Clinical Abstracts

September 2005

Editors:
Michael Bissell, MD, PhD, MPH
Ronald Domen, MD

Comparing methods for CSF immunoglobulin quantitation by nephelometry
Removing fibrinogen from serum protein electrophoresis specimens
PCR quality assurance
Glycation end products and dendritic cells in diabetes
Donation patterns and blood availability
Factors that influence a fetal lung maturity assay
Chlamydia pneumoniae and peripheral vascular disease

Comparing methods for CSF immunoglobulin quantitation by nephelometry

The simultaneous measurement of albumin and IgG in cerebrospinal fluid and serum samples plays a routine role in the diagnosis of multiple sclerosis. The quantitative determination of cerebrospinal fluid (CSF) IgM to show intrathecal production of IgM is an optional test for the diagnosis of multiple sclerosis. There are a number of situations in which CSF IgG measurements alone are not adequate and quantification of CSF IgA or IgM is useful. The quantitative determination of CSF IgA is of little value in the diagnosis of multiple sclerosis; however, strong intrathecal production of IgA may imply a different diagnosis. Intrathecal IgA production is the most sensitive CSF parameter for cerebral adrenoleukodystrophy and is not seen in patients with adrenomyeloneuropathy. An intrathecal IgA response can indicate a bacterial origin with a response rate of nearly 90 percent in neurotuberculosis and can provide information about a brain abscess. Intrathecal production of IgM can be seen in acute and chronic diseases, including neuroborreliosis, mumps meningitis, and parenchymatous neurosyphilis. Detection of an IgM response alone is not useful, but in combination with other CSF data, it can contribute to typical patterns seen in neuroborreliosis, mumps meningitis, or non-Hodgkin's lymphoma. However, it can be difficult to find automated methods with sufficient sensitivity to accurately determine IgA and IgM in CSF. The authors conducted a study to evaluate the performance characteristics of immunonephelometric assays for CSF IgA and IgM from two vendors. They evaluated for linearity, imprecision, method comparison, and reference interval verification the Dade Behring N Latex IgA and N Latex IgM tests on the BN II system and Beckman Coulter low-concentration IgA and IgM tests on the Immage immunochemistry system. Both IgA methods were linear from 1.4 mg/L to at least 50 mg/L. Both IgM methods were linear from 0.14 mg/L to more than 6 mg/L. The total imprecision of the BN II IgA and IgM methods and the Immage IgA method was less than 10 percent. The imprecision of the Immage IgM method was 10.2 percent at 0.49 mg/L and less than five percent at higher IgM concentrations. Method comparison studies indicated that IgA and IgM methods on both instruments showed good comparability. Reference interval studies demonstrated that both methods had similar reference intervals that agreed with published values of less than 6 mg/L for IgA and less than 1.3 mg/L for IgM. The authors concluded that methods for quantifying IgA and IgM in CSF on the BN II and Immage nephelometers perform well and give comparable results.

Owen WE, Roberts WL. Performance characteristics of four immunonephelometric assays for the quantitative determination of IgA and IgM in cerebrospinal fluid. Am J Clin Pathol. 2003;119:689-693.

Reprints: Dr. William L. Roberts, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108

Removing fibrinogen from serum protein electrophoresis specimens

Fibrinogen is found in serum specimens from patients who have congenital dysfibrinogenemias or acquired disorders of coagulation, such as anti-phospholipid syndrome, liver disease, and vitamin K deficiency, as well as in patients who are on anticoagulation therapy or who have an indwelling catheter kept open with low-dose heparin. The protein band for fibrinogen is located at the beta-gamma junction on serum protein electrophoresis and can be misinterpreted as a monoclonal protein. Some serum specimens from patients in the authors' institutions have exhibited such a discrete band on serum protein electrophoresis, but monoclonal immunoglobulins were not identified by serum immunofixation electrophoresis. It has long been known that fibrinogen is the least soluble of the major plasma proteins and is readily precipitated by salting out with sodium chloride or ammonium sulfate or by precipitation with ethanol. The authors hypothesized that treating serum with ethanol would be a simple, efficient method to eliminate fibrinogen from suspected specimens and that it would be simpler and more effective than the methods customarily used for presumptive identification. To illustrate the utility of this approach, they presented a case report from their laboratory. Pooled human plasma was mixed with absolute ethanol or saline (final concentrations of 40, 80, 100, 120, and 160 mL/L) and incubated at 4°C overnight or placed in an ice bath for 15 minutes. After centrifugation, the supernatants and resuspended pellets were used for protein electrophoresis and quantitative measurements of protein and fibrinogen. The fibrinogen band was eliminated from the electrophoretic pattern in the plasma samples treated with ethanol at 100 mL/L and incubated in an ice bath for 15 minutes without a significant change in immunoglobulin concentrations. The 100 mL/L of ethanol did not noticeably change the electrophoretic pattern of monoclonal immunoglobulins. The authors concluded that 100 mL/L of ethanol can selectively precipitate fibrinogen without significantly interfering with the immunoglobulins. The precipitation process can be completed in 15 minutes at 0°C to 4°C and can eliminate the need to obtain another blood sample.

