Journal articles: 'Hemolytic-assay'

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Yamada, S., T. Aiba, A. Hattori, T. Suzuki, S. L. Asa, and K. Kovacs. "Reverse Hemolytic Plaque Assay." Pathology - Research and Practice 187, no. 5 (June 1991): 546–51. http://dx.doi.org/10.1016/s0344-0338(11)80140-8.

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Xiao, Hang, Yu-bin Xu, Xia Liu, and De-qiang Dou. "Hemolytic assay for Huangqi Injection." Chinese Herbal Medicines 8, no. 1 (January 2016): 61–66. http://dx.doi.org/10.1016/s1674-6384(16)60009-6.

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Ekdahl, Kristina N., Dan Norberg, Anders A. Bengtsson, Gunnar Sturfelt, Ulf R. Nilsson, and Bo Nilsson. "Use of Serum or Buffer-Changed EDTA-Plasma in a Rapid, Inexpensive, and Easy-To-Perform Hemolytic Complement Assay for Differential Diagnosis of Systemic Lupus Erythematosus and Monitoring of Patients with the Disease." Clinical and Vaccine Immunology 14, no. 5 (March 7, 2007): 549–55. http://dx.doi.org/10.1128/cvi.00486-06.

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ABSTRACT We previously described a simplified quantitative hemolytic assay for classical pathway (CP) hemolytic function in serum that has been shown to correlate with the 50% hemolytic complement (CH50) assay. In the present study, we used this assay to compare CP functions; plasma levels of C3, C4, and C3dg; and ratios of C3dg to C3 in healthy individuals and patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) with different degrees of complement activation. A significant depression in CP function and levels of C4 and C3 and increased C3dg levels and C3dg/C3 ratios were observed in the SLE patients. In patients with RA, CP function was normal, whereas C3, C4, and C3dg levels and the C3dg/C3 ratio were elevated. The SLE results are compatible with systemic complement consumption, whereas the RA data suggest an acute-phase reaction with a normal C3 catabolic rate. To facilitate the handling of patient samples, we also developed a method to restore the hemolytic function of EDTA-plasma by transferring it to Veronal-buffered saline containing the thrombin inhibitor lepirudin. This process inhibits coagulation and enables complement activation, allowing a longer time lag between sample harvesting and testing. These results, combined with previous correlation studies, suggest that the CP hemolytic assay can effectively replace the CH50 assay for routine SLE differential diagnosis and monitoring of disease activity.

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Tenner, Andrea J., and Michael M. Frank. "A Sensitive Specific Hemolytic Assay for Proenzyme C1." Complement 4, no. 1 (1987): 42–52. http://dx.doi.org/10.1159/000463006.

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Yamada, Shozo. "The reverse hemolytic plaque assay in endocrine pathology." Endocrine Pathology 1, no. 3 (September 1990): 129–31. http://dx.doi.org/10.1007/bf02915389.

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Boyanton, Bobby L., Elizabeth M. Darnell, Anne E. Prada, Dana M. Hansz, and Barbara Robinson-Dunn. "Evaluation of the Lyra Direct Strep Assay To Detect Group A Streptococcus and Group C and G Beta-Hemolytic Streptococcus from Pharyngeal Specimens: TABLE 1." Journal of Clinical Microbiology 54, no. 1 (October 21, 2015): 175–77. http://dx.doi.org/10.1128/jcm.02405-15.

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The Lyra Direct strep assay was compared to culture for its ability to detectStreptococcusgroup A and β-hemolytic groups C/G using rapid antigen-negative pharyngeal specimens (n= 161). The Lyra assay correctly detected all β-hemolytic streptococci (group A,n= 19; group C/G,n= 5). In batch mode, the Lyra assay reduced intralaboratory turnaround time by 60% (18.1 h versus 45.0 h) but increased hands-on time by 96% (3 min 16 s versus 1 min 40 s per specimen).

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Mhatre, A., and W. P. Aston. "A simple hemolytic assay for bovine complement component C3." Veterinary Immunology and Immunopathology 15, no. 3 (June 1987): 239–51. http://dx.doi.org/10.1016/0165-2427(87)90086-9.

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Reis, Kathleen J. "A hemolytic assay for the measurement of equine complement." Veterinary Immunology and Immunopathology 23, no. 1-2 (November 1989): 129–37. http://dx.doi.org/10.1016/0165-2427(89)90115-3.

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Munkvad, S., J. Jespersen, J. Sidelmann, and J. Gram. "Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma." Clinical Chemistry 36, no. 7 (July 1, 1990): 1305–11. http://dx.doi.org/10.1093/clinchem/36.7.1305.

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Abstract We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated C1 by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes of activated C1 with C1-inh, and is not influenced by the concentration of C1-inh in the test sample. Concentrations of C1 correlated significantly with activities determined by a hemolytic assay (r = 0.55, t = 4.09, P less than 0.001). The estimated interassay CV was 5% and the intra-assay CV was 1%. The sensitivity, imprecision, and practical test performance of our assay are superior to those of conventionally used hemolytic assays.

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Naveed, Khalid, Aqeel Javeed, Muhammad Ashraf, Amjad Riaz, Aamir Ghafoor, and Adeel Sattar. "Effect of nabumetone on humoral immune responses in mice." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 72, no. 3 (May 2020): 915–20. http://dx.doi.org/10.1590/1678-4162-11460.

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ABSTRACT Nabumetone is used to reduce the pain and inflammation in rheumatoid arthritis. In the current study, immunomodulatory effect of Nabumetone is investigated in mice. The control group was administered normal saline orally as placebo. Nabumetone was administered orally via gavage in two treatment groups at 14mg/kg.b.w. doses and 28mg/kgb.w., respectively. Haemagglutination (HA) assay, Jerne hemolytic plaque and mice lethality assays were applied. In HA assay, the titer was significantly decreased in Nabumetone treatment groups (P< 0.001). In Jerne hemolytic plaque formation assay, there was a significant reduction (P< 0.001) in number of plaques in Nabumetone treated groups when compared with control. In mice lethality assay, there was a significant difference in mortality ratio of mice in control and Nabumetone treated groups (P< 0.001). Therefore, it is concluded that Nabumetone suppresses the humoral immune response in mice.

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Karış, Mert, Doğancan Şener, Hüsniye Tansel Yalçın, Ayşe Nalbantsoy, and Bayram Göçmen. "Major biological activities and protein profiles of skin secretions of Lissotriton vulgaris and Triturus ivanbureschi." Turkish Journal of Biochemistry 43, no. 6 (March 17, 2018): 605–12. http://dx.doi.org/10.1515/tjb-2017-0306.

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Abstract Objective The aim of this study was to determine the total protein amounts, protein profiles, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin secretions of the Lissotriton vulgaris and Triturus ivanbureschi. Methods Skin secretions were obtained, clarified, supernatants snap-frozen then lyophilized. Total protein amounts were determined by BCA assay kit. Protein profiles were revealed by the SDS-PAGE. The cytotoxicity and antimicrobial activity were determined by using MTT assay and minimum inhibitory concentration (MIC) method. Hemolytic effects were measured on rabbit red blood cells. Results Lissotriton vulgaris and T. ivanbureschi skin secretions have totally 18 and 20 protein fractions. IC50 values were detected between 1.40 and 40.28 μg/mL. The MIC results were found between 7.8 and 250 μg/mL. Lissotriton vulgaris skin secretion showed low hemolytic effect while T. ivanbureschi skin secretion showed high hemolytic effect. Conclusion This study is the first report showing the potential of L. vulgaris and T. ivanbureschi skin secretions for cytotoxicity, antimicrobial and hemolytic activity as an alternative therapeutic approach for traditional uses. Further studies need to focus on purification of the active components from these skin secretions and mode of action on cancer cell lines and microorganisms as anti-agents.

