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PubMed2000

A guide for diagnosis of patients with arterial and venous thrombosis.

Inasmuch as coagulation laboratories are involved in providing a diagnosis for underlying causes of venous and arterial thrombosis, we present a comprehensive review of the biological properties and functions of the components of the hemostatic system as they relate to the diagnosis of arterial and venous thrombosis. Moreover, as coagulation laboratories are necessary to evaluate the success of initial treatment modalities and to provide guidance for supplemental therapeutic intervention, we include information on antiplatelet and antithrombotic therapy. Included in clinical coagulation testing are assays that evaluate the potential of blood to form clots and tests for platelet numbers and platelet functions. Clot-based assays directly detect the biological activity of procoagulant factors and fibrinogen; chromogenic substrate assays evaluate proteolytic activities of clotting as well as fibrinolysis enzymes; and specific antibodies measure the concentrations of coagulation and fibrinolysis enzymes in plasma. Genetic testing is rapidly becoming incorporated into the clinical routine. The prothrombin time (PT), activated partial prothrombin time (APTT), and thrombin time (TT) are screening assays that measure the clotting times of recalcified whole blood or platelet-poor plasma. In addition to their function as screening assays, PT, APTT, and TT are the backbone of all the specialized clot-based assays for factor activities and for the indirect measurement of inhibitory antithrombin and protein C activities. Molecular markers related to hemostasis and fibrinolysis consist of proteins or peptides that indicate an ongoing physiological or abnormal process related to clot formation, thrombosis, vascular damage, or drug effect. Molecular markers are currently identified by means of specific antibodies prepared against them. The list of hemostatic molecular markers is rapidly growing. Most of the assays developed for molecular marker measurement, with the notable exception of the d-dimer assay, are typically used in clinical research.

Antifibrinolytic Agents↗

PubMed1996

Use of enzyme-linked immunosorbent assay and radioimmunoassay to determine serum and urine dexamethasone concentrations in thoroughbreds after intravenous administration of the steroid.

OBJECTIVE: To develop a simple and sensitive ELISA for detection of dexamethasone in horse serum and urine. SAMPLE POPULATION: Blood and urine samples from 3 thoroughbred mares. PROCEDURE: A dexamethasone oxime was prepared and conjugated to hemocyanin, bovine serum albumin and to horseradish peroxidase. One- and two-step double-antibody ELISA methods, as well as a radioimmunoassay method, were performed. The one-step ELISA was used to test urine from 3 Thoroughbred mares injected with 5 mg of dexamethasone, IV. RESULTS: The ELISA could detect dexamethasone in the range of 0.01 to 50 ng/ml, with intra- and interassay variations of 8.92 and 9.42%, respectively. Serum dexamethasone concentration reached a peak of 20 to 35 ng/ml 15 minutes after steroid administration and decreased to 1 ng/ml in 2.5 hours. Urine dexamethasone concentration 18 to 50 ng/ml 1 to 2 hours after drug administration and decreased to 1 ng/ml at 10 hours. CONCLUSION: The developed assay is sensitive as well as simple for detecting dexamethasone in horse serum and urine, and is comparable to radioimmunoassay. CLINICAL RELEVANCE: This method can be useful for screening samples from racehorses, because it is sensitive and does not require sample preparation or sophisticated equipment.

Animals↗

PubMed1998

Development of an assay for modulating anti-acetylcholine receptor autoantibodies using human rhabdomyosarcoma cell line.

