Identification and quantification of high fluorescence-stained lymphocytes as antibody synthesizing/secreting cells using the automated routine hematology analyzer XE-2100.

OBJECTIVES: The aim of this study was to classify and quantify the high fluorescence lymphocytes area (HFL-count) from the SYSMEX XE-2100 leucocyte differential channel as antibody-synthesizing or -secreting cells (ASC, plasma cells or lymphoplasmacytoid cells) in reactive diseases. To unequivocally identify the HFL cells, all possibly eligible cell populations have been investigated: activated B-lymphocytes, activated T-lymphocytes, large granular lymphocytes (LGL), activated monocytes, and immature granulocytes. METHODS: In total, 85 patients were analyzed on the XE-2100 and compared with the automated image analysis system Cellavision Diffmaster 96 based on artificial neural network and immunophenotyping method with the BD FACSCalibur. RESULTS: Reproducibility tests for HFL demonstrated a mean coefficient of variation of 13.9% for very low results and 1.5% for high results. The linearity data showed a good correlation (R(2) = 0.99) between expected and measured HFL. The comparison with possibly eligible cell populations showed no significant correlation between activated monocytes and immature granulocytes, with most immature granulocytes (promyelocyte I or II), natural killer cells or LGLs, activated T-lymphocytes, and sub-T-lymphocytes populations. However, for activated B-lymphocytes an excellent significant correlation with the peripheral blood smear, and the immunophenotyping method has been found with R(2) = 0.900, P < 0.001 and R(2) = 0.897, P < 0.001, respectively. The slope of 1.1 and intercept of minus 5 cells/microL of the regression equation between HFL-count and ASC (smear) do indicate an excellent quantification of the HFL-count, as well. CONCLUSION: The fully automated SYSMEX XE-2100 HFL-count identifies and quantifies the ASC cells (activated B-lymphocytes) with high precision and reliability in patients without hematology system diseases, thus providing a potential screening and monitoring tool for any patient with suspected infection. Additional studies are required to comprehend in more detail the full clinical utility of an HFL (ASC) count as a potential diagnostic indicator of inflammation, infection, or sepsis.

Comparison of the prevalence of APC-resistance in vascular patients and in a normal population cohort in Western Germany.

OBJECTIVE: To compare the prevalence of APC-resistance (APC-R) in patients with peripheral vascular disease and the general population. DESIGN: Prospective cohort examination. MATERIALS AND METHODS: Three hundred and eleven patients (group A) suffering from arterial occlusive disease or an abdominal aortic aneurysm were prospectively screened for APC-R. There were 228 men and 83 women with a mean age of 65 years (20-88 years). Two hundred and sixty patients underwent an open surgical or interventional procedure. A total of 306 patients were followed clinically for an average of 8 months (1-31 months). Two hundred and seven healthy volunteers (group B) served as a control group. RESULTS: The prevalence of a functional APC-R was 11% (33/311) and 8% in groups A and B, respectively, (p = 0.272). APC-R did not occur more frequently among patients who were treated primarily for a bypass occlusion (3/21 vs 30/290) (p = 0.476). None of five patients who had a postinterventional graft or vessel occlusion (1.9%) had an APC-R. Sixteen patients (5%) experienced an arterial occlusion during follow-up of which two had APC-R. CONCLUSIONS: Previously published increased prevalence rates of APC-R in patients with arterial disorders could not be confirmed in this study. A firm association between the presence of APC-R and previous bypass occlusion or postoperative failure of the vascular reconstruction could not be demonstrated.

Use of NT-proBNP in routine testing and comparison to BNP.