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Deftos, L. J. "Pituitary cells secrete calcitonin in the reverse hemolytic plaque assay." Biochemical and Biophysical Research Communications 146, no. 3 (August 1987): 1350–56. http://dx.doi.org/10.1016/0006-291x(87)90798-4.

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Khatri, Savita, Neetu Phougat, Mehak Dangi, and Anil Kumar Chhillar. "ANTIBACTERIAL, ANTIOXIDANT, CHEMICAL CONSTITUENTS, AND CYTOTOXICITY EVALUATION OF TERMINALIA ARJUNA (ROXB. EX DC.) WIGHT AND ARN." Asian Journal of Pharmaceutical and Clinical Research 10, no. 3 (March 1, 2017): 412. http://dx.doi.org/10.22159/ajpcr.2017.v10i3.16408.

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ABSTRACTObjective: The objective of this study is to evaluate the in vitro antibacterial and antioxidant prospective of Terminalia arjuna (leaves). The most activeextracts were examined for their chemical composition and cytotoxicity.Methods: The antibacterial activity of five different extracts were examined against 8 bacterial strains (5 Gram-positive and 3 Gram-negative) usingresazurin-based microtiter dilution assay (RMDA) and disk-diffusion assay. The antioxidant potential of five extracts was demonstrated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide radical scavenging assay. Chemical composition and cytotoxicity were assessed using gaschromatography-mass spectrometry (GC-MS) and hemolytic assay, respectively.Results: According to RMDA, the acetone extract (AE) exhibited highest antibacterial activity. The AE showed highest activity against Salmonellaenterica ser. typhi and Bacillus cereus with minimum inhibitory concentration, i.e., 195.31 μg/ml. In DPPH assay, AE showed the highest radicalscavenging activity with inhibition concentration50 23.09 μg/ml. In GC-MS analysis, the principal compound in AE was celidoniol (8.72 %). Accordingto the results of hemolytic assay, the AE showed non-toxic behavior upto 500 μg/ml.Conclusion: The present investigation represents T. arjuna as an incredible herb. The AE was found to possess promising antibacterial and antioxidantproperties.Keywords: Antibacterial, Antioxidant, Terminalia arjuna, Chemical composition, Cytotoxicity.

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Majhi, Arnab, Ajeya Nandi, Rana Adhikary, Sayantika Mahanti, and Biswadev Bishayi. "In vitro susceptibility of a penicillin-resistant and tolerable isolate of Streptococcus pneumoniae to combination therapy." Journal of Infection in Developing Countries 9, no. 07 (July 30, 2015): 702–9. http://dx.doi.org/10.3855/jidc.4711.

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Introduction: Preference for combination therapy to treat infection due to multidrug-resistant S. pneumoniae (MDRSP) has not been well elucidated in previous studies. Methodology: In the present study, 19 antibiotics in combinations were tested against an MDRSP isolate. In vitro susceptibility studies including minimum inhibitory concentration (MIC), minimal bactericidal concentrations (MBC) and disk agar diffusion (DAD), tolerance to resistant antibiotics, checkerboard assay, time-kill curve, hemolytic assay, and autolysis assay were performed on the test strain to study its in vitro susceptibility to combination therapy. Results: From the checkerboard assay and time-kill curve, it was observed that a combination of levofloxacin (MIC, 16 µg/mL) and ceftriaxone (MIC, 2 µg/mL), at sub-MIC concentration was synergistic and most effective against the MDRSP isolate (penicillin MIC, > 64 µg/mL). Hemolytic activities also increased significantly with combination therapy compared to monotherapy (p < 0.05). Moreover, the hemolytic activity of levofloxacin in combination with ceftriaxone was better than ciprofloxacin plus ceftriaxone or cefepime. The autolysis rate was also found to increase rapidly within one hour of exposure to levofloxacin plus ceftriaxone, and this was found to be significantly different from the other combinations at the fifth and sixth hour post incubation (p < 0.05). Conclusions: This data suggests that this combination is bactericidal in vitro, and requires further studies in in vivo models for treatment against MDRSP infections.

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Yoshida, Yoko, Xinping Fan, Yoshifumi Ohyama, Tetsuro Kokubo, Masanori Matsumoto, Hideo Yagi, Hiroko Shirotani-Ikejima, Toshiyuki Miyata, and Yoshihiro Fujimura. "Atypical Hemolytic Uremic Syndrome In Japan Characterized By The Inhibitory Antibody-Based Hemolytic Assay and The Gene Analysis." Blood 122, no. 21 (November 15, 2013): 2318. http://dx.doi.org/10.1182/blood.v122.21.2318.2318.

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Abstract Backgrounds and Aims Atypical hemolytic uremic syndrome (aHUS) is a life-threatening generalized disease, featured by a clinical triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Now it is well established that most of aHUS is caused by uncontrolled complement activation due to gene mutations involved in the alternative pathway, which includes C3, factor H (CFH), factor I (CFI), membrane cofactor protein (MCP), thrombomodulin (THBD) and factor B (CFB). Gene mutations in complement factor H- related proteins 1-5 (CFHR1-5) are also included in a category of aHUS. On the other hand, HUS and thrombotic thrombocytopenic purpura (TTP) are both categorized with a common pathological diagnosis, thrombotic microangiopathy (TMA). TTP, however, is now clearly defined by a deficient activity of ADAMTS13 Our laboratory of Nara Medical University has been functioning as a TMA referral center in Japan through analyzing ADAMTS13 since 1998. Through this study we identified 51 patients with hereditary deficiency of ADAMTS13 activity and 63 patients with aHUS with almost normal ADAMTS13 activity. To characterize these aHUS patients, as a first step we prepared 6 murine monoclonal antibodies (mAbs) against CFH, purified from normal plasma. One of anti-CFH mAbs, termed O-72 (IgG1-k), totally inhibited CFH function in the hemolytic assay described below. Epitope analysis of the mAb O-72 using yeast constructs clearly indicated that it resides on short consensus repeat 18 of CFH molecule. Patients and Methods (1) Patients: Of 63 patients with aHUS, 35 patients whose blood specimen were obtained within 3 months were extensively analyzed in this study. (2) Hemolytic assay: Using the mAb O-72, sheep red blood cells, and citrated plasmas, we were able to establish a quantitative hemolytic assay, according to the method of Sanchez-Corral et al (Mol Immunol 2004). The hemolysis obtained in the presence of the mAb O-72 (200 µg IgG/ml, final) was defined as a 100% hemolysis as the control. In this study, we consistently used citrated plasmas as test specimen, which were either freshly prepared or deep-frozen at -80oC within 3 months, and did not use sera. This is because our preliminary experiments clearly indicated that hemolytic activity using freshly prepared plasmas gives a consistent result, but that using sera was not. (3) Anti-CFH autoantibody: This was performed by western blot (WB) analysis using purified plasma derived CFH, (4) The comprehensive gene analyses on complement and complement regulatory factors, such as C3, CFH, CFI, MCP, THBD and CFB: These were performed as previously described [Fan et al. Molecular Immunology 2013], (5) Semi-quantitative WB analysis on antigens of CFHR1 and CFHR3, and (6) MLPA analyses for the exons of CFHR1 and CFHR3. Results and Discussion In the hemolytic assay, 3 unrelated patients with CFH-R1215Q mutation had a strong hemolysis (100% of the control). Interestingly, 4 individuals belonged to these 3 families, but without clinical signs of HUS, also showed an enhanced hemolysis as did the patients. Thus, it is important to note that our hemolytic assay can detect asymptomatic carriers of CFH-R1215Q mutation. To strengthen this observation, 3 patients with positive anti-CFH autoantibody but without CFH gene mutations also had an enhanced hemolysis (50-70% of the control), and one patient indeed had the homozygous deletion of CFHR1 gene. Fifteen patients carried C3 gene mutations, of which 13 patients had the same mutation of C3-I1157T (13/35, 37%). None of these patients, however, showed an enhanced hemolysis in our hemolytic assay. But, the C3-I1157T mutation can be easily detected with PCR followed by restriction fragments length polymorphism (RFLP) assay. Thus, using these combined assays of hemolysis, anti-CFH autoantibody detection, and RFLP for C3-I1157T, we can make a solid diagnosis on approximately 60% of Japanese aHUS patients within 5 days. But, the remaining 40% of the aHUS patients required the comprehensive gene analysis. A summary of the gene mutations in our 35 patients with aHUS is shown in Table 1. As consequence, here we have shown a major cause of aHUS (approximately 37% of the total) in Japan is a missense mutation of C3-I1157T, and a missense mutation of CFH (-R1215Q) is likely to be less (approximately 9% of the total) than those in Western countries-US. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees.

