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Effects of Pre-analytical Variables in Therapeutic Drug Monitoring
... The replacement of glass tubes by plastic tubes has minimized accidents due to broken glass, improved workplace safety and reduced safety concerns for health care professionals [64]. No differences in test interpretation were observed after switching from glass to plastic tubes for common clinical chemistry [65], therapeutic drug monitoring [66], coagulation [67], hormone, and tumor marker [68] tests. ...
In the clinical laboratory, knowledge of and the correct use of clot activators and anticoagulant additives are critical to preserve and maintain samples in optimal conditions prior to analysis. In 2017, the Latin America Confederation of Clinical Biochemistry (COLABIOCLI) commissioned the Latin American Working Group for Preanalytical Phase (WG-PRE-LATAM) to study preanalytical variability and establish guidelines for preanalytical procedures to be applied by clinical laboratories and health care professionals. The aim of this critical review, on behalf of COLABIOCLI WG-PRE-LATAM, is to provide information to understand the mechanisms of the interactions and reactions that occur between blood and clot activators and anticoagulant additives inside evacuated tubes used for laboratory testing. Clot activators-glass, silica, kaolin, bentonite, and dia-tomaceous earth-work by surface dependent mechanism whereas extrinsic biomolecules-thrombin, snake venoms, ellagic acid, and thromboplastin-start in vitro coagulation when added to blood. Few manufacturers of evacuated tubes state the type and concentration of clot activa-tors used in their products. With respect to anticoagulant additives, sodium citrate and oxalate complex free calcium and ethylenediaminetetraacetic acid chelates calcium. Heparin potentiates antithrombin and hirudin binds to active thrombin, inactivating the thrombin irreversibly. Blood collection tubes have improved continually over the years, from the glass tubes containing clot activators or anticoagulant additives that were prepared by laboratory personnel to the current standardized evacuated systems that permit more precise blood/additive ratios. Each clot activa-tor and anticoagulant additive demonstrates specific functionality, and both manufacturers of tubes and laboratory professional strive to provide suitable interference-free sample matrices for laboratory testing. Both manufacturers of in vitro diagnostic devices and laboratory professionals need to understand all aspects of venous blood sampling so that they do not underestimate the impact of tube additives on laboratory testing.
... Concentration differences when measuring different matrices can occur as a consequence of differential binding to a specific component of the blood (e.g. serum proteins or blood cells) [33] . The blood to plasma ratio is a measure to determine the concentration of a target analyte (in whole blood compared to plasma) and provides an indication of the analyte binding to erythrocytes. ...
The perspective discusses quantitative DBS analysis for anti‐doping testing in an athletic population and why only using volumetric sampling for this subgroup might not be enough. It presents examples to highlight where HCT variations occur, followed by a whole blood to plasma ratio and a HCT extraction bias discussion. Finally, options to correct for the HCT bias are presented.
... However, the most available commercial separator gels are expensive and they have several performance limitations including, instability for certain analytes, instability of the polymeric gel under extreme temperature conditions, presence of a part of gel or an oily film in the serum, absorption of specific drugs and some steroid hormones into the gel [23]. ...
Blood serum separator tubes (SST) are used for collecting blood samples for performing clinical chemistry assays. Some SST's have separator gels inside the tube which enable them better separation of the blood serum from packed cells during centrifugation. The cost, weak performance and interaction with blood ingredients are the most concerns of the available commercial gels. A commercial and cheap silicone oil as a polymeric base of the gel was chosen and formulated without and with several fillers. Subsequently, the compounds were crosslinked through a free radical crosslinking mechanism using dicumyl peroxide (DCP). The crosslinking took place in both, an oven and as well as under microwave irradiation in normal and under pressure conditions. The FTIR spectrometer analysis showed that both chain ends of the used silicone oil were terminated with a vinyl group. It also revealed that blood serum separator gel can be produced from selected silicone oil type. Among of different curing apparatus, curing in an oven was preferred due to less curing time and electrical energy consumption. The curing in normal pressure showed better results when compared with curing under pressure. Increasing the filler and DCP with various amounts had positive effect on gel densities. Silica was the most efficient filler among of the studied fillers. The cured compound filled with 10 and 8 phr silica and DCP, respectively, was chosen as appropriate gel for SST due to suitable density and thixotropy. The selected gel was cured in oven under normal pressure for 30 minutes at 160°C.
... Contamination from interstitial fluid, hemolysis and residual contaminations from Are capillary DBS applicable for therapeutic drug monitoring of common antipsychotics? A proof of concept future science group Research Article Patteet, Maudens, Stove et al. the surface of the skin can influence capillary concentrations too [15][16][17][18]. Therefore, a thorough clinical study was conducted to make a comparison between antipsychotic concentrations found in venous whole blood and capillary DBS samples. ...
DBS sampling has been proposed as an alternative for venous blood collection in therapeutic drug monitoring (TDM) of antipsychotics. For implementation in routine practice, a comparison between capillary and venous blood concentrations is mandatory.
A DBS method for quantification of antipsychotics was clinically validated. First, whole blood therapeutic ranges were calculated using the blood:serum ratio. Calculation of DBS:blood ratios and Passing-Bablok regression analysis demonstrated that concentrations obtained by DBS analysis were highly comparable to those obtained by conventional whole blood analysis. Clinical interpretation of serum, whole blood and DBS concentrations were highly identical (sensitivity 91.6-97.6%).
This is the first clinical study demonstrating the value of DBS sampling in TDM of antipsychotics.
