Automated determination of ABO/Rh in microplates

A semi-automated system for determining the ABO group and Rh type of blood samples has been developed using a commercially available automated microplate (ELISA) reader and a microcomputer. Optimization of serologic, measurement and interpretation parameters was accomplished without significantly changing an existing manual procedure. The first pass noninterpretation rate of this system in the laboratory prior to field trials is 7.1%. A commercial system of this type should be cost-effective as a primary instrument for small to medium sized blood centers and transfusion services.     

Allele-specific oligonucleotide polymerase chain reaction for the determination of Rh C/c and Rh E/e antigens in thalassaemic patients

Background

Thalassaemia is a genetic disease in which there is a relative or complete lack of alpha or beta globin chains. Patients with moderate to severe forms of thalassaemia need transfusions from the early years of life. Antibody production against blood group antigens may cause many problems in preparing compatible blood units for transfusion. The identification of definite blood group phenotypes by the haemagglutination method can be difficult because of the mixed population of red blood cells from the donor and recipient.

Materials and methods

Forty multiply transfused thalassaemic patients and ten healthy controls with no history of blood transfusion were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and haemagglutination methods were used to determine the presence of Rhesus (Rh) C, c, E and e antigens.

Results

In this study four primer sets were used for ASO-PCR amplification of RhC/c and RhE/e. Although PCR assays for RhC/c and RHE/e genotyping have been described previously, in this study we used a new condition for PCR by decreasing the annealing temperature from 63 °C to 58 °C in order to amplify all four genes in the same condition. In order to evaluate this single run molecular method, we used the haemagglutination test as the standard method and compared the results from the two methods. We found discrepancies between phenotype and genotype results among patients with beta thalassaemia, but complete agreement between phenotype and genotype in the control group.

Conclusions

The advantage of this new ASO-PCR method compared to a restriction fragment length polymorphism (RFLP) PCR method is that with the former all four genes can be amplified at the same time by PCR, and electrophoresis can be performed immediately to determine individual antigen profiles. The simplicity of the ASO-PCR method makes it suitable for routine use in medical centres and it is also cheaper than RFLP-PCR. Furthermore, as shown by previous studies, the results of haemagglutination and PCR tests often differ because the existence of donor red blood cells in the patient’s circulation can interfere with the interpretation of the haemagglutination test.     

An Automated Discontinuous Venous Blood Sampling System for Ex Vivo Glucose Determination in Humans

Background

Intensive insulin therapy reduces mortality and morbidity in critically ill patients but places great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A cost-effective solution to this resourcing problem could be provided by an effective and reliable automated blood sampling (ABS) system suitable for ex vivo glucose determination.

Method

The primary study aim was to compare the performance of a prototype ABS system with a manual reference system over a 30 h sampling period under controlled conditions in humans. Two venous cannulae were inserted to connect the ABS system and the reference system. Blood samples were taken with both systems at 15, 30, and 60 min intervals and analyzed using a Beckman glucose analyzer. During the study, blood glucose levels were altered through four meal ingestions.

Results

The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.976 (0.953−0.996). The system error was −3.327 ± 5.546% (−6.03−0.49). Through Clark error grid analysis, 420 data pairs were analyzed, showing that 98.6% of the data were in zone A and 1.4% were in zone B. Insulin titration error grid analysis revealed an acceptable treatment in 100% of cases. A 17.5-fold reduction in the occurrence of blood-withdrawal failures through occluded catheters was moreover achieved by the added implementation in the ABS system of a “keep vein open” saline infusion.

Conclusions

Our study showed that the ABS system described provides a user-friendly, reliable automated means for reproducible and accurate blood sampling from a peripheral vein for blood glucose determination and thus represents a promising alternative to frequent manual blood sampling.     

Anti-Wrb, and Other Autoantibodies Responsible for Positive Direct Antiglobulin Tests in 150 Individuals

SUMMARY Eluates from the red blood cells (and sera whenever free autoantibody was present) of 150 individuals with positive direct antiglobulin tests, have been studied for antibody specificity. Of 87 patients with AIHA, 64 had autoantibodies reacting with all red cell samples including Rh_null. Of these 64 anti-dl autoantibodies, two were, and 32 contained, auto-anti-Wr^b. Of 33 patients being treated with alphamethyldopa, who had developed positive direct antiglobulin tests, 23 had anti-dl autoantibodies four of which contained auto-anti-Wr^b. Of 30 haematologically normal donors with positive direct antiglobulin tests, 23 had anti-dl autoantibodies, two of which were, and six of which contained, auto-anti-Wr^b. The full specificities of autoantibodies, other than anti-Wr^b and anti-dl, in the 150 patients are described, as are the natures of the protein red cell coatings that caused the positive direct antiglobulin tests. The presence of free serum autoantibody as a correlate of the three clinical conditions is reported.
Several observations on auto-anti-Wr^b are documented. The antibody can cause gross red cell destruction in vivo , but can be benign on other occasions; it occurs with approximately the same frequency in AIHA patients and 'normal'donors with positive direct antiglobulin tests, but in fewer patients with alpha-methyldopa induced positive direct antiglobulin tests; it does not activate complement in vivo; , and finally it may eventually provide a clue to the aetiology of AIHA.     

