Optimizing d-mannose and glyceraldehyde concentrations as glucose preservatives without clinically affecting biochemical test results.

Objectives: The objectives were to evaluate blood additives that combined lithium heparin (LH)-salt with glyceraldehyde (GLY) or d-mannose (MAN) for preserving glucose levels in plasma samples and to simultaneously determine the compatibility of these additives with 14 other biochemical tests. Methods: Blood samples from 40 subjects, equally divided into healthy and diabetic groups, were collected using five different additives. The three most effective additives, LH/GLY, LH/MAN, and LH/GLY/MAN, were selected for ensuring the best preservation of glucose levels and compatibility with 14 biochemical tests. One-way analysis of variance was used to analyze the mean paired differences of glucose level and biochemical tests. Simultaneously, the clinical criteria from Johns Hopkins Hospital were used to guide the interpretation and set acceptable thresholds for measurements that exceeded the standards. Results: The combination of 160 mmol/L GLY, 8.4 mmol/L MAN, and LH, maintained glucose levels at approximately 93.4-93.7 % for healthy subjects and 91.3-92.8% for subjects with diabetes mellitus over 8 h. The mean paired differences of glucose levels in preservation were statistically insignificant. The biases in 14 biochemical tests for LH/GLY/MAN and LH/MAN remained within the acceptable clinical criteria during the 8 h. Conclusions: Combining 160 mmol/L GLY, 8.4 mmol/L MAN, and LH, proved more effective in maintaining glucose levels than individual additives or the conventional sodium fluoride preservative. It did not yield clinical discrepancies in the 14 biochemical tests during 8 h at room temperature.

Commutable Blood Materials from the Fixed-Cell Method for Performance Evaluation of Blood Glucose by a Glucose Meter.

Glucose meters provide a rapid blood glucose status for evidence-based diagnosis, monitoring, and treatment of diabetes mellitus. We aimed to evaluate the commutability of processed blood materials (PBMs) and their use in the performance evaluation of glucose meters. Two PBMs obtained by the fixed-cell method were analyzed for homogeneity, stability, and commutability. The compatibility of ten pairs between mass spectrometry and each glucose meter was categorized as compatible (mean paired difference ≤ 5%) and incompatible (mean paired difference > 5%). The performance of glucose meter 1 (n = 767) and glucose meter 2 (n = 266) was assessed. The glucose in the PBMs remained homogenized and stable for at least 180 days. Six out of ten pairs had commutable PBMs. Commutability of PBMs was observed in both well-compatible and incompatible glucose results. Target glucose values from mass spectrometry were significantly different (p ≤ 0.05) from consensus values in one group of glucose meters. When commutable PBMs were used, glucose meter 1 showed better performance than glucose meter 2, and the percentage of satisfaction was associated when using target values for glucose from mass spectrometry and consensus values, but the performance of glucose meter 2 was not associated. PBM from a fixed-cell method could be mass produced with acceptable homogeneity and stability. Commutability testing of PBMs is required prior to use in the performance evaluation of glucose meters, as the commutability of glucose in the PBMs obtained by a fixed-cell method was variable and depended on the individual glucose meter.

Commutability and Uncertainty of Blood Hemoglobin A1c Testing Materials.

BACKGROUND: Hemoglobin A1C (HbA1C) is used to evaluate glycemic control over a three-month period. Blood matrix-based HbA1C materials are needed for quality control and evaluation of HbA1C measurements. This study investigated the commutability of blood materials (BMs) and aimed to upgrade BMs for HbA1C testing for use as proficiency test (PT) material. METHODS: We measured BMs from a DM blood donor (n = 1), an in vitro glycation procedure (n = 3), and from commercial sources (n = 2) for HbA1C in parallel with fresh unprocessed BMs (n = 3) and clinical blood samples (n = 25). Two NGSP-certified methods, including a turbidimetric and an enzymatic immunoassay, were used for HbA1C determinations. Commutability as investigated according to CLSI EP14-Ed4 guidelines. RESULTS: The commutable BMs exhibited a mean paired difference of 0% to 9% when compared to reference methods, whereas the non-commutable BMs represented a mean paired difference of 3% to 11%. Fresh, unprocessed BMs with a low HbA1C of 6.0% were more commutable than BMs with a high HbA1C. The values of HbA1C in BMs (mean and uncertainty following ISO Guide 35 for RM production) were applied to upgrade the PT material to be used as a reference material. The relative uncertainty of BM-Ndm-1 and BM-Gcb-3 were 1 and 0.4%, respectively. CONCLUSIONS: The commutability of hemoglobin in BMs is dependent on the preparation process. Blood materials with a high HbA1C content are usually less commutable versus materials with low HbA1C content when prepared by the same process. Our study showed BMs can be successfully used as quality control or PT materials.

Effects of Simulated Adverse Environmental Conditions Related to Actual Conditions at Health Promoting Hospitals on the Performance of Blood Glucose Testing by Glucose Meters.