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Albaayit, Shaymaa Fadhel Abbas, Rukesh Maharjan, and Mariam Khan. "Evaluation of Hemolysis Activity of Zerumbone on RBCs and Brine Shrimp Toxicity." Baghdad Science Journal 18, no. 1 (March 10, 2021): 0065. http://dx.doi.org/10.21123/bsj.2021.18.1.0065.

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Zerumbone is a well-known compound having anti-cancer, anti-ulcer, anti-inflammatory and anti-hyperglycemic effects. During its use for the disease treatment, the membrane of erythrocyte can be affected by consumption of this bioactive compound. The current study was the first report of investigation of the hemolytic activities on human erythrocytes and cytotoxic profile of zerumbone. The toxicity of zerumbone on human erythrocytes was determined by in vitro hemolytic assay. Brine shrimp lethality assay was used to evaluate the cytotoxic effect of zerumbone at concentrations 10, 100 and 1000 μg/mL. The human erythrocyte test showed no significant toxicity at low concentrations, whereas hemolytic effect was amplified up to 17.5 % at the highest concentration. The half lethal concentration (LC50) value of zerumbone against brine shrimp was less than 1000 µg /mL (LC50=190 µg/ml) showing the significant toxic nature of this compound. These results provide a baseline in terms of the toxicity of therapeutic formulations from this compound to membrane erythrocytes with a great attention to the highest concentrations, which paves promise for drug development.

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Carey, R. M., K. M. Geary, M. K. Hunt, S. P. Ramos, M. S. Forbes, T. Inagami, M. J. Peach, and D. A. Leong. "Identification of individual renocortical cells that secrete renin." American Journal of Physiology-Renal Physiology 258, no. 3 (March 1, 1990): F649—F659. http://dx.doi.org/10.1152/ajprenal.1990.258.3.f649.

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Successful application of the reverse hemolytic plaque assay was developed to identify individual renocortical cells that secrete renin directly. The plaque assay was validated by a number of established criteria. Using this technique, we demonstrate an increase in renin secretion with beta-adrenergic stimulation and an inhibition of renin secretion with extracellular calcium in groups of renin-secreting cells. Transmission electron microscopy of the cell in the center of a hemolytic plaque demonstrated a modified vascular smooth muscle cell with densely packed secretory granules. Electron microscopy immunocytochemistry demonstrated the presence of renin in the secretory granules, confirming the identity of the cell as a renal juxtaglomerular cell. The technology developed here has allowed the precise identification and study of the individual renin-secreting juxtaglomerular cell.

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Gong, Yanshan, Yongsheng Yang, Yan Chen, Bingqing Sun, Yafei Xue, Xinxin Xu, Xi Wang, Nazrul Islam, Xiaoli Du, and Qinghai Hu. "Characterization of the hemolytic activity of Riemerella anatipestifer." Microbiology 166, no. 5 (May 1, 2020): 436–39. http://dx.doi.org/10.1099/mic.0.000896.

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Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R . anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba− strains created pores in the duck red blood cells, with 4.35–9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+ R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba- R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

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Yoshida, Yoko, Hideki Kato, Madoka Fujisawa, Yuka Sugawara, Yumiko Uchida, Masanori Matsumoto, Yoshihiro Fujimura, Toshiyuki Miyata, and Masaomi Nangaku. "Characterization of the patients with atypical hemolytic uremic syndrome by combination of hemolytic assay and gene analysis in Japan." Immunobiology 221, no. 10 (October 2016): 1175–76. http://dx.doi.org/10.1016/j.imbio.2016.06.115.

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Bendiar, Salma, Othman El Faqer, Naima Benjelloun, Souada Hsseini, Hicham Bellaoui, Samira Rais, Younes Zaid, El Mostafa Mtairag, and Mounia Oudghiri. "Immunomosuppressive Effect of Ziziphus Lotus L. (Desf.) Fruit’s Extract on Neutrophil Bactericidal Activity and on in Vivo Humoral Immune Response in Mice." Biomedical and Pharmacology Journal 15, no. 4 (December 20, 2022): 2331–41. http://dx.doi.org/10.13005/bpj/2572.

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Background: The fruit of Ziziphus Lotus L. (ZL) is rich in bioactive components. It is known for its high content in polyphenols which gives it its immunomodulatory, antioxidant, and antimicrobial properties. Objective: The intent of the current study was to evaluate, in vivo, the effect of the aqueous extract of ZL fruit’s pulp on humoral immune response as well as its effect on neutrophils’ bactericidal activities, hemolytic and antioxidant and activities. Methods: The antioxidant activity of ZL’s aqueous extract’s was evaluated using DPPH. Hemmagglutination titer assay was used to evaluate the effect of the extract on humoral immune response. ZL extract’s hemolytic activity was assessed by enumerating hemoglobin rates. The effect of ZL extract on the bactericidal activity of Neutrophils was evaluated using MTT colorimetric assay. Results / Discussion: A significant (P<0.05) immunosuppressive effect on humoral immunity (6-fold) was observed. Significant suppression (P<0.05) of the bactericidal activity of neutrophils treated with 0.5 and 1 g/ml of the extract was observed compared to untreated neutrophils. The extract exhibited a high antioxidant activity determined by DPPH test with an IC50 value 10-fold higher (P<0.05) than the IC50 of ascorbic acid. The highest hemolytic activity was found with the lowest concentration of the extract while the higher concentrations tested seem to have an anti-hemolytic activity with a dose dependent effect. Conclusion: The aqueous extract of ZL’s fruit pulp possesses an immunosuppressive activity on both the innate and adaptive immunity responses. Our results demonstrate an anti-oxidative activity as well as an ability to decrease neutrophil bactericidal hemolytic activities as well as humoral immune responses.

