reference ranges used for glucose, protein and lactate. There was concern about the rejection policies of some laboratories on these unrepeatable samples and the wavelengths used to measure bilirubin. The survey revealed the lack of spectrophotometric scanning for haem pigments and bilirubin in some hospitals. CONCLUSIONS: The current practice for the measurement of CSF samples in the UK is satisfactory in most laboratories responding to the questionnaire. National agreement on reference ranges for glucose, protein and lactate should be achievable. Those performing spectrophotometric scanning of the CSF were doing so in concordance with the national guidelines. Some hospitals in the UK may not have responded to the questionnaire because they did not offer spectrophotometric scanning.

Can opportunistic case-finding of paraproteins be clinically justified?

The addition of serum protein electrophoresis by laboratory staff upon finding an increased total protein or globulin appears to be practised widely in the UK. The criteria for assessing which samples are subject to electrophoresis vary considerably. They consist of initial objective laboratory data subsequently modified, somewhat subjectively, by other laboratory data and clinical details, but have often been chosen pragmatically. The aim of the practice is to identify patients with occult B-cell malignancies that warrant treatment. While it has been lent legitimacy in many cases by involving clinical haematologists in discussions, the views of other stakeholders, including other physicians and patients, have often not been considered, thus raising a number of ethical questions that need to be addressed. The practice is reviewed against both current knowledge of B-cell malignancies and monoclonal gammopathy of undetermined significance and criteria for screening, of which this forms a particular example. The arguments for and against addition of electrophoresis are finely balanced, partly because of the very limited outcome data available. We conclude that those currently following this practice should continue to do so, there is a need to establish outcome data as widely as possible according to standard criteria and there should be involvement of physicians, patients and national bodies in discussions about the practice so that the practical and ethical issues can be addressed.