Pemeriksaan asam urat adalah salah satu pemeriksaan yang dilakukan pada laboratorium klinik, untuk mendeteksi asam urat di dalam tubuh. Bahan pemeriksaan asam urat yang utama digunakan ialah sampel serum. Tabung yang akan digunakan untuk pemeriksaan asam urat yaitu tabung plain dan tabung SST adapun tujuan untuk mengetahui perbedaan kadar asam urat menggunakan tabung plain dan SST dengan menggunakan alat chemistry analyzer. Penelitian ini merupakan penelitian kuantitatif dengan rancangan penelitian observasional analitik dengan pendekatan cross sectional. Subjek pada penelitian ialah 35 mahasiswa/i yang telah memenuhi kriteria inklusi dan eksklusi. Uji statistik menggunakan uji Paired t Test dengan tingkat kepercayaan 95%. Rata-rata kadar asam urat menggunakan tabung plain adalah 5,33 mg/dL. Rata-rata kadar asam urat menggunakan tabung SST adalah 5,19 mg/dL. Dengan menggunakan uji paired t test diperoleh p = 0.000 (p < 0.05), yang berarti terdapat perbedaan hasil asam urat menggunakan tabung plain dan SST dengan chemistry analyzer. Terdapat perbedaan bermakna pada kadar asam urat menggunakan tabung plain dan tabung SST. Tabung plain disarankan sebagai tabung penampung darah untuk pemeriksaan asam urat dengan chemistry analyzer.
ISPEC 6th INTERNATIONAL CONFERENCE ON AGRICULTURE, ANIMAL SCIENCE and RURAL DEVELOPMENT May 16 -18, 2021 Siirt, Turkey
Laboratory diagnostics, a pivotal part of clinical decision making, is no safer than other areas of healthcare, with most errors occurring in the manually intensive preanalytical process. Patient misidentification errors are potentially associated with the worst clinical outcome due to the potential for misdiagnosis and inappropriate therapy. While it is misleadingly assumed that identification errors occur at a low frequency in clinical laboratories, misidentification of general laboratory specimens is around 1% and can produce serious harm to patients, when not promptly detected. This article focuses on this challenging issue, providing an overview on the prevalence and leading causes of identification errors, analyzing the potential adverse consequences, and providing tentative guidelines for detection and prevention based on direct-positive identification, the use of information technology for data entry, automated systems for patient identification and specimen labeling, two or more identifiers during sample collection and delta check technology to identify significant variance of results from historical values. Once misidentification is detected, rejection and recollection is the most suitable approach to manage the specimen.
Clin Chem Lab Med 2009;47:143–53.
The accuracy of gentamicin, netilmicin, and tobramycin concentration determinations by enzyme multiplied immunoassay technique (EMIT; Syva Corp., Palo Alto, Calif.), fluorescence polarization immunoassay (TDx; Abbott Diagnostics, Irving, Tex.), and radioimmunoassay were compared in the presence of 0 to 3,000 USP units of porcine heparin per ml. Gentamicin, netilmicin, and tobramycin concentrations determined by EMIT decreased by 10 and 50% in the presence of 75 and 1,000 USP units/ml, 2 and 5 USP units/ml, and 2 and 7.5 USP units/ml, respectively. Accuracy of the TDx and radioimmunoassay determinations, however, were not affected by the presence of heparin. Blood samples for the determination of gentamicin, netilmicin, and tobramycin by EMIT should not be collected in evacuated heparinized tubes.
The influence of various brands of evacuated blood collection systems (the old type, red stoppered Vacutainer®; the new type, blue stoppered Vacutainer®; Monoject® and Venoject®) on therapeutic drug monitoring was investigated. No interferences were found in the assay of ethosuximide, phenobarbital, phenytoin, valproic acid, digitoxin, digoxin, procainamide, gentamicin and theophylline.
Using Monoject® and old type Vacutainer® tubes, lower levels were found in the disopyramide assay: 91.3±4.6% (p<0.05) and 91.7±7.0% (not significant) respectively, and in the quinidine assay: 82.8±6.7% (p<0.02) and 83.9±4.4% (p<0.001) respectively as compared with glass tubes. In the carbamazepine assay a decrease was found in the Monoject® tubes only: 93.7±1.7% (p<0.01). The stoppers of Monoject® tubes and the old type Vacutainer® tubes contained the plasticizer tris(2-butoxyethyl)phosphate (tbep), which has been shown to be a potent inhibitor of the binding of several drugs to α1-acid glycoprotein.
Using the new type Vacutainer® and the Venoject®, no interferences were found.
Measurements of aminoglycosides by an agar disk diffusion assay are inhibited by heparin in a dose-dependent way. When assayed
by a homogeneous immunoassay, this was only evident for tobramycin. This indicates that specimens for aminoglycoside measurement
should not be obtained in heparinized tubes. When heparin is used clinically as an anticoagulant, the amount in blood does
not reach levels that affect the aminoglycoside activity.
Context.—Complementary and alternative medicine (herbal medicines) can affect laboratory test results by several mechanisms.
Objective.—In this review, published reports on effects of herbal remedies on abnormal laboratory test results are summarized and commented on.
Data Sources.—All published reports between 1980 and 2005 with the key words herbal remedies or alternative medicine and clinical laboratory test, clinical chemistry test, or drug-herb interaction were searched through Medline. The authors' own publications were also included. Important results were then synthesized.
Data Synthesis.—Falsely elevated or falsely lowered digoxin levels may be encountered in a patient taking digoxin and the Chinese medicine Chan Su or Dan Shen, owing to direct interference of a component of Chinese medicine with the antibody used in an immunoassay. St John's wort, a popular herbal antidepressant, increases clearance of many drugs, and abnormally low cyclosporine, digoxin, theophylline, or protease inhibitor concentrations may be observed in a patient taking any of these drugs in combination with St John's wort. Abnormal laboratory results may also be encountered owing to altered pathophysiology. Kava-kava, chaparral, and germander cause liver toxicity, and elevated alanine aminotransferase, aspartate aminotransferase, and bilirubin concentrations may be observed in a healthy individual taking such herbal products. An herbal product may be contaminated with a Western drug, and an unexpected drug level (such as phenytoin in a patient who never took phenytoin but took a Chinese herb) may confuse the laboratory staff and the clinician.
Conclusions.—Use of alternative medicines may significantly alter laboratory results, and communication among pathologists, clinical laboratory scientists, and physicians providing care to the patient is important in interpreting these results.
The protein binding of a number of basic drugs has been shown to be inhibited when blood is collected in Vacutainer tubes. We found that the plasticizer tris(2-butoxyethyl) phosphate (TBEP). present in plasma collected in Vacutainers. was a potent inhibitor of alprenolol and imipramine protein binding. Its concentration in the plasma could quantitatively explain the displacement phenomenon. Alprenolol binding to a solution of a physiologic concentration (0.67 gmlL. 0.015 mM) of α1-acid glycoprotein (orosomucoid) was decreased from 76% to 16% by addition of 10 uglml (0.026 mM) of TBEP. while imipramine binding was decreased from 69% to 13%. Alprenolol and imipramine binding to albumin and lipoproteins was virtually unchanged by TBEP. Due to its selective effect on binding to α1-acid glycoprotein. TBEP may be a useful tool for studying plasma protein binding of basic drugs.
Asian and Siberian ginsengs contain glycosides with structural similarities to digoxin. We studied potential interference of ginseng in 5 digoxin immunoassays in 3 Asian (2 liquid extracts, 1 capsule) and 3 Siberian ginseng preparations (1 liquid extract, 2 capsules). With the fluorescence polarization immunoassay (FPIA), we observed apparent digoxin activity in 1 Asian liquid preparation and in the liquid extract and 1 capsule form of Siberian ginseng. In mice fed ginseng, we observed digoxin activities in the serum (Asian, 0.48–0.68 ng/mL [0.6–0.9 nmol/L]; Siberian, 0.20–0.47 ng/mL [0.3–0.6 nmol/L]), indicating that such interferences also occur in vivo. Serum pools prepared from samples from patients receiving digoxin and then supplemented with Asian or Siberian ginseng showed falsely increased digoxin values using the FPIA (eg, for Asian ginseng, 1.54 ng/mL [2.0 nmol/L] vs control value, 1.10 ng/mL [1.4 nmol/L]) and falsely decreased values using the microparticle enzyme immunoassay (MEIA; 0.73 ng/mL [0.9 nmol/L] vs control value, 1.04 ng/mL [1.3 nmol/L]). Digoxin-like immunoreactive substances (DLISs) showed synergistic effects with ginsengs in interfering with the FPIA and MEIA for digoxin. No interference was observed with 3 other digoxin assays, even in the presence of elevated DLISs.
A separator or barrier gel is a common component of serum and plasma collection tubes. Despite their advantages, the use of these tubes is not universally accepted, especially for therapeutic drug monitoring (TDM). The aim of this study was to evaluate whether the polyacrylester separator gel in Sarstedt S-Monovette\® tubes influences the concentration of 10 selected parameters (amikacin, vancomycin, valproic acid, acetaminophen, cortisol, free thyroxine, thyroid-stimulating hormone, transferrin, prealbumin and carcinoembryonic antigen) in a clinically significant way.
Results from patient samples collected in plastic Sarstedt S-Monovette® tubes with separator gel were compared with those from plain serum sample tubes. Analytes were measured in both tubes on 4 consecutive days to study the influence of prolonged contact with the separator gel. Between analyses tubes were stored at 4°C. Stability was also evaluated over 72 h for each collection tube. When statistical differences were detected, the clinical significance was evaluated based on the total allowable error (TEa).
On day 1 no statistically significant differences were observed between samples collected in Sarstedt S-Monovette® tubes with and without separator gel. Statistical differences were present from day 2 on, but were not clinically significant. All evaluated parameters were clinically stable over 72 h at 4°C based on TEa, except for transferrin en fT4.
The separator gel in Sarstedt S-Monovette® tubes did not show statistically significant differences on the day of phlebotomy. Later on statistically significant differences appeared but except for the stability of fT4 and transferrin they all remained clinically insignificant.
Although current abuse of barbiturates is low compared with other classes of abused drugs, their narrow margin of safety, risk of dependence, and abuse liability remain a health concern. Limited information is available on the disposition of barbiturates in different biologic matrices.
The authors conducted a clinical study of the disposition of barbiturates in oral fluid, plasma, and urine after single-dose administration to healthy subjects.
Three parallel groups of 15 subjects were administered a single oral dose of one barbiturate: butalbital (50 mg), Phenobarbital (30 mg), or sodium secobarbital (100 mg). Subjects remained at the clinic for two confinement periods; the first was -1 to 36 hours postdose and again at 48 to 52 hours. Oral fluid specimens were collected by bilateral collection (Intercept; one on each side of the mouth simultaneously). Blood specimens were obtained by venipuncture and urine specimens were collected through separate collection pools of varying periods. Oral fluid specimens were analyzed for barbiturates by liquid chromatography-tandem mass spectroscopy with a limit of quantitation of 8 ng/mL. Plasma and urine specimens were analyzed by gas chromatography-mass spectroscopy with a limit of quantitation of 100 ng/mL.
