Routine Hospital Laboratory Research Articles

Determination of 11 minerals in children using inductively coupled plasma mass spectrometry

BackgroundMinerals play an important role in children’s growth and cognition. In this study, our aim was to establish reference intervals of mineral level in Children of Liaoning province (China) and provide a reliable basis for future preventive healthcare guidelines.MethodsRandom samples of 2217 healthy children aged 0–15 years who were referred for routine hospital laboratory examinations in the cities of Shenyang, Fushun, Fuxin, Benxi, Chaoyang and Lingyuan were invited to participate in the study. Serum levels of Calcium (Ca), Iron (Fe), Zinc (Zn), Magnesium (Mg), Copper (Cu), Manganese (Mn), Selenium (Se), Mercury (Hg), Nickel (Ni), Cobalt (Co), and Lithium (Li) were determined by inductively coupled plasma mass spectrometer(ICP-MS).ResultsSex-related significant differences in the serum concentrations of the Mg and Cu (P < 0.05). Age-related significant differences were found in serum levels of Ca, Fe, Zn, Mg, Cu and Mn (P < 0.05) and the concentrations of 9 minerals (Ca, Mg, Cu, Mn,Se,Hg,Ni,Co and Li in serum varied from season to season (P < 0.05). The Spearman correlation matrix of 11 minerals was shown as a heat map, the correlations between Ca-Zn, Ca-Mg, Fe-Zn, Fe-Se, Mn-Ni, Se-Hg, Se-Ni, Hg-Ni, and Ni-Co, Ni-Li were the strongest compared with others.ConclusionsThe reference intervals of serum 11 minerals for children should considering the sex, age and season, which may be useful for decisive diagnoses of abnormality of the 11minerals and the related diseases.

Detection of MET Exon 14 Skipping Alterations in Lung Cancer Clinical Samples Using a PCR-Based Approach.

The receptor tyrosine kinase (RTK) c-MET plays important roles in cancer, yet despite being frequently overexpressed, clinical responses to targeting this receptor have been limited in the clinical setting. A singular significant challenge has been the accurate identification of biomarkers for the selection of responsive patients. However, recently mutations which result in the loss of exon 14 (called METex14 skipping) have emerged as novel biomarkers in non-small cell lung carcinomas (NSCLC) to predict for responsiveness to targeted therapy with c-MET inhibitors. Currently, the diverse genomic alterations responsible for METex14 skipping pose a challenge for routine clinical diagnostic testing. Next generation sequencing (NGS) is the current gold standard for identifying the diverse mutations associated with METex14, but the cost for such a procedure remains to some degree prohibitive as often NGS is requested on a case-by-case basis, and many hospitals may not even have the capacity or resources to conduct NGS.However, PCR-based approaches to detect METex14 have been developed which can be conducted in most routine hospital laboratories and may therefore allow a cost-effective approach to pre-screen patients that may respond to c-MET inhibitors prior to conducting NGS, or until all patients will have NGS conducted as routine practise. In this chapter, we describe one such PCR-based approach for screening samples for the detection of METex14 in NSCLC.

A review of biomarker utilization in the diagnosis and management of acute pancreatitis reveals amylase ordering is favored in patients requiring laparoscopic cholecystectomy

BackgroundDespite widespread recommendations to favor lipase over amylase in the diagnosis and management of acute pancreatitis, many routine hospital laboratories still offer amylase testing. This study sought to evaluate and compare ordering patterns of amylase and lipase in patients with acute pancreatitis. MethodsWe analyzed 438 patients with acute pancreatitis admitted to our hospital. Data collection included pancreatitis etiology and management as well as biochemical profiles of amylase and lipase. We compared serial ordering patterns, degree of biomarker elevation, and normalization kinetics. ResultsAll patients had at least one lipase ordered during their admission, and only 51 patients (12%) had at least one amylase ordered. On average, lipase was elevated 5 times higher above its respective upper reference limit than amylase at admission. Pancreatitis etiology was skewed toward gallstones in the amylase group as compared to the lipase only group (69% vs. 43%), and surgical patients (laparoscopic cholecystectomy) were more likely to have amylase ordered and/or trended. ConclusionsAmylase measurement was not necessary in the diagnosis and management of 88% of patients with acute pancreatitis. Of patients for whom amylase was ordered, it was common for these patients to be those referred to surgical procedures, possibly because amylase normalization may be documented faster than that of lipase.