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Kapil, Arona, Bijoy Kundu, Sanjay K. Khare, and Manisha Shukla. "Synthetic peptide as inhibitors of human complement activation." Protein & Peptide Letters 4, no. 6 (December 1997): 405–8. http://dx.doi.org/10.2174/092986650406221017164542.

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Abstract: Several structurally diverse hexapeptides related to IgE have been evaluated for their ability to inhibit complement activation by using hemolytic assay. Out of the compounds tested, four hexapeptides exhibited total suppression of human complement.

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Robertson, Kirstin P., C. Jeffrey Smith, Andrea M. Gough, and Edson R. Rocha. "Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB." Infection and Immunity 74, no. 4 (April 2006): 2304–16. http://dx.doi.org/10.1128/iai.74.4.2304-2316.2006.

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ABSTRACT This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.

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GONZÁLEZ-RODRÍGUEZ, NIEVES, JESÚS A. SANTOS, ANDRÉS OTERO, and MARÍA-LUISA GARCÍA-LÓPEZ. "Cell-Associated Hemolytic Activity in Environmental Strains of Plesiomonas shigelloides Expressing Cell-Free, Iron-Influenced Extracellular Hemolysin." Journal of Food Protection 70, no. 4 (April 1, 2007): 885–90. http://dx.doi.org/10.4315/0362-028x-70.4.885.

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Hemolysis is a means of providing pathogenic bacteria with heme iron in vivo. In a previous work, iron-influenced hemolytic activity against sheep erythrocytes was detected in cell-free supernatants, but not in the cell fraction of two environmental Plesiomonas shigelloides strains incubated without shaking. Both strains have the hugA gene, which encodes an outer membrane receptor required for heme iron utilization. The present study was undertaken to investigate the expression of a second hemolytic activity detected during aerated incubation in normal and iron-depleted tryptone soya broth (id-TSB). An agar overlay procedure and doubling dilution titrations were employed to detect the hemolytic activity against several erythrocyte species. The kinetics of growth and hemolytic activity were assayed at 35°C in aerated normal and id-TSB and salmon extract. Overlaid colonies showed a cell-associated beta-hemolytic activity within 4 h. For aerated cell-free supernatants, titers above 16 were not attained until 30 to 48 h of incubation; the best activity was noted with dog and mouse erythrocytes. After 24 h of aerated incubation, sonicated cells yielded high hemolytic activity against dog erythrocytes without activity in supernatants, but after 48 h, only 28 to 30% of the total activity remained cell associated. The hemolytic factor was released in broths during the death phase. Hemolytic activity was not detected in fish extract. This and other studies suggest that P. shigelloides may produce at least two hemolytic factors, their expression and detection being influenced by environmental growth conditions and testing procedures. The overlay assay appears to be the best routine method for detecting hemolytic activity in P. shigelloides.

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Diaz, T. M., I. Moscoso, A. Centeno, E. Lopez-Pelaez, D. Ortega, and N. Doménech. "Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay." Transplant Immunology 13, no. 4 (December 2004): 313–17. http://dx.doi.org/10.1016/j.trim.2004.09.001.

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Ayyar, Manikandan, Mohan Prasath Mani, Saravana Kumar Jaganathan, and Rajasekar Rathanasamy. "Preparation, characterization and blood compatibility assessment of a novel electrospun nanocomposite comprising polyurethane and ayurvedic-indhulekha oil for tissue engineering applications." Biomedical Engineering / Biomedizinische Technik 63, no. 3 (June 27, 2018): 245–53. http://dx.doi.org/10.1515/bmt-2017-0022.

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AbstractElectrospun polyurethane based nanocomposite scaffolds were fabricated by mixing with indhulekha oil. Scanning electron microscope (SEM) portrayed the nanofibrous nature of the composite and the average diameters of the composite scaffold were smaller than the pristine scaffolds. The fabricated scaffold was found to be hydrophobic (114°) due to the inclusion of indhulekha oil, which was displayed in contact angle measurement analysis. The fourier transform infrared spectroscopy (FTIR) results indicated that the indhulekha oil was dispersed in PU matrix identified by formation of hydrogen bond and peak shifting of CH group. The PU/indhulekha oil nanocomposite exhibits a higher decomposition onset temperature and also residual weight percentage at 900°C was more compared to the pure PU. Surface roughness was found to be increased in the composite compared to the pristine PU as indicated by the atomic force microscopy (AFM) analysis. In order to investigate the blood compatibility of electrospun nanocomposites the activated partial thromboplastin time (APTT) assay, prothrombin time (PT) assay and hemolytic assay were performed. The blood compatibility results APTT and PT revealed that the developed nanocomposites demonstrated delayed clotting time indicating the anticoagulant nature of the composite in comparison with the pristine PU. Further, it was also observed that the hemolytic index of nanocomposites was reduced compared to pure PU suggesting the non-hemolytic nature of the fabricated scaffold. Hence, the fabricated nanocomposites might be considered as a potent substitute for scaffolding damaged tissue due to their inherent physicochemical and blood compatibility properties.

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Bosco, D., M. Chanson, R. Bruzzone, and P. Meda. "Visualization of amylase secretion from individual pancreatic acini." American Journal of Physiology-Gastrointestinal and Liver Physiology 254, no. 5 (May 1, 1988): G664—G670. http://dx.doi.org/10.1152/ajpgi.1988.254.5.g664.

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To assess the secretion of individual rat pancreatic acini, we developed a reverse hemolytic plaque assay that allows for a direct visualization of amylase release. This release was detected around secreting cells by the presence of hemolytic plaques that resulted from the complement-mediated lysis of red blood cells bearing amylase-antiamylase complexes bound to protein A. Controls showed that these plaques reflected specifically the active secretion of amylase. Quantitation of hemolytic plaques showed that after a 30-min incubation approximately 50% of the acini secreted under basal conditions. Stimulation of amylase release by increasing concentrations of carbamylcholine resulted in a dose-dependent recruitment of secreting acini as well as in a time-dependent enhancement in the response of individual acini. Under all conditions, the wide distribution of hemolytic plaque sizes indicated large differences in the secretory output of individual acini. Thus, using a new method to directly visualize and quantitate amylase secretion, we have provided evidence for a functional heterogeneity of pancreatic acini.

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Abdulwahhab, Aqeel M., and Khawlah J. Khalaf. "Effect of cultivation conditions on hemolysin production from clinical isolates of Serratia marcescens." Al-Mustansiriyah Journal of Science 33, no. 1 (March 10, 2022): 6–14. http://dx.doi.org/10.23851/mjs.v33i1.1080.

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Sixty-four isolates were collected from clinical sources in Baghdad and Mosul city. The identification of S. marcescens confirmed by using API 20E and VITEK-2 systems. Twelve isolates of S. marcescens produced hemolysin, which were detected by two ways, qualitative hemolytic assay and quantitative hemolytic assay. The optical density of hemolysin producing isolates at standard condition (37oC, pH=7, 24h) showed that Serratia marcescens (SmU9) isolate gave the highest absorbance of hemolysin production (0.7) but at (25°C, pH=7,24h) the absorbance of hemolysin production turn into (3.0), compared with the absorbance at 571nm for Control without hemolysin (0.060) and completely hemolysis by Triton x-100 (6.0). However, the optimum optical density of the best hemolysin producing isolate (SmU9) at the optimum cultivation conditions (25°C, pH=9, 24h, 150 rpm, 1% inoculum size in Nutrient broth) was (6.0).

