[Using thromboelastography to measure coagulation following massive blood loss]. / Stolling meten met trombo-elastografie bij massaal bloedverlies.

Thromboelastography is becoming increasingly important for diagnosing coagulation disorders in patients with massive blood loss. This whole-blood measurement provides information about the speed of clot formation, clot strength, and degree of fibrinolysis. The result can be used as a basis for making a faster and better choice of a suitable blood product for the patient with severe blood loss. This technique can be carried out simply and quickly as a rapid test ('point-of-care test') or in a central laboratory. Use of thromboelastography in patients undergoing cardiac surgery results in reduced use of blood products and is proven to be cost effective. A reduction in the use of blood products was also seen in trauma patients and patients undergoing liver transplantation when this technique was used. Studies on other groups of patients with massive blood loss are being conducted at the moment.

[Confusion caused by dietary supplement lithium orotate]. / Verwarring bij behandelaars door voedingssupplement lithiumorotaat.

A 38-year-old woman was referred to our hospital with pelvic inflammatory disease. She had been diagnosed as having a tubo-ovarian abscess, for which antibiotic therapy with metronidazole and levofloxacin was indicated. Because the patient was already taking lithium she was prescribed clindamycin instead of metronidazole, a drug that interacts with lithium. For the same reason, naproxen could not be prescribed to reduce pain. However, the lithium taken by the patient turned out to be a dietary supplement containing lithium orotate which she had obtained via the Internet. Because the serum lithium level proved to be below 0.05 mmol/l clindamycin was still replaced by metronidazole, and naproxen could be started.

Anti-Emm in a pregnant patient--case report.

A 23-year-old primigravida of North African origin presented with a positive antibody screen at booking at 15 weeks of gestation. An antibody to a high-frequency antigen (HFA) of unknown identity was detected, which was reactive with the red blood cells of the father. This led to several challenges including antibody identification, clinical monitoring to detect signs of haemolytic disease of the foetus and newborn (HDFN) and compatible blood in case perinatal transfusion was needed. Anti-Emm was identified 2 months post-partum. This is the first published case which describes a pregnant patient with anti-Emm.

Optimizing blood pigment analysis in cerebrospinal fluid for the diagnosis of subarachnoid haemorrhage--a practical approach.

BACKGROUND: Patients presenting with sudden severe headache may have a subarachnoid haemorrhage (SAH). After a normal head computer tomography (CT), a lumbar puncture is routinely performed to rule out SAH. Photospectrometry is then used to detect bilirubin in cerebrospinal fluid (CSF). Photospectrometric analysis of CSF reaches a high sensitivity, but a low specificity for SAH. This low specificity necessitates extensive additional research to rule out cerebral aneurysm accompanied by high costs and risk of complications. OBJECTIVE: The objective of this study was to retrospectively evaluate two different CSF interpretation methods using photospectrometry in patients presenting with acute headache. The first of these is the Leiden method, an iterative model using a standard calculation. The second is the UK NEQAS guideline, which uses the original spectrum in combination with a decision tree. Our goal was to obtain retrospective data on patients screened with both methods to improve specificity of CSF research. RESULTS: We included 361 patients in this study; 47 of these had a raised bilirubin concentration in the CSF according to the Leiden method. In only nine of these 47 patients was an aneurysm found; in the other patients the Leiden test was positive for other reasons (viral meningitis, hyperbilirubinaemia, etc.). Of the 47 patients with raised bilirubin, 24 could be re-evaluated using the UK NEQAS. Of these 24 patients, five had an aneurysm. No aneurysms were found in patients with a negative result according to the UK NEQAS guideline. CONCLUSION: Our data show that a raised bilirubin calculated using the Leiden method seems to have a lower specificity than the UK NEQAS guideline. For practical reasons, it seems advantageous to use the Leiden method as a screening method and use the UK NEQAS guideline if a positive result is found.

Hb Haaglanden: a new nonsickling ß7Glu>Val variant. Consequences for basic diagnostics, screening, and risk assessment when dealing with HbS-like variants.

INTRODUCTION: To report a new hemoglobin variant undistinguishable from the common HbS on HPLC. To show the efficiency of the simplest confirmation method for HbS and to discuss the implications that may occur if HbS-like variants are wrongly reported as HbS. METHODS: Basic hematology, separation and measurement of the Hb fractions, 'sickle test,' and molecular analysis. RESULTS: The abnormal Hb fractions were eluting in the HbS window on HPLC, sickle test was however negative, and DNA sequencing of the beta globin gene revealed an unclassified variant HBBc.23A>T, p.Glu8Val in heterozygous form. CONCLUSIONS: Although the amino acid substitution of this new variant is identical to that of HbS and shifted of a single amino acid position, no polymerization occurs in vitro. The sickle test is a valid method to confirm or exclude HbS trait in individual cases. Whenever the case is part of a possible couple at risk, then one has to use full DNA analysis in both partners not to miss hidden concomitant defects important for genetic risk predictions.

