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September 2002

Quality control of the sedimentation rate
The erythrocyte sedimentation rate is widely used for the diagnosis and followup of patients with inflammatory diseases, rheumatoid arthritis in particular. The test has a long-standing history and tradition in clinical laboratories. Several new techniques have emerged in recent decades for measuring the ESR. These have attempted to address the need to guarantee the safety of operators, automate the measurement itself to optimize workflow, and create a unique workstation to measure the ESR and perform other hematologic tests on a single specimen. This can be accomplished using the recommended specimen—undiluted blood with tripotassium EDTA—which is more reliable than the traditional sodium citrate. The authors evaluated and validated a new measurement procedure, the so-called Test 1, which allows automated, safe, and precise measurements of the ESR in specimens collected in sodium citrate and tripotassium EDTA. They undertook the present study to describe and evaluate a procedure based on using fresh human whole blood for daily quality control of the ESR in clinical labs. The purpose of the procedure is to achieve daily quality control of the ESR and establish limits of agreement between working and reference methods. The authors used data from routine patient samples to calculate the daily cumulative mean and monitor its reproducibility over time. They compared results from the measurement of the ESR in specimens stored at 4°C for 24 hours and results obtained in fresh samples. The authors concluded that, based on their results, which were obtained in fresh blood samples, quality control for measuring the ESR with the Test 1 apparatus is feasible, inexpensive, and reliable.

Plebani M, Piva E. Erythrocyte sedimentation rate. Am J Clin Pathol. 2002;117: 621-626.

Reprints: Dr. M. Plebani, Servizio di Medicina de Laboratorio, Azienda Ospedaliera de Padova, via N. Giustiniani 2, 35128, Padova, Italy

Biochemical markers of bone metabolism in serum and femur aspirates
Biochemical markers of bone metabolism in peripheral blood have been studied in various bone diseases. Among the unambiguously accepted indicators of osteoblastic activity are alkaline phosphatase, bone-specific alkaline phosphatase, and osteocalcin. Osteocalcin and bone-specific alkaline phosphatase, among other parameters, have been shown in peripheral blood and urine to be of value in evaluating postmenopausal osteoporosis. The carboxy-terminal cross-linked telopeptide of type 1 collagen and the amino-terminal propeptide of type 1 procollagen are established markers of collagen type 1 metabolism. Measurements of these biochemical markers in direct samples of cancellous bone have not been reported. The authors conducted a study to measure biochemical markers of bone metabolism directly in healthy cancellous bone of the distal femur. This was done in 27 patients undergoing arthroscopy of the knee. Bone-specific alkaline phosphatase, osteocalcin, and collagen type 1 metabolites were studied simultaneously in serum and the distal femur using radioimmunoassay. The levels of bone-specific alkaline phosphatase and osteocalcin did not differ between the serum and bone. Concentrations of type 1 collagen metabolites were, however, elevated significantly in healthy bone. The mean bone-to-serum ratio for alkaline phosphatase and osteocalcin were 1.1 and 1.2, respectively. Collagen type 1 metabolites were 2.2 for the carboxy-terminal cross-linked telopeptide and 2.3 for the procollagen type 1 N-terminal peptide. Collagen type 1 metabolites appear to be formed locally and released into the circulation. And bone seems to be a major contributor of these analytes to the serum pool.

Berger CE, Kröner A, Thomas E, et al. Comparison of biochemical markers of bone metabolism in serum and femur aspirates. Clin Orthop. 2002;395:174-179.

Reprints: Dr. Christian Berger, Dept. of Orthopaedics, Danube Hospital, Langobardenstr. 122, 1220 Vienna, Austria

Understanding subclinical thyroid disease
The debate continues regarding to what extent subclinical thyroid disease should be treated. Minor abnormalities in thyroid function that indicate subclinical hypothyroidism or hyperthyroidism are common. Both conditions have been associated with an increased risk of disease as well as abnormalities in other biochemical and physiological measures that are often abnormal in patients with overt thyroid disease. Subclinical thyroid disease is defined by high or low serum thyroid-stimulating hormone levels with T 4 and T 3 within laboratory reference ranges. An important issue is whether an individual's serum concentrations of T 4 and T 3 are normal in subclinical thyroid disease. Population-based reference ranges for these two analytes are quite wide because of large differences in thyroid function tests among healthy subjects. The differences are caused by analytical and biological variation. The authors estimated biological and analytical variation in thyroid function tests in 16 healthy men in a routine laboratory setting using monthly sampling for 12 months. This included estimating within—and between—individual variation. This was used to assess the utility of population-based reference ranges for serum TSH, total T 3 , total T 4 , and free T 4 index. The authors also calculated the number of tests needed to estimate the individual set point with certain levels of confidence and the difference required between two hormone values for significance in repeated testing of an individual. All of the test subjects had different variations of thyroid function tests around individual mean values or set points. The width of the individual 95 percent confidence intervals was approximately half that of the group for all variables. Therefore, the index of individuality was low: T 4 equaled 0.58; T 3 equaled 0.54; FTI equaled 0.59; TSH equaled 0.49. A single test result described the individual set point only to within ± 25 percent for T 4 , T 3 , and free T 4 index and only to within ± 50 percent for TSH. The differences that were requred to be 95 percent confident of significant changes in repeated testing were 28, with a range of 11 to 62 nmol/L for T 4 ; 0.55, with a range of 0.3 to 0.9 nmol/L for T 3 ; 33, with a range of 15 to 61 nmol/L for free T 4 index; and 0.75, with a range of 0.2 to 1.6 mU/L for TSH. The data indicated that each individual had a unique thyroid function set point. The individual reference ranges for test results were narrow compared with the group reference ranges. Accordingly, a test result for an individual may not correspond to a test result within laboratory reference limits. These data indicate that the distinction between subclinical and overt thyroid disease (abnormal serum TSH and abnormal T 4 or T 3 ) is somewhat arbitrary. For the same degree of thyroid function abnormality, the diagnosis depends to a considerable extent on the position of the patient's normal set point for T 4 and T 3 .