Using the hemoglobin-binding Staphylococcus aureus protein IsdH to enable plasma analysis of hemolyzed blood samples.

Background Intravascular hemolysis and in vitro hemolysis are prevalent contributors to failed blood sample analysis in the routine hospital laboratory. Interferences by hemoglobin in spectrophotometric and certain enzyme activity assays is the major causative factor. Methods By exploiting the hemoglobin-binding properties of the iron-regulated surface determinant H (IsdH) protein from Staphylococcus aureus we have developed a new method to instantly remove hemoglobin and hemoglobin-haptoglobin complexes from plasma in vitro thereby enabling the measurement of hemoglobin-sensitive analytes in hemolyzed plasma. In the present study we used an engineered IsdH mutant form conjugated to Sepharose for the efficient removal of plasma hemoglobin in concentrations up to 15 mg/mL. The high abundance of haptoglobin, which forms a tight complex with hemoglobin in plasma, did not affect the hemoglobin removal by IsdH Sepharose. Results Applying the method on plasma samples that beforehand were spiked with blood hemolysate re-enabled measurement of the hemolysis sensitive parameters: alkaline phosphatase, conjugated bilirubin, iron, ferritin, γ-glutamyltransferase, total thyroxine and troponin T. IsdH Sepharose-mediated hemoglobin removal also enabled measurement of hemolysis sensitive parameters in hemolyzed samples from anonymized patients. Conclusions In conclusion, IsdH Sepharose is a simple cost-effective pretreatment of hemolyzed samples correcting and enabling the measurement of several important hemoglobin-sensitive parameters in a way compatible with standard procedures in routine laboratories.

Determination of trace elements in Ethiopian, Vietnamese, and Japanese women using high-resolution IC-PMS

ObjectivesHumans and other living organisms require small quantities of trace elements throughout life. Both insufficient and excessive intakes of trace elements can have negative consequences. However, there is little information on serum level of trace elements in different populations. This study examines serum levels of trace elements in Ethiopian, Japanese, and Vietnamese women. MethodsRandom samples of healthy women who were referred for routine hospital laboratory examinations in the cities of Hanoi, Sapporo, and Gondar were invited to participate in the study. Serum levels of magnesium, zinc, copper, iron, selenium, and calcium were determined using an inductively coupled plasma mass spectrometer. Furthermore, body mass index of each study participant was determined. ResultsThe mean ± SD serum concentrations of zinc (μg/dL), copper (μg/dL), iron (μg/dL), selenium (μg/dL) and calcium (mg/dL), respectively, were 76.51 ± 39.16, 152.20 ± 55.37, 385.68 ± 217.95, 9.15 ± 4.21, and 14.18 ± 3.91 in Ethiopian women; 111.49 ± 52.92, 105.86 ± 26.02, 155.09 ± 94.83, 14.11 ± 3.41, and 11.66 ± 2.51 in Vietnamese women; and 60.69 ± 9.76, 107 ± 156, 268 ± 128, 8.33 ± 3.65, and 11.18 ± 0.68 in Japanese participants. Ethiopian women had significantly higher level of serum calcium than Vietnamese and Japanese women (both P < 0.05). Although the mean calcium concentration in Vietnamese women was higher than in women from Japan, the difference was not statistically significant (P > 0.05). Furthermore, compared with Japanese women, Ethiopian women had significantly high iron and copper concentrations (P < 0.05). Serum selenium and zinc levels were higher in Vietnamese than Ethiopian women. ConclusionThe study revealed a remarkable difference in serum concentrations of trace elements in women from different countries, implying differences in trace elements in the food or soil.

Plakoglobin immunolocalization as a diagnostic test for arrhythmogenic right ventricular cardiomyopathy.