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Lindbäck, Toril, Indira Secic, and Liv Marit Rørvik. "A Contingency Locus inprfAin a Listeria monocytogenes Subgroup Allows Reactivation of the PrfA Virulence Regulator during Infection in Mice." Applied and Environmental Microbiology 77, no. 10 (April 1, 2011): 3478–83. http://dx.doi.org/10.1128/aem.02708-10.

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ABSTRACTA nonhemolyticListeria monocytogenesstrain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 109CFU were intraperitoneally injected into mice. HemolyticL. monocytogenesbacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing ofprfAfrom the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation ofprfAand that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. NonhemolyticL. monocytogenesstrains with identical duplications inprfAwere isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.

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Jaskowski, Troy D., Thomas B. Martins, Christine M. Litwin, and Harry R. Hill. "Comparison of Three Different Methods for Measuring Classical Pathway Complement Activity." Clinical Diagnostic Laboratory Immunology 6, no. 1 (January 1, 1999): 137–39. http://dx.doi.org/10.1128/cdli.6.1.137-139.1999.

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ABSTRACT The complement system plays an important role in host defense against infection and in most inflammatory processes. The standard 50% hemolytic complement (CH50) assay is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. Our objective in this study was to compare two newer methods (the enzyme immunoassay and the liposome immunoassay) to a commercial CH50 assay for measuring total classical complement activity. We conclude that both newer methods compare well with a CH50 assay and are equally sensitive in screening routine clinical sera.

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Wu, Tung-Kung, Yu-Kuo Wang, Yi-Chin Chen, Jen-Min Feng, Yen-Hsi Liu, and Ting-Yi Wang. "Identification of a Vibrio furnissii Oligopeptide Permease and Characterization of Its In Vitro Hemolytic Activity." Journal of Bacteriology 189, no. 22 (September 14, 2007): 8215–23. http://dx.doi.org/10.1128/jb.01039-07.

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ABSTRACT We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.

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Ahmed, Najwa Sh, Amal G. S. Fayyad, Wan Mohter Yusoff, Subhi Jawad Hamza, Abdul Hussen Al-Faisal, and Adeeb A. AL-Shami. "Detection of Perforin in suspension of leukemia lymphocyte culture." Journal of Biotechnology Research Center 8, no. 1 (January 1, 2014): 20–24. http://dx.doi.org/10.24126/jobrc.2014.8.1.293.

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This work aimed to study the concentrations and hemolytic assay of perforin in serum and culture suspension of lymphocyte for acute lymphocyte leukemia ALL; chronic lymphocyte leukemia CLL, and control. To achieve this goal, blood samples were collected from 20 leukemic cases 10 ALL and 10 CLL, and isolation of lymphocyte done then proliferation of leukemia lymphocyte in culture for the detection of perforin concentration by ELISA kit and hemolytic activity of perforin in suspension lymphocyte culture and serum were impacted. The results, showed significant increase in the level of concentrations of perforin in both CLL and ALL as compared with the control, however, showed significant lower in level of hemolytic activity of perforin in these groups as compared to the control. The conclusion is that expression of perforin is strongly associated with leukemia patient in Iraqi population.

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Beyer, C., L. W. Statius van Eps, J. J. Kastelein, D. P. Brandjes, W. M. Mairuhu, and A. van den Ende. "Urinary creatine: biochemical indicator for evaluation of sickle cell crises." Clinical Chemistry 31, no. 7 (July 1, 1985): 1232–34. http://dx.doi.org/10.1093/clinchem/31.7.1232.

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Abstract In a patient with known sickle cell beta 0-thalassemia we measured serum lactate dehydrogenase (LD) activity and 24-h urinary creatine excretion rate as markers to evaluate sickle cell crises. We believe that a distinction based on biochemical findings can be made between hemolytic and painful vaso-occlusive sickle cell crises with muscular involvement. To assess hemolytic crises by objective biochemical measures, we have used assay of LD activity, and to assess painful crises with muscular involvement objectively, the 24-h urinary creatine excretion rate. We conclude that hemolytic crises are characterized by high serum LD activities. Furthermore, we conclude that--at least in this patient--painful crises are accompanied by high 24-h urinary creatine excretion rates. Our findings suggest that muscle involvement may play an important role in painful vaso-occlusive sickle cell crises.

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Nofiani, Risa, Rio Kurniadi, and Puji Ardiningsih. "Antimicrobial, Antioxidant, Hemolytic Activities and Toxicity of Ethyl Acetate Extract From an Unidentified Coral-Associated Fungus, Aspergillus brevipes RK06." Indonesian Journal of Cancer Chemoprevention 2, no. 2 (June 28, 2011): 212. http://dx.doi.org/10.14499/indonesianjcanchemoprev2iss2pp212-216.

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Marine fungi are one of the potential and prolific sources to produce unique and novel structure of bioactive compounds. The aim of this research was to explore the biological activities potency from ethyl acetate extract of Aspergillus brevipes RK06. A. brevipes RK06 was successfully isolated from an unidentified coral from Randayan Island, Kalimantan Barat. The extract inhibited the oxidation of linoleic acid (Ferric thiocyanate assay) with a lipid peroxidation inhibition value of 28.44%. The IC50 value of the extract for brine shrimp lethality test was 34.19ug/mL. The hemolytic percentage of the extract for hemolysis on cow erythrocytes was 5.21%. The extract showed a growth inhibition against Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus. Based on the assays, the extract showed a potential citotoxity and both low antioxidant and hemolytic activities.Keywords: antioxidant, hemolytic activity, antimicrobial, toxicity, Aspergillus brevipes RK06

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Cairns, E., J. St Germain, and D. A. Bell. "The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients." Journal of Immunology 135, no. 6 (December 1, 1985): 3839–44. http://dx.doi.org/10.4049/jimmunol.135.6.3839.

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Abstract Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.

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35

Luna-Vázquez-Gómez, Roberto, María Evarista Arellano-García, Juan Carlos García-Ramos, Patricia Radilla-Chávez, David Sergio Salas-Vargas, Francisco Casillas-Figueroa, Balam Ruiz-Ruiz, Nina Bogdanchikova, and Alexey Pestryakov. "Hemolysis of Human Erythrocytes by Argovit™ AgNPs from Healthy and Diabetic Donors: An In Vitro Study." Materials 14, no. 11 (May 24, 2021): 2792. http://dx.doi.org/10.3390/ma14112792.

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The use of nanomaterials is becoming increasingly widespread, leading to substantial research focused on nanomedicine. Nevertheless, the lack of complete toxicity profiles limits nanomaterials’ uses, despite their remarkable diagnostic and therapeutic results on in vitro and in vivo models. Silver nanoparticles (AgNPs), particularly Argovit™, have shown microbicidal, virucidal, and antitumoral effects. Among the first-line toxicity tests is the hemolysis assay. Here, the hemolytic effect of Argovit™ AgNPs on erythrocytes from one healthy donor (HDE) and one diabetic donor (DDE) is evaluated by the hemolysis assay against AgNO3. The results showed that Argovit™, in concentrations ≤24 µg/mL of metallic silver, did not show a hemolytic effect on the HDE or DDE. On the contrary, AgNO3 at the same concentration of silver ions produces more than 10% hemolysis in both the erythrocyte types. In all the experimental conditions assessed, the DDE was shown to be more prone to hemolysis than the HDE elicited by Ag+ ions or AgNPs, but much more evident with Ag+ ions. The results show that Argovit™ is the least hemolytic compared with the other twenty-two AgNP formulations previously reported, probably due to the polymer mass used to stabilize the Argovit™ formulation. The results obtained provide relevant information that contributes to obtaining a comprehensive toxicological profile to design safe and effective AgNP formulations.

