Therapeutic drug monitoring of anti-epileptic drugs - a clinical verification of volumetric absorptive micro sampling.

Background Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) can serve as a valuable tool in optimising and individualising epilepsy treatment, especially in vulnerable groups such as pregnant women, the elderly and children. Unfortunately, TDM is often performed suboptimally due to limitations in blood collection. Therefore, we investigated volumetric absorptive micro sampling (VAMS) - a new home-sampling technique. We aimed to evaluate VAMS to determine and quantify the different AEDs and concentrations of 16 different AEDs in whole blood collected by VAMS. Methods Patient blood samples (n = 138) were collected via venepunctures at the Academic Centre for Epileptology Kempenhaeghe. AED concentrations were determined, and these concentrations were used to compare the VAMS method (whole blood) with the conventional method (serum). In addition, the recovery was examined as well as the impact of haematocrit. Finally, AED-spiked blood was used to test the stability of the AEDs inside the micro-sampler devices over a period of time and whether temperature had an effect on the stability. Results VAMS allows for an accurate detection of 16 different AEDs within 2 days after sampling. Deviation in recovery was less than 10% and high correlations were found between VAMS and conventional sampling. Moreover, haematocrit does not have an effect with values between 0.3 and 0.5 (L/L). Finally, although storage temperature of VAMS does affect some AEDs, most are unaffected. Conclusions VAMS enables an accurate detection of a wide variety of AEDs within 2 days after sampling.

Inconclusive decisions and error rates in forensic science.

In recent years, there has been discussion and controversy relating to the treatment of inconclusive decisions in forensic feature comparison disciplines when considering the reliability of examination methods and results. In this article, we offer a brief review of the various viewpoints and suggestions that have been recently put forth, followed by a solution that we believe addresses the treatment of inconclusive decisions. We consider the issues in the context of method conformance and method performance as two distinct concepts, both of which are necessary for the determination of reliability. Method conformance relates to an assessment of whether the outcome of a method is the result of the analyst's adherence to the procedures that define the method. Method performance reflects the capacity of a method to discriminate between different propositions of interest (e.g., mated and non-mated comparisons). We then discuss implications of these issues for the forensic science community.

Denitrification of wastewater containing high nitrate and calcium concentrations.

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO3- -N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO3- -N/gVSSh and very low residual COD in the effluent.

Reconstruction of defects involving the Achilles tendon and local soft tissues: a quick solution for a lingering problem.

A total of seven patients (six men and one woman) with a defect in the Achilles tendon and overlying soft tissue underwent reconstruction using either a composite radial forearm flap (n = 3) or an anterolateral thigh flap (n = 4). The Achilles tendons were reconstructed using chimeric palmaris longus (n = 2) or tensor fascia lata (n = 2) flaps or transfer of the flexor hallucis longus tendon (n = 3). Surgical parameters such as the rate of complications and the time between the initial repair and flap surgery were analysed. Function was measured objectively by recording the circumference of the calf, the isometric strength of the plantar flexors and the range of movement of the ankle. The Achilles tendon Total Rupture Score (ATRS) questionnaire was used as a patient-reported outcome measure. Most patients had undergone several previous operations to the Achilles tendon prior to flap surgery. The mean time to flap surgery was 14.3 months (2.1 to 40.7). At a mean follow-up of 32.3 months (12.1 to 59.6) the circumference of the calf on the operated lower limb was reduced by a mean of 1.9 cm (sd 0.74) compared with the contralateral limb (p = 0.042). The mean strength of the plantar flexors on the operated lower limb was reduced to 88.9% of that of the contralateral limb (p = 0.043). There was no significant difference in the range of movement between the two sides (p = 0.317). The mean ATRS score was 72 points (sd 20.0). One patient who had an initial successful reconstruction developed a skin defect of the composite flap 12 months after free flap surgery and this resulted in recurrent infections, culminating in transtibial amputation 44 months after reconstruction. These otherwise indicate that reconstruction of the Achilles tendon combined with flap cover results in a successful and functional reconstruction.

Hematological advantage of a membrane oxygenator over a bubble oxygenator in long perfusions.

