Bacterial antigen detection in cerebrospinal fluid of patients with meningitis

This study compared the sensitivity and specificity of four test systems in detecting Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, and gram-negative organisms in cerebrospinal fluid (CSF), versus culture. The tests used on CSF from 155 patients with meningitis were the Phadebact coagglutination (CoA) test, the Directigen latex agglutination (LA) test, counterimmunoelectrophoresis (CIE), and the Limulus amebocyte lysate (LAL) test. The sensitivity for patients with bacterial meningitis was 78% (18/23) for LA, 78% (25/32) for CoA, and 67% (18/27) for CIE for detection of H. influenzae type b; 71% (10/14) for CoA, 100% (6/6) for LA, and 50% (6/13) for CIE in detecting S. pneumoniae; and 33% (1/3) for LA and 50% (2/4) for CIE in detecting N. meningitidis. LAL had a sensitivity of 77% (37/48) in detecting CSF gram-negative endotoxin. The specificities of those with bacterial meningitis for H. influenzae, S. pneumoniae, and N. meningitidis tested by LA were, respectively, 100% (35/35), 96% (50/52), and 100% (54/54); for H. influenzae and S. pneumoniae using CoA 97% (62/64) and 96% (80/83); for H. influenzae, S. pneumoniae, and N. meningitidis using CIE 67% (18/27), 50% (6/12), and 50% (2/4). The specificity of LAL was 86% (38/44). The detection of bacterial antigen from CSF in patients with meningitis by commercial agglutination tests is more sensitive than CIE and is highly specific.     

Subcellular localization of human heart atypical creatine kinase

Human heart subcellular fractions were obtained using method A described by Scholte et al [7,8]. It was found that 3.5 +/- 2.5%, 3.4 +/- 1.9%, 8.5 +/- 2.9%, 2.5 +/- 1.3%, and 88 +/- 21% of the total creatine kinase activity was associated with the myofibrillar, nuclear, mitochondrial, microsomal and particle-free soluble fractions respectively. Using DEAE-sephacel chromatography it was determined that the atypical creatine kinase (CK-Z) accounted for approximately 90% of the creatine kinase activity associated with the mitochondrial fraction.     

Bleeding by the numbers: The utility and the limitations of bleeding scores,bleeding prediction tools,and bleeding case definitions

A patient’s history of bleeding, whether spontaneous or in response to challenges, provides important information about both the likelihood of that patient having a biochemically-defined hemostatic defect, and that patient’s risk of future bleeding. Other variables including age, comorbidities and medications influence these probabilities. Scoring systems have been devised in an effort to make the estimates quantitative in specific populations. An example of a bleeding score is the MCMDM1-VWD questionnaire, which was developed to predict the likelihood of a patient having von Willebrand disease. It sums standardized details of the bleeding history, weighted by severity. The HAS-BLED score typifies bleeding prediction tools, developed to predict bleeding during anticoagulant therapy. Although prior bleeding is one item in this score, other comorbidities like hypertension or a history of stroke count for more. A third and related concept is that of bleeding case definitions, which are critical to standardize the reporting of outcomes in trials of antithrombotic agents, and which have entrenched the recognition of different severities of bleeding. We advocate that future efforts should blend some of these features. Information about comorbidities and medication use could refine the interpretation of bleeding events in a bleeding score. So could the introduction of a denominator reflecting the number and duration of challenges to which the patient has been exposed when bleeding might have been expected. More detailed information about the type, frequency and severity of prior bleeding could improve the prognostic power of bleeding prediction tools. More detailed history-based scores might ultimately supersede biochemical testing in many cases.     

Specific method for serum creatinine determination based on ion exchange chromatography and an automated alkaline picrate reaction -- a proposed reference method.

A proposed reference method for serum creatinine has been developed under the auspices of the Committee on Reference Methods and Reference Materials of the Canadian Society of Clinical Chemists. A serum ultrafiltrate at pH 2.0 is applied through a closed sample loop injection system to a short column containing cationic resin of high resolving power. Elution with sodium citrate buffer by means of minipump at constant rate passes the eluate into alkaline picrate reagents in a continuous flow system (Technicon AAIII pump, AAII colorimeter 50 mm x 1.5 mm flow cell, narrow band width filter). The colour reaction peak is monitored visually to verify specificity and the area is calculated electronically. Specificity has been demonstrated by use of Jaffé-reactive substances such as glucose, sodium acetoacetate, L-ascorbic acid, pyruvic acid, L-dopa and glycocyamidine and also by use of an alternate colour reaction, sodium 3,5-dinitrobenzoate in place of alkaline picrate in the analysis of serum pools. Routine methods in common use, i.e., manual and automated alkaline picrate procedures, demonstrated a statistically significant high bias in interlaboratory studies in which this procedure was used for reference.     

A rapid manual method for routine assay of ascorbic acid in serum and plasma.

