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The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP.

Sera from 16 of 23 (69.6%) patients with endometriosis, a potential autoimmune disease, and 2 of 17 (11.8%) control individuals had autoantibodies against the bovine carbonic anhydrase (CA) molecular weight marker, as determined by the Western blot technique. The reactivity of these antibodies to purified human CA I, human CA II, and two preparations of bovine CA II were investigated. Of the 16 endometriosis patients who were reactive to the bovine CA molecular weight marker, 14 were reactive to at least one purified human CA isoenzyme tested, 8 were reactive to at least one purified bovine CA II, and 2 did not react with any of the CA isoenzymes tested. Variation in cross-reactivity between species and in the biochemical characteristics of various CA isoenzyme preparations may partially explain these findings. Autoantibodies to CA isoenzymes have recently been reported in other autoimmune diseases. Further investigation is required to determine the significance of CA autoantibody production in patients with endometriosis.

Nitric oxide is generated from the terminal guanidino nitrogen of L-arginine yielding citrulline. This reaction is catalyzed by two major types of nitric oxide synthase: inducible and constitutive. Nitric oxide is a gaseous mediator responsible for a variety of physiologic phenomena. Its short half-life in biologic systems has created problems in its direct determination. Many experiments depend on the use of inhibitors of nitric oxide synthase to provide indirect evidence for the involvement of nitric oxide. Spectroscopic and electrochemical methods are the best for the direct measurement of nitric oxide; however, the advantages and disadvantages of each technique should be considered carefully before a specific method is selected.

The HemoCue beta-glucose photometer (Angelholm, Sweden) was evaluated for use in monitoring blood glucose in both diabetic and nondiabetic patients undergoing open heart surgery. Because occasional discrepancies were noted in patients with low total proteins when the Reflotron (Boehringer Mannheim, Indianapolis, IN) was used for this purpose, the effects of protein and hematocrit on glucose results from both instruments were investigated and compared with plasma values from a Paramax 720 ZX (Baxter Healthcare, Irvine, CA). Linear-regression analysis of the HemoCue results (y) versus Paramax (x) yielded y = 0.956x + 0.35, r2 = 0.980, with Sy/x = 0.57 mmol/L (10.3 mg/dL). Results from the Reflotron (y) versus Paramax (x) yielded y = 1.075x - 0.10, r2 = 0.964, with Sy/x = 0.99 mmol/L (17.8 mg/dL). Bias plots of (HemoCue-Paramax) or (Reflotron-Paramax) versus glucose, hematocrit, or protein showed no effect of glucose on the results from either instrument and no effect of protein or hematocrit on the HemoCue findings. The Reflotron, however, showed a positive bias of up to 3.5 mmol/L (63 mg/dL) at protein concentrations between 30-40 g/L (3.0-4.0 g/dL) and a possible positive bias at low hematocrit levels.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by the presence of anti-mitochondrial antibodies specifically directed against the M2 group of mitochondrial antigens. Recently, the E-1, the E-2, and protein X components of pyruvate dehydrogenase enzyme complex have been identified as the major antigens within the M2 group of autoantigens. An immunoassay using pyruvate dehydrogenase enzyme complex as a specific antigen for the diagnosis of PBC was developed. Pyruvate dehydrogenase enzyme complex was attached to polystyrene microbeads, incubated with sera from PBC patients (n = 18), normal controls (n = 50), or patients with other autoimmune diseases (n = 26), followed by incubation with a second fluorescein isothiocyanate conjugated goat anti-human immunoglobulin and then analyzed by flow cytometry. High numbers of fluorescence channels (mean, 1,693 +/- 846) were obtained for all PBC sera except for two patients. Compared to the conventional anti-mitochondrial antibody assay, the assay had a sensitivity rate of 94% and a specificity rate of 100%. The reactive antibodies are predominantly of the immunoglobulin G3 subclass. Their levels could be correlated with the histopathologic stages of PBC. These results were corroborated by immunoblotting. Sera from patients with later stages of PBC strongly reacted with pyruvate dehydrogenase enzyme complex components, E1 alpha, and protein X.