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Amrutha, Peneremmal, Balakrishnan Sathya Priya, Shanmugaasokan Lakshmanasenthil, Antonythiraviam Anne Jenifer, Laxmi Satheesh Pillai, Gunasekaran Suja, and Thirumalairaj Vinothkumar. "Pyrethrin from Tanacetum cineriifoliun as repellent against mosquitoes." International Current Pharmaceutical Journal 2, no. 10 (September 14, 2013): 170–76. http://dx.doi.org/10.3329/icpj.v2i10.16411.

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The present study is conducted to find the efficacy of plant extracts to act as mosquito repellents. Initially, the larvicidal activity of the extracts and the toxicity study was conducted. From the results, the extracts with maximum larvicidal activity and the minimum toxicity were used for the further analysis. The least toxicity was observed with the ethanol extracts of Lantana camara, Tagetes erecta and Tanacetum cineriifolium. The Lanatana camara and Tanacetum cineriifolium showed the maximum larvicidal activity. The DNA sugar damage assay is performed and read at 532 nm. The hemolytic activity of the extract is conducted and the results indicate that the extracts showed no hemolytic activity at the concentration of 100 and 200mg/L. Only the flower extracts of Tagaetes erecta, Lantana camara and Tanacetum cineriifolium showed moderate hemolytic activity at 500 and 1000 mg/L. Finally, the HPLC analysis of the extracts showed that the ethanol extracts of Lantana camara and Tanacetum cineriifloium revealed the presence of marker compound. Therefore the ethanol extracts of Lantana camara and Tanacetum cineriifolium were taken up for the membrane stabilization assay. The skin irritation potential of the cream prepared using the ethanol extract of Tanacetum cineriifolium was analysed, the cream showed no irritation.DOI: http://dx.doi.org/10.3329/icpj.v2i10.16411 International Current Pharmaceutical Journal, September 2013, 2(10): 170-176

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Mouffouk, Chaima, Leila Hambaba, Hamada Haba, Soumia Mouffouk, and Chawki Bensouici. "Evaluation of Cytotoxic Effect, Anti-Cholinesterase, Hemolytic and Antibacterial activities of the Species Scabiosa stellata L." Current Bioactive Compounds 16, no. 1 (February 20, 2020): 72–79. http://dx.doi.org/10.2174/1573407214666180730102338.

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Objective: In this study, cytotoxic effect, anticholinesterase, hemolytic and antibacterial activities of crude extracts (petroleum ether, ethyl acetate and n-butanol) obtained from the plant Scabiosa stellata L. were evaluated. Methods: The cytotoxicity of extracts was tested by Brine shrimp lethality method; the acetylcholinesterase inhibitory activity was performed using Ellman's colorimetric method and the hemolytic activity was assessed by spectrophotometric method towards human erythrocytes. Furthermore, the antibacterial activity was estimated by agar disk diffusion assay against ten bacterial strains. Results: The phytochemical screening of the extracts revealed the presence of several types of secondary metabolites. A significant cytotoxic effect was observed for the n-butanolic extract with 57.2 ± 0.2 % of mortality at 80 μg/mL, the ethyl acetate extract had a moderate anticholinesterase activity at 200 μg/mL. The hemolytic assay exhibited that n-butanolic and ethyl acetate extracts induce hemolysis in dose-dependent manner with values of EC50 at 37.3 ± 0.5 and 106.6 ± 0.3 μg/mL, respectively. All the crude extracts showed antibacterial activity against most tested strains, with zones of inhibition ranging from 9 to 20 mm. Conclusion: The results indicate that the extracts obtained from S. stellata can be an important source of therapeutic agents against pathological damage due to free radicals inducing neurodegenerative and infectious diseases, while n-butanolic extract could be used as a good source of alternative natural antiproliferative compounds.

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Levina, Eleonora V., Anatoly I. Kalinovsky, Pavel S. Dmitrenok, Ekaterina A. Martyyas, and Valentin A. Stonik. "Two New Steroidal Saponins, Hylodoside A and Novaeguinoside Y, from the Starfish Leptasterias Hylodes Reticulata and Culcita Novaeguineae (Juvenile)." Natural Product Communications 5, no. 11 (November 2010): 1934578X1000501. http://dx.doi.org/10.1177/1934578x1000501107.

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New steroid glycosides, hylodoside A (1) and novaeguinoside Y (2), along with previously known polyhydroxylated steroids (3-7) were isolated from the ethanolic extracts of the starfish Leptasterias hylodes reticulata and Culcita novaeguineae (juvenile). The structures have been elucidated using 1D and 2D NMR spectra (1H, 13C, DEPT, COSY-45, 1D-TOCSY, HSQCGP, ROESY, HSQC and HMBCGP) and mass spectrometry. Compound 3 was shown to inhibit the growth of Staphylococcus aureus up to 10% from the control at a concentration of 1 mg/mL. Steroids 1, 2 and 5 showed moderate hemolytic activity in the mouse erythrocytes assay. Compounds 3, 4 and 6 displayed pH-depended hemolytic properties.

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YAN, TINGTING, LILI TAN, DANGSHENG XIONG, BINGCHUN ZHANG, and KE YANG. "A MANGANESE OXIDE CONTAINED COATING FOR BIODEGRADABLE AZ31B MAGNESIUM ALLOY." Surface Review and Letters 16, no. 04 (August 2009): 533–38. http://dx.doi.org/10.1142/s0218625x09012937.

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A manganese oxide contained coating was prepared on biodegradable AZ31B magnesium alloy to control the degradation of AZ31B and improve its biocompatibility. Morphology, composition, and corrosion resistance of the coating were studied. The SEM observations showed that the coating was approximately 4–6 μm in thickness with net-like microcracks. The XPS analysis indicated that the coating was mainly composed of MgO , Mg ( OH )2, MnO 2, Mn 2 O 3, and Mn 3 O 4. It was found that AZ31B with such coating showed better corrosion resistance in simulated blood plasma through electrochemical and immersion tests. The hemolytic assay indicated that the treated AZ31B had no hemolytic effect.

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Nussenzveig, Roberto H., Nikhil Sangle, Robert D. Christensen, Mohamed E. Salama, Josef Prchal, Hassan Yaish, and Archana M. Agarwal. "The Clinical Utility Of Next-Generation Sequencing In The Diagnosis Of Hereditary Hemolytic Anemias." Blood 122, no. 21 (November 15, 2013): 3421. http://dx.doi.org/10.1182/blood.v122.21.3421.3421.