The efficacy of laboratory diagnosis of Helicobacter pylori infections in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes.

A total of 500 consecutive patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by the Campylobacter-like organism (CLO) test, culture, histology, and PCR. Serum samples were tested by two different serological assays. Patients were considered H. pylori positive if at least two of the four biopsy specimen-based methods yielded positive results. PCR had the highest diagnostic sensitivity (99.4%), followed by histology (92.2%), culture (89.5%), and the CLO test (89.0%). The specificities of all methods were higher than 98%. Of the organisms from the 181 PCR-positive patients, the vacA (s and m regions), cagA, and iceA genotypes were determined by reverse hybridization (line probe assay) or an allele-specific PCR. Organisms that were detected by PCR but that remained undetected by the CLO test were significantly more often vacA s1 (P = 0.006), m1 (P = 0.028), and cagA positive (P = 0.029) than vacA s2, m2, and cagA negative, respectively. Organisms that were detected by PCR but that remained undetected by culture or histology more often contained iceA1 (P = 0. 034 and P = 0.029, respectively) than iceA2. Higher H. pylori density was associated with vacA s2 (P = 0.024), vacA m2 (P = 0.050), and cagA-negative (P = 0.035) genotypes. Also, the diagnostic results of the CLO test (P = 0.001) and culture (P = 0.031) but not those of the PCR (P = 0.130) were significantly associated with the H. pylori density. The rate of detection by the four biopsy specimen-based tests was lower for patients who used proton pump inhibitors, but this was independent of the H. pylori genotypes. These observations may be explained by different bacterial densities, as established by the distinct genotypes of H. pylori, and confirm that the biologies of strains with such genotypes are considerably different.

Detection and identification of monoclonal gammopathies by capillary electrophoresis.

Capillary electrophoresis (CE) and immunosubtraction capillary electrophoresis (IS-CE) were compared with the conventional methods agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE) for detection and identification of paraproteins. In total, 74 paraproteins out of 468 serum samples were detected by both methods. Seventy-three monoclonal bands with concentrations ranging from 0.6 to 50.9 g/L were detected by the routine method. With CE, 70 paraproteins were detected and quantified on the electropherogram. Four paraproteins were not detected by CE; three of these were IgG (0.6, 1.1, and 2.2 g/L, respectively), and one was a IgM paraprotein (20.3 g/L) that could be visualized by minor changes in the running conditions. In comparison with IFE, 69 paraproteins were typed identically using IS-CE; only one paraprotein (IgMkappa, 14.9 g/L) could not be identified. On the other hand, a monoclonal IgA band that had not been detected by AGE was identified by CE and IS-CE. We conclude that, in general, CE could be a useful method for detection of paraproteins and that IS-CE is a good alternative to IFE. Additional studies are required to investigate the ionic strength and pH of the running buffer, because these prove to be the most crucial factors for routine CE separation of paraproteins.

A Dutch family with Hb Atlanta [beta 75(E19)Leu-->Pro].

We have investigated four members of a three-generation Dutch family for a suspected hemoglobinopathy. Chronic hemolysis and a moderate macrocytic normochromic anemia with slight morphological abnormalities of the red cells was observed in all four. Hemoglobin chain synthesis in vitro and separation of the globin chains by reversed phase high performance liquid chromatography revealed an abnormal beta-globin species in addition to the normal alpha and beta chains. The decreased amount of normal beta-globin and the low amount of unidentified protein suggested an unstable beta-globin variant. An abnormal band was detected by isoelectrofocusing. In one family member tested, the hemoglobin in an erythrocyte lysate had decreased heat stability. All carriers were positive in the isopropanol hemoglobin instability test. Treatment of erythrocytes with methylviolet gave rise to microgranular inclusions. Nucleotide sequencing of the polymerase chain reaction-amplified beta-globin gene revealed a heterozygous single base pair T-->C mutation at codon 75, which changes the normal CTG codon for leucine to a CCG codon for proline. This variant has previously been identified as Hb Atlanta or beta 75(E19)Leu-->Pro. The mutation creates a new Msp I restriction site, which was used to confirm the diagnosis in all four family members. A quantitative reverse transcriptase polymerase chain reaction procedure for determining the relative amounts of mRNA transcripts for the normal and abnormal globin chain showed a comparable stability for both transcripts.

