A rapid, inexpensive and disposable point-of-care blood test for sickle cell disease using novel, highly specific monoclonal antibodies.

Sickle cell disease (SCD) is a significant healthcare burden worldwide, but most affected individuals reside in low-resource areas where access to diagnostic testing may be limited. We developed and validated a rapid, inexpensive, disposable diagnostic test, the HemoTypeSC™ , based on novel monoclonal antibodies (MAbs) that differentiate normal adult haemoglobin (Hb A), sickle haemoglobin (Hb S) and haemoglobin C (Hb C). In competitive enzyme-linked immunosorbent assays, each MAb bound only its target with <0·1% cross-reactivity. With the HemoTypeSC™ test procedure, the sensitivity for each variant was <5·0 g/l. The accuracy of HemoTypeSC™ was evaluated on 100 whole blood samples from individuals with common relevant haemoglobin phenotypes, including normal (Hb AA, N = 20), carrier or trait (Hb AS, N = 22; Hb AC, N = 20), SCD (Hb SS, N = 22; Hb SC, N = 13), and Hb C disease (Hb CC, N = 3). The correct haemoglobin phenotype was identified in 100% of these samples. The accuracy of the test was not affected by Hb F (0-94·8% of total Hb) or Hb A2 (0-5·6% of total Hb). HemoTypeSC™ requires <1 µl of whole blood and no instruments or power sources. The total time-to-result is <20 min. HemoTypeSC™ may be a practical solution for point-of-care testing for SCD and carrier status in low-resource settings.

Hemoglobin variants identified in the Uganda Sickle Surveillance Study.

The Uganda Sickle Surveillance Study analyzed dried blood spots that were collected from almost 100 000 infants and young children from all 10 regions and 112 districts in the Republic of Uganda, with the primary objective of determining the prevalence of sickle cell trait and disease. An overall prevalence of 13.3% sickle cell trait and 0.7% sickle cell disease was recently reported. The isoelectric focusing electrophoresis technique coincidentally revealed numerous hemoglobin (Hb) variants (defined as an electrophoresis band that was not Hb A, Hb F, Hb S, or Hb C) with an overall country-wide prevalence of 0.5%, but with considerable geographic variability, being highest in the northwest regions and districts. To elucidate these Hb variants, the original isoelectric focusing (IEF) gels were reviewed to identify and locate the variant samples; corresponding dried blood spots were retrieved for further testing. Subsequent DNA-based investigation of 5 predominant isoelectric focusing patterns identified 2 α-globin variants (Hb Stanleyville II, Asn78Lys; Hb G-Pest, Asp74Asn), 1 ß-globin variant (Hb O-Arab, Glu121Lys), and 2 fusion globin variants (Hb P-Nilotic, ß31-δ50; Hb Kenya, Aγ81Leu-ß86Ala). Compound heterozygotes containing an Hb variant plus Hb S were also identified, including both Hb S/O-Arab and HbS/Kenya. Regional differences in the types and prevalence of these hemoglobin variants likely reflect tribal ancestries and migration patterns. Algorithms are proposed to characterize these Hb variants, which will be helpful for emerging neonatal hemoglobinopathy screening programs that are under way in sub-Saharan Africa.

Superantigens and chronic rhinosinusitis: skewing of T-cell receptor V beta-distributions in polyp-derived CD4+ and CD8+ T cells.

BACKGROUND: Recent studies have suggested that Staphylococcus aureus secrete superantigenic toxins that play a role in the etiology of chronic rhinosinusitis with nasal polyposis (CRSwNP). Twenty S. aureus superantigens (SAg's) have been identified, each of which bind the V beta-region of the T-cell receptor (TCR) outside the peptide-binding site. Approximately 50 distinct V beta-domains exist in the human repertoire, and distinct SAg's will bind only particular domains generating a pattern of V beta-enrichment in lymphocytes dependent on the binding characteristics of a given toxin. The aim of this study was to analyze the pattern of V beta-expression in polyp-derived lymphocytes from CRSwNP patients. METHODS: Polyps were harvested from 20 patients with CRSwNP and 3 patients with antrochoanal polyps. Flow cytometry was used to analyze the V beta-repertoire of polyp-derived CD4+ and CD8+ lymphocytes. Data were analyzed in light of the known skewing associated with SAg exposure in vivo and in vitro. Skewing was defined as a percentage of V beta-expression >2 SD of that seen in normal blood. RESULTS: Seven of 20 subjects exhibited skewing in V beta-domains with strong associations with S. aureus SAg's. The three antrochoanal polyps failed to show any significant V beta-skewing. CONCLUSION: This study establishes evidence of S. aureus SAg-T-cell interactions in polyp lymphocytes of 35% of CRSwNP patients. Although these results are consistent with intranasal exposure of polyp lymphocytes to SAg's, additional study is necessary to establish the role of these toxins in disease pathogenesis.

Superantigens and chronic rhinosinusitis II: analysis of T-cell receptor V beta domains in nasal polyps.

BACKGROUND: The superantigen (SAg) hypothesis of chronic rhinosinusitis (CRS) suggests that toxins within the nose stimulate massive oligoclonal expansion of T-cell populations with subsequent eosinophil recruitment and tissue inflammation. SAgs are capable of activating 1 x 10(4) more lymphocytes than conventional antigens by binding specific Vbeta-domains of the T-cell receptor (TCR). The net effect is skewing from the normal Vbeta-profile by oligoclonal expansion of specific domains. This study will assess polyp tissue for evidence of an SAg response in CRS with nasal polyps (CRSwNP). METHODS: This study consists of a prospective analysis of 18 CRSwNP subjects undergoing sinus surgery. Flow cytometry was used to analyze the distribution of 24 specific TCR V beta-domains in lymphocytes from polyp tissue and blood. Evidence of oligoclonal expansion was tabulated for each specimen and defined as mean normative percentage + 2 SD. RESULTS: Eighteen of 18 CRSwNP subjects showed expansion of polyp lymphocytes expressing TCRs with specific V beta-domains. The average number of V beta-clones per CRSwNP subject was seven in polyp lymphocytes but only two in blood lymphocytes. CONCLUSION: The current results indicate that polyp lymphocytes exhibit significant oligoclonal expansion of specific V beta-domains. These data are considered diagnostic of a SAg effect. The corresponding blood profile is much less, suggesting that the nose is the primary source of stimulus. Although the trigger(s) for this expansion are unknown, these data are consistent with the hypothesis that staphylococcal toxins are central to the development of CRSwNP.