College of American Pathologists
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November 2003

The genetics of susceptibility to disseminated Mycobacterium bovis infection
In countries where anti-tubercular vaccine-that is, the bacille Calmette-Guérin (BCG) vaccine-is given at birth, disseminated Mycobacterium bovis BCG infections (BCGitis) occur. This condition occurs in the context of primary immunodeficiencies causing specific susceptibility to these weakly pathogenic mycobacteria. In some cases, susceptibility to other intracellular pathogens, such as Salmonella, has been associated with these disorders. Well-defined immunodeficiencies account for about half of the cases of BCGitis. The other cases were considered, until recently, to be idiopathic. Recent studies have demonstrated, however, that underlying molecular defects involve key elements of the interferon (IFN)-γ-dependent pathway of macrophage activation. The authors studied the clinical and genetic heterogeneity of this disorder in five Tunisian patients from four unrelated families who presented with BCGitis. They found that two unrelated patients had different homozygous interleukin-12 receptor β1 subunit gene splice-site mutations (64+5G∅A and 550-2A∅G). Two siblings and one unrelated patient, all of whom were from the same town, carried the same mutation (297 del8) within the IL-12p40 gene. This is the first reported description of a familial cytokine deficiency. All patients had profound deficiency of in vitro interferon-g production. Clinical severity of the mycobacterial infection was heterogeneous, even among affected members of the same family, suggesting the intervention of modifying genes.

Elloumi-Zghal H, Barbouche MR, Chemli J, et al. Clinical and Genetic Heterogeneity of Inherited Autosomal Recessive Susceptibility to Disseminated Mycobacterium bovis Bacille Calmette-Guérin Infection. J Infect Dis. 2002;185:1468-1475.

Reprints: Dr. Sonia Abdelhak, Institut Pasteur de Tunis, BP 74, 13 Place Pasteur, Tunis Belvédère 1002, Tunisia;

Using the induced sputum technique to identify sarcoidosis in patients with uveitis
The cause of up to a third of uveitis is related to systemic diseases, of which sarcoidosis is one of the most common. Sarcoidosis, a multiple-system granulomatous disease of unknown origin, is usually diagnosed using a combination of clinical, radiologic, histologic, and laboratory findings and is supported by abnormal serum levels of angiotensin-converting enzyme and a CD4/CD8 ratio greater than 2.5 for alveolar lymphocytes obtained by bronchoalveolar lavage. BAL is a noninvasive yet unpleasant procedure, and patients often are reluctant to undergo it. The only entirely noninvasive alternative is to examine sputum. It is far easier to conduct the induced sputum procedure because it is entirely noninvasive and can be performed frequently to follow the dynamic course of pulmonary inflammatory disease as well as the effect of treatment. The authors reported on their experience using the induced sputum procedure to identify sarcoidosis as the cause of uveitis. They studied 17 patients (13 men and four women) with uveitis and compared this group with two control groups. The first control group consisted of 10 patients with sarcoid disease without uveitis and the second consisted of five healthy volunteers. Sputum was induced by a 20-min. inhalation of 3.5 percent saline using an ultrasonic nebulizer. Differential counts of 200 cells on cytopreps stained by Giemsa were used as the catalyst. T-lymphocyte subset analyses were done by fluorescence-activated cell sorter using the monoclonal antibodies CD4 and CD8. ACE serum levels were also obtained. The authors found that the difference in the CD4/CD8 ratios in the induced sputum examination between the patients with elevated ACE levels and the patients with normal ACE levels was statistically significant (P=0.0001). They also found that a CD4/CD8 ratio greater than 2.5 and an ACE level greater than 145 µL/mL per minute were considered to be abnormal. The authors concluded that induced sputum examination showed increased CD4/CD8 ratios in patients with uveitis who also had elevated ACE levels, suggesting the presence of sarcoidosis.

Neudorfer M, Leibovitch I, Onn A, et al. Induced Sputum for Identifying Sarcoidosis in Patients with Uveitis. Ophthalmology. 2002;109:858-861.

