Evaluation and implementation of the gel test for indirect antiglobulin testing in a community hospital laboratory.

BACKGROUND: The gel test, developed by Lapierre in 1984, was designed to standardize antiglobulin testing while improving sensitivity and specificity of the method. PRINCIPLE: Anti-human serum immunoglobulin G (IgG) mixed with Sephadex G100 (gel phase) in a microtube traps red cell-IgG agglutination complexes during migration through the gel in a centrifugation step. Agglutination complexes are visibly detectable at various levels in the microtube as an inverse function of antibody coated on red cells. Unsensitized red cells form a cell pellet at the base of the microtube. OBJECTIVE: To determine if indirect anti-human globulin testing could be standardized and simplified by replacing the tube test with the gel test without compromising quality or increasing costs. SETTING: A medium-sized community hospital. RESULTS: In a blinded retrospective study, we used patient sera (n = 40), which included 10 positive specimens containing 18 known antibodies. Sixteen antibodies were detected and identified with the tube method (1 anti-D and 1 anti-C not detected). By the gel test, 18 antibodies were detected and identified. All negative samples showed 100% concordance. Favorable results were obtained in a nonblinded prospective correlation study (n = 121). Our technologists found the gel test easier to read and more reproducible and reliable than the tube method; they also found increased sensitivity for detecting weakly reacting antibodies. We successfully introduced the gel test into our laboratory as the standard method for indirect antiglobulin testing. Following implementation, improved personnel management was achieved. CONCLUSIONS: The gel test is a reliable and advantageous method and is appropriate for routine use for detection and identification of alloantibodies in a community hospital transfusion service laboratory.

Two sensitive immunometric assays for serum thyroid stimulating hormone evaluated.

This study was conducted to evaluate the analytical performance (functional sensitivity, reproducibility, parallelism, and accuracy) of two recent commercial kits marketed as third generation immunometric assays for measuring serum thyroid stimulating hormone (TSH). One assay is automated; the other is manual. Accuracy by method comparisons was evaluated using 86 patient samples assayed by an established third generation immunometric assay as the comparative method. The new assays met the third generation criterion for functional sensitivity (CV < or = 20 percent at TSH < or = 0.02 mIU/L), were reproducible (CVs < 11 percent), and measured serum TSH in parallel with the calibrator curves. Linear regression analysis of the intermethod comparison data showed highly correlated (R > .095) results; however, the regression slopes were non-unity, indicating patient sample results were not transferable between methods. Clinical laboratories choosing a third generation TSH assay should validate the performance characteristics of the selected method to ensure reliable results for patient care.

Identification and quantification of hemoglobins A, F, S, and C by automated chromatography.

The Bio-Rad Variant Hemoglobin Testing System is an automated HPLC analyzer marketed with a Beta-thalassemia Short Program to quantify hemoglobins (Hbs) F and A2 and assist in detecting Hbs A, S, C, D, and E. We evaluated this system to replace several traditional methods for Hb in our hospital laboratory. Analytical performance relevant to quantifying Hbs A, S, C, and F was assessed with blood samples obtained from our local patient population. Studies of precision (CVs < 3%) and analytical limits (% of total Hb) of Hbs A (2-86%), F (1-89%), S (5-90%), and C (3-92%) demonstrated results comparable with or exceeding those of traditional methods. Results for patients' samples (n) for Hbs A (107), F (157), S (128), and C (27) correlated well (r > 0.93) with results by traditional methods. The satisfactory performance and efficiency led us to implement this system for routine quantification of clinically significant Hbs.

Norepinephrine and epinephrine levels in affected versus unaffected limbs in sympathetically maintained pain.

OBJECTIVE: To test the hypothesis that there is relative sympathetic hyperactivity in the affected limb in patients with sympathetically maintained pain syndromes by measuring serum norepinephrine and epinephrine in the affected versus the unaffected sides. DESIGN: Venous pool samples were drawn just proximal to the affected area and from an identical site on the unaffected side. Serum norepinephrine and epinephrine were measured by high-pressure liquid chromatography with electrochemical detection. SUBJECTS: Sixteen women and seven men with a mean age of 44.4 years diagnosed as having sympathetically maintained pain on the basis of a positive response to paravertebral block and a criteria-based diagnostic scheme. RESULTS: The serum norepinephrine level was significantly lower in the affected limbs than the unaffected limbs (p = 0.024). The serum epinephrine level was not significantly different. CONCLUSIONS: These results are not consistent with the hypothesis of segmental sympathetic hyperactivity in the affected limb in sympathetically maintained pain and support a hypothesis of peripheral receptor upregulation with pathologic response to circulating catecholamines. Other possible explanations are discussed.

Analytical evaluation of methods for serum creatine kinase-MB. Electrophoresis, immunoinhibition and solid phase separation.

The purpose of this study was to evaluate immunoassay methods for the measurement of serum cardiac creatine kinase isoenzyme (CK-MB) with respect to sensitivity and specificity. The CK-MB electrophoretic assay (Helena Laboratories) was used as the reference. Two principles of immunoassay were included in the evaluation,--immunoinhibition and solid phase separation. The direct immunoinhibition techniques were from Beckman Instruments (CKMB reagent) and DuPont Medical Products (CKMB). Three solid phase separation techniques were from Abbott Laboratories (IMx CKMB), DuPont (acaPlus MCKMB), and Tosoh Medics Inc. (AIA-Pack CKMB). The electrophoretic method for separation of the CK isoenzymes has good specificity but lacks sensitivity for CK-MB in low concentrations. The immunoinhibition methods lack specificity and correlate poorly with the electrophoresis method and with the solid phase methods. The solid phase separation techniques are highly sensitive and show an excellent correlation with electrophoresis when based on specificity. The solid phase separation methods correlate well with each other.