Qiu LL, Levinson SS, Keeling KL, et al. Convenient and effective method for removing fibrinogen from serum specimens before protein electrophoresis. Clin Chem. 2003;49:868-872.

Reprints: Ronald J. Elin, Dept. of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202; rjelin01@gwise.louisville.edu

PCR quality assurance

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has included participation in external quality assurance schemes as a key tool for quality assessment of molecular amplification methods applied to clinical diagnostics. However, external quality assurance (EQA) programs for many diagnostic tests based on nucleic acid amplification have not been implemented on a regular basis. A valuable alternative to this limitation is the development of EQA trials based on methodologic proficiency testing and directed at evaluating analytical aspects of the majority of PCR-based tests. The authors developed an EQA program for evaluating DNA extraction and amplification and analyzing products after PCR. Their program involved three control levels: DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of the results after electrophoresis. The scheme was carried out under the auspices of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) and was included in the National Concerted Action for Quality Control in Oncological Laboratories of the Italian Ministry of Health. Two rounds of studies were performed. The first involved 16 laboratories and the second involved 39 Italian laboratories. In this article, the authors presented the results of the second pilot EQA study. Study participants received a package containing primers and reference materials to evaluate the aforementioned three controls. All participants were asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation. Results varied in all phases of the experimental procedure: pre-amplification, amplification, and post-PCR interpretation. To give a general estimation of the quality of performances for each laboratory, the authors designed a score scheme in which the results of any action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, the authors created a qualitative ranking list that provided the final interpretation of results as excellent (more than 63 points; n=4 laboratories), good (53 to 63 points; n=13), sufficient (42 to 52 points; n=15), poor (31 to 41 points; n=3), and not acceptable (fewer than 31 points; n=4). The authors concluded that this survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed at evaluating analytical aspects of the majority of PCR-based tests.

Raggi CC, Pinzani P, Paradiso A, et al. External quality assurance program for PCR amplification of genomic DNA: an Italian experience. Clin Chem. 2003;498:782-791.

Reprints: Claudio Orlando, Clinical Biochemistry Unit, Dept. of Clinical Physiopathology, University of Florence, viale Pieraccini 6, 50139 Florence, Italy; c.orlando@dfc.unifi.it

Glycation end products and dendritic cells in diabetes

Advanced glycation end products, a complex and heterogeneous group of post-translational modifications of proteins in vivo, have been widely studied for their involvement in diabetic complications; these complications are largely vascular and accompanied by inflammation. Reducing sugars and their metabolites react with free amino groups (the Maillard reaction) and sequentially alter to produce advanced glycation end products (AGEs). Carbonyl compounds from glycolytic intermediates of Maillard reactions, including glyoxal and methylglyoxal (MG), are major contributors to AGE formation. MG is the most reactive AGE precursor, and its concentration is increased in diabetics. MG reacts irreversibly with lysine to form glycosylamine protein cross-links and with arginine to give imidazolone derivatives. AGEs are associated with complications of rheumatoid arthritis, Alzheimer's disease, and diabetes. Hyperglycemia in diabetes increases the rate of formation of AGEs. Because dendritic cells initiate and modulate inflammatory responses, the authors hypothesized that AGEs might exert immunomodulatory effects via antigen-presenting dendritic cells. To test this hypothesis, they investigated effects of the AGE peptide, compared with the naked peptide, on maturation, co-stimulatory molecule expression, and the function of dendritic cells in peripheral blood. From flow cytometry, the authors found a dose-dependent inhibition in CD83 expression on dendritic cells exposed for 2.5 hours to each of two synthetic AGE peptides. A similar culture for 24 hours additionally produced an inhibition of CD80 expression, whereas exposure to AGEs for three days induced a large increase in the number of dendritic cells and a concomitant loss of monocyte/macrophages. Exposure of dendritic cells to AGEs results in a dose-dependent loss in their capacity to stimulate primary proliferation of allogeneic T-cells. The authors concluded that AGEs promote the development of dendritic cells but that these cells fail to express maturation markers and lose the capacity to stimulate primary T-cell responses. Effects of AGEs on dendritic cells could be instrumental in the immunological changes associated with diabetes.