Three types of autoantibodies against the acetylcholine receptors (AChR) of skeletal muscle are detectable in patients with myasthenia gravis including binding, blocking, and modulating anti-AChR antibodies. Modulating autoantibodies correlate best with the severity of the disease, but are also technically most difficult to measure because the assay generally requires fresh human muscle cells. We have developed an assay for the modulation of anti-AChR antibodies using a rhabdomyosarcoma (RD) cell line expressing AChR on the cell surface. By decreasing the FetalClone III serum from 10% to 0.5% in Eagles Minimal Essential Medium (EMEM) we were able to increase the number of AChR on RD cells to meet the need of sensitivity of the assay. The extent of modulation was determined as the percent of AChR internalized in the presence or absence of modulating autoantibodies. Less than 6% modulation was found with the normal serum (n = 42). The CVs of both the intra- and day-to-day precision were less than 20%. When clinical samples (n = 105) were assayed in our laboratory and also at Nichols Institute, a correlation coefficient of 0.816 was obtained. The selection of RD cell line, the success of increasing the expression of the AChR on RD cells and the use of 125I alpha-bungarotoxin of high specific activity allowed the establishment of an assay which can be used in routine clinical laboratory for the measurement of modulating anti-AChR autoantibodies for the management of patients with myasthenia gravis.

Autoantibodies↗

PubMed2003

Introducing a molecular test into the clinical microbiology laboratory: development, evaluation, and validation.

CONTEXT: In the mid-1980s, the polymerase chain reaction methodology for the amplification of minute amounts of target DNA was successfully developed and then introduced into clinical use; such technology has led to a revolution in diagnostic testing. Despite enormous advances in the detection of infectious agents by amplification methods, there are also limitations that must be addressed. OBJECTIVE: To highlight the pertinent steps and issues associated with the introduction of an amplification assay into a clinical microbiology laboratory as well as the subsequent ongoing activities following its introduction into routine laboratory use. DATA SOURCES: Data were obtained from literature searches from 1990 through September 2002 using the subject headings "polymerase chain reaction," "molecular assays," and "amplification" as well as publications of the National Committee for Clinical Laboratory Standards. DATA EXTRACTION AND SYNTHESIS: Using the findings obtained from these studies and publications, the process of introducing a molecular assay into the clinical microbiology laboratory was broken down into 4 major components: (1) initial phase of assay development, (2) polymerase chain reaction assay verification in which analytic sensitivity and specificity is determined, (3) assay validation to determine clinical sensitivity and specificity, and (4) interpretation of results and ongoing, required activities. The approach, as well as the advantages and limitations involved in each step of the process, was highlighted and discussed within the context of the published literature. CONCLUSIONS: The application of molecular testing methods in the clinical laboratory has dramatically improved our ability to diagnose infectious diseases. However, the clinical usefulness of molecular testing will only be maximized to its fullest benefit by appropriate and careful studies correlating clinical findings with assay results.

Bacteria↗

PubMed2000

Immature uterotrophic assay is more sensitive than ovariectomized uterotrophic assay for the detection of estrogenicity of p-nonylphenol in Sprague-Dawley rats.

Many efforts have been made to develop assays for detecting endocrine disrupters (EDs). Among them, uterotrophic assay has been known efficient for detecting EDs, especially estrogenic compounds. This study was performed to compare the immature uterotrophic assay with an ovariectomized assay using p-nonylphenol (NP), a weakly estrogenic compound. NP was given to either immature or ovariectomized rats subcutaneously or orally (only immature) at doses of 10, 100, and 1000 mg/kg for 3 days. After treatment with NP, the rats were examined for parameters such as uterine weight, uterine weight per body weight ratio, luminal epithelial height of uterus and vagina, diameter of uterine ducts, and number of uterine glands. Both systems were shown to increase uterine weight in a dose-dependent manner. In the immature system (subcutaneous injection), uterine weight, diameter of uterine duct and vaginal luminal epithelial height were significantly increased at 100 mg/kg/day, while in the ovariectomized system these parameters were not significant at the same dose (except for vaginal luminal epithelial height). These results suggest that the immature system (subcutaneous injection) might be most sensitive to detecting a weakly estrogenic compound and that the measurement of vaginal epithelium is a good end-point.

Animals↗

PubMed1998

New testing strategy to detect early HIV-1 infection for use in incidence estimates and for clinical and prevention purposes.