Irradiation effect on aging red blood cells.

Irradiation of stored red blood cells (RBC) is increasingly utilized for patients who are immunosuppressed or on chemotherapeutic regimens. With the growing demand for irradiated cellular blood products, there has been an increasing need for transfusion services to store previously irradiated blood until needed for transfusion. The effect of irradiation on aging stored RBC has not been studied to date. Five units each of group A, RBC collected in CPD-Adsol (AS-1) with a prior shelf-life of 10, 20, 30, and 40 days, respectively, were divided equally utilizing a sterile docking device and stored at 1 to 6 degrees C. Baseline samples from each bag were obtained for the measurement of extracellular potassium (K+), plasma free hemoglobin (PFH), total lactate dehydrogenase (LD), and erythrocyte 2,3-DPG activity. One of each pair received 2,000 rads of gamma irradiation. Samples were obtained at 3 and 7 days post-irradiation, and multiples of 7 days until expiration. All irradiated units reached a state of K+ equilibrium at 60 to 70 mmol per L irrespective of the length of previous storage with an inverse relationship of RBC age at irradiation and the time required to reach the state of equilibrium. Increased K+ leakage from irradiated aging RBC suggests the need for including in vivo studies of cell survival to establish a post-irradiation storage life. Length of storage prior to irradiation had no effect on PFH, LD activity, and 2,3-diphosphoglycerate (2,3-DPG) activity compared to paired controls.

Serum alpha-fetoprotein: II. Clinical evaluation of two automated immunoassays for maternal serum screening.

A prospective clinical evaluation is reported for analysis of maternal serum alpha fetoprotein (s.AFP) testing by recently developed enzymeimmunoassay automated systems. The reference method for the study was a manual competitive binding radioimmunoassay which has been utilized by us in the Maternal Serum AFP Screening Program at the Medical University of South Carolina for the past five years. The patients were classified by weeks of gestation, 16 to 20. Mean concentrations of s.AFP increased with the increase in gestational week for each method; calculated multiples of median (MoM) values remained relatively constant regardless of the week of gestation. Mean and median concentrations of s.AFP measured by the radioimmunoassay and the coated tube enzymeimmunoassay methods showed close agreement, indicating the direct transferability of the methods for clinical purposes. The microparticle capture enzymeimmunoassay yielded higher results of s.AFP indicating the necessity for a correction if the method transfer is to be considered for sequential patient testing. (Multiples of median values for each method were uncorrected for maternal age, weight, or diabetes mellitus; lack of correction would not affect comparison of methods for clinical application.

Serum alpha-fetoprotein: I. Evaluation of quantitative assays adapted to automated immunoassay systems.

Serum alpha-fetoprotein (s.AFP) has been established as a useful tool in monitoring of high-risk pregnancies, as an indicator of fetal neural tube defects, and has been used as an adjunct tumor marker and for monitoring therapeutic efficacy in the treatment of certain tumors. To date, the methods for measuring s.AFP are based upon the immunologic principle and are manual methods. The purpose here is to relate the evaluation of two automated systems for the assay of s.AFP. The automated systems are based upon the following immunoassay methods: a microparticle capture enzyme separation and final quantitation by reflectance fluorescence, and a solid phase 'sandwich' separation coupled with enzyme activity measurement (EIA). The reference method is a competitive binding radioimmunoassay. It has been found by us that the automated methods directly transfer analytically with the manual assay. All methods are referenced to the same standard (WHO 1st Intl. Std. for AFP 72/225).

Screening for macroamylase in a community hospital.

We evaluated the polyethylene glycol precipitation test (Gastroenterology 83: 378-382, 1982), looking for macroamylase in the serum of 66 patients whose values for serum amylase were above normal. Three patients (4.5%) were identified by this method as having macroamylase , and this was confirmed by gel-filtration chromatography and electrophoresis. We find this to be the test of choice as a screening procedure for macroamylasemia because of its speed, simplicity, and apparent reliability. Diagnosis of macroamylasemia is important in preventing needless treatment and investigation for pancreatitis.

Carbamazepine--erythromycin interaction leading to carbamazepine toxicity in four epileptic children.

We report four cases of carbamazepine toxicity in children associated with the concurrent administration of erythromycin. They all developed clinical toxicity (ataxia, dizziness, nausea, and vomiting) when erythromycin administration was begun; symptoms disappeared after erythromycin was discontinued. Serum carbamazepine levels were measured before, during, and, in most cases, after the toxic episodes. In all cases, there was a sharp increase in carbamazepine concentration after erythromycin therapy was begun and a rapid fall once erythromycin was discontinued. Our data support the previous suggestion that erythromycin interferes with the liver microsomal metabolism of carbamazepine with a subsequent increase in blood levels of the drug.

Simplified fluorometry of total metanephrines in urine.

We describe a simplified fluorometric method for quantitatively determining metanephrines in urine. The method is based on the elution of metanephrines from an ion-exchange column followed by production of a fluorescent derivative that has excitation and emission maxima of 405 nm and 505 nm, respectively. Metanephrine concentrations as great as 7.0 mg/L were within the linear range of the assay. Analytical recovery of metanephrines was 82-87%. Cross contamination by catecholamines was insignificant. The upper range of normal was 520 micrograms/24 h. Within-run and between-run CV's were 5% and about 9%, respectively. Twenty-four-hour urine specimens from five patients with pheochromocytoma showed above-normal amounts of the metanephrines by this method.