Andersen S, Pedersen KM, Bruun NH, et al. Narrow individual variations in serum T4 and T3 in normal subjects: a clue to the understanding of subclinical thyroid disease. J Clin Endocrinol Metab. 2002;87:1068-1072.

Reprints: Dr. Stig Andersen, Dept. of Endocrinology and Medicine, Aalborg Hospital, DK-9000 Aalborg, Denmark;

Storage of red blood cells for 11 weeks
Cumulative events decrease the quality and utility of red blood cells that are stored. Hemolysis and loss of viability can be partially offset by cell washing or metabolic rejuvenation, or both, but better RBC storage systems are required. The authors previously described nine- and 10-week RBC storage systems using white blood cell reduction and storage in 200- or 300-mL volumes of hypotonic alkaline experimental solutions. These solutions contained saline, adenine, glucose, mannitol, and disodium phosphate. The systems appear to work by supporting an initial increase in RBC adenosine triphosphate synthesis that provides metabolic energy for processes that sustain cell viability during longer storage. The systems also suppress red blood cell shape changes and membrane microvesiculation, which contribute substantially to hemolysis. Replacing some of the sodium chloride with sodium bicarbonate was shown to achieve greater rates of ATP synthesis during prolonged storage. In an attempt to further increase the time these solutions can be stored, the authors conducted three studies. First, they conducted a human trial measuring RBC recovery after 11 weeks using a solution containing bicarbonate. This study showed a mean 24-hr RBC recovery of 79 percent but a 1.35 percent hemolysis rate. Second, they measured the effect of increasing the concentrations of mannitol to reduce hemolysis during the 11-week storage. Third, they measured hemolysis during 12 weeks of RBC storage in one of the solutions containing a higher mannitol concentration. In the first study, 24-hr in vivo recovery of 51 chromium-labeled autologous RBCs was measured in nine volunteers after storing their RBCs in EAS-67 for 11 weeks. In the second study, four units of blood were divided and stored in aliquots with an experimental AS (EAS) containing 0, 15, 30, or 45 mmol/L of mannitol. Hemolysis, red cell morphology, and microvesicle protein were measured in the specimens. In the third study, six full units were stored for 12 weeks in the EAS containing 30 mmol/L of mannitol, with weekly sampling of morphologic and biochemical measures of RBC quality. Increasing mannitol content of the EAS reduced hemolysis but increased microvesiculation. EAS-76 with 30 mmol/L of mannitol allowed 11 weeks of storage with 0.48 ± 0.10 percent hemolysis at 11 weeks and 0.62 ± 0.14 percent hemolysis at 12 weeks. The authors concluded that it is possible to store RBCs in EAS for 11 weeks with more than 75 percent recovery and less than one percent hemolysis.

Hess JR, Rugg N, Gormas JK, et al. RBC storage for 11 weeks. Transfusion. 2001;41: 1586-1590.

Reprints: John R. Hess, Blood Research Detachment, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910-7500;

Acid steatocrit as a screening tool for steatorrhoea
Poor growth in infancy and childhood may be due to fat malabsorption. The gold standard for the diagnosis of fat malabsorption is the 72-hr fecal fat determination. In 1981, the steatocrit method was described in the medical literature as a simple procedure for estimating stool fat content in newborns. Acidifying the fecal homogenate was shown to improve the reliability of steatocrit results. The acid steatocrit has been shown in several clinical studies to yield a clinically useful sensitivity and specificity for detecting steatorrhoea. Reports in the literature on the acid steatocrit method have dealt principally with healthy infants, cystic fibrosis infants with pancreatic insufficiency, and adults. The authors compared the acid steatocrit results of healthy children with those of sick children with and without gastrointestinal involvement. They took stool samples from 166 children, which consisted of 50 healthy children, 26 asthmatic patients, and 90 patients with gastrointestinal problems. The latter group was composed of 34 treated cystic fibrosis patients with exocrine pancreatic insufficiency, 16 unrelated celiac disease patients, and 40 patients with various gastrointestinal problems. The median values and ranges of the acid steatocrit results were 3.3 percent (range, zero to 21 percent) for healthy children, 4.5 percent (range, 1.8 to 22.5 percent) for asthma patients, 24.7 percent (range, 2.6 to 68.2 percent) for treated cystic fibrosis patients with exocrine pancreatic insufficiency, 19.8 percent (range, three to 77.7 percent) for untreated celiac disease patients, and 5.5 percent (range, 1.8 to 29 percent) for patients with various gastrointestinal diseases. The authors concluded that the acid steatocrit results of treated cystic fibrosis patients with exocrine pancreatic insufficiency and untreated celiac disease patients were similar and significantly higher than those of healthy children and asthma patients. Acid steatocrit can be considered a reliable tool in screening for steatorrhoea in pediatric populations.