CONTEXT: Differentiating individuals with early human immunodeficiency virus 1 (HIV-1) infection from those infected for longer periods is difficult but important for estimating HIV incidence and for purposes of clinical care and prevention. OBJECTIVE: To develop and validate a serologic testing algorithm in which HIV-1-positive persons with reactive test results on a sensitive HIV-1 enzyme immunoassay (EIA) but nonreactive results on a less sensitive (LS) EIA are identified as having early infection. DESIGN: Diagnostic test and testing strategy development, validation, and application. Specimens were tested with both a sensitive HIV-1 EIA (3A11 assay) and a less sensitive modification of the same EIA (3A11-LS assay). SETTINGS AND PARTICIPANTS: For assay development: 104 persons seroconverting to HIV-1 comprising 38 plasma donors, 18 patients of a sexually transmitted disease clinic in Trinidad, and 48 participants in the San Francisco Men's Health Study (SFMHS); 268 men without the acquired immunodeficiency syndrome (AIDS) in the SFMHS who had been infected for at least 2.5 years; and 207 persons with clinical AIDS; for testing strategy validation: 488 men in the SFMHS from 1985 through 1990 and 1275449 repeat blood donors at 3 American Red Cross blood centers from 1993 through 1995; and for HIV-1 incidence estimates: 2717910 first-time blood donors. We retrospectively identified persons eligible for a study of early infection. MAIN OUTCOME MEASURE: Ability to identify early HIV infection. RESULTS: Estimated mean time from being 3A11 reactive/3A11-LS nonreactive to being 3A11 reactive/3A11-LS reactive was 129 days (95% confidence interval [CI], 109-149 days) [corrected]. Our testing strategy accurately diagnosed 95% of persons with early infection; however, 0.4% (1/268) of men with established infection and 2% (5/207) of persons with late-stage AIDS were misdiagnosed as having early HIV-1 infection. Average yearly incidence estimates in SFMHS subjects were 1.5% per year vs observed average incidence of 1.4 per 100 person-years. Incidence in repeat blood donors using the sensitive/less sensitive assay testing strategy was 2.95 per 100000 per year (95% CI, 1.14-6.53/100000) vs observed incidence of 2.60 per 100000 person-years (95% CI, 1.49-4.21/100000). Overall incidence in first-time blood donors was 7.18 per 100000 per year (95% CI, 4.51-11.20/100000) and did not change statistically significantly between 1993 and 1996. Use of the sensitive/less sensitive testing strategy alone would have identified all 17 persons with antibodies to HIV-1 eligible for a study of early HIV-1 infection and would have increased enrollment. CONCLUSIONS: The sensitive/less sensitive testing strategy provides accurate diagnosis of early HIV-1 infection, provides accurate estimates of HIV-1 incidence, can facilitate clinical studies of early HIV-1 infection, and provides information on HIV-1 infection duration for care planning.

AIDS Serodiagnosis↗

PubMed2000

Nefopam enantiomers: preclinical pharmacology/toxicology and pharmacokinetic characteristics in healthy subjects after intravenous administration.

Nefopam (NEF) is a potent analgesic compound administered as a racemic mixture. Previous in vitro and in vivo studies with nefopam enantiomers have shown that (+)nefopam [(+)NEF] is substantially more potent than (-)nefopam [(-)NEF]. Differences between enantiomers have also been suggested in metabolic studies in vitro. The impact of these differences in vivo is not known because there is little or no information on the relative plasma concentrations of the enantiomers or on their kinetics. In this study, individual enantiomers of nefopam were synthesized and examined for acute toxicity in male and female rats and mice. Pharmacologic properties of enantiomers were examined using in vitro binding assays and antinociceptive tests in rats and mice. Additionally, a pharmacokinetic study was conducted in human volunteers. Subjects were administered 20 mg nefopam as Acupan(R) either as a 5- or 20-min intravenous infusion. In a control phase, subjects were administered only vehicle. Blood samples were collected through the following 24 h. Plasma samples were analyzed for individual enantiomers using a chiral assay developed for this purpose. The pharmacologic differences of previous studies were confirmed in receptor binding assays and in the hot plate and the formalin tests in mice. Neither enantiomer demonstrated substantial activity in the tail flick test in rats. No significant differences were revealed between LD(50) values of nefopam enantiomers after oral or intravenous administration in male and female rats or mice. There were no significant differences in AUC(0-infinity), C(max), or half-life between enantiomers following intravenous administration. Based on these findings, there is currently no compelling rationale to justify administering or monitoring individual enantiomers.