OBJECTIVES: B-type natriuretic peptide (BNP) is a strong diagnostic predictor of left-ventricular (LV)-dysfunction. Recently, the aminoterminal portion of pro-BNP (NT-proBNP) has been introduced, which could be even more sensitive because of its longer half-life. The aim of this study was to evaluate the new marker NT-proBNP within a large, heterogeneous population of patients with suspected cardiovascular disease at risk of cardiovascular dysfunction and to compare it with the established diagnostic parameter BNP. SUBJECTS AND METHODS: NT-proBNP and BNP were measured in 339 hospitalised patients undergoing diagnostic angiography (median age 66 years, 244 male vs. 95 female). RESULTS: Median values of NT-proBNP increased with worsening LV-dysfunction and higher NYHA class. The area under the receiver operator characteristics curve (AUC) of NT-proBNP for detecting severe systolic dysfunction or for detecting any systolic LV-dysfunction was 0.83 and 0.77, respectively. The latter improved (AUC=0.81) when patients with clinically relevant heart disease like valvular dysfunction were included, independent of the haemodynamic values. Compared to BNP, NT-proBNP tended to be more accurate in identifying lesser degrees of LV-dysfunction. CONCLUSIONS: Even after optimisation of target criteria, there was still a substantial overlap of NT-proBNP values between patients with and without relevant heart disease. Therefore, NT-proBNP is not suitable as a screening test for LV-dysfunction in the community. Nevertheless, because of its good negative predictive value, NT-proBNP could be an easy and effective tool to rule out severe systolic LV-dysfunction in high risk patients. No clinically significant advantage of BNP testing could be found.

[The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients]. / Stellenwert der natriuretischen Peptide NT-pro-BNP und BNP für die Beurteilung der linksventrikulären Grösse und Funktion. Eine prospektive Untersuchung an 150 Patienten.

BACKGROUND AND OBJECTIVE: An early detection of patients with left-ventricular (LV) dysfunction is essential for effective treatment of congestive heart failure. The B-type natriuretic peptide (BNP) was described as a strong diagnostic parameter of LV-dysfunction. Aim of this study was to compare the diagnostic value of BNP and the aminoterminal part of pro-BNP (NT-pro-BNP) within an heterogenous patient population. SUBJECTS AND METHODS: NT-pro-BNP and BNP were measured in 150 hospitalized cardiologic patients (age-median 64 years, 109 male versus 41 female). The values were correlated with clinical and haemodynamic parameters of the invasively determined LV-function. RESULTS: Patients with pathologic haemodynamic values of the LV-function had significantly higher NT-pro-BNP and BNP levels than patients with normal haemodynamic parameters. In our study population a severe systolic LV-dysfunction (ejectionfraction EF<40 %) could be detected with a sensitivity and specifity of 100 % and 64 % by NT-pro-BNP and 100 % and 45 % by BNP. Sensitivity and specifity for the detection of any systolic dysfunction (EF<60 %) and of a systolic or diastolic dysfunction, respectively, were 94 %, 37 % and 88 %, 41 % for NT-pro-BNP (94 %, 40 % and 84 %, 44 % for BNP). The corresponding negative predictive values were 100 %, 96 % and 71 % for NT-pro-BNP and 100 %, 96 % and 68 % for BNP. CONCLUSION: NT-pro-BNP and BNP were highly sensitive diagnostic parameters with a very good negative predictive value for LV-dysfunction. Because of the uncomplicated measurement, they could be used effectively to rule out LV-dysfunction in cardiovascular high risk patients by general practitioners.

Apolipoprotein B-100: employing the electrochemiluminescence technology of the Elecsys systems for the detection of the point mutation Arg(3500)Gln.

Apolipoprotein B-100 (apo B-100) plays an essential role in lipoprotein metabolism where it is involved in the clearance of LDL particles from the bloodstream. The mutation Arg3500Gln in the apo B-100 gene impairs the binding of the LDL particles to the LDL receptor, resulting in elevated LDL-cholesterol levels in the blood which, in turn, fuel the development of premature atherosclerosis. Here we describe a rapid, automated test for the detection of the most frequent mutation in the apo B-100 gene. This PCR-based test employs electrochemiluminescence as detection technology and allows the reliable discrimination of all genotypes. The assay has been especially developed for the non-specialized routine clinical chemistry laboratory by employing an analyzer and chemistry often present in this type of labof1tory. Because of its low costs and easy handling the assay can be performed on a daily basis.

Apolipoprotein E polymorphism: automated determination of apolipoprotein E2, E3, and E4 isoforms.