Price CL, Sharp PS, North ME, et al. Advanced glycation end products modulate the maturation and function of peripheral blood dendritic cells. Diabetes. 2004;53:1452-1458.

Reprints: S.C. Knight, Antigen Presentation Research Group, Faculty of Medicine Imperial College London, Northwick Park and St. Marks Campus, Level 7W, St. Marks Hospital, Watford Rd., Harrow, Middlesex, HA1 3UJ, United Kingdom; s.knight@imperial.ac.uk

Donation patterns and blood availability

Each year, only five percent of the eligible U.S. population donates blood. The key to ensuring blood availability is encouraging repeat donors to give more regularly. An estimated 63 percent of repeat whole blood donors give less than two donations per year, about one-quarter of the maximum donations permitted. Targeting repeat donors has two main advantages: repeat donors have consistently been shown to have a lower incidence of infectious diseases and deferrable risks than first-time donors; and, encouraging already committed repeat donors to modify their donation habits is likely to be more cost-effective than trying to attract lapsed donors or those who have never donated. The primary goal of this study was to use the donation histories of donors from five geographically diverse blood centers to predict the number of additional donations that could be collected if five percent, 15 percent, or 25 percent of donors who did not return within 12 months of donating could be persuaded to donate one more time in that period. In addition, the authors examined the frequency with which first-time donors with different demographic profiles returned to donate to help identify donor groups more likely to become repeat donors. First-time donors giving between 1991 and 1994 were followed for three to six years. The impact of decreasing the number of non-returning donors in a 12-month period was projected using the Poisson distribution to model the donation patterns of 539,063 donors who gave in 1995. Repeat donors were classified as returning first-time (gave a first-time donation within 12 months of their index donation), established (gave a repeat donation), and inactive (didn't donate in the 12 months before their index donation). Forty-nine percent of first-time donors did not return within six years. Returning first-time donors tended to be U.S. born, white, and had more than a high school education. In 1995, 30 percent of donors were first-time, 31 percent were established, seven percent were returning first-time, and 32 percent were inactive, giving 1.5, 2.4, 1.9, and 1.7 donations in 12 months, respectively. Reducing non-returning donors by five percent could increase blood collections by 2.7 percent. Similarly, 15 percent or 25 percent reductions in non-returns could increase collections by 8.6 percent or 16 percent, respectively. The authors concluded that most donors are not giving near the donation limit. A substantial increase in donations could be achieved by a relatively small decrease in donor non-returns. Research is needed to understand why approximately half of first-time donors donate only once.

Schreiber GB, Sanchez AM, Glynn SA, et al. Increasing blood availability by changing donation patterns. Transfusion. 2003;43: 591-597.

Reprints: George B. Schreiber, Westat Inc., 1650 Research Blvd., Rockville, MD 20850; georgeschreiber@westat.com