Van den Neucker AM, Kerkvliet EM, Theunissen PMVM, et al. Acid steatocrit: a reliable screening tool for steatorrhoea. Acta Paediatr. 2001;90:873-875.

Reprints: A.M. Van den Neucker, Dept. of Pediatrics, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, Netherlands;

Interference in immunoassays
Serious problems may be caused by interference in immunoassays. Heterophilic antibodies and human anti-mouse antibodies (HAMA) are important sources of positive and negative interference, particularly in sandwich, or two-site, immunoassays. Heterophilic antibodies are human antibodies of any subclass against any part of a murine antibody where the human antibodies are of sufficient titer and affinity to have an analytically significant effect and the immunogen has not been identified. HAMAs with a known immunogen have been distinguished from heterophilic antibodies because they may give different types of interference. The authors chose carcin-oembryonic antigen assays for a study of assay interference. CEA is a well-characterized, stable protein. The concentrations of CEA in serum are usually very low, making CEA assays prone to interference. The authors tested 11,261 samples with a CEA assay with bovine immunoglobulin but no murine immunoglobulins in the buffer. They ran these in parallel to their routine CEA assay using 15 mg/L of heat-treated, nonspecific murine immunoglobulin (MAK33) in the buffer and with the Fc fragments removed from the capture antibody. They estimated the frequency of interference to be four percent (95 percent confidence interval, 3.3 to 4.7 percent). Adding 15 mg/L of native MAK33 had little effect, whereas adding 15 mg/L of heat-treated MAK33 reduced interference to 0.86 percent (95 percent confidence interval, 0.61 to 1.12 percent). Adding 50 mg/L reduced interference to 0.06 percent (range, zero to 0.13 percent). Removing the Fc fragments by itself reduced interference to 0.10 percent (95 percent confidence interval, 0.02 to 0.19 percent). No statistically significant differences for age or gender were noted between 210 patients with interference and a randomly selected interference-negative control group of 186 patients. Fifteen of 25 individuals positive for interference who had four or more samples screened for interference had an interference-negative sample before or after the peak of interference. Interference, therefore, was not constant in patients. The authors concluded that in a two-site, two-step immunometric assay using mouse monoclonal antibodies, performance could be improved by using a heat-treated nonspecific murine immunoglobulin in the buffer or removing the Fc fragments from the capture antibody.

Bjerner J, Nustad K, Norum LF, et al. Immunometric assay interference: incidence and prevention. Clin Chem. 2002;48: 613-621.

Reprints: Dr. Johan Bjerner, Central Laboratory, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway; johan.bjerner!

Repeatability of BAL differential cell counts
Nonbronchoscopic lavage is a technique that is safely used to obtain bronchoalveolar lavage samples from children who are being intubated for elective surgical procedures. The literature contains no reports on the repeatability of the cell counts that are obtained from these specimens. The authors, therefore, decided to undertake a study of the repeatability of the cell counts. They studied 43 children who were admitted for elective surgery. Cell counts were obtained following nonbronchoscopic lavage. In each case, two samples were obtained with either the catheter wedged in the same position (n=21) or the catheter reinserted and wedged again (n=22). Thirty slides from nonbronchoscopic lavage samples were selected at random. Two independent observers counted 500 cells on each slide on two occasions. The authors then assessed the repeatability of the lavage sampling and cell counting for different cell types. They assessed inter- and intra-observer repeatability for the differential cell counting. The demonstrated reproducibility was good for all cell types except lymphocytes (interobserver: Lin's concordance coefficient, 0.42; repeatability coefficient, 0.66). The eosinophil differential count was highly repeatable using either method (Lin's concordance coefficient, 0.99 and 0.95; repeatability coefficient, 0.58 and 1.36). The authors concluded that variation in the sampling method for nonbronchoscopic quantitation of eosinophils can be reduced by taking two separate samples and averaging the differential cell counts. Furthermore, increasing the number of cells counted should ensure more accurate quantification of lymphocytes.

Warke TJ, Kamath S, Fitch PS, et al. The repeatability of nonbronchoscopic bronchoalveolar lavage differential cell counts. Eur Respir J. 2001;18:1009-1012.

Reprints: M. Ennis, Dept. of Clinical Biochemistry, The Queen's University of Belfast, Institute of Clinical Science, Grosvernor Rd., Belfast BT12 6BJ, Northern Ireland, United Kingdom