A recent study using an anti-plakoglobin antibody and immunofluorescence methods in endomyocardial tissue specimens found that a marked reduction in plakoglobin staining was highly sensitive and specific for the diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC). The purpose of our study was to determine the diagnostic utility of plakoglobin immunolocalization using more standard immunoperoxidase methods suitable for clinical laboratories. Between January 2007 and October 2010, all patients at our center with suspected ARVC underwent noninvasive and genetic testing, right ventricular (RV) angiography, electrophysiologic studies, and endomyocardial biopsy from the RV septum. Several studies using anti-plakoglobin antibodies were performed using standard immunoperoxidase methods at concentrations of 1:50,000 and 1:75,000 after serial dilutions. Among 16 patients, nine patients fulfilled the clinical criteria for ARVC, and seven patients were found to have other cardiac diagnoses. In the initial study (1:50,000) only one of nine ARVC patients showed reduced plakoglobin signal while the others had normal staining. On repeat staining (1:75,000), reduced signal was observed in three of five of the ARVC patients compared to none in controls (four patients did not have adequate tissue for the repeat experiment). These results confirm that abnormal plakoglobin staining can differentiate biopsies from patients with ARVC from those with other myopathies, but with low sensitivity. Further, each specimen must be studied at a particular concentration due to variable antibody reactivity. The necessity for such fine-tuning of the reaction, as well as the subjectivity involved in interpretation of the results, would make this method difficult to utilize in routine hospital laboratories.

Chapter Two - Free Testosterone: Clinical Utility and Important Analytical Aspects of Measurement

Testosterone, the most abundant androgen in men, is a steroid hormone that is synthesized predominantly by the testes. In women, minor amounts are synthesized in the ovaries. Androgen precursors are also produced and secreted from the adrenal glands in both sexes, where they undergo peripheral conversion to testosterone. Circulating concentrations are approximately 15-25 times higher in adult men compared to women. Maintenance of these levels is necessary for development and maintenance of secondary sexual characteristics, libido, growth, prevention of osteoporosis, and most importantly in men, spermatogenesis. Most testosterone circulates tightly bound to sex hormone-binding globulin (SHBG) or weakly bound to albumin. A minor amount circulates as free testosterone, and it is believed that this is the metabolically active fraction. Measurement of free testosterone is important in the diagnosis of many diseases, most importantly disorders of androgen deficiency in men (i.e., hypogonadism) and androgen excess in women (i.e., polycystic ovary syndrome and hirsutism). Many methodologies are available for free testosterone measurement including the reference methods (equilibrium dialysis and ultrafiltration), analog immunoassay, and calculated free testosterone based on measurement of total testosterone, SHBG, and albumin. Moreover, measurement of bioavailable testosterone, a combination of albumin-bound and free testosterone, also has clinical utility and can be measured by selective protein precipitation or calculation. In this review, the advantages and limitations of each of these methods will be discussed in the context of clinical utility and implementation into a routine hospital laboratory. Furthermore, up and coming methodologies for free testosterone measurement, including liquid chromatography-tandem mass spectrometry, will also be discussed.

The evaluation of two automated soluble fibrin assays for use in the routine hospital laboratory

The soluble fibrin monomer (sFM) assay, like the D-dimer (DDi) assay, has the potential to be used both as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and as a thrombotic marker. It differs from DDi in that it is a much earlier produced fragment produced only by thrombin action on fibrinogen, whereas DDi is a much later produced fragment formed by plasmin cleavage of cross-linked fibrin. In our study, we compared two commercially available automated sFM assays in the routine hospital setting using samples obtained from the general hospital ward and the emergency room. The results obtained with the two automated assays (Stago LIA sFM assays and the LPIA-Iatro SF assay) were compared with each other and with the results obtained using the routine semiquantitative hemagglutination assay. The study showed that both automated assays were comparable with each other. No patient sample previously classified as positive would be missed, but with the higher sensitivity in the automated tests, more samples are positive. In conclusion, we suggest that both automated tests are suitable for routine laboratory use. Both assays had the advantage over the hemagglutination assay in that previously frozen samples could be used, and the assays are easier and quicker to perform. The LIA sFM Stago has slightly better sensitivity but has a tendency to lower specificity than the Iatro SF test.

Estimating rotavirus gastroenteritis hospitalisations by using hospital episode statistics before and after the introduction of rotavirus vaccine in Australia