Analgesics, Non-Narcotic↗

PubMed1981

Radioimmunoassay for urinary prostaglandins E and F alpha: normal values in different age groups.

A new radioimmunoassay for urinary prostaglandins E and F alpha is described which allows the extraction and chromatographic separation of urinary prostaglandins with an overall recovery of 80.5% and 89.3% for prostaglandins E2 and F2 alpha respectively. The antibodies raised in the rabbit could not distinguish prostaglandins of series 1 and 2 but were found highly specific towards their metabolites and other primary prostaglandins. The sensitivity of the assay developed was at 2.2 pg for prostaglandin E and 2pg for prostaglandin F alpha. This assay was applied to normal adults of both sexes; it was shown that the daily urinary excretion of prostaglandins F alpha and E was greater in men than in women. In children aged from 1 to 114 months, the urinary excretion of prostaglandins increased with age. This increase was more marked during the first two years of life and was significantly related to age.

Adult↗

PubMed1990

Detection of neuron specific enolase concentrations in cerebrospinal fluid from patients with neurological disorders by means of a sensitive enzyme immunoassay.

An enzyme linked immunosorbent assay (ELISA) for the detection of neuron specific enolase (NSE) in cerebrospinal fluid (CSF) was developed. The sensitivity of the ELISA was less than 1 microgram/ml. This sensitivity is comparable with radioimmunoassays which have the disadvantage that radiolabelled products are used. The developed assay was used to measure cerebrospinal fluid neuron specific enolase (CSF-NSE) levels in 1178 patients with neurological disorders to establish its potential usefulness and clinical application. CSF-NSE levels in this group of patients were independent of sex and no correlation with age was found. CSF-NSE was significantly increased in Creutzfeldt-Jacob disease, meningeal hemorrhage, thrombosis, Guillain-Barré syndrome and in schizophrenia.

Animals↗

PubMed2004

Mosaic eyes, genomic instability mutants, and cancer susceptibility.

We now know that genomic instability contributes to cancer. The zebrafish mosaic eye assay developed by George Streisinger takes advantage of the organism's transparency to provide an excellent assay for detecting somatic mutation. This assay allowed us to identify zebrafish mutants with increased frequencies of somatic mutation and spontaneous cancer. Here, we have described details of mutagenesis, the basis and practical use of the mosaic eye assay, and the histological methods used to study genomic instability mutants and cancer susceptibility. These techniques should prove useful to other zebrafish researchers, as they are broadly applicable to many other biological investigations of embryos, larvae, and adult zebrafish.

Animals↗

PubMed2003

High-performance liquid chromatographic method for the quantification of anthranilic and 3-hydroxyanthranilic acid in rat brain dialysate.

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) have attracted considerable attention as two of the L-tryptophan kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of ANA and 3-HANA has been developed using reversed-phase high performance liquid chromatography (HPLC) with fluorimetric detection. The HPLC assay was carried out using a C(18) column (5 microm, 250 x 4.6 mm i.d.). The mobile phase consisted of a mixture of 25 mM sodium/acetic acid buffer (pH 5.5) and methanol (90:10 v/v). Fluorimetric detection at lambda(ex)=316 nm and lambda(em)=420 nm was used. The assay was applied to the measurement of ANA and 3-HANA acid in rat brain dialysate following administration of L-tryptophan or L-kynurenine. 3-HANA and ANA levels were progressively increased during 90 min following administration of L-tryptophan, then decreased progressively to basal levels. 3-HANA levels were significantly higher than ANA levels after L-kynurenine administration. These findings suggest that the assay developed should provide an improved means for investigation of neurobiology of kynurenine pathway.

3-Hydroxyanthranilic Acid↗

PubMed1988

The OVEIA assay, a simultaneous measurement of oestrone and pregnanediol glucuronides, for studying the treatment of infertility patients.

