OBJECTIVE: To investigate the effectiveness and safety of low concentration dithiothreitol (DTT) in removing the interference of monoclonal anti-CD38 on transfusion compatibility testing, and develop a reasonable clinical transfusion strategy. METHODS: The blood type, direct antiglobulin testing (DAT) and antibody screening were tested according to standard methods. Antibody screening cells and donor's red blood cells were treated by DTT 0.2, 0.1, 0.05, 0.02, 0.01 and 0.005 mol/L, and antibody screening and cross-matching of serums after monoclonal anti-CD38 treatment were performed by anti-human globulin card. RESULTS: The 0.01 mol/L DTT at 37℃ for 30 minutes could remove the effect of monoclonal anti-CD38 on antibody screening and cross-matching, meanwhile retain their effectiveness in detecting anti-K, anti-LW, anti-JMH, anti-Lu^b, anti-e, anti-Di^a and anti-Jk^a alloantibodies. All the 10 patients had no acute or delayed haemolytic transfusion reactions and their routine blood tests showed that the red blood cells transfusion was effective. CONCLUSION The 0.01 mol/L DTT is a safe and effective method for removing the interference of monoclonal anti-CD38 with transfusion compatibility testing, while retaining the ability to detect most alloantibodies.
Antibodies, Monoclonal/pharmacology* ; Blood Grouping and Crossmatching ; Blood Transfusion ; Dithiothreitol/pharmacology* ; Erythrocytes ; Humans ; Isoantibodies/pharmacology*

OBJECTIVE: To investigate the procedure of pre-transfusion testing and transfusion strategy of patients with multiple myeloma (MM) treated by daratumumab (DarA). METHODS: The blood samples of MM patients before and after DarA treatment from the Fifth Affiliated Hospital of Sun Yat-sen University were collected, and the ABO/Rh blood group antigen identification and DAT test results were compared. The results of antibody screening and cross matching of the patients before and after inactivation of red blood cells with 0.2 mol/L dithiothreitol (DTT) were compared and analyzed. RESULTS: ABO/Rh blood group antigen typing showed no affecting in patients after treated by DarA; the result of DAT test showed negative. Irregular antibody screening showed that all the three cells(Ⅰ, Ⅱ and Ⅲ) were positive(1+~2+) and the self-control was negative. By microcolumn agglutination method, the main side of the multi-bag of blood showed no matched, while the secondary side showed all identical. After treated by DTT solution, the cross matching results in reagent red blood cells and the red blood cells of blood donors were both consistent, and the irregular antibody screening was negative. The K(+)O type erythrocytes used in parallel control were transformed into K(-)O type erythrocytes after DTT treatment. However, there was no significant changes in E(+) O type erythrocytes before and after DTT treatment. There was no condensation on the primary and secondary side of the condensed amine method. The primary and secondary sides of blood matching by saline method showed negative. CONCLUSION After treated by DarA, cross matching results from microcolumn agglutination method can be interfered by the residual drug antibody in MM patients, while the interference was eliminated in the presence of 0.2 mol/L DTT solution. However, no disturbance was observed when using condensed amine method or saline method. Therefore, corresponding transfusion procedures should be selected according to the emergency degree of blood transfusion to ensure the safety and timeliness of blood transfusion.
Antibodies, Monoclonal ; Blood Transfusion ; Dithiothreitol ; Humans ; Multiple Myeloma/therapy*

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.
Complement System Proteins ; Dithiothreitol ; Edetic Acid ; Fluorescence ; Humans ; Immunoglobulin G ; Transplant Recipients

Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
Acinar Cells* ; Animals ; Antioxidants ; Calcium ; Catalase ; Cell Membrane* ; Cytosol ; Dithiothreitol ; Extracellular Fluid ; Hand ; Hydrogen Peroxide* ; Hydrogen* ; Inositol 1,4,5-Trisphosphate ; Ions ; Manganese ; Mice* ; Perfusion ; Plasma Membrane Calcium-Transporting ATPases ; Plasma* ; Reactive Oxygen Species

LW antigens are expressed in higher intensities in D-positive blood cells than D-negative cells, which can result in false identification of anti-D in pretransfusion testing. Although several cases of anti-LW have been reported abroad, to the best of our knowledge, none have been reported in Korea. Herein, we report a case of anti-LW in a 58 year-old RhD positive patient with non-Hodgkin's lymphoma with a positive direct Coombs test and a suspicion of the presence of passive anti-D antibodies because of a history of intravenous immunoglobulin administration. However, during a 5-month follow up, the antibody was confirmed as anti-LW on grounds that it showed weakened reaction in dithiothreitol treated cells and enforced reaction in cord O+ cells when compared to the results from antibody identification panel cells.
Antibodies ; Blood Cells ; Coombs Test ; Dithiothreitol ; Follow-Up Studies ; Humans ; Immunoglobulins ; Korea ; Lymphoma, Non-Hodgkin ; Sensitivity and Specificity*