A method is described for the assay of ascorbic acid in either serum or heparinized plasma. 1. The assay is based on the reduction of ferric chloride by ascorbic acid with the resulting ferrous ion quantitated by the addition of 2,4,6-tripyridyl-s-triazine to form a purple colour with a maximum absorbance at 595 nm. 2. Uric acid interference has been eliminated by the use of a high molarity acetate buffer and by optimising the amount of TPTZ and ferric chloride used. 3. Protein was found to cause rapid fading of the final colour; it was therefore necessary to remove the protein, by addition of 10% trichloroacetic acid, from the specimen prior to the final assay. This had the added advantage of assisting to stabilize the ascorbic acid prior to final assay. 4. All reagents used are easily obtained and no special equipment is required.     

A simplified assay for serum 25-hydroxycalciferol

The measurement of serum 25-hydroxycalciferol (25-OH D) has proved to be a reliable index of the vitamin D nutritional status in man providing the renal 25-hydroxycalciferol-1 alpha-hydroxylase is functional. Since it is known that 25 OHD binds to alpha-globulins for its transport to the kidney, we have developed a competitive binding assay using an alpha-globulin enriched fraction (Cohn's fraction IV) as the ligand protein. A detection limit of 0.1 ng 25-OHD/tube was achieved and the non specific binding amounted to less than 3% of the initial binding. The intra- and inter-assay variations were 8.9 and 8.4% respectively. The reference values (nmol/1 +/- 2 s.d.) obtained from samples of 40 children were 50.5 +/- 21.5 with extremes of 34.8 and 95.2. The method thus proved to be a reliable and sensitive tool to assess serum 25-OHD levels.     

A comparison of the "EMIT" assay with two iodinated radioimmunoassays for diphenylhydantoin.

In the treatment of epilepsy, it is important for the laboratory to provide blood diphenylhydantoin levels accurately and quickly. However, physicians have often obtained laboratory results which have been inconsistent with the patient's clinical condition. We examined the "Enzyme Multiplied Immuno Technique" (EMIT) and two iodinated radioimmunoassay procedures for their suitability to provide quick and accurate blood diphenylhydantoin levels in laboratories that perform relatively few assays. The results indicated that all three procedures can be used with reasonable success. We found that the simplicity and the long shelf-life of the EMIT procedure made it very desirable for institutions that have a small volume of diphenylhydantoin assays.     

Falsely-elevated serum creatinine values in diabetic ketoacidosis -- clinical implications

Unusual elevations of serum creatinine (S-CR) out proportion to increases in serum urea nitrogen (S-UN) are frequently observed in patients with diabetic ketoacidosis when S-CR is measured by the Jaffe end-point reaction. This has been ascribed to interference from acetoacetate but this is not however observed with kinetic DuPont ACA methodology. Eighteen patients with diabetic ketoacidosis were studied: SCR measurements were done using the end point Technicon SMA 6/60 method (Group a, 10 patients) or the kinetic DuPont ACA method (Group B, 8 patients). The values for S-CR in Group A patients (mean value and S-D were 3.3 +/- 1.1 mg/dl) were significantly different from Group B patients (1.6 +/- 0.24 mg/dl) (P less than 0.01). A significant positive correlation was obtained between the "excess anion gap" and S-CR in Group A patients (r = + 0.81, p less than 0.01). The results from two patients in whom serial measurements of S-UN, S-CR and the anion gap were carried out further demonstrate the analytical interference. The study demonstrates that in diabetic ketoacidosis elevated creatinine values measured by an end-point method should not necessarily be interpreted as evidence of significant renal impairment and if possible should be verified by a kinetic method which is free of "ketone" interference.     

An investigation of factors influencing the measurement of creatine phosphokinase activity in serum using coupled enzymatic methods.

1. The factors influencing the measurement of creatine phosphokinase (CPK) activity in serum by coupled enzymatic methods were investigated to establish optimum conditions for this type of assay. Such a study was indicated following observations by the authors of poor performance of commerically produced reagent kits together with the failure of most of the established an well accepted methods to operate under true optimum zero order kinetics in the reaction phase state. 2. The factors invested were the effects of pH, substrate concentrations (creatine phosphate, glucose and NADP+), added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes, dithiothreitol (DTT) as an activator and conditions of storage of substrate stability. DTT was found to be a suitable activator but not a reactivator of the reaction. The optimum concentrations of creatine phosphate, glucose and NADP+ were found to be 20.0, 20.0 and 2.0 mmol/litre, respectively. Optimum activieies of the enzymes, glucose-6-phosphate dehydrosenase and hexokinase were 1000 and 2000 units/litre, respectively. 3. The between-day precision of the method for measuring serum at pH 6.8 and 30 degrees C at three activity levels under the optimum conditions developed was excellent yielding coefficients of variation ranging from 2.0 to 2.7%.     

Determination of serum bile acids by glass capillary gas-liquid chromatography.

Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gas-liquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxycholic and deoxycholic acids in a group of healthy fasting women was 2.14 mumol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 mumol/l and at 38-41 weeks of gestation 2.10 mumol/l. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 mumol/l.