A new simple assay developed for establishing the limits of the fertile period was evaluated in infertility patients. This assay is a colorimetric method for measuring the oestrone glucuronide/pregnanediol glucuronide ratio. Steroid levels were assayed simultaneously and an index of their ratio was obtained in this dual analyte assay (DAA). Twenty subfertile women, receiving clomiphene citrate for ovulation induction, were monitored for the DAA index using early morning urine samples. Ten of these women were also monitored in a pretreatment cycle. A further 10 patients on an in-vitro fertilization (IVF) programme were assessed with the same assay. DAA ratios were found to correlated with follicular phase serum oestradiol levels. Peak DAA ratios dropped dramatically when significant rises in urinary luteinizing hormone concentrations occurred prior to ovulation. An association between high DAA ratios and adequate luteal phase activity was also established. In addition, the total volume of the three largest ovarian follicles, as measured by ultrasound, in IVF patients was found to correlate with DAA measurements.

Adult↗

PubMed1989

Preparation of filamentous hemagglutinin from Bordetella pertussis and assay for serum antibodies to filamentous hemagglutinin and pertussis toxin for clinical and public health laboratories.

A procedure that is sufficiently simple and economical for use in clinical and public health laboratories for producing and purifying filamentous hemagglutinin (FHA) and determining antibodies to this major antigen of Bordetella pertussis in serum is described. High yields of FHA (40 to 80 mg/liter) were obtained in the supernatant by cultivating B. pertussis in modified CL medium. The FHA antigen was separated from pertussis toxin (PT) and other antigens by chromatography on hydroxylapatite. Removal of residual PT activity in the FHA fraction was effected by affinity absorption of PT with Fetuin immobilized to Sepharose 4B. The FHA was used as the antigen for determining titers of immunoglobulin G (IgG), IgA, and IgM to FHA in sera of patients with pertussis by an improved enzyme-linked immunosorbent assay. Development of the interfering background color commonly observed in conventional FHA enzyme-linked immunosorbent assay procedures was eliminated by washing the reaction wells with a buffer of high ionic strength before adding the peroxidase conjugates. In the absence of nonspecific background color, the reaction endpoints were easy to read. The FHA prepared by the procedure described was identical to a reference preparation of purified FHA in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and serological specificity assays. High yields of FHA were obtained from all four strains of B. pertussis tested in this study, indicating that the procedure for enhanced production of FHA may be generally applicable to other strains of B. pertussis. Results from tests of 50 serum specimens with clinical information on pertussis for FHA and PT antibodies by the assay procedures described exemplified the usefulness and caveats of serodiagnosis for pertussis.

Antibodies, Bacterial↗

PubMed1990

Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei.

Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.

Animals↗

PubMed1997

Salivary melatonin as a circadian phase marker: validation and comparison to plasma melatonin.

There are many situations in which it would be useful to know the phase state of the biological clock. It is recognized that measurement of melatonin levels can provide this information, but traditionally blood has been used for the analysis, and there are many problems in extending the measurements into the home or field situations. The aim of this study was to develop and validate a salivary melatonin radioimmunoassay and to compare results obtained against a plasma assay for determining the onset of melatonin secretion. The assay developed was sensitive (4.3 pM) and required only 200 microliters of sample. A rhythm in melatonin was detected in saliva, peaking at approximately 120 pM or 30% of the plasma levels. Using an objective criterion for determining the onset of secretion (mean +/- 2 standard deviations of three daytime samples), the time of onset was shown to exhibit low intraindividual variability (coefficient of variation = 1.5%-4.3%). The time of onset determined using saliva was significantly correlated with the plasma onset (r = .70, p < .05). The onsets determined were 22:30 h +/- 22 min for the saliva and 21:50 h +/- 16 min for plasma for 17 subjects. Similarly, the acrophases of the saliva and plasma melatonin rhythms were significantly correlated. Neither posture alone nor changes in posture affected the calculation of the onset of melatonin secretion using the saliva approach. Very high saliva flow rates induced by citric acid resulted in lower melatonin concentrations compared to the gentle chewing on parafin film. These results firmly establish the use of salivary melatonin measurements for phase typing of the melatonin rhythm in humans.