Factors that influence a fetal lung maturity assay

Respiratory distress syndrome ranks sixth among the leading causes of death in newborn infants. Also referred to as hyaline membrane disease, respiratory distress syndrome results from an insufficient amount of pulmonary surfactant being produced by the neonatal lung and released into the amniotic fluid. Pulmonary surfactant is composed primarily of phospholipids and is contained within lamellar bodies that are synthesized and extruded by the type II cells of the alveolar epithelium. The Abbott TDx Fetal Lung Maturity II (FLM II) assay uses fluorescent polarization to assess the lung maturity of unborn infants by measuring the ratio of surfactant to albumin in filtered, uncentrifuged amniotic fluid. The FLM II assay has been shown to correlate with fetal outcome. Although numerous studies have examined the clinical performance of the FLM II assay, few have analyzed the pre-analytical variables that can potentially influence the results of this assay. The authors evaluated three commonly occurring pre-analytical factors that impact FLM II results: centrifugation, sample storage (at -20°C, 4°C, and room temperature), and contamination by whole blood. They tested 18 specimens after centrifugation and after resuspension by vortex mixing. They also analyzed 23 specimens stored at -20°C for up to 448 days and then thawed (duplicate measurements), 20 specimens stored at 4°C, and 24 specimens stored at room temperature. In addition, they evaluated the effects of whole blood diluted into 19 different specimens. The authors found that centrifugation significantly decreased FLM II results from baseline (P<0.0001) and resuspension returned results to baseline values (P=0.286). Storage at -20°C produced highly variable results that demonstrated a nonsignificant negative trend associated with storage time. Specimens were stable for 24 hours when stored at 4°C and 16 hours at room temperature. Blood contamination produced significantly positive differences in results only in specimens with baseline values of 39 mg/g or less with a 5.8 mg/g increase in FLM II in every 0.1 x 10¹²/L increase in erythrocyte count (slope, 58.4). The authors concluded that resuspension of centrifuged specimens produces clinically valid FLM II results. Results from specimens stored at -20°C can be highly variable and decrease over time. Results from specimens stored at 4°C and at room temperature are stable for 24 and 16 hours, respectively. Blood contamination up to 0.03 x 10¹² erythrocytes/L is acceptable for FLM II analysis.

Grenache DG, Parvin CA, Gronowski AM. Pre-analytical factors that influence the Abbott TDx Fetal Lung Maturity II assay. Clin Chem. 2003; 49:935-939.

Reprints: Ann M. Gronowski, Washington University School of Medicine, Dept. of Pathology and Immunology, Division of Laboratory Medicine, Box 8118, 660 S. Euclid Ave., St. Louis, MO 63110; gronowski@pathbox.wustl.edu

Chlamydia pneumoniae and peripheral vascular disease

The first seroepidemiologic study that reported high titers of circulating antibodies against Chlamydia pneumoniae in patients with coronary atherosclerosis was published in 1988. Several subsequent studies have suggested that chronic infection with C. pneumoniae may contribute to the pathogenesis of atherosclerosis. The authors conducted a case-control study to assess the relationship between C. pneumoniae and peripheral artery occlusive disease (PAOD) involving the carotid, femoral, and aortoiliac arteries. The study was divided into three parts: a clinical study (inflammation markers), a study of immune response, and the detection of C. pneumoniae DNA in samples of arterial wall from patients and control subjects. The study included 64 patients with peripheral artery occlusive disease and 50 control subjects who underwent varicose vein surgery, matched to the patient group for age, gender, and tobacco use. The fibrinogen level in all study subjects was measured as a marker of inflammation. Blood samples were taken from all subjects to determine immunoglobulin G elementary bodies against C. pneumoniae with microimmunofluorescence and enzyme-linked immunosorbent assay, and of IgA elementary bodies with ELISA. The cutoff titers were 1:32 for microimmunofluorescence and 1.1 for ELISA. Biopsy specimens of arterial atheromatous plaque were obtained from patients and of pudendal artery and saphenous vein from control subjects, and they were studied with hemi-nested polymerase chain reaction. No differences in fibrinogen level were noted between patients and controls. The prevalence of IgG anti-elementary bodies with microimmunofluorescence was 78 percent in patients and 24 percent in control subjects (P=.0001; odds ratio [OR], 11.3; 95 percent confidence interval [CI], 4.7-27.2). The prevalence of IgG anti-elementary bodies with ELISA was 75 percent in patients and 16 percent in control subjects (P=.0001; OR, 15.7; 95 percent CI, 6.1-40). No differences were noted in IgA anti-elementary bodies titers. Bacterial DNA was detected in 67 percent of atheromatous plaques versus 12 percent of pudendal arteries (P=.0001) and four percent of saphenous veins. A weak correlation was found between seropositivity and the presence of intravascular DNA. The authors concluded that C. pneumoniae is related to the pathogenesis of atherosclerotic peripheral artery occlusive disease.

Linares-Palomino JP, Gutiérrez J, Lopez-Espada C, et al. Genomic, serologic, and clinical case-control study of Chlamydia pneumoniae and peripheral artery occlusive disease. J Vasc Surg. 2004;40:359-366.

Reprints: Dr. Jose Linares-Palomino, Departamento de Cirugia Vascular, Hospital Universitario St. Cecilio, Avda Dr Oloriz No. 16, E-18012 Granada, Spain; jlinaresp@seacv.org