Apolipoprotein E (apo E) plays an essential role in lipoprotein metabolism, where it is involved in the clearance of chylomicrons and very low density lipoproteins. Apart from some rare variants, apo E exists in three common isoforms (E2, E3, and E4). The different isoforms have not only been associated with different plasma lipid levels but have also been correlated with certain pathological conditions, such as lipid disorders (dysbetalipoproteinemia, hypercholesterolemia), cardiovascular diseases, and Alzheimer's disease. Here we describe a rapid, automated test for the determination of the most frequent polymorphisms (E2, E3, and E4). This polymerase chain reaction-based test allows the reliable discrimination of all six genotypes. The assay has been developed especially for the nonspecialized routine clinical laboratory by employing an analyzer and chemistry often present in this type of laboratory. Because of its low costs and easy handling, the assay can be performed on a daily basis.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 917 analysis system.

The new selective access analysis system BM/Hitachi 917 was evaluated in an international multicentre study, mainly according to the ECCLS protocol for the evaluation of analysers in clinical chemistry. Forty-three different analytes, covering 56 different methods--enzymes, substrates, electrolytes, specific proteins, drugs and urine applications--were tested in seven European clinical chemistry laboratories. Additionally, the practicability of the BM/ Hitachi 917 was tested according to a standardized questionnaire. Within-run CVs (median of 3 days) for enzymes, substrates and electrolytes were <2% except for creatine-kinase MB isoform and lipase at low concentration. For proteins, drugs and urine analytes the within-run CVs were < 4% except for digoxin and albumin in urine. Between-day median CVs were generally < 3% for enzymes, substrates and electrolytes, and < 6% for proteins, drugs and urine analytes, except for lipase, creatine kinase and MB isoform, D-dimer, glycosylated haemoglobin, rheumatoid factors, digoxin, digitoxin, theophylline and albumin in urine in some materials. Linearity was found according to the test specifications or better and there were no relevant effects seen in drift and carry-over testing. The interference results clearly show that also for the BM/Hitachi 917 interference exists sometimes, as could be expected because of the chemistries applied. It is a situation that can be found in equivalent analysers as well. The accuracy is acceptable regarding a 95-105% recovery in standard reference material, with the exception of the creatinine Jaffé method. Most of the 160 method comparisons showed acceptable agreement according to our criteria: enzymes, substrates, urine analytes deviation of slope +/- 5%, electrolytes +/- 3%, and proteins and drugs +/- 10%. The assessment of practicability for 14 groups of attributes resulted in a grading of one-three scores better for the BM/Hitachi 917 than the present laboratory situation. In conclusion, the results of the study showed good analytical performance and confirmed the usefulness of the system as a consolidated workstation in medium-sized to large clinical chemistry laboratories.

Haemochromatosis: automated detection of the two point mutations in the HFE gene: Cys282Tyr and His63Asp.

Hereditary haemochromatosis (HH) is one of the most common inherited diseases among Caucasians. Two mutations in the HFE gene have been implicated in HH: 80 to 90% of the patients with HH are homozygous for the point mutation CYS282Tyr, while the majority of the remaining patients displays either a compound heterozygosity for the mutation CYS282Tyr and the point mutation HIS63Asp, or are homozygous for HIS63Asp. Though the disease can be treated easily, symptoms are non-specific, and onset and severity are influenced by environmental factors, and therefore the disease can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, which may result in their failure. In order to detect patients with HH, simple and cost-effective tests are needed. We have developed a rapid, automated, PCR-based test which makes use of a diagnostic restriction site in each of two amplified fragments. The test employs off-the-shelf chemistry and uses the automated detection process of an immunoassay analyzer that is available in many clinical laboratories, thus avoiding an additional investment in a more specialized PCR analyzer. Because of its low costs and easy handling, the assay is particularly suited for the routine clinical laboratories.

Activated protein C resistance: automated detection of the factor V Leiden mutation by mismatch hybridization.

BACKGROUND: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient's genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization. METHODS: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 degrees C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics). RESULTS: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance. CONCLUSIONS: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.

[The Cologne Guidelines Conference: computer-assisted clinical practice guidelines in clinical diagnosis]. / Die Kölner Leitlinien-Konferenz: Computergestützte Leitlinien zur klinischen Diagnostik.