To determine whether the large volumes of cardiotomy suction which occur during long perfusions can obscure the hematological advantage of the membrane oxygenator (MO) over the bubble oxygenator (BO), we studied 23 patients undergoing a coronary artery bypass grafting operation with an expected perfusion time of 3 hours (MO group, N = 10, SciMed spiral coil; BO group, N = 13, Shiley 100-A). During MO perfusion we found significantly higher platelet numbers, better platelet function (adenosine diphosphate-induced platelet aggregation), and less hemolysis (plasma hemoglobin), than during the BO perfusion. After the MO perfusion we measured significantly shorter bleeding times (Simplate II) and fewer transfusions of blood products. However, blood loss and whole-blood transfusions 18 hours after perfusion did not differ significantly between both groups. So in coronary artery bypass grafting operations with long perfusion times (mean, 3 hours), the MO still causes significantly less platelet and erythrocyte damage than the BO, despite the large volumes of cardiotomy suction known to occur during these operations.

Isoelectric focusing and hybridisation experiments on creatine kinase (EC 2.7.3.2).

In the case of increased creatine kinase levels, the MM isozyme in human serum can often be subdivided by electrophoresis into three isozymes: MM1, MM2 and MM3. This paper presents the results of hybridisation experiments, which prove the existence of two different M-subunits. The MM isozyme fractions used in the hybridisation experiments were isolated by isoelectric focusing of human sera. The experiments were completed by hybridisation of mixtures of BB and MM isozymes, leading to the formation of two MB isozymes.

Mitochondrial creatine kinase (EC 2.7.3.2) in the brain.

No CK MM was found in human brain tissue. Both human and rat brain tissue, however, contain a non MM, non BB CK isozyme. The protein is membrane bound. Evidence is presented that it is a mitochondrial variant of the enzyme. This mitochondrial brain CK occurs in two forms. Both a low molecular mass form (Mr = 65,000) and a high molecular mass form are detectable. The isoelectric point of the low molecular mass form, Br-CKm1, is similar to the isoelectric point of CK MM. Complications may arise when techniques are used that try to separate the mitochondrial CK forms from CK MM on basis of differences in net change. The two mitochondrial brain CK forms are called Br-CKm1 and Br-CKm2. Whether these two enzymes are identical to the mitochondrial heart CK forms remains unclear.

Mitochondrial CK (EC 2.7.3.2) in the human heart.

CKm, a mitochondrial form of CK, can be isolated from human cardiac muscle. It occurs in two forms, separable on the basis of the difference in net charge. Both molecules have a dimeric structure and consist of two identical subunits. Upon incubation in a human serum, the CK isozyme with the faster cathodal mobility is rapidly converted into the slower form, which has an isoelectric point of 6.94. This makes it impossible to separate this slow CKM form from CK MM (isoelectric point: 6.86) by means of any method that separates on a basis of differences in net charge. Furthermore the two molecules have an almost identical molecular weight. The only difference between the two molecules we have been able to detect is the different behaviour against anti-M antibodies.

Identification of the carboxypeptidase responsible for the post-synthetic modification of creatine kinase in human serum.

The enzyme responsible for the post-translational modification of creatine kinase-MM isoenzyme was purified from human plasma. The enzymatic activity of this enzyme (modifying protein) on the synthetic substrates hippuryl-L-arginine, hippuryl-L-lysine, 3-(2-furylacryloyl)-L-arginine and 3-(2-furylacryloyl)-L-alanyl-L-lysine and the ratio of activities on these substrates are in good agreement with the enzymatic activity of the human serum carboxypeptidase N. The effect of metal ions, chelating agents, proteolytic inhibitors and carboxypeptidase N inhibitor could not differentiate the modifying protein from human serum carboxypeptidase N. Affinity chromatography on Concanavalin-A-Sepharose demonstrated the glycoprotein nature of the modifying protein. The difference in molecular weight observed between modifying protein and carboxypeptidase N can be explained by known instability characteristics and the influence of proteolytic enzymes during purification. Double immunodiffusion analysis with purified antiserum to human carboxypeptidase N confirmed the identity of the modifying protein and carboxypeptidase N.

Post-synthetic changes in creatine kinase isozymes (EC 2.7.3.2).

1. An in vivo change from creatine kinase MM3 into MM2 and finally into MM1 was described earlier in sera of patients during a post-myocardial infarction period. The same change takes place in vitro. 2. Parallel to this change, a turn-over from the MB2 isozyme into the MB1 form can be detected in vitro. 3. Immediately after the mixing of various extracts from heart tissue and other muscles with a serum with low creatine kinase activity, only MM3 and MB2 can be detected. MM1, MM2 and MB1 are not present in muscle cells. From these three observations it can be concluded that the phenomenon of the three MM and two MB isozymes is postsynthetic (i.e. epigenetic). Furthermore evidence is presented that the change from MM3 into MM1 is brought about by a thermolabile substance. This factor transforms one of the two M2 chains, present in the MM3 form, into a M1 chain. This results in the MM2 isozyme. Later on the second M2 chain is also transformed, resulting in the MM1 isozyme. The same mechanism is proposed for the observed change in MB pattern.