Haemolytic disease of the newborn due to anti-K antibodies.

We describe a case of intrauterine foetal death at 32 weeks gestation with signs of hydrops foetalis without erythroblastosis. The hydrops foetalis appeared to be caused by blood group incompatibility. Irregular antibodies of anti-K specificity were found in the serum of the mother, while the father was positive for the K-antigen. The antibody dependent cell-mediated cytotoxicity (ADCC) test with serum of the mother was positive. This case shows the characteristics of haemolytic disease of the newborn by anti-K. Moreover, it underlines the necessity of both adequate follow-up of individual cases, and screening for irregular antibodies in all pregnancies as well as central registration of screening results.

Regular physical activity and changes in risk factors for coronary heart disease: a nine months prospective study.

This study reports the non-acute effects of a long-term training programme of increasing intensity on some cardiovascular risk factors and the interrelation between these risk factors. Twenty sedentary men and 14 sedentary women were trained 3 to 4 times a week for nine months. After 36 weeks all individuals ran a half marathon run. The Wmax, weight, body mass index, systolic and diastolic blood pressure were recorded. The concentrations of fibrinogen, tissue plasminogen activator, plasminogen activator inhibitor, triacylglycerols, total cholesterol, LDL cholesterol, HDL cholesterol and lipoprotein(a) were measured. The training programme induced a median increase in Wmax of 12% in the male group (from 226 to 251.5 Watt) and of 18% in the female group (from 160 to 188.5 Watt). These increases inn Wmax did not correlate with any other property under investigation in this study. Blood pressure was not altered, but body weight and body mass index were significantly decreased in the male group (from 74.6 to 72.2 kg and from 23.1 to 22.0 kg/m2, respectively) at the end of the training programme and decreased non-significantly in the female group (from 63.0 to 60.7 kg and from 21.6 to 21.5 kg/m2, respectively). In the male group total cholesterol, low density lipoprotein cholesterol and triacylglycerols decreased significantly under the influence of the training sessions. Furthermore, in both groups, a great decrease in plasma plasminogen activator inhibitor concentrations was noticed: in men from 22.5.10(3) AU/l to 4.5.10(3) AU/l and in women from 18.7 x 10(3) AU/l to 5.1 x 10(3) AU/l. However, the changes in the lipid and fibrinolytic quantities were not correlated with each other. Initial total cholesterol, LDL cholesterol and triacylglycerol levels correlated significantly with systolic blood pressure, while diastolic pressure was correlated to tissue plasminogen activator. Since tissue plasminogen activator also was significantly related to triacylglycerols, a trias existed between primary risk factors like blood pressure, lipid levels and fibrinolysis. In contrast, the changes in these properties under the influence of physical training were not interrelated. Median serum lipoprotein(a) concentrations were significantly increased in both men and women five days before the half marathon run: from 32 mg/l to 39 mg/l in men, and from 65 mg/l to 125.5 mg/l in women. Concomitantly, median fibrinogen concentrations were significantly elevated in men (from 2.32 g/l to 3.10 g/l) and non-significantly in women (from 2.62 g/l to 2.93 g/l), although no correlation existed between the changes in these properties. In conclusion, the nine months exercise programme increased the aerobic fitness in both men and women as indicated by the Wmax increase. This improvement coincided but was not correlated with beneficial changes in several anthropometric, lipid and fibrinolytic properties. Improvement in the risk factors under investigation was more pronounced in men than in women. The changes in lipid and haemostasis properties did not correlate with each other. The increases in lipoprotein(a) and fibrinogen concentrations, both atherogenic indices, could actually present a normal physiological response to the physical strain of exercise training of increasing workload.

Long-term physical exercise and lipoprotein(a) levels in a previously sedentary male and female population.

We investigated the effect of long-term physical exercise on serum lipoprotein(a) levels. 21 sedentary men and 15 sedentary women were trained three to four times a week with increasing intensity during 9 months. After 24 weeks all subjects ran a 15 km race and after 36 weeks a half marathon run (21 km). Blood samples were drawn before the training programme, 5 days before both races and 5 days after the half marathon run. Median (interquartile range) pre-training values in the male group were 32 (11-63) mg/L and in the female group 65 (23-199) mg/L. After 24 weeks of training, serum lipoprotein(a) concentrations had risen significantly in both male and female groups. Moreover, after 36 weeks of training, in preparation for the half marathon competition, median serum lipoprotein(a) rose almost twofold in both groups and was still elevated 5 days later. This study demonstrates that an exercise programme which includes running of increasing distances significantly increases serum lipoprotein(a) concentration.