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Abstract Hereditary hemolytic anemia encompasses a diverse group of genetically and phenotypically heterogeneous disorders that are characterized by increased red cell destruction, with consequences ranging from relatively harmless to severe life-threatening anemia. Moreover, red cell hemolysis leads to increased production of bilirubin, a breakdown product of hemoglobin, which in neonates places them at risk for extreme jaundice and its consequences. Two of the more common genetic causes of hereditary hemolytic anemia, excluding hemoglobinopathies, can be attributed to defects in either the red cell cytoskeleton or enzyme deficiency (e.g. G6PD, PKLR). Morphological and biochemical diagnosis of hereditary hemolytic anemia due to defects in RBC cytoskeleton or enzyme deficiency is routinely performed in many laboratories. However, routine studies can be challenging, particularly in transfusion-dependent infants and children since these patients have mostly transfused RBCs. Molecular diagnosis has also been challenging not only due to molecular heterogeneity but also due to the number and size of the genes involved. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of the molecular causes of the two major types of hereditary hemolytic anemia described above. Our diagnostic panel includes 25 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA). One hundred base-pair paired-end sequencing was done on a HiSeq 2000 system (Illumina, San Diego, CA). Bioinformatic analysis was based on an “in house” pipeline using standard open-source software. A total of 19 patients with unexplained hemolytic anemia, and 30 normal controls were tested in our assay. Mutations in the appropriate genes were identified in 17/19 patients, many of these being novel. All identified mutations were confirmed by Sanger sequencing. In silico prediction of the impact resulting from the novel mutations was performed using two web-based software packages, Sift and Polyphen. Where possible, inheritance of pathogenic mutations was determined in immediate relatives. One of the cases we investigated involved a neonate with unexplained jaundice and subsequent, significantly compensated, anemia without family history of a hemolytic disorder. Routine studies were suggestive for hereditary spherocytosis due to the presence of microspherocytes on the proband’s blood film, increased osmotic fragility, and decreased eosin-5-maleimide stained red cells. Two pathogenic mutations, in compound heterozygosity, were identified in the SPTA1 gene (α-spectrin). A previously reported mutation αLEPRA, known to be associated with recessive spectrin-deficient HS, and a novel mutation in intron 45 +1 (c.6530+1G>A) disrupting the consensus splice site. Screening of other relevant genes failed to reveal additional mutations. Studies of his parents revealed both to be heterozygous carriers with the asymptomatic mother harboring the αLEPRA mutation and the asymptomatic father harboring the novel mutation. Our results demonstrate the clinical utility of this assay for molecular diagnosis and genetic counseling for parents at risk of having affected children. Next-generation sequencing provides a cost-effective and rapid approach to molecular diagnosis, especially in cases where traditional testing has failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in several cases with no family history. Furthermore, we have validated its clinical utility in neonates risk for hyperbillirubinemia, as well as, in patients with transfusion dependent hemolytic anemia. Disclosures: No relevant conflicts of interest to declare.

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Ho, K. Y., D. A. Leong, Y. N. Sinha, M. L. Johnson, W. S. Evans, and M. O. Thorner. "Sex-related differences in GH secretion in rat using reverse hemolytic plaque assay." American Journal of Physiology-Endocrinology and Metabolism 250, no. 6 (June 1, 1986): E650—E654. http://dx.doi.org/10.1152/ajpendo.1986.250.6.e650.

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It is not known whether enhanced growth hormone (GH)-releasing factor (GRF)-stimulated GH release observed in the male reflects differences in somatotrope numbers and/or secretory response to GRF. We addressed this question by using the hemolytic plaque assay which allows quantification of hormone secretion by single pituitary cells. Time-course studies and GRF-GH concentration-response relationships (0.01, 0.1, 1, 10, 100, 1,000 nM GRF) in age-matched male and diestrous day 2 female rats were compared by quantitating the percent of GH plaque-forming cells, and measuring the plaque areas. The male pituitary contained a greater percent (P less than 0.05) of somatotropes (% of plaque-forming cells 45 +/- 2 vs. 27 +/- 4% in the female; mean +/- SE). GRF induced a greater concentration-dependent increase in plaque areas in the male. Maximal responses were attained at 10 nM GRF in both sexes. However, mean maximal plaque area was significantly greater (P less than 0.001) and the EC50 was significantly lower (P less than 0.05) in the male (0.25 +/- 0.09 vs. 1.78 +/- 0.64 nM in the female). The data suggest that the greater percent of somatotropes in the male and greater secretory capacity and sensitivity to GRF may contribute to sex-related differences in GH secretion in the rat.

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Conte, Roberto, Cristina Tassi, Andrea Bontadini, Daniela Belletti, and Pier Luigi Lazzari. "T Lymphocyte Colony Assay in Patients with Idiopathic Autoimmune Hemolytic Anemia: Preliminary Results." Vox Sanguinis 60, no. 3 (1991): 189–90. http://dx.doi.org/10.1159/000461282.

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Brovedani, Valentina, Silvio Sosa, Mark Poli, Martino Forino, Katia Varello, Aurelia Tubaro, and Marco Pelin. "A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels." Toxicon 119 (September 2016): 225–33. http://dx.doi.org/10.1016/j.toxicon.2016.06.013.

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Conte, Roberto, Cristina Tassi, Andrea Bontadini, Daniela Belletti, and Pier Luigi Tazzari. "T Lymphocyte Colony Assay in Patients with Idiopathic Autoimmune Hemolytic Anemia: Preliminary Results." Vox Sanguinis 60, no. 3 (April 1991): 189–90. http://dx.doi.org/10.1111/j.1423-0410.1991.tb00902.x.

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Novaretti, Marcia Cristina Zago, Eduardo Jens, Thiago Pagliarini, Silvia Leão Bonifácio, Pedro Enrique Dorlhiac-Llacer, and Dalton de Alencar Fischer Chamone. "Hemolytic disease of the newborn due to anti-U." Revista do Hospital das Clínicas 58, no. 6 (2003): 320–23. http://dx.doi.org/10.1590/s0041-87812003000600006.

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Anti-U is a rare red blood cell alloantibody that has been found exclusively in blacks. It can cause hemolytic disease of the newborn and hemolytic transfusion reactions. We describe the case of a female newborn presenting a strongly positive direct antiglobulin test due to an IgG antibody in cord blood. Anti-U was recovered from cord blood using acid eluate technique. Her mother presented positive screening of antibodies with anti-U identified at delivery. It was of IgG1 and IgG3 subclasses and showed a titer of 32. Monocyte monolayer assay showed moderate interaction of Fc receptors with maternal serum with a positive result (3.1%). The newborn was treated only with 48 hours of phototherapy for mild hemolytic disease. She recovered well and was discharged on the 4th day of life. We conclude that whenever an antibody against a high frequency erythrocyte antigen is identified in brown and black pregnant women, anti-U must be investigated.

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Takahashi, Sumio. "Growth hormone secretion in old female rats analyzed by the reverse hemolytic plaque assay." Acta Endocrinologica 127, no. 6 (December 1992): 531–35. http://dx.doi.org/10.1530/acta.0.1270531.

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Growth hormone (GH) release was studied in young (3–4-month-old) and old persistent diestrous (20– 21-month-old) female rats using the reverse hemolytic plaque assay. A bimodal distribution of reverse hemolytic plaque area was observed in both young and old female rats. The mean and median of the plaque area of GH cells from old females were smaller than those from young female rats. The percentage of plaque-forming GH cells in old female rats was lower than in young female rats. The percentage of large plaque-forming GH cells (plaque area, more than 8 × 103 μm2) was lower in old female rats than in young female rats. GH-releasing hormone (GHRH) increased the mean and median of plaque areas in both young and old female rats. However, responsiveness to GHRH was reduced in old female rats. These results indicate that the amount of GH released from individual GH cells decreases with age in female rats, resulting in diminished GH secretion.

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Vibhuti, Raj Kamal, and Avijit Pramanik. "Investigations of hemolytic activity and iron utilization sources of Vibrio alginolyticus ATCC 17749." Research Journal of Biotechnology 18, no. 1 (December 15, 2022): 100–106. http://dx.doi.org/10.25303/1801rjbt1000106.