Reprints: Dr. Meira Neudorfer, Dept. of Ophthalmology, Tel-Aviv Sourasky Medical Center, Tel-Aviv University, 6 Weitzman St., Tel Aviv, 64239, Israel

Diaspirin-crosslinked hemoglobin reduces blood transfusion in noncardiac surgery
Diaspirin-crosslinked hemoglobin (DCLHb) is a purified human hemoglobin derivative that binds oxygen and has the potential to reduce the need for red blood cell transfusion in patients undergoing surgery. The authors conducted a multicenter, randomized, prospective, double-blinded clinical trial to study whether DCLHb could prevent or reduce allogeneic blood transfusion in major orthopedic, abdominal, and vascular surgery. A total of 181 patients were randomized to receive DCLHb (92 patients) or packed red blood cells (PRBCs; 89 patients). Eighty-nine DCLHb patients (97 percent) received DCLHb, and 84 control patients (94 percent) received PRBCs. During the first 36 hours of the study period, 63 percent of DCLHb patients (versus 40 percent of PRBC patients) received three units of study infusion. By post-operative day seven, 21 of 89 (24 percent) DCLHb-treated patients still had not received allogeneic transfusion of PRBCs. Over the seven-day study period, the median amount of red cells transfused per DCLHb patient was 700 mL (range, 250 to 1,000 mL), compared with 820 mL (range, 500 to 1,137 mL) per patient in the control group. In the DCLHb group, 23 percent of patients completely avoided blood exposure. Mortality (four percent versus three percent) and incidence of at least one serious adverse event (21 percent versus 16 percent) were similar in the DCLHb and PRBC groups, respectively. Six DCLHb patients experienced serious adverse events-four developed amylase or lipase levels three times normal or greater and two had evidence of pancreatitis. The study was terminated early because of adverse events and safety concerns in the DCLHb group. The authors concluded that although the safety profile of DCLHb needs to be improved, there was demonstrated efficacy in reducing exposure to allogeneic blood for elective surgery.

Schubert A, Przybelski RJ, Eîdt JF, et al. Diaspirin-crosslinked Hemoglobin Reduces Blood Transfusion in Noncardiac Surgery: a Multicenter, Randomized, Controlled, Double-blinded Trial. Anesth Analg. 2003;97: 323-332.

Reprints: Armin Schubert, MD, Dept. of General Anesthesiology, Cleveland Clinic Foundation, 9500 Euclid Ave., E31, Cleveland, OH 44195;

Onchocerciasis: targets for a new therapeutic approach
Onchocerciasis, or river blindness, caused by the filaria Onchocerca volvulus, affects more than 17 million people in Africa, Latin America, and Yemen. It is the second most common cause of preventable blindness in sub-Saharan Africa, with an estimated prevalence of 500,000 with visual impairment and 270,000 with blindness. The vector is Simulium spp blackflies, which transfer infective larvae during a meal of blood. Within a year, the larvae mature to female and male worms located in subcutaneous nodules (onchocercomas). The female worms produce millions of microfilarias (first stage larvae) during their lifetime of up to 14 years. The microfilarias induce the pathology characteristic of the disease, which includes chronic dermatitis and skin atrophy, lymphadenitis and fibrosis, and the ocular inflammation that can lead to blindness. Endosymbiotic bacteria (Wolbachia spp) in filarias have emerged as a new target for treatment with drugs. Recent experimental findings also indicate the endotoxin-like molecules from Wolbachia have a role in the pathogenesis of the disease and in adverse reactions after treatment.

Hoerauf A, Büttner DW, Adjei O, et al. Onchocerciasis: Endosymbiotic Wolbachia Bacteria in the Worms are Targets for a New Therapeutic Approach. BMJ. 2003; 326: 207-210.

Reprints: A. Hoerauf, Bernhard Nocht Institute for Tropical Medicine, Dept. of Helminthology, Bernhard-Nocht-Strasse 74, 20359, Hamburg, Germany;