We evaluated three different methods for measuring lactate dehydrogenase (EC 1.1.1.27) isoenzyme LD-1 activity--agarose gel electrophoresis and two immunoassays, Isomune-LD (Roche) and LD-1 Immuno (Seragen)--in patients' samples for which measurement of creatine kinase-MB was ordered. Regression analyses of the comparisons gave the following results: LD-1 (%) from Isomune-LD (y) vs electrophoresis (x) (n = 51), y = 1.05x + 1.99, r = 0.92; LD-1 (%) from LD-1 Immuno (y) vs electrophoresis (n = 27), y = 1.05x + 3.94, r = 0.88; LD-1 (%) from LD-1 Immuno (y) vs Isomune-LD (x) (n = 41), y = 1.06x + 0.48, r = 0.95. Comparison by Student's paired t-test yielded significant differences between the mean values by electrophoresis and both Isomune-LD (P less than 0.005) and LD-1 Immuno (P less than 0.001), but no significant difference between the two immunoassays (P greater than 0.2). Analyzing these results by the overlap index, we conclude that electrophoresis shows the best clinical correlation followed, in order, by the Isomune-LD and the LD-1 Immuno methods. Both immunoassays are simpler and more rapid than electrophoresis, but in our hands the Isomune-LD method demonstrated greater precision and better correlation with electrophoretic values.

The source of the abnormally high concentration of lactate in the cerebrospinal fluid in patients with bacterial meningitis is not known. It may represent a bacterial metabolite. To determine whether cerebrospinal fluid possesses appropriate substrates to support the growth of pathogenic aerobic bacteria, three Gram-positive and nine Gram-negative bacteria were separately inoculated in pooled normal cerebrospinal fluid. After incubation for 24 h, all Gram-positive bacteria increased lactate, eight Gram-negative bacteria decreased lactate, and one Gram-negative bacteria failed to significantly change the lactate concentration. We conclude that lactate produced in cerebrospinal fluid in patients with aerobic bacterial meningitis is not necessarily a bacterial metabolite.

Phosphatidylglycerol is one component of amniotic fluid that is unaffected by several of the reported interferences with conventional thin-layer chromatographic measurement of phospholipid. To compare a rapid immunological agglutination test, "Amniostat-FLM", with two-dimensional thin-layer chromatography, we determined phosphatidylglycerol by both methods in 41 amniotic fluid specimens obtained at 31 to 40 weeks of gestation. We also assayed phosphatidylglycerol in 14 of these specimens by an enzymic, colorimetric procedure. The agglutination test is rapid and simple but relatively insensitive; it yielded positive results for only seven of 23 specimens in which phosphatidylglycerol was detected by thin-layer chromatography. In 18 specimens in which no phosphatidylglycerol was detected by thin-layer chromatography, results by Amniostat-FLM were also negative; however, eight of these specimens, assayed by the enzymic method, had phosphatidylglycerol present. Apparently, the Amniostat-FLM detects phosphatidylglycerol only at concentrations exceeding 25 mumol/L, or more than 15% of the total phospholipid composition.

Phosphatidylglycerol (PG) in amniotic fluid is recognized as a good indicator of fetal lung maturity and is unaffected by moderate amounts of blood or meconium contamination. A rapid immunologic agglutination assay, Ultrasensitive AmnioStat-FLM (FLM), was compared with two-dimensional thin-layer chromatography (TLC) and an enzymic, colorimetric procedure (E-PG). Eighty amniotic fluid specimens were analyzed. FLM results were reported as high (H), intermediate (I), or low positive (L). TLC was compared with FLM:H (n = 27), mean 0.14 (fraction of total phospholipids); I (n = 7), mean 0.11; L (n = 9), mean 0.03; negative results had no detectable PG by TLC. In 33 cases E-PG was compared with FLM:H (n = 9), mean 7.0 mumol/L; I (n = 5), mean 8.1 mumol/L; L (n = 3), mean 3.0 mumol/L; negative (n = 16), mean 3.2 mumol/L. Records were reviewed in 70 cases. Thirty cases were excluded: sample to delivery time was greater than 72 hours; steroids were given or sepsis was documented. Fetal lung immaturity was clinically present in six cases: respiratory distress syndrome in three cases and transient tachypnea of the newborn (TTN) in three cases. One false positive result was identified (TTN, FLM:H). FLM sensitivity for fetal lung maturity was 85.3%, specificity was 83.3%, and the positive predictive value for fetal lung maturity was 96.7%. FLM is a fast, reliable indicator of fetal lung maturity.

Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin. This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes. Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band. Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.