Fluorimetric determination of cefuroxime in body fluids.

A simple and accurate fluorimetric procedure for measuring cefuroxime in body fluids is described. A fluorescent product was produced by addition of hydrochloric acid, heating, and cooling, followed by addition of sodium hydroxide and further heating at 100 degree C. The fluorescence intensity of the final solution was measured in a fluorimeter at an excitation wavelength of 375 nm and an emission wavelength of 440 nm and related to the antibiotic concentration. Thin-layer chromatography of the final solution showed a single fluorescent spot (Rf value, 0.6). Freedom from interference from other therapeutic agents and endogenous substances, as well as the close correlation between this method and the standard microbiological assay method, was demonstrated. The simplicity and reliability of the fluorimetric assay method make it particularly suitable for clinical use.     

Protection of staff from body fluids.

The problem solving approach to decision making is a logical and useful tool to use. Tracy Scales illustrates the use of this approach by discussing the problem of protecting staff from the ever increasing risk of infection from the patient's body fluids.     

Pregnancy-associated globulin in body fluids of pregnant women.

Pregnancy-associated globulin (PAG) in saliva, urine and amniotic fluid was investigated just before and after delivery by Ouchterlony's method and immunoelectrosyneresis. In saliva, the incidence was more than 60 percent; 12 out of 18 samples by immunoelectrosyneresis and 11 out of 18 by Ouchterlony's method. In urine, 8 (33 percent) and 6 (25 percent) out of 24 samples were positive by immunoelectrosyneresis and Ouchterlony's method, but 19 out of 21 samples (91 percent) were positive by immunoelectrosyneresis. Of 12 pregnant women in whom serum, saliva, urine and amniotic fluid were tested by immunoelectrosyneresis, four showed positive reaction in the above four samples and 11 in three samples except urine.     

Lysozyme in human body fluids

The activity of lysozyme was studied in the following body fluids using the diffusion method on agarose gel: blood serum of adults and children, umbilical cord blood serum, cerebrospinal fluid, amniotic fluid, urine, saliva, gastric juice, bile, tears, mothers' milk, sperm of a healthy subject, and exudates. From the results obtained it was found that lysozyme is present in almost all body fluids, though in different amounts. In serum its activity is normally from 7 to 13 mg/l. The highest content was found in tears, about 120 times higher than in serum. A considerable amount of lysozyme was found in gastric juice, about 8 times more than in normal serum. Lysozyme activity is lowest in or absent from urine, bile and cerebrospinal fluid. In other body fluids the activity of lysozyme was found to be the same as in serum.     

Approach to the analysis of body fluids for the detection of infection

Sterile body fluids represent an important source for the diagnosis of infectious diseases because they can be sampled by sterile methods that bypass the normal bacterial flora so heavily colonizing the body surface. Thus when these specimens are received, full advantage should be taken to perform complete microscopic and cultural tests for viral, bacterial, mycobacterial, fungal, and parasitic diseases. In many cases the evaluation can be tailored to the types of organisms that are likely to infect particular body cavities. Ideally, the workup could also be based on the history, clinical presentation, and preliminary examination of the patient, but under most circumstances it may be more appropriate for the laboratory to proceed with a more complete workup of these vital specimens than physicians request. Specimens should be transported promptly to the laboratory and should be viewed quickly by Gram's or acridine orange stain and, in selected situations, also by acid-fast stain, direct fluorescence for legionellosis, and direct wet mount for parasites. Results of these studies should be called in without delay to the responsible physician. Cultures should also be inoculated as soon as possible. Nonspecific tests, including the cell count and protein, glucose, lactic acid, and LDH levels, may provide valuable clues to the presence of infection. Direct antigen detection does not replace traditional microscopic and cultural evaluation of these specimens but may have supplemental value.     