IntroductionHospital discharge records and laboratory data have shown a substantial early impact from the rotavirus vaccination program that commenced in 2007 in Australia. However, these assessments are affected by the validity and reliability of hospital discharge coding and stool testing to measure the true incidence of hospitalised disease. The aim of this study was to assess the validity of these data sources for disease estimation, both before and after, vaccine introduction. MethodsAll hospitalisations at a major paediatric centre in children aged <5 years from 2000 to 2009 containing acute gastroenteritis (AGE) ICD 10 AM diagnosis codes were linked to hospital laboratory stool testing data. The validity of the rotavirus-specific diagnosis code (A08.0) and the incidence of hospitalisations attributable to rotavirus by both direct estimation and with adjustments for non-testing and miscoding were calculated for pre- and post-vaccination periods. ResultsA laboratory record of stool testing was available for 36% of all AGE hospitalisations (n=4948) the rotavirus code had high specificity (98.4%; 95% CI, 97.5–99.1%) and positive predictive value (96.8%; 94.8–98.3%), and modest sensitivity (61.6%; 58–65.1%). Of all rotavirus test positive hospitalisations only a third had a rotavirus code. The estimated annual average number of rotavirus hospitalisations, following adjustment for non-testing and miscoding was 5- and 6-fold higher than identified, respectively, from testing and coding alone. Direct and adjusted estimates yielded similar percentage reductions in annual average rotavirus hospitalisations of over 65%. ConclusionDue to the limited use of stool testing and poor sensitivity of the rotavirus-specific diagnosis code routine hospital discharge and laboratory data substantially underestimate the true incidence of rotavirus hospitalisations and absolute vaccine impact. However, this data can still be used to monitor vaccine impact as the effects of miscoding and under-testing appear to be comparable between pre and post vaccination periods.

Development and validation of a quantitative assay based on tandem mass spectrometry

Many routine hospital and clinical research assays have relied upon immunoassay procedures to achieve sensitive measurements of a range of important analytes. Some of the methods have been developed in-house but increasingly commercial kits and automated analysers have become commonplace. The accuracies of these methods are under question in health care. Mass spectrometry (MS) is potentially a more accurate technique with the ability to demonstrate specificity. An introduction of the basic analytical aspects of liquid chromatography (LC)-MS/MS leads on to the validation of the method before general use. LC coupled with MS and tandem mass spectrometry (MS(n)) is being adopted in a number of hospital laboratories for the quantitative analysis of a number of analytes from physiological matrices, but standards for development and validation of such assays are not easily available. Most assays can be regarded as in-house methods and herein may lay the failure so far for mass spectrometric methods to improve quality of results between laboratories for an analyte using the same technology. Manufacturers are taking on board the experience of clinical laboratories with kits containing all or most of the disposable items and reagents. A number of documents and guidelines have been consulted. These documents are expensive to purchase, are often very long and not easy to read. This review highlights the specific requirements for introduction of a tandem mass spectrometric test for small molecules into a routine hospital laboratory. A number of experiments need to be planned and executed in order to describe a new quantitative method in terms of selectivity, accuracy, imprecision, sensitivity and stability. The introduction of a quantitative method based on tandem MS requires careful validation. This review has distilled out important points from a number of key documents in order to provide a working validation guideline for clinical laboratories. In a supplementary file a working document for assembling the assay validation is proposed.

Pleural fluid analysis of lung cancer vs benign inflammatory disease patients

Background:Correct diagnosis of pleural effusion (PE) as either benign or malignant is crucial, although conventional cytological evaluation is of limited diagnostic accuracy, with relatively low sensitivity rates.Methods:We identified biological markers accurately detected in a simple PE examination. We analysed data from 19 patients diagnosed with lung cancer (nine adeno-Ca, five non-small-cell Ca (not specified), four squamous-cell Ca, one large-cell Ca) and 22 patients with benign inflammatory pathologies: secondary to trauma, pneumonia or TB.Results:Pleural effusion concentrations of seven analysed biological markers were significantly lower in lung cancer patients than in benign inflammatory patients, especially in matrix metalloproteinase (MMP)-9, MMP-3 and CycD1 (lower by 65% (P<0.000003), 40% (P<0.0007) and 34% (P<0.0001), respectively), and in Ki67, ImAnOx, carbonyls and p27. High rates of sensitivity and specificity values were found for MMP-9, MMP-3 and CycD1: 80 and 100% 87 and 73% and 87 and 82%, respectively.Conclusion:Although our results are of significant merit in both the clinical and pathogenetic aspects of lung cancer, further research aimed at defining the best combination for marker analysis is warranted. The relative simplicity in analysing these markers in any routine hospital laboratory may result in its acceptance as a new diagnostic tool.

Immunostimulation and growth faltering in UK infants.