A study on post-synthetic modifications in alfa-alfa enolase (EC 4.2.1.11) brought about by a human serum protein.

Human alfa-alfa enolase is modified in the blood circulation by a serum protein for which the name 'modifying protein' is proposed. The protein occurs in every human serum tested and appears to be the same protein that is responsible for the post-synthetic modification in the M subunit of creatine kinase. Three alfa-alfa enolase forms, the original one plus two modified forms can be found in serum both in vivo and after in vitro incubation. The original alfa-alfa enolase is modified completely within a few hours in vitro in a pH-controlled human serum matrix at 37 degrees C. As the modification also takes place in vivo it is theoretically possible to acquire information about the activity of a disease process by doing one single determination of the amount of circulating alfa-alfa 3 enolase. A mechanism is proposed for the modification, where at first one of the two alfa chains is modified resulting in the alfa-alfa 2 form. Ultimately the second alfa chain is also modified. The alfa-alfa 1 form seems to be the completely modified alfa-alfa form. The three enolase forms differ in their isoelectric points but have similar Michaelis-Menten constants.

A study on the dimeric structure of creatine kinase (EC 2.7.3.2).

The generally accepted dimeric structure of creatine kinase (CK) is one in which M subunits and B subunits can occur. This paper reports the existence of three MM-bands and two MB-bands in human sera with increased CK-activity. These findings and supplementary data gathered from hybridisation experiments have led to the conclusion that there are two different M subunits, which can both occur in the enzyme in vivo. Further, findings are reported on the different CK-isozyme patterns of extracts from the cerebellum and from the cortex and the medulla of the cerebrum.

Indices for the age of the creatine kinase M-chain in the blood.

The apparent activation energy of the CK reaction as well as the Michaelis-Menten constants and the isoelectric point of CK MM can be used as indices for the mean age of the CK M-chain in the blood in vivo and in vitro. Modifications in the CK M-chain take place in vivo in the blood and in vitro in a serum matrix. Gradual increases in the apparent activation energy are also observed both in vivo and in vitro. It is confirmed that the modification in the CK M-chain causes a rise in the apparent activation energy, mu. A gradual increase in apparent activation energy, due to the ageing process of the CK M-chain, was observed after myocardial infarction. A significantly increased value for u was observed at the time that total CK activity already had returned to reference values. In spite of the normal CK value, the apparent activation energy still indicated that there had been myocardial damage. The Michaelis-Menten constants for creatine phosphate and ADP are also influenced significantly by the modification in the M-chain. While the apparent activation energy increases, the Michaelis constants decrease in the order MM3, MM2, MM1. The Michaelis-Menten constants for both ADP and CrP can be used as an index for the mean age of the enzyme in the blood. The Michaelis-Menten constants for CrP and ADP show significant variations with the measuring temperature for virtually all CK MM forms.

Purification and determination of the modifying protein responsible for the post-synthetic modification of creatine kinase (EC 2.7.3.2) and enolase (EC 4.2.1.11).

The purification of a serum protein, responsible for the postsynthetic modification of CK and enolase, is described. A purification of about 1300-fold could be reached after subsequent chromatography of human serum on DEAE cellulose and Sephacryl S-200 Superfine followed by affinity chromatography using antibodies against human serum albumin, C3 and C4 and against total human serum proteins. A recovery of 160% of modifying activity was found. The molecular mass and the isoelectric point of the modifying protein have been determined. It is concluded that the concentration of the modifying protein in human serum is less than 210 mg/l.

Immunological measurements on the disappearance of creatine kinase MM from the circulation.

Both a two-site immunoradiometric assay and a two-site enzyme-linked immunosorbent assay for creatine kinase MM are described. Linearity, reproducibility and cross-reactivity of the assays are satisfactory. Creatine kinase MM incubated in a pH-controlled serum matrix loses its activity, but has its antigenic determinants affected as well. Of all the techniques used, only the immunoradiometric assay is capable of measuring part of the inactivated enzyme. Measurements with this assay on the sera of patients after a myocardial infarction show identical results for enzyme activity and creatine kinase protein quantity. The in vitro disappearance rate of creatine kinase activity is slow compared with the in vivo half-life of the enzyme. These two observations lead to the conclusion that creatine kinase is removed from the circulation long before it is inactivated in the blood stream.