Effects of long-term exercise of moderate intensity on anthropometric values and serum lipids and lipoproteins.

The influence of endurance training on serum lipids and lipoproteins was investigated in 20 sedentary males and 14 sedentary females. The total group was trained 3 to 4 times a week for 9 months. After 24 weeks all subjects ran a 15 km-race and after 36 weeks a half-marathon (21 km) race. Anthropometric values were determined before and after the training programme. Blood samples were drawn before the start of the training programme and, in order to avoid the measurement of acute effects, 5 days before both races. In the male group, median body weight and body mass were significantly decreased (p < 0.01) after nine months of training, while in the female group body weight and body mass index remained essentially unchanged. Percentage body fat, measured by skinfold thickness was significantly decreased in both groups at the end of the training programme. During the training period, median serum total cholesterol, low density lipid cholesterol and triacylglycerol concentrations decreased significantly (p < 0.01) in the male group, while in the female population the median serum lipid- and lipoprotein concentrations did not differ from pre-training values. The changes in serum lipids or lipoproteins did not correlate significantly with changes in body weight, body mass index or percentage body fat. Stepwise multiple regression showed that these changes were mostly dependent on initial concentrations in serum. Finally, no significant increase in median high density lipid cholesterol was observed in either the male or female group.(ABSTRACT TRUNCATED AT 250 WORDS)

High plasma antithrombin levels in acute hepatitis A.

BACKGROUND: Laboratory testing of antithrombin serves to demonstrate a decreased concentration that is indicative of a thrombotic tendency. We report unexpected high levels of antithrombin in a patient with hepatitis A. METHODS: A 37-year-old woman with a 4-week history of acute hepatitis A was studied. The diagnosis of cholestatic hepatitis A was confirmed by positive serology and liver biopsy. The patient recovered completely within 6 months. RESULTS: Antithrombin activity was increased (234%; reference range, 80-120%). This high activity was confirmed by an antithrombin antigen of 210%. On recovery antithrombin activity returned to normal. CONCLUSIONS: We are unaware of other reports of such high levels of antithrombin, in particular in patients with hepatitis A. It is not clear whether the high concentration observed in this case is due to an acute-phase reaction or to impaired clearance by the liver. The presence of a molecular variant with different secretion characteristics is unlikely, since antithrombin activity returned to normal on recovery, suggesting base-line values within the normal range.

Androgen turnover during marathon running.

The purpose of the study was to investigate the effect of prolonged physical stress on peripheral androgen turnover. Venous blood samples were taken from 18 athletes 24 h before finishing a competitive marathon run and directly after running the race. Serum cortisol, testosterone (T), dehydroepiandrosteronesulfate (DHEAS), sex hormone binding globulin (SHBG), and 5 alpha-androstane- 3 alpha, 17 beta-diolglucuronide (3 alpha-AdiolG) were determined and corrected for hemoconcentration. Marathon running caused a rise in serum cortisol concentration in all athletes. Furthermore, a significant (P < 0.01) rise in serum T and T-index (index of free T) was observed. The significant (P < 0.01) rise in serum DHEAS concentration, a mainly adrenal cortical androgen, pointed toward a stimulation of the adrenal cortex or a reduced hepatic metabolic clearance rate. Finally, 3 alpha-AdiolG, an androgen metabolite exclusively formed in peripheral tissues, was increased in the sera of all athletes. These results suggest that marathon running leads to increased concentrations of serum adrenal and gonadal androgens. The simultaneously increased 3 alpha-AdiolG levels may be caused by increased androgen turnover in peripheral tissues containing 5 alpha-reductase.

Prolonged physical conditioning and blood platelet release markers: a longitudinal study.