Eighteen clinical practice guidelines on interdisciplinary diagnostic issues were developed at the University Hospital of Cologne, Germany. The guideline committee is organized and directed by the quality control program, which also includes the local Cochrane initiative and a wide range of organizational topics. During guideline development, questions of differential diagnosis were addressed to the same extent as the organizational and financial realization. Broad consideration was given to medicolegal implications, but need for interspecialty cooperation was judged to be more critical and even more relevant in this regard. Guidelines were primarily developed as algorithms and translated to text versions secondarily. Critical steps in the decision tree were supported by rated literature and recommendations weighte by criteria as used in evidence-based medicine. For implementation, guidelines were presented to colleagues in a series of short lectures, as print versions containing all literature used in the developing process, and in hypertext format, which is accessible via intranet. Three levels of presentation were chosen in the html-version: algorithm, decision, and information. The former is due to orientation in the guideline, the second displays the binary question, and the latter makes the scientific background available, together with literature and links for more information. Efforts to check effectiveness are currently been made, questions of efficiency will be addressed in future.

Activated protein C (APC)-resistance: automated detection of the point mutation at position 1691 in the factor V gene.

Proteolytic cleavage of factor Va, caused by activated protein C, is an important mechanism in limiting clot formation in normal haemostasis. A single point mutation in the factor V gene has been demonstrated to cause resistance of factor Va to proteolytic cleavage by activated protein C. With an 8-fold increased risk of thrombosis and a 2 to 13% prevalence in the Caucasian population for the heterozygous state of this mutation, knowledge of the patient's genetic disposition is of great importance in conditions such as pregnancy, surgery, use of oral contraceptives and immobilization. Therefore we have developed an automated test for the detection of the factor V mutation. This PCR based test makes use of the disappearance of an Mnl 1 restriction site if the mutation is present. The assay has been developed for the widely used ES-systems of Boehringer Mannheim. The test discriminates between the heterozygous and the homozygous state. Because of its low costs and easy handling the assay can be used as a screening test and can be performed in routine clinical laboratories.

Mutation analysis of exon 3 of the LDL receptor gene in patients with severe hypercholesterolemia.

Single-strand conformation polymorphism analysis was used to screen for mutations in exon 3 of the low density lipoprotein receptor gene in a group of 218 unrelated patients with severe hypercholesterolemia (low density lipoprotein cholesterol > 6.7 mmol/l) living in the Cologne area of Germany. Including the complementary primers the fragment studied had a length of 176 bp. An abnormal single-strand conformation polymorphism pattern was observed in eight patients, four of whom had an identical abnormal fragment pattern indicating that five different mutations were present. By direct DNA sequencing, the underlying mutations could be confirmed (Cys54-->Tyr, Trp66-->Gly, Glu80-->Lys, 2 bp insertion (AT between codon 44 and 45, 9 bp deletion (codons 65 to 67)). The analysis of the pathogenicity indicates that all mutations were causative for the low density lipoprotein cholesterol elevation. The Trp66-->Gly and Glu80-->Lys mutations were previously described in a French-Canadian population and in an English population, respectively. The 2 bp insertion was detected in four unrelated patients and is one of the most frequent mutations detected up to now in the German population.

Synthesis and cytotoxic activity of C-glycosidic nicotinamide riboside analogues.

The C-glycosidic nicotinamide riboside analogue (2) was prepared by reaction of ribonolactone 24 with the lithiated oxazoline 19 followed by triethylsilane reduction to 26 and deprotection. Selective phosphorylation to the pseudonucleotide 34 was effected via the isopropylidene compound 33. In contrast to the benzoic acid riboside (28) the benzamide riboside (2) showed extremely high cytotoxicity at nanomolar concentrations to S49.1 lymphoma cells but only slightly increased dexamethasone toxicity.

Glucocorticoid-induced lymphoma cell growth inhibition: the role of leukotriene B4.