A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 µg/mL. The inter- and intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.
Administration, Oral ; Ascorbic Acid* ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Dehydroascorbic Acid* ; Dithiothreitol ; Humans* ; Plasma* ; Tablets ; Volunteers

BACKGROUND: ABO antibody titration is important in cases such as ABO incompatible hemolytic disease of the fetus and newborn (HDFN), ABO incompatible bone marrow, or solid organ transplantation. This study was conducted in order to evaluate usability of ORTHO VISION (Ortho Clinical Diagnostics, Raritan, USA) designed automated ABO antibody titration equipment. METHODS: The isoagglutination titers were determined in 80 subjects (20 A, 20 B, 40 O (anti-A 20, anti-B 20)) using a conventional tube technique, including a 30 minute room temperature phase (CTT), Dithiothreitol treated manual column agglutination technique (MCAT), and automated column agglutination technique (ACAT) by ORTHO VISION. The concordance of titer was compared within one dilution step between the two methods. RESULTS: The isoagglutinin titers measured by the ACAT with anti-human globulin poly cassette (ACAT_Poly) and anti-human globulin IgG cassette (ACAT_IgG) were the highest and the isoagglutinin titer measured by the MCAT was also higher than that by the CTT. The isoagglutinin titer measured by the ACAT with reverse diluents cassette (ACAT_Reverse) was similar to that measured by the CTT. The concordance of anti-A and anti-B titers between CTT and ACAT_Reverse was 83% and 68%. The concordance of anti-A and anti-B titers between MCAT and ACAT_Poly was 100% and 83%. The concordance of anti-A and anti-B titers between MCAT and ACAT_IgG was 98% and 88%. CONCLUSION: Automated isoagglutinin titration using ACAT_Poly or ACAT_IgG without DTT showed reliable concordance with DTT treated MCAT, and it appears to be a possible replacement for the conventional MCAT method.
ABO Blood-Group System ; Agglutination ; Automation ; Bone Marrow ; Dithiothreitol ; Fetus ; Humans ; Immunoglobulin G ; Infant, Newborn ; Organ Transplantation ; Transplants

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca2+]i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate (H2DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca2+ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca2+ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca2+-sensitive fluorescence dye fura-2, the [Ca2+]i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.
Animals ; Calcium ; Calcium-Transporting ATPases ; Central Nervous System Sensitization ; Dithiothreitol ; Fluoresceins ; Fluorescence ; Fura-2 ; Humans ; Membranes ; Microscopy, Confocal ; Neurons ; Nociception ; Perfusion ; Rats ; Reactive Oxygen Species ; Substantia Gelatinosa ; Thapsigargin ; Tissue Donors

Several approaches have been introduced to detect allo-antibodies in the presence of warm auto-antibodies, and these methods include warm autoadsorption, cysteine-activated papain and dithiothreitol (ZZAP), and polyethylene glycol (PEG) and dilution of the patient's serum. Among them, the dilution technique is a simple and rapid method. During pretransfusion testing of a 33 year-old systemic lupus erythematosus (SLE) patient with warm auto-antibodies, antibody identification was done by the dilution technique with using serum diluted 1-in-8. The patient demonstrated an anti-Fy(b) pattern of reactivity in his sera. Contrary to our expectations, the phenotype of the erythrocytes was Fy(a+/b+) and the genotype, as assessed by performing PCR-restriction fragment length polymorphism (RFLP), was FY*A/FY*B. These results suggest that the antibody is an autoantibody showing anti-Fy(b) specificities. An antibody identification test using undiluted serum showed the same result when 40 days had passed. We report here on a case with auto-anti-Fy(b) proven by the dilution method in the presence of warm autoantibodies.
Autoantibodies ; Dithiothreitol ; Erythrocytes ; Genotype ; Humans ; Indicator Dilution Techniques ; Lupus Erythematosus, Systemic ; Papain ; Phenotype ; Polyethylene Glycols ; Sensitivity and Specificity*

BACKGROUND: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. OBJECTIVES: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. METHODS: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. RESULTS: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. CONCLUSION: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice.
Acrylic Resins ; Animals ; Antibodies ; Candida ; Candida albicans ; Cell Wall ; Chromatography ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Chromatography, Reverse-Phase ; Dithiothreitol ; Endopeptidase K ; Ethanolamines ; Fungi ; Glucan Endo-1,3-beta-D-Glucosidase ; Immunization ; Immunization, Passive ; Mice ; Multienzyme Complexes ; Peptide Hydrolases ; Phospholipases ; Proteins ; Survival Rate