Free κ and free λ immunoglobulin light chain levels in serum
Monoclonal gammopathies are characterized by the clonal expansion of plasma cells. The monoclonal immunoglobulin secreted by the cells in these conditions is an indication of this state. The monoclonal immunoglobulin can be quantitatively measured to monitor the course of the disease. Monoclonal gammopathies include multiple myeloma, light-chain myeloma, Waldenstrom macroglobulinemia, nonsecretory myeloma (NSMM), smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, primary systemic amyloidosis (AL), and light-chain-deposition disease (LCDD). Monoclonal light-chain diseases and NSMM often do not have sufficiently high concentrations of serum monoclonal light chains to be detected by serum protein electrophoresis or immunofixation. In addition, when immunofixation detects a monoclonal light chain, the amount of protein may be too low to be quantified and monitored by SPEP. New sensitive nephelometric assays specific for κ and λ free light chains but that do not recognize light chains bound to immunoglobulin heavy chains have recently been described. The authors undertook a study to determine serum reference intervals and diagnostic ranges for these assays. They used an automated immunoassay for free light chains and sera from a population that was 21 to 90 years of age. They used the calculated reference and diagnostic intervals to compare free-light chain results with those obtained by immunofixation to detect low concentrations of monoclonal κ and λ free light chains in the sera of patients with monoclonal gammopathies. The authors found that these concentrations increased with population age, with an apparent change for those older than 80 years. The ratio of κ free light chains to λ free light chains did not exhibit an age-dependent trend. The diagnostic interval for free light chain κs and λs was 0.26 to 1.65. The 95 percent reference interval for κ free light chains was 3.3 to 19.4 mg/L, and that for λ free light chains was 5.7 to 26.3 mg/L. Detection and quantification of monoclonal free light chains by nephelometry were more sensitive than IFE in serum samples from patients with primary systemic amyloidosis and light-chain-deposition disease. The authors concluded that the serum-free light-chain assay complements immunofixation and allows quantification of free light chains in light-chain-disease patients who do not have detectable serum or urine M-spike.

Katzmann JA, Clark RJ, Abraham RS, et al. Serum Reference Intervals and Diagnostic Ranges for Free κ and Free λ Immunoglobulin Light Chains: Relative Sensitivity for Detection of Monoclonal Light Chains. Clin Chem. 2002;48:1437-1444.

Reprints: Jerry A. Katzmann, Mayo Clinic, Hilton Bldg., Rm. 920, 200 First St. SW, Rochester, MN 55905;

Prevalence and partitioning of reference range subgroups
Population-based reference intervals are the predominant method for interpreting clinical biochemical laboratory measurements in medical diagnosis and screening. Subgroup partitioning of reference ranges improves the specificity of these intervals. Demographic descriptors, such as age, gender, or race, as well as lifestyle factors, should be considered as potential partitioning categories. In large-scale reference interval studies involving up to thousands of reference individuals, the usefulness of partitioning is even more evident because large reference samples make partitioning into increasingly smaller subgroups feasible. Existing partitioning models, however, do not seem to give reliable estimates of the proportions of the subgroup distributions falling outside the reference limits of the combined distribution. The authors constructed a new model based on correlating these proportions to distance between reference limits of the subgroup distributions, with one pair of reference limits at a time being considered. When applying the new model to various partitioning problems, the authors discovered a complication: unequal prevalences seem to make the critical distances used as partitioning criteria shorter compared with equal prevalences. Moreover, it appears that subgroup prevalences should also be considered when using proportion criteria or, in practice, whenever reference data are being partitioned. The authors created a report that was meant to be a generalization of their previous work, covering the situation of unequal subgroup prevalences. Dramatic shrinkage of the critical distances between reference limits of the subgroups needed for partitioning was observed as the ratio of prevalences, the larger one divided by the smaller one, was increased from unity. The prevalences of subgroups in the reference population should be known and observed in the calculations for every reference interval study, irrespective of whether distance or proportion criteria are being used to determine the need for reference interval partitioning. The authors presented detailed methods to account for the prevalences when applying each of these types of criteria. Analytical expressions for the distance criteria, to be used when high precision is needed, and approximate distances, to be used in practical work, were derived. General guidelines for partitioning gaussian distributed data were presented. The authors concluded that the new model offers an improved correspondence between the critical distances and the critical proportions and accounts for the prevalence effect.

Lahti A, Petersen PH, Boyd JC. Impact of Subgroup Prevalences on Partitioning of Gaussian-distributed Reference Values. Clin Chem. 2002;48:1987-1999.

Reprints: Ari Lahti, Dept. of Clinical Chemistry, Rikshospitalet University Hospital of Oslo, N-0027 Oslo, Norway;