Glucocorticoid-sensitive murine S49.1 lymphoma cells respond in a biphasic way to the steroid challenge. The first effect of corticosteroids is to induce a reversible growth inhibition, which is probably permissive for the following cytolysis. Distinct mechanisms for the two effects are likely. Since dilution of S49.1 lymphoma cultures resulted in a drastic reduction of the proliferation rate, which could be overcome by the addition of conditioned medium, the proliferation appears to depend on the presence of autocrine growth factors. Therefore, the cytostatic effect of corticosteroids could possibly be attributable to an interference with the production of endogenous growth factors. Analysis of the growth-promoting activity in culture supernatant showed that the critical growth factor in diluted cultures is an arachidonic acid metabolite, the leukotriene B4. The role of leukotriene B4 in S49.1 cell proliferation received further support from the finding that while nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway which is necessary for leukotriene formation blocked lymphoma multiplication, indomethacin, an inhibitor of cyclooxygenase activity, did not affect proliferation. Quantitation of the leukotriene B4 content of dexamethasone-treated vs. untreated cultures revealed an almost complete inhibition of leukotriene production, pointing to the significance of this mechanism for the glucocorticoid-induced lymphoma growth inhibition. Moreover, these findings offer a new approach to increase the therapeutic effectiveness of glucocorticoid therapy of steroid-sensitive leukemias and lymphomas.

Reduction of DNA-polymerase beta activity of CHO cells by single and combined heat treatments.

The effect of single and combined heat treatments on the activity of DNA polymerase beta was studied in CHO cells. The activity of polymerase beta was determined by measuring the amount of [3H]TTP incorporated into activated calf thymus DNA in the presence of aphidicolin, a specific inhibitor of DNA polymerase alpha. Biphasic response curves were obtained for all temperatures tested (40-46 degrees C) showing the sensitivity to decrease during heating. A constant activation energy of Ea = 120 +/- 10 kcal/mole was found for the initial heat sensitivity, whereas the Arrhenius plot for the final sensitivity is characterized by an inflection point at 43 degrees C with Ea = 360 +/- 40 kcal/mole or Ea = 130 +/- 20 kcal/mole for temperatures below or above 43 degrees C, respectively. The observed decrease of the polymerase activity is not due to a decrease in the number of active enzyme molecules but to a change in its affinity, since the inhibition is reversible when increasing concentrations of TTP are applied. When acute or chronic thermo-tolerance was induced by a priming heat treatment at 43 degrees C for 45 min followed by a time interval at 37 degrees C for 16 h or by a preincubation at 40 degrees C for 16 h, respectively, the thermal sensitivity of polymerase beta was lowered by a factor of up to 5. By contrast, pretreatment at a higher temperature followed by a lower temperature (step-down heating) did not alter the sensitivity of polymerase beta to the second treatment. The results indicate that heat-induced cell death cannot be the consequence of the reduction of the polymerase beta activity, confirming earlier studies on this subject.

Glucocorticoid-induced lymphoma cell death: the good and the evil.

Glucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the production of growth mediators and glucocorticoid-induced cell death. The mechanism of glucocorticoid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuclease appears to initiate the lytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.

Glucocorticoid-induced cell death and poly[adenosine diphosphate(ADP)-ribosyl]ation: increased toxicity of dexamethasone on mouse S49.1 lymphoma cells with the poly(ADP-ribosyl)ation inhibitor benzamide.

Treatment of S49.1 mouse lymphoma cells with the synthetic glucocorticoid dexamethasone resulted in a delayed cell death. During the 24-h latency period, DNA, RNA, and protein synthesis fell to about 50%, 60%, and 30% of control values, respectively, without a change in ATP levels, the latter suggesting cellular integrity. The onset of cellular suicide was characterized by the occurrence of DNA strand breaks, finally leading to the total digestion of internucleosomal DNA. Concomitant with the appearance of DNA fragmentation, poly(ADP-ribosyl)ation was activated, a process probably involved in DNA repair. Activation of poly(ADP-ribose)synthetase was paralleled by a fall in the level of the substrate NAD. An antagonistic role of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death was suggested by the observation that low concentrations of the potent poly(ADP-ribose)synthetase inhibitor benzamide enhanced the toxicity of dexamethasone several-fold and shortened the interval between steroid addition and the onset of cell death. In addition, the fall in NAD was prevented by benzamide. The antagonistic function of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death is, therefore, comparable to the role of the poly(ADP-ribose)synthetase in cells treated with alkylating agents, suggesting involvement of a DNA repair phenomenon in opposition to the mechanism of glucocorticoid-induced cell death.