Adult↗

PubMed1978

Bone marrow acid phosphatase in staging of prostatic cancer: how reliable is it?

To evaluate the reliability of bone marrow acid phosphatase in the staging of prostatic carcinoma we analyzed 50 bone marrow samples collected at random from the hematology service at this hospital. The samples were assayed for acid phosphatase content by a colorimetric method using sodium thymolphthalein monophosphate as a substrate and by 2 immunochemical assays developed at our laboratory (counter immunoelectrophoresis and radioimmunoassay). We found a high percentage (61 per cent) of falsely positive results in patients with various hematological diseases without evidence of prostatic carcinoma by the colorimetric evaluation. All of these patients except 1 had negative immunochemical assay. Until a specific assay for prostatic acid phosphatase is developed for clinical use we caution the use of a single elevation of bone marrow acid phosphatase as a parameter of metastatic disease.

Acid Phosphatase↗

PubMed1996

Comparative analysis of three nucleic acid-based detection systems for hepatitis C virus RNA in plasma from liver transplant recipients.

The early detection of hepatitis C viraemia (HCV) following liver transplantation is important for monitoring disease recurrence and planning antiviral chemotherapy. In the current study, the sensitivity, specificity, and concordance of three HCV RNA assays were compared using a random sample of 84 plasma specimens from 23 transplant recipients. Two of the assays were prototype commercial tests: Roche Molecular Diagnostic's RT-PCR HCV Amplicor system; and Chiron's Quantiplex HCV-RNA assay. The third was a 'home brew' PCR-liquid hybridization/gel retardation assay developed at the University of Pittsburgh Medical Center (UPMC). On all criteria the PCR-based assays out-performed the Quantiplex assay and displayed an overall concordance of 87%. A high percentage of specimens in the Quantiplex assay gave indeterminate results (12%) or high coefficients of variance (13%). The specificities of all RNA assays were determined using HCV serostatus as the gold standard. Both of the PCR-based assays had specificities of 100%, whereas the Chiron Quantiplex HCV assay had a specificity of 88%, if indeterminates were counted as negatives, and a specificity of 64% if indeterminates were counted as positives. The calculated sensitivities of the PCR-based assays were 56% and 48% for the 'home brew' and the Roche assays, respectively. The UPMC HCV assay, however, was determined to be capable of reproducibly detecting four or fewer chimpanzee infectious doses, suggesting that HCV viraemia was not present in the PCR-negative cases. The sensitivity of the Quantiplex assay was 41% counting indeterminates as negatives and 46% counting them as positives. The high cost of the Quantiplex assay combined with the number of uninterpretable results, the lack of sensitivity, and reduced specificity may limit the usefulness of this assay for monitoring HCV recurrence.

Hepacivirus↗

PubMed1991

Techniques used to define human MHC antigens: serology.

The classical, routine test employed for definition of HLA antigens expressed in humans (tissue typing) is the complement-mediated cytotoxicity assay developed by Terasaki and McClelland in the early 1960s. In both healthy persons and patients, the assay target cells are usually lymphocytes obtained from peripheral blood, but when typing cadaver donors, splenic or lymph node lymphocytes can be used. HLA-A, B, Cw (class I) antigens are expressed on all nucleated cells while HLA-DR, DQ (class II) are restricted to B lymphocytes and immune activated cells. Tissue typing has been achieved using culture cells from amniocentesis and typing of cell lines is possible with small modifications to the standardised cytotoxicity assay. Usually, target cells are incubated under oil with typing antisera at 22 degrees C in a 60- or 72-well Terasaki tray. After 30 min rabbit serum is added as a source of complement. After a further 60 min incubation the test is stained. A positive reaction results in target cell death. There are local variations to this test. Automation of the assay is now commonplace, from reagent dispensing to automated reading of finished assay. The use of antibody-coated magnetisable microspheres has enabled separation of pure B lymphocyte samples for class II typing and has reduced incubation times through antigen modulation. It is possible to define antibodies to HLA antigens in the same assay using target cells with known HLA phenotypes.

Complement System Proteins↗