To study the long-term non-acute effect of endurance physical exercise on blood platelet activation, 20 sedentary males and 14 sedentary females were trained 3 to 4 times a week for 9 months. After 24 weeks all subjects ran a 15-km race; and after 36 weeks a half-marathon (21 km) race. Blood samples were drawn before the training programme and 5 days after both races. Median (interquartile range) platelet factor 4 and beta-thromboglobulin pretraining values for the total group were 9 (5-35) and 69 (40-495) IU/ml, respectively. During the course of the training programme, plasma platelet factor 4 concentrations rose steadily and significantly in both the male and female group (p < 0.05), together with a non-significant rise in plasma beta-thromboglobulin. At the end of the training procedure, 5 days after the half-marathon run, median (interquartile range) plasma factor 4 and beta-thromboglobulin concentrations for the total group were 150 (62-198) and 156 (84-288) IU/ml, respectively. No difference existed in median platelet factor 4 and beta-thromboglobulin concentrations of the male and female population before or during the training programme. In summary, the results of this study demonstrate that prolonged physical conditioning of increasing intensity is mainly associated with an elevation of the platelet protein platelet factor 4.

Prolonged endurance exercise and blood coagulation: a 9 month prospective study.

To study the long-term overall effect of physical exercise on blood coagulation, 20 sedentary males and 15 sedentary females were trained three to four times a week with increasing intensity for 9 months. After 24 and 36 weeks all subjects ran a 15 km and a half-marathon (21 km) race, respectively. Blood samples were drawn before the training programme, 5 days before both races and 5 days after the half-marathon run. Plasma factor VIII coagulant activity and von Willebrand factor antigen concentration did not increase during the training programme. In both males and females plasma fibrinogen concentration was not enhanced after 24 weeks of training but increased in preparation for the 21 km race and was still raised significantly (P < 0.01) 5 days later. No significant changes in plasma thrombin-antithrombin III concentrations were observed in either group during the training programme. The results of this study demonstrate that an exercise programme of increasing intensity induces physical stress which has significant effects on plasma fibrinogen concentrations, even at rest. However, in contrast to acute post-exercise effects, a regular physical fitness programme does not induce a long-term activation of the haemostatic system.

Inhibition of protein phosphorylation by opioid-inhibited adenylyl cyclase in rat brain membranes.

Opioid inhibition of adenylyl cyclase is a major second messenger system associated with opioid receptors in brain. To identify membrane phosphoproteins whose phosphorylation state is modulated by opioid inhibition of adenylyl cyclase, rat striatal membranes were preincubated with opioid agonists in the presence of 500 microM 5'-adenylyl-imidodiphosphate (which acted as a substrate for adenylyl cyclase, but not for protein kinase) before addition of [gamma-32P]ATP. Under these conditions, adenylyl cyclase in the membranes formed cyclic AMP, which stimulated cyclic AMP-dependent protein kinase. This process was confirmed by observing forskolin-stimulated phosphorylation of two bands of MW 85 and 63 kDa, which were also stimulated directly by cyclic AMP. Forskolin-stimulated phosphorylation of these two bands was inhibited by 15 to 30% by opioid agonists such as D-Ala2-Met5-enkephalinamide. This inhibition of phosphorylation was mediated by opioid receptors, because it required both sodium and GTP, and was blocked by naloxone. These results suggest that these two proteins may be primary targets of opioid-inhibited adenylyl cyclase in striatal membranes.

One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application.

We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.

Androstanediolglucuronide: a parameter for peripheral androgen activity before and during therapy with cyproterone acetate.

Serum 5 alpha-androstane-3 alpha,17 beta-diolglucuronide (3 alpha-AdiolG) levels were measured and the degree of hirsutism was scored in female outpatients complaining of excessive hair growth before and during treatment with cyproterone acetate. In a group of 16 patients with idiopathic hirsutism and in a group of 9 patients with either polycystic ovary syndrome and hirsutism or 21-hydroxylase deficiency and hirsutism, the serum 3 alpha-AdiolG levels were significantly increased (p less than 0.01) as compared with the serum 3 alpha-AdiolG level in a control group of 13 apparently healthy women: 3 alpha-AdiolG levels, median (range), being 5.3 (2.3-7.8) nmol/l, 8.5 (4.1-10.4) nmol/l, and 2.9 (1.5-5.2) nmol/l, respectively. In contrast to a previous report, no correlation was found between the serum 3 alpha-AdiolG levels and the Quetelet Index (N = 18, R = 0.42, p greater than 0.05), indicating an apparent ineffectiveness of the excessive androgen turnover in fat tissue. The use of the anti-androgen drug cyproterone acetate alone or in combination with ethinylestradiol in reverse sequential therapy did lower the 3 alpha-AdiolG levels significantly (p less than 0.01) together with a significant decrease (p less than 0.01) in hirsutism score. From the results of this study we therefore conclude that 3 alpha-AdiolG can be used as a parameter for peripheral androgen action before and during treatment with anti-androgens.