Barbiturate side effects included dizziness, drowsiness, and somnolence. All effects resolved spontaneously without medical intervention. The three barbiturates were detectable in oral fluid and plasma within 15 to 60 minutes of administration and in the first urine pooled collection at 2 hours. Butalbital and Phenobarbital remained detectable in all specimens through 48 to 52 hours, whereas secobarbital was frequently negative in the last collection. Oral fluid to plasma ratios appeared stable over the 1- to 48-hour collection period.
This study demonstrated that single, oral therapeutic doses of butalbital, Phenobarbital, and secobarbital were excreted in readily detectable concentrations in oral fluid over a period of approximately 2 days. Oral fluid patterns of appearance and elimination were similar to that observed for plasma and urine.
The considerable variability in the warfarin dose–response relationship between individuals, is explained mainly by genetic variation in its major metabolic (CYP2C9) and target (VKORC1) enzymes. Despite the predominance of pharmacogenetics, environmental factors also affect the pharmacokinetics and pharmacodynamics of warfarin, and are often overlooked. Among these factors, dietary and supplemental vitamin K consumption is a controllable contributor to within‐, and between‐patient variability of warfarin sensitivity. In this commentary we review the current role of vitamin K in warfarin anticoagulation therapy, with emphasis on the following:
1 The effect of dietary and supplemental vitamin K on warfarin anticoagulation, beyond the impact of genetic variability in CYP2C9 and VKORC1. We deal separately with the effects of vitamin K on warfarin dose requirements during the induction of therapy, as opposed to its effect on stability of anticoagulation control during maintenance therapy.
2 The role of vitamin K supplementation in warfarin treated patients with vitamin K deficiency as well as in patients with unstable warfarin anticoagulation, and
3 The role of therapeutic vitamin K in cases of warfarin over‐anticoagulation.
The term pharmacogenetics was first used in the late 1950s and can be defined as the study of genetic factors affecting drug response. Prior to formal use of this term, there was already clinical data available in relation to variable patient responses to the drugs isoniazid, primaquine and succinylcholine. The subject area developed rapidly, particularly with regard to genetic factors affecting drug disposition. There is now comprehensive understanding of the molecular basis for variable drug metabolism by the cytochromes P450 and also for variable glucuronidation, acetylation and methylation of certain drugs. Some of this knowledge has already been translated to the clinic. The molecular basis of variation in drug targets, such as receptors and enzymes, is generally less well understood, although there is consistent evidence that polymorphisms in the genes encoding the beta-adrenergic receptors and the enzyme vitamin K epoxide reductase is of clinical importance. The genetic basis of rare idiosyncratic adverse drug reactions had also been examined. Susceptibility to reactions affecting skin and liver appears to be determined in part by the HLA (human leucocyte antigen) genotype, whereas reactions affecting the heart and muscle may be determined by polymorphisms in genes encoding ion channels and transporters respectively. Genome-wide association studies are increasingly being used to study drug response and susceptibility to adverse drug reactions, resulting in identification of some novel pharmacogenetic associations.
Important scientific principles of pain medicine pharmacology affect urine drug testing (UDT). This paper reviews sources of variability in pharmacokinetics, pharmacodynamics, pharmacogenetics, and issues relating to the collection, handling, and assay of urine and how these factors may affect test interpretation and application.
Articles concerning the pharmacokinetics, pharmacodynamics, and pharmacogenetics of opioids are reviewed and interpreted for pain clinicians who treat patients with chronic opioid therapy. These data are applied to the use of UDT.
Intraindividual and interindividual variability in drug metabolizing enzyme activity due to genetic polymorphisms or environmental effects can result in day-to-day and patient-to-patient variability in drug exposure. Transporters, also under genetic and environmental control, can play an important role in opioid response and contribute to the significant variability in opioid pharmacokinetics and response. The use of urine creatinine concentration to adjust urine drug concentrations, discussion of UDT assays, and application of UDT in light of an understanding of pharmacokinetics, pharmacodynamics, and pharmacogenetics are reviewed. In addition, the methodology used for testing has an important role in accuracy. Because of these factors, UDT cannot be used to determine patient compliance with a specific opioid dose.
UDT, when used with an understanding of the principles of pharmacokinetics, pharmacodynamics, and pharmacogenetics of opioids, can be a useful tool in chronic pain management. Clinicians must keep in mind the limitations, purpose, and value of UDT, and the inability to predict patient compliance with a drug dosage using commercial algorithms.
We measured the concentrations of phenobarbital, phenytoin, primidone, ethosuximide, antipyrine, and caffeine in paired samples of saliva and plasma by gas chromatograph-mass spectrometer-computer (GC/MS/COM) and enzyme immunoassay. Mixed saliva was collected for the antipyrine and caffeine studies, parotid saliva for the phenobarbital, primidone, ethosuximide and phenytoin studies. The saliva/plasma (S/P) ratios (by weight) obtained by GC/MS/COM were: phenobarbital, 0.31-0.37; phenytoin, 0.11; ethosuximide, 1.04; antipyrine, 0.83-0.95; caffeine, 0.55. The S/P ratio obtained by enzyme immunoassay were: phenobarbital, 0.32; phenytoin, 0.12; primidone, 0.85. The concentrations of phenytoin, primidone, ethosuximide and antipyrine in saliva correspond to the free fraction of the drug in plasma. When we analyzed samples containing phenobarbital or phenytoin (plasma or saliva) by both techniques, we found that the enzyme immunoassay values were generally higher than GC/MS/COM values, suggesting that the metabolites as well as the parent drug were measured in the immunoassay.
In 25 clinical samples serum lidocaine concentrations fell from a mean of 6.5 +/- 2.1 mg/L (mean +/- SD) to 4.9 +/- 1.8 mg/L (p less than 0.001) when the blood sample was allowed to make contact with the stopper of the Vacutainer collection tube. In vitro experiments showed that this effect of the stopper occurred only with whole blood and was dependent on sample concentration. The plasma binding of lidocaine decreased from a normal value of 56% +/- 2.2 (mean +/- SD) to 28% +/- 2.2 (p less than 0.001) when exposed to the Vacutainer stopper. We conclude that a chemical leached from such stoppers displaces lidocaine from its plasma-binding sites and that the drug is then redistributed into the erythrocytes, producing spuriously low lidocaine concentrations in plasma or serum. Such artifacts are important in therapeutic drug monitoring and can lead to erroneous clinical decisions.
With the use of glass syringes without heparin and all glass equipment, the percent of unbound quinidine was measured by ultrafiltration and a double-extraction assay method after addition of 2 microgram/ml of quinidine sulfate. Compared to the all-glass method, collection of blood using Vacutainers resulted in an erroneous and variable decrease in quinidine binding related to blood to rubber-stopper contact. With glass, the unbound quinidine fraction was (mean +/- standard error) 10 +/- 1% in 10 normal volunteers, 8.5 +/- 1.5% in 10 patients with congestive heart failure, and 11 +/- 2% in 11 patients with chronic renal failure (although in 8 of the latter 11 patients the percent of unbound quinidine was 4 or more standard errors from the mean of the normal group). During cardiac catheterization, patients had markedly elevated unbound quinidine fractions: 24 +/- 2% (p less than 0.001). This abnormality coincided with the addition of heparin in vivo and was less apparent after the addition of up to 10 U/ml of heparin in vitro (120% and 29% increase in unbound quinidine fractions, respectively). Quinidine binding should be measured with all glass or equivalent equipment.
The committee on pediatric clinical chemistry (CPCC) has published selected techniques for collecting blood from newborns and older infants; the national committee for clinical laboratory standards (NCCLS) proposed 'standards' for collecting blood for patients of all ages. The authors of this article present comprehensive information on this subject, which intends 'to strengthen the NCCLS in its efforts to establish standards... in this routine but critical manipulation. It is stated that 75 to 95% of all requests for clinical chemical tests on blood (excluding P(O 2) (?)) in a pediatric institution are performed on samples, obtained by skin puncture. Skin puncture; blood collection, transport, centrifugation; a discussion; and 59 references are included. (van Kampen, Groningen).
The protein binding of a number of basic drugs has been shown to be inhibited when blood is collected in Vacutainer tubes. We found that the plasticizer tris(2-butoxyethyl) phosphate (TBEP), present in plasma collected in Vacutainers, was a potent inhibitor of alprenolol and imipramine protein binding. Its concentration in the plasma could quantitatively explain the displacement phenomenon. Alprenolol binding to a solution of a physiologic concentration (0.67 gm/L, 0.015 mM) of alpha 1-acid glycoprotein (orosomucoid) was decreased from 75% to 16% by addition of 10 microgram/ml (0.026 mM) of TBEP, while imipramine binding was decreased from 69% to 13%. Alprenolol and imipramine binding to albumin and lipoproteins was virtually unchanged by TBEP. Due to its selective effect on binding to alpha 1-acid glycoprotein, TBEP may be a useful tool for studying plasma protein binding of basic drugs.
This article has no abstract; the first 100 words appear below.
Most drugs are carried from their sites of absorption to their sites of action and elimination by the circulating blood. Some drugs are simply dissolved in serum water, but many others are partly associated with blood constituents such as albumin, globulins, lipoproteins and erythrocytes. For the great majority of drugs binding to serum albumin is quantitatively by far the most important and often accounts for almost the entire drug binding in plasma.¹²³ Albumin binding influences the fate of drugs in the body. Only the unbound or free drug diffuses through capillary walls, reaches the site of drug action, and is . . .
*Part two will appear in the March 4 issue.
Source Information
From the Centre de Recherche Merrell International, Strasbourg, France, and the Division of Clinical Pharmacology, the Clinical Institute, Addiction Research Foundation and the Toronto Western Hospital, Toronto, Canada (address reprint requests to Dr. Koch-Weser at the Centre de Recherche Merrell International, 16, rue d'Ankara, 67000 Strasbourg, France).
Spuriously low values of plasma propranolol concentrations resulted when one commercial brand of collection tube was used. The mechanism of this effect was established and was due to the presence of the stopper containing substances that reduced plasma propranolol binding and resulted in a redistribution of the drug in whole blood so that more was present in the cells and less in the plasma. A similar phenomenon was noted some years ago when a needle for intravenous infusion was used as an indwelling cannula. Th mechanism in this case could not be established as in the interim the phenomenon was no longer detectable. Although it was established that these findings do not apply to all drugs, the data suggest that collection methods should be routinely checked at intervals unless separate venipuncture and all glass collection tubes are used.
We evaluated a plastic evacuated blood-drawing tube containing an integral serum-separating barrier gel, by direct comparison with a glass counterpart. The plastic tube demonstrated no differences when compared for common clinical chemistry analytes with multiple types of instruments and systems. A total of 260 such different combinations were studied with emphasis on tests sensitive to drawing and handling indexes such as lactate dehydrogenase and potassium. A total of six separate blood drawings were tested with no significant differences noted in these tests. The total study included subjective evaluations of the plastic tube's use as a blood-drawing device and objective studies based on quantitative test results from normal and hospitalized patients and use of the primary sampling tubes (both plastic and glass) for 48-h storage.
The effect of serum separator tubes (SSTs) on free and total serum phenytoin and carbamazepine concentrations was determined by comparing standard no-additive tubes with SSTs (Becton Dickinson SST and Terumo Autosep). The influence of time prior to centrifugation, sample volume, and initial drug concentration on the effects were also studied. Results were analyzed using repeated measures two-way analysis of variance with tube type and either time, sample volume, or concentration as main effects. The most significant reductions noted were with Becton Dickinson SSTs in free and total serum phenytoin and total carbamazepine concentrations, where all reductions were less than 10%. The only factor to significantly influence extent of reduction was the effect of time on total serum phenytoin concentration in Becton Dickinson SSTs. Terumo Autosep tubes caused no major reductions in free or total phenytoin or carbamazepine serum concentrations. Autosep tubes should provide accurate measurements of total and free serum phenytoin and carbamazepine concentrations. With Becton Dickinson SSTs, the reductions noted in free and total phenytoin and total carbamazepine concentrations were not large enough to preclude their clinical use. Becton Dickinson SSTs should not be used for determining free or total phenytoin or total carbamazepine concentrations for purposes of research.
The furan dicarboxylic acid 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (5-propyl FPA) accumulates in uraemic plasma and is a potent inhibitor of the binding of other anionic ligands to albumin. The interaction of 5-propyl FPA with human albumin has been investigated by equilibrium dialysis at 37 degrees and pH 7.4. Analysis of the binding data on the basis of a two-site model gave binding parameters of n1 = 0.6 and K1 = 4.8 x 10(6) M-1 for the primary binding site. 5-Propyl FPA binding was observed to decrease as the pH was raised from 6.4 to 8.3 which emphasizes the need for pH control of whole plasma or serum. Temperature, however, had little effect on binding as assessed by equilibrium dialysis at 10 degrees, 25 degrees and 37 degrees. The high affinity of 5-propyl FPA for albumin explains its retention in uraemic plasma, its potency as a binding inhibitor and points to active tubular secretion as the mechanism by which it is normally excreted by the kidney.
The purpose of this study was to determine the suitability of four collection tubes containing separation gels for collection of specimens for 15 therapeutic drugs. To blood tubes from drug-free volunteers, selected drugs were added and measured after centrifugation and storage for 6, 24, and 72 h in the collection tube. We found that the Becton Dickinson SST tube extracted phenytoin even with immediate separation of serum. For prolonged storage, the SST tube was unsuitable for quinidine (greater than 24 h) and lidocaine (greater than 6 h); the Monoject Corvac was unsuitable for lidocaine (any storage). Partially filled tubes caused additional errors. Completely filled Terumo Autosep and Monoject Corvac tubes are satisfactory for all drugs tested if serum is removed promptly.
The free fractions of five drugs were determined in plasma treated with EDTA and in serum. The free fractions of phenytoin, sodium valproate, and phenobarbital in serum were significantly higher than in plasma (P less than 0.05). In contrast, the free fractions of carbamazepine and theophylline were significantly lower in serum than in plasma. Although we observed minor differences in the protein patterns obtained with two-dimensional electrophoresis, these did not have an important influence on protein binding. No significant differences were observed between plasma and serum in the physiological data, except for pH. Binding of these drugs by protein was shown to be pH-dependent. We conclude that serum may be better than plasma for determination of the free fraction, because the free fraction in plasma is affected by anticoagulants such as EDTA salts and heparin.
The plasma (or serum) protein binding of disopyramide (DSP) in five haemodialysis patients was studied using an ultrafiltration technique. There was an increase in the free fraction of DSP in the plasma on dialysis days in comparison to the levels on interdialysis days, which was associated with an elevation of the free fatty acid levels in the plasma together with the increase of the free fraction of DSP. The inhibitory effect of free fatty acids on DSP binding in an in vitro study was enhanced in proportion to their concentrations, and was shown to be due to competition at one binding site by experiments with oleic acid as a representative displacer.
Certain endogenous organic acids, such as indoxyl sulphate, 2-hydroxyhippuric acid and hippuric acid, which are characteristically elevated in chronic renal failure, scarcely affected the protein binding of DSP. The findings indicate that free DSP should be monitored in patients with elevated plasma free fatty acid levels, such as those on haemodialysis therapy.
The terms "serum concentration" and "plasma concentration" are often used interchangeably in therapeutic drug monitoring, but few studies have addressed the comparability of serum and plasma drug concentrations. Before implementing a change in the procedure for measuring aminoglycoside concentrations in our institution, we prospectively compared values for tobramycin and gentamicin concentrations measured in serum and plasma. For the 208 samples that were tested, plasma aminoglycoside concentrations were significantly lower than those in serum. This relationship was similar for tobramycin and gentamicin, and was unaffected by the presence of concurrent therapy with ticarcillin. This difference, while statistically significant, was not, in our estimation, of sufficient magnitude to be of clinical importance. Plasma aminoglycoside concentrations may be considered equivalent to serum concentrations and, owing to the shorter processing time involved, appear to be the preferred medium for measuring these values.