BACKGROUND: Annual peaks in temperature and humidity exceed manufacturers' specifications for blood glucose test strip storage in Thailand. Health Promoting Hospitals (HPH) do not always provide the same level of health services that hospitals do since they often only turn on air-conditioning units during working hours. METHODS: The blood glucose testing performance of 4 glucose meters were investigated for short and long terms stress at 5 simulated conditions. Temperature and relative humidity (RH) at 5 HPHs in different regions of Thailand were monitored for 9 weeks during April to July 2019. The use of air conditioning in storage rooms for storing test strips was surveyed at 131 HPHs using questionnaires. RESULTS: Median-paired differences of glucose measurements with stressed test strips in 5 simulated conditions significantly differed (P < 0.05) both in the short term (3 days) and in the long term (30 days) with 4 glucose meters when compared to unstressed test strips. The average temperature of all HPHs exceeded 30°C (86°F). The average RH was 84%. There was only one HPH that occasionally turned on its air conditioners. Most HPHs kept both opened and unopened vials of test strips in rooms without air conditioners. Further, 21.4-32.0% of HPHs kept test strips at room with air conditioners. CONCLUSIONS: This study provides evidence for poor performance of blood glucose testing by glucose meters that are affected by adverse environmental conditions. The environmental for test strips storage at HPHs should be considered to prevent analytical errors of glucose measurement.

Sandwich antibody-based biosensor system for identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

Mycobacterial infection, leading to pulmonary disease, remains a world health problem. Clinical symptoms of pulmonary disease caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) are very similar. A rapid method for the differentiation of MTBC and NTM infection is essential for appropriate therapy. In this study, we aim to establish an antibody-based biosensor system for the identification of MTBC and NTM infection. Monoclonal antibodies (mAbs) specific for Ag85B proteins of mycobacteria were generated and characterized. The generated anti-Ag85B mAb clones AM85B-5 and AM85B-8 reacted to Ag85B of Mycobacterium spp.; in contrast, clone AM85B-9 specifically reacted to Ag85B of MTBC. By employing the produced mAbs, single and sandwich antibody-based biosensors using bio-layer interferometry were established for determination of Ag85B proteins. The sandwich antibody-based biosensor system was demonstrated to be suitable for detection of Ag85B protein and identification of MTBC and NTM. Using anti-Ag85B mAbs AM85B-8 and AM85B-9 as immobilized antibodies on sensor chips and using mAb AM85B-5 as secondary antibody, the established sandwich antibody-based biosensor could discriminate MTBC and NTM. The developed biosensor system can be used for culture confirmation of mycobacteria and speciation to MTBC and NTM.

Rapid diagnosis of tuberculosis by identification of Antigen 85 in mycobacterial culture system.

The standard culture for identification of Mycobacterium tuberculosis takes a long time to perform. We introduce here a method for fast identification of M. tuberculosis in mycobacterial culture system. Antibodies to Antigen (Ag) 85 of M. tuberculosis were produced and subsequently used to develop enzyme-linked immunosorbent assay (ELISA) for detecting Ag85 in the culture filtrate. By this detection, rapid tuberculosis (TB) diagnosis was achieved in comparison to the standard culture system with 89.6% sensitivity and 94% specificity. We thus suggest a new TB diagnosis strategy in which clinical samples are cultured in mycobacteria liquid culture medium. The culture filtrates are taken for detection of the Ag85 by ELISA. Using this strategy, 25%, 50%, 80%, and 90% of TB patients will be detected within day 3, week 1, 2, and 4, respectively. The established assay will enable a faster diagnosis of TB, leading to more efficient treatment of TB patients and control of disease transmission.

Non-mitogen containing conditioned medium for hybridoma production and single cell cloning.

BACKGROUND: The hybridoma technique is the standard method for production of monoclonal antibodies of interest. However, the newly formed hybridomas and cells at low density often grow poorly or die. This is the major obstacle to production of monoclonal antibodies. OBJECTIVE: The aim of this study was to establish a method for preparation of conditioned medium in the absence of mitogen for promoting the growth of hybridomas after cell fusion and during single cell cloning. METHODS: Culture supernatants were obtained from the cultures of BW5147 mouse thymoma cells without mitogen stimulation. Novel conditioned mediums were investigated for their ability to support hybridoma single-cell cloning and hybridoma production. RESULTS: We demonstrated that these conditioned mediums could support hybridoma single-cell growth, both for stable and newly generated hybridomas, at a level equal to the commercial conditioned medium BM-Condimed H1. The conditioned medium was most effective at a final concentration of 20% with the basal medium supplemented with 10% fetal calf serum. The novel conditioned medium could also be effectively employed for generation of hybridomas secreting various monoclonal antibodies. Interestingly, fibroblast overgrowth in the post-fusion wells was markedly reduced when using the novel conditioned medium, as compared to the commercial BM-Condimed H1, likely due to the absence of mitogen in the conditioned medium. CONCLUSION: We describe the method for production of a novel conditioned medium for hybridoma technology. This method is simple, needing no special technology or sophisticated equipment. The novel conditioned medium is, therefore, recommended for use in place of expensive commercial conditioned medium for hybridoma technology, especially in resource-limited countries.

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes.

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM(+) spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG(+) spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.