Enzymic analysis for rapid detection of microbial infection in human body fluids: an overview

One possible means of rapidly detecting microorganisms in patients with suspected infectious diseases is the direct measurement of microbial enzymes in body fluids. This technique is based on the fact that bacterial, fungal, and viral organisms possess enzymes that are not produced by mammalian cells and are thus not found in uninfected human body fluids. Detection of one of these microbial enzymes in blood, cerebrospinal fluid, or other body fluids would thus be indicative of microbial infection. Potentially useful enzymes for this purpose include bacterial beta-lactamases, fungal adenine deaminases, and viral thymidine kinases. In addition, glycosidases such as neuraminidases and galactosidases can be used as markers for microbial infection, provided that the enzymic activity can be appropriately identified as being of microbial origin. The direct measurement of microbial enzymes offers great potential for the rapid diagnosis of infectious diseases.     

Electron microscopy of body fluids

Transmission electron microscopy and scanning electron microscopy are complementary techniques; the former is useful for examining the fine internal structural detail of tissue and cell surfaces and the latter is useful for studying three-dimensional configurations. The methods and findings of transmission and scanning electron microscopic study of effusions are summarized, and their practical application in routine clinical work is assessed.     

Quantitation of ciprofloxacin in body fluids by high-pressure liquid chromatography.

We describe a reverse-phase high-pressure liquid chromatography method for the quantitation of a new quinoline carboxylic acid antimicrobial agent, ciprofloxacin (Bay o 9867). This assay utilizes the intrinsic fluorescence of ciprofloxacin for primary detection but employs UV absorption as a secondary detection system. Mobile phases contained methanol and phosphate buffer and used a common C18 mu Bondapak column. A single precipitation step of a 50-microliter specimen was the only sample preparation necessary. The assay is linear from 2,000 to 10 ng/ml and sensitive to 5 ng/ml. The mean recovery of ciprofloxacin from serum was 105.7%. The coefficient of variation was less than or equal to 3.1% for same-day precision and less than or equal to 6.3% for assay-to-assay precision. Because the assay requires only small specimen volumes and minimal sample preparation and because of its defined characteristics, this assay would be ideal for clinical trials and pharmacokinetics studies of ciprofloxacin.     

Mass spectrometric analysis of human transferrin in different body fluids.

In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.     

Radioimmunoassay of dibenzazepines and dibenzcycloheptanodienes in body fluids and tissues.

In an attempt to establish a radioimmunoassay (RIA), imipramine and amitriptyline immunogens were prepared; desmethyl derivatives were converted into hemisuccinates, conjugated with bovine serum albumin and used for rabbit immunization. [3H]Amitriptyline (4.3 TBq/mmol) and [3H]imipramine (2.9 TBq/mmol) were prepared by catalytic dehalogenation or reductive alkylation. Dibenzazepines and dibenzcycloheptanodienes were determined in biological fluids by a direct method without deproteinization (lower detection limit of 0.5 microgram.l-1); using high-yield methods they were extracted from cell membranes. Assay of tricyclic antidepressants in humans showed that these substances disappear from plasma much earlier than from cell membranes. Dissociation of the antidepressants bound to cell membranes is slow and their plasma concentrations are not influenced by standing for 2 h at 4 degrees C. During preparing the membranes for binding studies these substances are not removed, and they may affect the results of the binding studies.     

Measurement of fibronectin in human body fluids

Two facts must be taken into consideration when quantifying fibronectin in biological samples: the sensitivity of the assay method and the appropriate handling of samples. A three-antibody, non-competitive ELISA was used in the present study. This procedure offers a simple, inexpensive and very sensitive technique for evaluating fibronectin: reproducible standard curves in the range 5-100 micrograms/l, 3% variability in the assay, and a detection limit of 0.5 ng/well that allows accurate measurement of fibronectin in segmental bronchoalveolar lavage and in ascites liquid samples. Typical dilution ranged from 1/10000 for plasma to 1/100 for bronchoalveolar lavages. For long-term storage of samples two procedures are recommended. First, a rapid freezing with liquid nitrogen, storage at -20 degrees C and thawing once at 37 degrees C avoiding refreezing. Losses of immunoreactive fibronectin after 35 days storage were 15, 15 and 22% in plasma, bronchoalveolar lavages and ascites fluid, respectively. Alternatively, samples can be stored as ammonium sulphate precipitates. This allows the easy availability of the samples when assays have to be repeated, and avoids fractionation in vials and freeze-thawing cycles. Using this storage procedure, losses are, however, slightly higher (22, 25, 25%, respectively, after 35 days).