The influence of storage conditions on the total and free concentration of valproic acid (VPA) was studied in six normal male subjects who ingested 750 mg of VPA (3 X 250 mg Depakene capsules; Abbott Laboratories). Blood samples were collected in various types of Vacutainer tubes (red top, no additives; green top, sodium heparin; blue top, sodium citrate; and purple top, EDTA) 2 h post administration of VPA. Either these samples were centrifuged immediately or stored for various periods of time at room temperature or refrigerated, or the supernate was frozen prior to analysis. Free VPA samples were obtained utilizing the Amicon ultrafiltration system. All VPA samples were analyzed by gas-liquid chromatography. Total VPA concentrations obtained from plasma collected with sodium citrate were lower (p less than 0.05) than either serum or plasma collected with other anticoagulants. There were no differences (p greater than 0.05) in total or free VPA concentrations between samples collected in serum or in plasma collected with heparin or EDTA. Storing samples for 96 h at room temperature did not alter the total VPA concentrations but was found to increase the free fraction of VPA (p less than 0.05). The refrigeration or freezing of the supernate from the blood samples for 7 days did not alter (p greater than 0.05) the total or the free fraction of VPA. The results of this study demonstrate that total and/or free VPA may be collected from either serum or plasma, provided sodium citrate is not used to collect plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
The stability of therapeutic concentrations of 11 drugs (amikacin, carbamazepine, digoxin, gentamicin, lithium, methotrexate, phenobarbital, phenytoin, quinidine, theophylline, tobramycin) and two trace elements (copper and zinc) in plasma stored in serum separator ("Corvac" brand) blood collection tubes was investigated over a 1 week period of storage in the refrigerator. No significant change in concentration was noted for any analyte during the study period. Concentrations were also not significantly different from those observed during concurrent storage of the same plasma samples in nonserum separator ("Vacutainer" brand) blood collection tubes.
We have investigated the change in amiodarone and desethylamiodarone concentrations in blood sampled from three different Vacutainer tubes: (a) sodium heparin, (b) gel separator (SST), and (c) no additive (plain tube). Amiodarone and desethylamiodarone concentrations were determined by a reverse-phase high pressure liquid chromatography technique in samples from 12 subjects on chronic amiodarone therapy. Amiodarone concentrations were significantly lower in plasma compared with serum from either gel separator (11.5%, p = 0.05) or no additive (13.5%, p = 0.01) tubes. Desethylamiodarone concentrations were significantly lower in plasma compared with serum from gel separator tubes (8.5%, p = 0.04) and were slightly lower compared with no additive tubes (4.4%, p = 0.41). Serum amiodarone and desethylamiodarone concentrations from either no additive or gel separator tubes yielded similar results. We conclude that significant amiodarone and desethylamiodarone concentration differences occur between serum and plasma, and that no binding of amiodarone and desethylamiodarone to the separator gel occurs.
The binding of disopyramide (DSP) to plasma (or serum) proteins was determined using an ultrafiltration technique in three patients undergoing haemodialysis.
An increase in the free fraction (FF) of DSP during dialysis occurred together with elevation of the free fatty acid (FFA) level in plasma. The effect of FFA on protein binding in vitro was examined using DSP- and FFA-spiked solutions containing human α1-acid glycoprotein and serum albumin. The FF of DSP rose in proportion to increasing FFA levels, supporting the in vivo observations.
The findings suggest that the free concentration of DSP should be routinely monitored, especially in haemodialysis patients.
The stability and availability of cyclosporine from the oral dosage form when stored in plastic syringes were examined.
Solutions of cyclosporine were stored in plastic syringes for 28 days at 25°C. Half of the solutions remained in room light, and samples were obtained at 3, 6, and 12 hours and 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 days. The remaining samples were protected from light, and samples were obtained at 1, 3, 5, 7, 14, 21, and 28 days. Samples stored in plastic were compared with control solutions stored in the original amber bottle. All samples were analyzed for unchanged cyclosporine by high-pressure liquid chromatography.
Cyclosporine was stable and completely available from the oral dosage form when stored in plastic syringes for up to 28 days. There was no difference in the stability of cyclosporine between the solutions exposed to room light and those protected from light.
Solutions of the oral dosage form of cyclosporine may be stored in plastic syringes without protection from light for up to 28 days.
The Corvac integrated serum separator tube (TM Monoject Scientific) has been examined for use in processing specimens for drug analysis. This study was undertaken because the SST serum separator tube (Becton-Dickinson) was shown to cause decreases in drug levels of lidocaine, pentobarbital, and phenytoin, which were dependent upon blood volume and time of contact with the gel. Pools of donor blood were spiked with lidocaine or phenytoin and processed in Corvac tubes and red top tubes. Lidocaine levels were depressed by 5.2% when Corvac was used. Phenytoin levels were unchanged. The lidocaine depression was independent of blood volume, time of gel contact, or tube lot number. Paired patient specimens for quinidine, lidocaine, or theophylline analysis were obtained in Corvac and red top tubes. Lidocaine levels were depressed by 6.0% (p less than 0.01). Quinidine and theophylline levels were unchanged. All drug analyses were carried out using the Syva enzyme multiplied immunoassay technique. The Corvac tube has been shown to cause decreased levels of lidocaine but not of the other drugs tested. Corvac (silicone gel) results differ from SST (polyester gel) results because of the different construction of these tubes.
The effects were tested of eight common types of blood collection tubes and two types of "plasma separators" on the stability of the tricyclic antidepressants amitriptyline, imipramine, clomipramine, and their monodemethylated metabolites in venous blood samples. Although EDTA-containing Venoject lavender and Vacutainer lavender tubes seemed to give the most stable plasma samples, and Venoject red the most stable serum samples, the differences were too small to have practical consequences. Vacutainer royal blue collection tubes gave significant losses of greater than 20% of some of the substances. The tubes with serum separator gel or filter proved unsuitable, since they were responsible for losses of greater than 40%. The losses were not caused by redistribution between blood cells and plasma but occurred mainly as a result of contact between the contents and the caps of the tubes. Experiments with freezing, thawing, and storage of samples showed that freshly sampled blood could be stored at room temperature for 24 h in Venoject green tubes without significant losses. Serum samples could be stored at refrigerator temperature for 4 weeks without important losses. Freezing, thawing, and storage at -20 degrees C did not influence the serum or plasma concentrations.