The purpose of the study was to determine whether chronic immunostimulation could explain growth faltering in disadvantaged children in the UK, as it does in developing countries such as The Gambia. In all, 216 infants, age 10-21 months, were recruited when blood samples were taken for the routine or clinical purposes of a longitudinal study tracking a larger cohort of children. Aliquots of blood were collected on Guthrie cards to determine blood concentrations of albumin (Alb), alpha(1)-antichymotrypsin (ACT), and immunoglobulin G (IgG). Haemoglobin concentrations were determined by routine hospital laboratory analysis. Heights and weights were measured and converted to z-scores; birth weights were used with recruitment weight to calculate a 'thrive index' for each child. Age-corrected plasma IgG concentration was negatively associated with both height- and weight-for-age z-scores (P = 0.042 and 0.038, respectively) but not with the thrive index or body mass index z-scores. Blood haemoglobin levels were positively related to height- and weight-for age z-scores, as well as to the thrive index (P = 0.026, 0.014, and 0.007, respectively). Although significant, these relationships could only account for a small part the observed growth variation. Although the relationships were weak, the results suggest that some of the observed variation in growth of these UK infants may be explained on the basis of persistent immunostimulation or poor iron status. In terms of markers of immunostimulation (Alb, ACT, ACT:Alb ratio, IgG), both absolute levels and relationships with height-for-age are substantially different than those previously observed in cohort studies of infants in The Gambia.

Field-testing and validation in a primary care setting of a point-of-care test for C-reactive protein.

Use of point-of-care testing (POCT) in primary care has increased. There is a need for high-quality field evaluation of POCT before deployment can be considered. A POCT system for C-reactive protein was evaluated in a routine general practice setting. The standard laboratory method was a dry slide method based in a routine hospital laboratory. Scatterplots for both venous and capillary blood POCT system results versus the standard laboratory assay produced correlation coefficients of greater than 0.96. Bland-Altman plots indicated that 95% of venous and capillary POCT results fell within +/-10 mg/L of the mean value with no clinically significant difference from laboratory results. The POCT system performed reliably in a routine general practice setting with satisfactory performance against an accepted laboratory method.

Vitamin Analysis in Routine Hospital Laboratory

Vitamin Analysis in Routine Hospital Laboratory

Ribavirin quantification in combination treatment of chronic hepatitis C.

Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C(18)-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4 degrees C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 +/- 1.06 micro g/ml (n = 24) at week 12 of treatment (W12) and 3.24 +/- 1.35 micro g/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 μg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1–150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.

Assessment of homocysteine as a cardiovascular risk factor in clinical practice.

Elevated plasma total homocysteine concentrations are a marker of vitamin deficiency and a risk factor for cardiovascular disease. It is possible that vitamin supplementation with folic acid and other B vitamins, which lower plasma homocysteine concentrations, may reduce the risk of cardiovascular disease. Large-scale clinical trials are currently underway to assess the homocysteine hypothesis of cardiovascular disease. Pending the outcome of such trials, measurement of plasma homocysteine concentrations in people at high risk of cardiovascular disease may help to identify patients who could benefit from more intensive treatment of classical cardiovascular risk factors. The introduction of immunoassays for homocysteine determination has made assessment of homocysteine status accessible to most routine hospital laboratories, and this review summarizes the evidence on why and how to assess homocysteine as a risk factor for cardiovascular disease in clinical practice.

PERFORMANCE OF THE ANALYTICAL ASSAYS OF PACLITAXEL, DOCETAXEL, AND CYCLOSPORIN A IN A ROUTINE HOSPITAL LABORATORY SETTING

The taxanes paclitaxel and docetaxel are important anticancer agents. To optimize therapy of these drugs, many studies have been performed by us with pharmacokinetic monitoring of the compounds. The numerous determinations of paclitaxel and docetaxel in our laboratory enabled us to monitor performance of the bioanalytical assays over a prolonged period of time. In addition, we analyzed the performance of the bioanalytical assay of cyclosporin A, a compound co-administered to enhance absorption of orally administered paclitaxel and docetaxel. Here, we report our experience with these assays over the past four years. Paclitaxel and docetaxel were analyzed by validated high-performance liquid chromatography (HPLC) assays developed at our Institute. Cyclosporin A was analyzed with use of a specific fluorescence polarization immunoassay (s-FPIA) developed and validated by Abbott Laboratories. For acceptance of an analytical run, we used the criteria for calibration and quality control samples issued by the conference on Analytical Methods Validation (1990). Quality control samples have been used to monitor performance of the assays. In the past four years, all three analytical assays showed excellent performance. In this period, we performed 84 analytical runs of paclitaxel, 19 runs of docetaxel, and 131 runs of cyclosporin A. Accuracies of the paclitaxel, docetaxel, and cyclosporin A assays were 92–102%, 103–112%, and 103–105%, respectively. Precisions of the paclitaxel and docetaxel assays were less than 10% for all concentrations. For the cyclosporin A assay, the coefficients of variation were always less than 12%. It can be concluded that the validated analytical assays of paclitaxel, docetaxel, and cyclosporin A showed very good performance in a routine hospital laboratory setting for a prolonged period of time.

