Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (Coombs-negative AIHA) is characterized by laboratory evidence of in vivo hemolysis, together with a negative DAT performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The immunoradiometric assay (IRMA) for redblood-cell-bound immunoglobulin G (RBC-IgG) can be used to diagnose patients in whom CTT does not detect low levels of red cell autoantibodies. We investigated the diagnostic cutoff value of the IRMA for RBC-IgG in Coombs-negative AIHA and calculated its sensitivity and specificity. Of the 140 patients with negative DAT by CTT referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 64 patients (Coombs-negative AIHA). The numbers of Coombs-negative AIHA and non-AIHA patients changed with age and gender. The cutoff values were determined from receiver operating characteristic (ROC) curve according to age and gender. The IRMA for RBC-IgG proved to be sensitive (71.4%) and specific (87.8%) when using these cutoffs. Using these cutoffs for 41 patients with negative DAT referred to our laboratory in 2006, all the pseudonegative cases were treated with steroids before the test. The 31 untreated cases could be grouped using one cutoff value of 78.5 and showed 100% sensitivity and 94% specificity, independent of gender and age. Results indicate that RBC-IgG could become a standard approach for the diagnosis of Coombs-negative AIHA, when measured before treatment. Am. J. Hematol. 84:98-101, 2009. V

Direct antiglobulin test (DAT)-negative (DAT-)autoimmune hemolytic anemia (AIHA) is empirically thought to show the same clinical conditions as DAT-positive (DAT1)AIHA, with the exception of an adequate amount of red blood cell (RBC)-bound immunoglobulin (Ig)G. We investigated the clinical characteristics of DAT2AIHA in comparison with DAT1AIHA. Of the 582 patients referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 216 patients (DAT2AIHA, n 5 154; DAT1AIHA, n 5 62). The percentage of reticulocytes, mean corpuscular volume, RBC-IgG levels, white blood cell count, and total protein (TP) levels were significantly higher in patients with DAT1AIHA than patients with DAT2AIHA. The hemoglobin level was significantly lower in patients with DAT1AIHA. No significant differences between patients with DAT2AIHA and DAT1AIHA existed with respect to age, gender, idiopathic/secondary nature, complications such as Evans syndrome, effectiveness of steroid treatment, or survival rate at 1 year following diagnosis. Patients with DAT2AIHA required significantly lower doses of steroids for maintenance therapy. Based on multivariate analysis of idiopathic DAT2AIHA (n 5 110), TP and Evans syndrome were associated with the effectiveness of steroids (adjusted odds ratio [aOR] IntroductionSteroid therapy is the first choice in treatment of patients with autoimmune hemolytic anemia (AIHA) with 80% effectiveness [1]. Without proper steroid therapy, patients with AIHA have a poor prognosis and a 31-53% mortality rate [2]. Since the 1950s, investigators have used steroids to treat AIHA and reported very high effectiveness through several case-series studies [3][4][5]. There are few case-control studies involving AIHA, which show higher evidence than case-series studies.The detection of red blood cell-bound immunoglobulin G (RBC-IgG) and complement by a direct antiglobulin test (DAT) remains the main serologic assay in the diagnosis of AIHA [6]. Several methodologies have been investigated for the detection and evaluation of these autoantibodies. A DAT using the conventional tube technique (CTT) is the method most commonly used in blood centers and is still considered the gold standard [7]. A positive DAT almost always exists in association with AIHA [8] and forms the basis for the serologic diagnosis of AIHA [9]. However, it has also been shown that a negative DAT does not exclude the diagnosis of AIHA [9] and 1-10% of patients with AIHA have been reported to have a negative . These patients may carry a lower number of IgG molecules per RBC, yielding a negative tube DAT and in vivo hemolysis [13]. In fact, Petz and Garratty [14] reported that autoantibodies were detected in the eluates from 11 of 27 patients with acquired hemolytic anemia and a negative DAT, and concentrated elutes reacted with RBC of common Rh phenotypes, but did not react with Rh-null RBC. The immunoradiometric assay (IRMA) [15] for RBC-IgG is a representative method to quantitatively detect RBC-IgG. We previously reported [16] t...

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Cold-induced autoinflammatory syndrome 1 (CIAS1) gene is a member of the NALP subfamily of the CATERPILLER protein family that is expressed predominantly in peripheral blood leukocytes, which is to regulate apoptosis or inflammation through the activation of NF-jB and caspase. Recent genetic analyses suggested an association between inflammation and oxidative stress-related genes in the development of hypertension. This is the first genetic study indicating an association between the CIAS1 gene and susceptibility to essential hypertension (EH). The frequency of subject with the homozygote of 12 repeat allele was significantly higher in patients with hypertension compared with control subjects (987 cases, 924 controls) (P ¼ 0.030; odds ratio ¼ 1.24) at a novel VNTR polymorphism of CIAS1 intron 4 loci. We also found that the mean of systolic blood pressure of homozygotes of 12 repeat allele was 6.4 mmHg higher than those of homozygotes of non-12 repeat allele in male random population (P ¼ 0.009). The frequency of six SNPs spanning of the CIAS1 gene was not significantly between patients and controls. The real-time PCR analysis showed that among healthy young adults, 12-12 subjects expressed CIAS1 mRNA in peripheral leukocytes significantly more abundantly than homozygote of non-12 repeat alleles subjects (Po0.05). Reporter gene assay of the CIAS1-VNTR in HL60 stimulated by lipopolysaccharides showed that the intronic sequence involving 12 repeat increased the expression of luciferase compared with 9, 7, and 6 repeats.

Background: Direct antiglobulin test (DAT)-negative warm autoimmune hemolytic anemia (AIHA) is mainly caused by three mechanisms: red blood cell (RBC)-bound immunoglobulin (Ig)G below the detection limit of routine DAT; RBC-bound IgA or IgM; or low-affinity autoantibodies. Although most cases of DAT-negative AIHA are thought to be caused by RBC-bound IgG, and combinatory serological analyses are recommended, the relative ratios of each mechanism have not been clarified. Methods: Two groups of patients with undiagnosed hemolytic anemia and negative conventional tube method-DAT (TM-DAT) were investigated using anti-IgA and anti-IgM sera, or column agglutination method-DAT (CM-DAT), respectively, in addition to radioimmunological quantitation of RBC-bound IgG. Results: Three of 73 patients with DAT-negative AIHA showed positive RBC-bound IgA and normal amounts of RBC-bound IgG. Another group of 3 patients were RBC-bound IgM-positive, but only one of these showed normal amounts of RBC-bound IgG. In another group of patients with DAT-negative AIHA, 4 of the 20 showed positive CM-DAT and negative CM-DAT after washing RBCs. Three of these patients had normal amounts of RBC-bound IgG. Five patients with positive CM-DAT both before and after washing RBCs had high amounts of RBC-bound IgG. Conclusion: Relative ratios of patients with DAT-negative AIHA resulting from RBC-bound IgG, RBC-bound IgA, RBC-bound IgM, and low-affinity IgG were estimated as 80, 4, 1 and 15%, respectively. A new classification and diagnostic algorithm for DAT-negative AIHA were proposed.