Evaluation of red blood cell size distribution histograms: the fraction of microcytes.

A method is described for the assessment of the fraction of microcytes from the red blood cell (RBC) size distribution histogram. For quantification of the fraction of microcytes an upper threshold corresponding with a RBC volume of 70 fL is established. The reference interval for the fraction of microcytes covers a range from 0.01 to 0.09. In order to examine the relationship between the fraction of microcytes and the mean cell volume (MCV) values computer simulation studies were performed. The computer simulations are based on a Gaussian distributed reference RBC histogram as generated on a Technicon H 6000/H 601 Hematology Analyser. From our studies it is shown that determination of the fraction of microcytes yields a higher sensitivity than MCV values for detecting small populations of microcytes. In particular, measurement of the fraction of microcytes is very sensitive to minor changes in the MCV values if a normocytic RBC population combined with a microcytic RBC fraction shows a wide dispersion in their cell volumes.

Changes in size and zinc protoporphyrin/haemoglobin ratio in red cells of infants during the first months of life.

A longitudinal investigation of changes in red blood cell (RBC) size distribution and zinc protoporphyrin/haemoglobin (ZPP/Hb) ratio was performed. In the course of the first few weeks after birth RBC size distribution histograms show a loss of macrocytic RBCs demonstrated by a rapid decrease of the fraction of macrocytes, whereas the degree of RBC volume dispersion decreases simultaneously. Within this period a significant change in the fraction of microcytes was not observed. At about 6 months of age, a marked shift of RBC size distribution histograms towards lower volumes occurs. Concomitant with the lower MCV values, the values for the absolute distribution width at half peak height (ADW0.5) also showed an obvious decrease. At birth, ZPP/Hb ratios are about three times higher than those of adult subjects and decrease slowly during infancy. This suggests that iron deficiency is unlikely in the first months of life.

Interference of plasma fluorophores on the red blood cell zinc protoporphyrin: haemoglobin ratio as determined on a haematofluorimeter.

Direct measurement of the zinc protoporphyrin:haemoglobin ratio (ZPP:Hb ratio) in blood samples is performed by using a haematofluorimeter. Interference by non-specific fluorophores can be eliminated by removing the plasma and making the measurement on washed red blood cells (RBCs). After re-suspending RBCs in isotonic saline, haematofluorimeter readings for the ZPP:Hb ratios revealed higher stability in the course of time whereas a good relationship was found with results obtained by application of an extraction method. Separate reference ranges were established for adult male and female subjects. After washing, the mean values calculated for ZPP:Hb ratios of subjects belonging to the reference groups demonstrated a reduction of 0.04 mumol ZPP mol Hb, corresponding with approximately 30%. In the patients' group, application of washing resulted in a variable decrease of ZPP:Hb ratios.

Some effects on light scattering intensity and red blood cell size distribution histograms due to sphering.

At present most haematology blood cell analysers routinely provide red blood cell (RBC) size distribution histograms. Sophisticated improvements of the instruments have re-awakened interest in the study of size histograms. The quantitative information derived from the histograms may be applied more fruitfully if insight is available, with respect to some essential principles of sizing technology and methods for treatment of RBCs before measurement. In this study the consequences of sphering RBCs are investigated in relation to the generation of size distribution histograms by means of methods based on light scattering intensity (LSI). Sphering of RBCs results in considerably narrower histograms than upsphered RBCs. The overall signal to noise ratio increases and there is a broader gap between large platelets and microcytic RBCs. Narrower size distribution ranges will enable closer modes to be separated. Compared to unsphered RBCs, microcytic sphered RBCs yield increased LSI whereas macrocytic sphered RBCs yield decreased LSI.

Similar alterations of lymphocyte subpopulations in type I and type II diabetes.

Peripheral blood lymphocytes of diabetes mellitus patients were analyzed by flow-cytometry using monoclonal antibodies directed against cell surface markers present in T- and B-cells, monocytes and natural killer cells. The lymphocyte subsets were quantified and expressed in an absolute amount. The study included 17 patients with type I (insulin-dependent), 21 patients with type II (non-insulin-dependent) diabetes and 40 age-matched control subjects. Quantification of the cells present within different lymphocyte subsets revealed a general increase in both patient groups compared to their controls, with the exception of activated T-cells. However, no significant difference was found in the relative amount of T-helper cells and T-suppressor/cytotoxic cells of the diabetes patients when they were compared with their control groups. The fact that we found similar changes in lymphocyte subsets in both type I and type II diabetes suggests that the altered immunological state is secondary to the diabetes mellitus in general.