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.

Characteristics of a rapid, point-of-care lateral flow immunoassay for the diagnosis of sickle cell disease.

Sickle cell disease (SCD) is a common and life-threatening hematological disorder, affecting approximately 400,000 newborns annually worldwide. Most SCD births occur in low-resource countries, particularly in sub-Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a prototype immunoassay as a novel, rapid, and low-cost point-of-care (POC) diagnostic device (Sickle SCAN) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3-100%) and specificity (92.5-100%) to detect the presence of HbA, HbS, and HbS were excellent. The test demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC were accurately detected at concentrations as low as 1-2%. Dried blood spot samples yielded clear positive bands, without loss of sensitivity or specificity, and devices stored at 37°C gave reliable results. These analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high-HbF phenotypes, suggests that this POC device is suitable for large-scale screening and potentially for accurate diagnosis of SCD in limited 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.

Bcl-2 regulatory pathway is functional in chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal, malignant CD5(+), CD23(+) B cells. In vivo, these cells have an antiapoptotic phenotype (high levels of Bcl-2 and low levels of proapoptotic Bcl-2 family proteins, such as Bax). Abnormal B cells accumulate due to altered apoptosis regulation rather than to increased proliferation. However, it is unclear whether there are inherent Bcl-2 apoptotic pathway defects. With in vitro culture, these B cells rapidly apoptosis. METHODS: To investigate apoptosis regulation, Bcl-2, Bax, mitochondrial membrane potential, annexin V, and caspase activation were simultaneous monitored in individual cells during in vitro apoptosis. RESULTS: With in vitro culture, 30% to 50% of B cells were apoptotic at 24 h compared with fewer than 10% of T cells. Apoptotic B cells showed dramatic Bax upregulation and slight Bcl-2 decreases accompanied by decreased mitochondrial membrane potential and increased activated caspase-3 protein levels. Caspase-3 and caspase-9 activities were increased 18- to 51-fold and 6- to 11-fold, respectively, after 24 h of culture. Caspase-8 showed limited or no activation (less than fourfold). CONCLUSIONS: These data show that in vitro apoptosis of CLL B cells occurs through a well-characterized Bcl-2 regulatory pathway consistent with that pathway being functional. Further, these cells' antiapoptotic phenotype is dependent on the in vivo environment, potentially involving paracrine/autocrine interactions.

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.

Negative regulation of monocyte adhesion to arterial elastic laminae by signal regulatory protein alpha and Src homology 2 domain-containing protein-tyrosine phosphatase-1.

Elastic laminae are extracellular matrix constituents that not only contribute to the stability and elasticity of arteries but also play a role in regulating arterial morphogenesis and pathogenesis. We demonstrate here that an important function of arterial elastic laminae is to prevent monocyte adhesion, which is mediated by the inhibitory receptor signal regulatory protein (SIRP) alpha and Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1. In a matrix-based arterial reconstruction model in vivo, elastic laminae were resistant to leukocyte adhesion and transmigration compared with the collagen-dominant arterial adventitia. The density of leukocytes within the elastic lamina-dominant media was about 58-70-fold lower than that within the adventitia from 1 to 30 days. An in vitro assay confirmed the inhibitory effect of elastic laminae on monocyte adhesion. The exposure of monocytes to elastic laminae induced activation of SIRP alpha, which in turn activated SHP-1. Elastic lamina degradation peptides extracted from arterial specimens could also activate SIRP alpha and SHP-1. The knockdown of SIRP alpha and SHP-1 by specific small interfering RNA diminished the inhibitory effect of elastic laminae, resulting in a significant increase in monocyte adhesion. These observations suggest that SIRP alpha and SHP-1 potentially mediate the inhibitory effect of elastic laminae on monocyte adhesion.

Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity.

BACKGROUND: Stat5(1) (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. METHODS: Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4',6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies. RESULTS: Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was approximately 70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity. CONCLUSION: We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines.