The challenging blood cross-matching and resolution for multiple myeloma patients in different disease stages / 中国输血杂志

【Objective】 To explore the challenging blood cross-matching and resolution for multiple myeloma (MM) patients in different disease stages. 【Methods】 For a patient who was first diagnosed as MM and scheduled for blood transfusion, his blood was cross matched with donors’ blood by microcolumn gel method and tube test. When the major side of cross-matching was agglutinated, the patient’s plasma was cross matched with donors’ red blood cell (RBC) by polybrene test, then plasma dilution cross matched with donors’ RBC by microcolumn gel method. For a patient who was diagnosed as recurrent refractory MM and scheduled for blood transfusion, his blood was cross matched with donors’ blood by microcolumn gel method. 【Results】 1) Case 1 was a first-visit outpatient. The major side of microcolumn cross-match test was agglutinated with the shape of fine line. The result of tube method also showed agglutination of major sides, and the rouleaux were detected by the microscopy. Then polybrene method and microcolumn gel method (after plasma diluted) were applied for cross-matching again with the above two donors’ blood and showed compatibility. 2) Case 2 was a recurrent refractory MM patient. The major and minor sides of microcolumn cross-match test were both agglutinated with the shape of granular. The patient was treated with anti-CD38 monoclonal antibody. The RBCs, after treated with dithiothreitol (DTT) was used to cross match with patient plasma by microcolumn test, and the result was compatible. 【Conclusion】 Polybrene method and microcolumn gel method after plasma diluted are suitable for blood cross-matching of newly diagnosed MM patients, also for those treated with CD38 monoclonal antibody, as the drug interference with cross-matching can be eliminated by DTT.

Daratumomab interference with pre-transfusion examination of patients with multiple myeloma / 中国输血杂志

【Objective】 To solve daratumomab interference with blood compatibility testing in multiple myeloma (MM) patients treated by daratumomab(DARA). 【Methods】 The irregular antibodies screening before and after the DARA treatment, and the major side crossmatch via coombs' test and polybrene method, respectively, were performed to resolve the nonspecific interference in a MM patient’s cross-matching test, produce by DARA. 【Results】 The initial panreactivity on the major side with agglutination (3+ ~4+ ), produce by DARA, was overcome by dithiothreitol (DTT) treatment, and turner out to be none agglutination. Otherwise, DARA had no effect on the crossmatch using polybrene method. 【Conclusion】 Antibody screening and identification should be conducted before DARA treatment in MM patients, and DARA interference with blood compatibility testing can be resolved by DTT treatment or the crossmatch using polybrene method.

Interference of CD38 monoclonal antibody in blood compatibility testing and its countermeasures: A general consensus among experts / 中国输血杂志

With continuous discovery of tumor immune targets and continuous changes in antibody research and development technology, antibody drugs are becoming more and more widely used in clinical practice. However, some targets are not only expressed on tumor cells, but also on red blood cells. Therefore, the clinical application of antibodies against the corresponding targets may interfere with the detection of blood transfusion compatibility, resulting in difficulty in blood matching or delay of blood transfusion. This consensus summarizes the current solutions for the interference of CD38 monoclonal antibody (CD38 mAb) in transfusion compatibility testing. After analyzing the advantages and disadvantages of different methods, polybrene and sulfhydryl reducing agents [dithiothreitol (DTT) or 2-mercaptoethanol (2-Me)], as a solution for CD38 mAb interference in blood compatibility testing, are recommended for Chinese patients, so as to eliminate blood transfusion interference produce by CD38 mAb and further provide a pre-transfusion workflow for clinicians and technicians in Department of Blood Transfusion.

Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins

Members of the proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.

Anti-LW Showing Anti-D Mimicking Specificity: A Case Report / 대한수혈학회지

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.

HPLC-UV method for the simultaneous determinations of ascorbic acid and dehydroascorbic acid in human plasma

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.

A Case of Panagglutination on Antibody Identification in a Multiple Myeloma Patient Receiving Daratumumab / 대한수혈학회지

Herein, we report a patient showing panagglutination in the unexpected antibody identification test after the administration of daratumumab. The patient was a 66-year-old woman who had undergone multiple cycles of chemotherapy and autologous peripheral blood stem cell transplantation for treating multiple myeloma; however, despite treatment, she had relapsed. Therefore, daratumumab, on clinical trials in Korea, started to be administered. After administration of daratumumab, the result of antibody screening test was positive, on the contrary to the result prior to the administration. Moreover, all positive reactions were shown in the antibody identification to the panel cells. After destroying CD38 antigens on the surface of RBCs using DTT, negative results were obtained. Daratumumab—a novel therapeutic human CD38 monoclonal antibody that can be used as targeted immunotherapy—is an FDA-approved drug for treating multiple myeloma. Because CD38 is expressed not only on myeloma cells, but also on red blood cells (RBCs), the use of daratumumab might lead to RBC agglutinations, and thereby resulting in false-positive results on the pre-transfusion tests. Therefore, caution is needed in case of a patient receiving daratumumab. Furthermore, additional test using DTT is required, especially when panagglutination was shown in the antibody identification test, as in this case.

A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca2+, Mn2+ and Mg2+. Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca2+ than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Comparison of Total and IgG ABO Antibody Titers in Healthy Individuals by Using Tube and Column Agglutination Techniques

BACKGROUND: Most immune reactions related to transfusion and transplantation are caused by IgM ABO antibodies. However, IgG also plays an important role in these reactions. Therefore, a method to measure antibodies, including IgG, is necessary. We investigated ABO antibody titers of healthy individuals using a column agglutination technique (CAT) with or without dithiothreitol (DTT) and compared them with titers obtained using a conventional tube method. METHODS: Among healthy adults who underwent a medical examination, 180 individuals (60 with blood group A, 60 with group B, and 60 with group O) were selected. Antibody titrations were performed using the immediate spin (IS) tube, anti-human globulin (AHG) tube, and CAT with or without DTT methods. RESULTS: Higher median values of anti-B and anti-A titers in groups A and B individuals, respectively, were obtained using the IS method than using the AHG method. Higher values for group O individuals were obtained using the AHG method. Higher median titers of anti-B and anti-A in group O individuals were obtained using CAT without DTT than using the AHG method. Median titers of anti-B and anti-A in all blood groups were higher in CAT without DTT than in CAT with DTT, especially for group O individuals. CONCLUSIONS: We recommend CAT with and without DTT for titration of anti-A and anti-B, especially in group O individuals, to provide more sensitive results that include IgG data. Adjustment of insurance coverage of fees associated with antibody titration might be necessary, considering the actual cost of reagents and personnel.

Inhibitory action of dithiothreitol on selenite-induced cataract in rats / 第二军医大学学报

Objective: To study the inhibitory action of self-designed dithiothreitol(DTT) eye drops on selenite-induced rat cataract(simulating the senile cataract). Methods: Twelve-day-old SD rats (body weight about 25 g) were injected with Na2SeO3 subcutaneouly, quaque die alterna, for 3 times (0.2 ml/time, with a total amount of 210 μg). Six days later over 90% of rats developed nuclear cataracts. The right eyes of the rats were treated with 25 mmol/L DTT eye drops (4 times/day, 1 drop/ time) for 3 weeks. The left eyes were taken as control and were treated with normal saline at the same dosage. The lens were examined using slit lamp and the diameter of the cataract plaques were measured using vernier cursor. The eyes of cataract models were also treated with 25 mmol/L DTT (n = 89), pirenoxine eye drops (PDE, Japan, n = 22), pirenoxine sodium eye drops (PSED, China, n = 24, 2 died during the experiment), and normal saline (n = 45). Each eye was administered with 1 drop/time, 4 times/day for 3 weeks. Then slit lamp was used to examine the cortical lesions of both eye and the lens opacity. Results: The DTT eye drops significantly reduced the diameter of the cataract plaques and significantly reduced the rate of the cortical opacity after the nuclear cataract. The inhibitory action of DTT eye drops on the selenite-induced cataract was superior to pirenoxine eye drops(Japan) and pirenoxine sodium eye drops(China). Conclusion: Our study shows that the DTT eye drops has satisfactory inhibitory action on selenite-induced cataract in rats.

The Inhibition of TREK2 Channel by an Oxidizing Agent, 5,5'-dithio- bis (2-nitrobenzoic acid), via Interaction with the C-terminus Distal to the 353rd Amino Acid

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

Sulfhydryl modification affects coronary artery tension by changing activity of delayed rectifier K+ current

It has been reported that a change in the cellular redox state may be involved in the regulation of vascular tone, but the underlying mechanism is not fully understood. The present study was designed to investigate the cellular effect of sulfhydryl modifying agents in the coronary artery of rabbit using the tension measurement and whole cell clamping method. The application of diamide, a sulfhydryl oxidizing agent, relaxed the endothelium denuded coronary arteries in a dose dependent manner. The fact that this diamide-induced relaxation was significantly attenuated by a pretreatment of 4-AP, and the coronary arteries precontracted with 100 mM K+ instead of histamine, suggests the involvement of 4-AP sensitive K+ channels in the diamide-induced relaxation of coronary arteries. Whole cell patch clamp studies revealed that the 4-AP sensitive IdK was significantly enhanced by the membrane permeant oxidizing agents, diamide and DTDP, and were reversed by subsequent exposure to the reducing agent, DTT. Neither the membrane impermeant oxidizing or reducing agents, GSSG or GSH, had any effect on the activity of IdK, indicating that intracellular sulfhydryl modification is critical for modulating IdK activity. The Diamide failed to significantly alter the voltage dependence of the activation and inactivation parameters, and did not change the inactivation process, suggesting that diamide increases the number of functional channels without altering their gating properties. Since IdK has been believed to play an important role in regulating membrane potential and arterial tone, our results about the effect of sulfhydryl modifying agents on coronary arterial tone and IdK activity should help understand the pathophysiology of the diseases, where oxidative damage has been implicated.