Laboratory values for specimens from a case of intravascular hemolysis showed that hemoglobin was significantly increased and thus could interfere with the determination of other analytes. We studied this problem by adding increasing amounts of purified hemoglobin (to a maximum concentration of 19.3 mg/L) to aliquots of pooled serum samples. The hemoglobin significantly interfered with the determination of only five analytes: albumin, aspartate aminotransferase, direct bilirubin, and total protein on the SMAC, and creatinine on the Astra. We propose that for cases of proven intravascular hemolysis, values for only the analytes not affected by hemoglobin should be reported. We find lactate dehydrogenase activity useful in assessing the components of in vivo hemolysis; the differences between serum and plasma values for potassium, lactate dehydrogenase, and hemoglobin are related to in vitro hemolysis. Criteria for specimen collection and assessment of type of hemolysis are proposed.
Two blood-sampling techniques, capillary and venous, were compared using the EMIT method. No difference was found in reproducibility. However, persistent positive bias of 0.628 micrograms/ml (p less than 0.01) was noted with the capillary sampling method. Any hemolysis that occurred in the capillary sampling technique did not significantly affect the assay.
Using the EMIT assay, we tested clinical samples from patients receiving 12 commonly monitored drugs to evaluate the effect of serum separator gel contained in serum separator blood collection tubes (SST) (Becton-Dickinson). There were significant concentration decreases for lidocaine, pentobarbital, and phenytoin. In vitro experiments demonstrated that this effect on phenytoin was dependent on time of exposure to the gel and volume of whole blood, but was not dependent on the presence of red blood cells or initial concentration. Bias attributed to the use of SST could interfere with the usefulness of clinical results at the upper and lower limits of the therapeutic range. This problem can be minimized by processing samples of at least 2 ml within 1 h.
The suitability of serum and plasma anticoagulated with heparin, EDTA, citrate, or oxalate was assessed for analysis of free and total phenytoin, carbamazepine, and valproic acid. The free fraction was isolated by ultrafiltration through FreeLevel devices (Syva, Palo Alto, CA). Serum, heparin, and EDTA plasma were satisfactory for both free and total phenytoin and carbamazepine. EDTA could not be used for EMIT (Syva) analysis of valproate. Citrate and, to a lesser degree, oxalate cause a significant negative interference in the concentration of these three drugs as measured both by EMIT and gas-liquid chromatography.
It has previously been shown that saliva phenytoin concentration bears a constant relationship to plasma free concentration whether protein binding of phenytoin is normal or disturbed by other drugs, pregnancy, renal failure, or hypoalbuminaemia. The present work examines the relationship between saliva (S), plasma free (F), and plasma total (P) concentrations of other anticonvulsants in 100 epileptic patients. Mean S/P ratios were for phenobarbitone 0.37 (r = 0.95), primidone 0.95 (r = 0.87), and carbamazepine 0.27 (r = 0.94). A highly significant correlation of S with F was found for these drugs, more significant than the correlation of S with P for carbamazepine in patients receiving multiple anticonvulsant drugs. Saliva valproate, however, had no predictive value for P or F. No binding to saliva proteins was demonstrated for any drug. Data for in vitro binding to plasma proteins was in good agreement with ex vivo data. Saliva is therefore a valid medium for monitoring treatment with phenobarbitone, primidone, and carbamazepine, as well as phenytoin.
The binding of valproic acid (VPA) to human serum albumin (HSA) and to pooled human plasma has been investigated by using equilibrium dialysis with three different dialysis solutions: phosphate buffer (solution I), Krebs solution (solution II), and Krebs solution without calcium (solution III). The effect of pH variation from 6.4 to 8.2 has been also investigated. VPA free fraction increased by increasing pH with all the dialysis solutions (from 4.1% at pH 6.4 to 9.4% at pH 8.2 with solution I, from 8.1% to 11.3% with solution II, and from 10.6% to 14.3% with solution III, in plasma). At each pH value, free fraction obtained with solution III was the highest and that obtained with solution I was the lowest. Data in plasma and HSA solution were similar. In a separate experiment we compared (at pH 7.4, with plasma) the three more frequently used dialysis solutions: phosphate buffer, phosphate buffer with NaCl, and Krebs solution. They gave, respectively, a mean VPA free fraction of 7.8, 10.3, and 12.7%. These findings can explain the wide range of VPA free fraction values reported in the literature. Researchers intending to determine VPA free concentration by equilibrium dialysis should take into account these methodological aspects.
We evaluated the effect on drug concentration of the duration of sample storage in three different tubes for blood collection: SST (Becton Dickinson), AutoSep (Terumo), and Microvette (Sarstedt), all of which contain an inert barrier (e.g., gel) for cleaner separation of serum from coagulum during centrifugation. With two of the tubes (SST and AutoSep) we examined samples taken from patients undergoing treatment with phenobarbital, phenytoin, and carbamazepine. We found significant decrease in the drug, concentration after 24 h with the serum standing over the gel barrier in the SST tubes for phenobarbital and both the SST and AutoSep tubes for carbamazepine. Adding aqueous drug solutions or serum samples supplemented with the above drugs in therapeutic concentrations to the three types of tubes resulted in a more pronounced decrease of concentration in the SST and Auto-Sep tubes than in the Microvette tubes.