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As iron plays an indispensable role in the various physiological process with the exception of Borrelia burgdorferi, it is a vital but constrained micronutrient. It is anticipated that the capability of this possible human pathogenic organism to use iron will be crucial for both the establishment of an infection in its hosts and for survival in the habitat. The current study aimed to determine the iron utilization spectrum and hemolytic activity of Vibrio alginolyticus ATCC 17749. The ability of V. alginolyticus ATCC 17749 to utilize iron-containing compounds was tested by agar plate growth promotion assay. It was also confirmed by liquid growth promotion assay where iron was restricted by 400 μM 2,2’-Bipyridyl. After that various iron sources of concentrations ranging from 0.1 μM to 10 μM were added for growth promotion of V. alginolyticus. Ferrous sulfate, ferrioxamine and hemin stimulated the growth of V. alginolyticus as a sole iron source while catechol and ferric dicitrate did not stimulate growth. Vibrio alginolyticus supernatant was found to be hemolytic and on the blood agar plate, V. alginolyticus ATCC 17749 showed a hemolytic zone. According to our research, hemolysin synthesis is strongly influenced by the iron concentration of the growth medium. Hemin is used as the only source of iron and hemolysis might represent virulence traits of Vibrio alginolyticus. Iron utilization systems might be a potential target for antibiotics or a controlling point for hemolysin expression.

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48

Begtrup, Amber Hogart, Neha Dagaonkar, Suvarnamala Pushkaran, Katie M. Giger, Ammar Husami, Diane Kissell, Clinton H. Joiner, Mehdi Keddache, Kejian Zhang, and Theodosia A. Kalfa. "Development Of a Comprehensive Rapid Next-Generation Sequencing Assay For The Diagnosis Of Inherited Hemolytic Anemia." Blood 122, no. 21 (November 15, 2013): 949. http://dx.doi.org/10.1182/blood.v122.21.949.949.

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Abstract Hereditary hemolytic anemia is caused by defects in hemoglobin, in the red blood cell (RBC) cytoskeleton proteins, or by deficiencies in RBC enzymes. RBC cytoskeletal disorders include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt), which are typically inherited as autosomal dominant disorders but can also present as recessive forms, frequently severe. The RBC enzymopathies, such as glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiency, are X-linked or recessively inherited disorders causing hemolytic anemia. Although rare, congenital dyserythropoietic anemias (CDA) are inherited red cell lineage disorders which occasionally, especially CDA-II, can be misdiagnosed as HS. Diagnosis of an inherited hemolytic anemia not due to a hemoglobin disorder is based upon morphology of the RBCs, functional analysis of the RBCs through ektacytometry or osmotic fragility, and enzymatic assays. The diagnosis of severely affected patients is complicated by transfusion dependence. Diagnosis based on genetic mutations is attractive, especially in transfused patients, and provides additional insight into the mechanisms of disease. Despite these advantages, genetic diagnoses have been limited by expense and long turn-around times for clinical results. To address these issues, we have developed a rapid comprehensive clinical next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. The protein-coding exons plus 25 bases of exon/intron junction as well as promoter sequences with known relationship to clinical phenotypes were included in the design. Genomic DNA was digested with a panel of 8 restriction enzymes and oligonucleotide probes were used to enrich the target regions. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Enrichment and sequencing were completed within 48 hours. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. Initial validation included 5 affected probands, 1 affected sibling of a proband, 4 parental samples, and 2 unrelated control individuals with no history of hemolytic anemia. Overall, > 99% of all nucleotides in the regions of interest had at least 20X sequencing coverage. Our assay confirmed a previously identified maternally inherited nonsense mutation, Y1089X, and a paternally inherited A970D missense variant in SPTA1 in a patient (SPCA) with severe HS. The A970D SPTA1 missense change was also seen in two unaffected parents and an unaffected control, further validating that this missense variant alone does not cause dominant HS. The common SPTA1 allele, αLELY was seen in the clinically unaffected mother of SPCA, but was not found in the affected child, suggesting that this allele must be inherited in trans with a pathologic SPTA1 mutation to have clinical effect. Analysis of additional probands revealed both dominant and recessive forms of HS due to mutations in ANK1. Patient AAHS2 presented with typical dominant HS, and was found to have a novel ANK1 frameshift mutation, c.3464delG. Patient HEEM presented with severe transfusion-dependent anemia and was found to have one reported ANK1 missense mutation, T1075I (ankyrin Tubarao) in the spectrin-binding domain, and one novel nonsense mutation, Y735X. In addition, we identified novel amino acid changes in the newly identified dehydrated HSt gene PIEZO1. HEGR, a patient with hemolytic anemia, splenomegaly and portal vein thrombosis in infancy was found to have a novel 6 base pair insertion at R1462 in PIEZO1. Thal1 was previously diagnosed with dominant β-thalassemia, however had a more severe presentation than expected. We found a PIEZO1missense mutation R2302H in Thal1 that likely explains the severe phenotype. The initial validation of this comprehensive sequencing assay has demonstrated that this is a robust and rapid diagnostic tool for patients with severe hereditary hemolytic anemia. Simultaneous investigation of the key protein-coding genes involved in proper RBC function and survival, provides new insight into the variable phenotypes of patients with hemolytic anemia and ultimately will improve the management of patients with severe disease. Disclosures: No relevant conflicts of interest to declare.

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49

Wang, Y., T. A. McAllister, P. R. Cheeke, and K. J. Cheng. "Assessment of inhibitory effects of ruminal fluid on biological activity of steroidal saponins using hemolytic assay." Canadian Journal of Animal Science 79, no. 4 (December 1, 1999): 561–64. http://dx.doi.org/10.4141/a98-051.

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In vitro studies demonstrated that cell-free ruminal fluid inhibits the saponin-based hemolytic activity of yucca extract, and that the inhibitory factor isolatable with the protein fraction of ruminal fluid is not heat labile. Hemolysis assay is not reliable for assessing saponin activity in ruminant nutrition applications. Key words: Yucca extract, ruminal fluid, hemolysis, saponins

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50

Alhurayri, Fatimah, Edith Porter, Rachid Douglas-Louis, Emi Minejima, Juliane Bubeck Wardenburg, and Annie Wong-Beringer. "Increased Risk of Thrombocytopenia and Death in Patients with Bacteremia Caused by High Alpha Toxin-Producing Methicillin-Resistant Staphylococcus aureus." Toxins 13, no. 10 (October 14, 2021): 726. http://dx.doi.org/10.3390/toxins13100726.

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Alpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited. We examined the link between in vitro Hla activity and outcome. Study isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production by Western immunoblotting in a subset of isolates and Hla activity by hemolysis assay in all isolates. Relevant demographics, laboratory and clinical data were extracted from patients’ medical records to correlate Hla activity of the infecting isolates with outcome. Hla production strongly correlated with hemolytic activity (rs = 0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs. 16.55 HU/mL in normal platelet count, p = 0.012) and from non survivors (median 30.96 vs. 14.87 HU/mL in survivors, p = 0.014) but hemolytic activity of MSSA strains did not differ between patient groups. In vitro Hla activity of MRSA strains obtained from patients with bacteremia is significantly associated with increased risk for thrombocytopenia and death which supports future studies to evaluate feasibility of bedside phenotyping and therapeutic targeting.

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Journal articles on the topic 'Hemolytic-assay'

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