With regard to glycohemoglobin measurement: are we sure that high-performance liquid chromatography currently works in the clinical routine?

The clinical usefulness of glycated hemoglobin (HbA1C) depends crucially on the accuracy and precision of its assay. When we compared an immunological bench-top analyzer (DCA 2000, Bayer Diagnostici, Milan) to the high-performance liquid chromatography (HPLC) reference method used in a routine hospital laboratory (Diamat and Fast Diamat, Bio-Rad Lab., Milan) by assaying multiple control sera, we found so many sources of systematic analytical errors in the routine use of HPLC as to compromise between-assay precision. DCA 2000 showed intra- and interassay coefficients of variation (CV) of 1.1% and 2.3% with the normal standard serum, 1.0% and 4.2% with the pathological one; Diamat yielded CVs of 1.3% and 7.0%, 1.3% and 5.7%, respectively. Although the measurement of 161 blood samples showed that Diamat usually overestimated HbA1C (paired t-test, P<0.001), a great variability of Diamat performance became evident when the relationship Diamat vs DCA was evaluated day by day over 17 days of observation (analysis of variance, ANOVA, P<0.001). Intra- and interassay CVs of Fast Diamat initially (new instrument still on approval) were 0.6% and 2.5% (normal standard serum), 0.3% and 1.9% (high standard serum), yet after 6 months of routine laboratory use, they became 3.1% and 3.2%, 1% and 12.3%, respectively. Main sources of error were: inaccurate autodilution, unsuitable parameter settings, disregard of the maintenance schedule. We conclude that the tendency to overlook major critical aspects in the routine use of HPLC is detrimental to the quality of HbA1C determination and implies the loss of HbA1C value in clinical practice. Both carefully supervising laboratory quality and checking the likelihood of the analytical result with the clinical setting appear even more important.

MOLECULAR DIAGNOSIS OF HAEMOPHILIA A AND OTHER COAGULATION DISORDERS 35

Molecular diagnosis of gene defects that cause the various phenotypes of inherited deficiencies of coagulation factors depends on the mapping and cloning of the respective genes and the analysis of mutations that cause these phenotypes. Direct and indirect molecular diagnoses (using DNA polymorphisms) are the methods currently used for the most common coagulation disorders. Haemophilia A is due to defects on the X-linked gene of factor 8. The most common mutation in patients with severe haemophilia A is a partial inversion of factor 8 mediated by a 9.5 kb sequence, present in the large intron 22 of factor VIII and twice more about 400 kb telomeric to it. Studies of more than 2000 patients with severe haemophilia A showed that different types of the partial inversion account for 43% of the molecular defects in those patients. Almost all mothers of these patients are carriers of the inversion and the majority of these inversions occur in male germ cells. This inversion could be easily diagnosed by Southern blot analysis. Besides the common inversion, a large number of different molecular defects have been found in patients with haemophilia A. As a rule, each unrelated family shows a private molecular lesion. Missense mutations are the predominant defects in patients with mild-to-moderate disease (≈ 87%). Large deletions account for about 5% of patients, while frameshifts due to small deletion/insertions and nonsense codons are found in 46% of patients with severe disease. The current methodology to detect these defects is laborious and not suitable for a routine hospital laboratory. Therefore, the majority of these defects are diagnosed indirectly using DNA polymorphisms to mark the abnormal gene in a given family. The development of “DNA-chips” for hemophilia A would likely provide a solution to the problem of direct detection of these mutations. The molecular diagnostic approaches for factor 9 deficiency(haemophilia B), von Willebrand disease and recent progress in the understanding of the pathophysiology of these disorders using transgenic mice and the study of potential regulatory molecules will be presented.