Disseminated intravascular coagulation associated with acute hemoglobinemia or hemoglobinuria following Rh(0)(D) immune globulin intravenous administration for immune thrombocytopenic purpura

The Food and Drug Administration (FDA) licensed Rh(o)(D) immune globulin intravenous (anti-D IGIV) on March 24, 1995, for treatment of immune thrombocytopenic purpura (ITP). A previous review described data on 15 patients who experienced acute hemoglobinemia or hemoglobinuria following anti-D IGIV administration for ITP or secondary thrombocytopenia. Eleven of those patients also experienced clinically compromising anemia, transfusion with packed red blood cells, renal insufficiency, dialysis, or death. That review suggested that patients receiving anti-D IGIV be monitored for those and other potential complications of hemoglobinemia, particularly disseminated intravascular coagulation (DIC). Through November 30, 2004, the FDA received 6 reports of DIC associated with "acute hemolysis" (or similar terms), 5 of which involved fatalities. The attending or consulting physicians assessed that acute hemolysis or DIC caused or contributed to each death. This review presents the first case series of DIC associated with acute hemoglobinemia or hemoglobinuria following anti-D IGIV administration for ITP. The purpose of this review is to increase awareness among physicians and other health care professionals that DIC may be a rare but potentially severe complication of anti-D IGIV treatment. Increased awareness of DIC as a diagnostic possibility may enable prompt recognition and medical intervention in affected patients.     

Monitoring Therapy with Vitamin K Antagonists in Patients with Lupus Anticoagulant: Effect on Different Tests for INR Determination

Background: Lupus anticoagulant (antiphospholipid antibodies) is associated with venous and arterial thrombosis in patients with and without autoimmune disorders. Vitamin K antagonists are the treatment of choice in patients with thrombosis, of which the dose is titrated by INR monitoring. Several recent reports suggest that the presence of the lupus anticoagulant disturbs the INR test and may lead to unreliable results with a large variation in INR values, dependent on the reagents used.Methods: We studied 11 lupus anticoagulant positive patients and 11 lupus anticoagulant negative patients, all using vitamin K antagonists. The INR value was determined using seven different tests and the variation in INR values was compared between the two groups. The amidolytic Factor X levels were used as an phospholipid independent measure for intensity of warfarin therapy. Factor VII and X activity were measured to assess the stability of warfarin therapy.Results: The variation of the results with different INR tests within one patient was minimal and comparable in the two groups for INR's in the therapeutic range. The coefficient of variation for the cases and control group was 10.43 and 9.35, respectively. Variation in both groups increased at supratherapeutic levels of anticoagulation and when the anticoagulation was unstable (measured with Factor X/Factor VII ratio). The relationship between INR values and Factor X analysis revealed no influence of the lupus anticoagulant.Conclusions: In this study, lupus anticoagulant antibodies do not disturb INR laboratory tests. Differences in INR measurements are seen in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved.Abbreviated Abstract. This study investigates the influence of lupus anticoagulant on INR determination tests in patients treated with warfarin. Eleven cases and eleven lupus anticoagulant negative control patients, also on warfarin therapy, were included. Seven INR results per patient were obtained using different laboratory tests. A factor X assay was performed to obtain an independent measure for the intensity of warfarin therapy.The variation of INR results between the cases and controls revealed no difference in these groups. In addition, the relationship between INR values and Factor X analysis indicated no influence of the lupus anticoagulant. What was observed was an increased difference in INR values in patients with a high intensity of anticoagulation and in patients who either just started or in whom no stable anticoagulation has been achieved     

Systematic Performance Comparison of Fe3+/Fe0/Peroxymonosulfate and Fe3+/Fe0/Peroxydisulfate Systems for Organics Removal

Activated zero-valent iron (Ac-ZVI) coupled with Fe³⁺ was employed to activate peroxymonosulfate (PMS) and peroxydisulfate (PDS) for acid orange 7 (AO7) removal. Fe³⁺ was used to promote Fe²⁺ liberation from Ac-ZVI as an active species for reactive oxygen species (ROS) generation. The factors affecting AO7 degradation, namely, the Ac-ZVI:Fe³⁺ ratio, PMS/PDS dosage, and pH, were compared. In both PMS and PDS systems, the AO7 degradation rate increased gradually with increasing Fe³⁺ concentration at fixed Ac-ZVI loading due to the Fe³⁺-promoted liberation of Fe²⁺ from Ac-ZVI. The AO7 degradation rate increased with increasing PMS/PDS dosage due to the greater amount of ROS generated. The degradation rate in the PDS system decreased while the degradation rate in the PMS system increased with increasing pH due to the difference in the PDS and PMS activation mechanisms. On the basis of the radical scavenging study, sulfate radical was identified as the dominant ROS in both systems. The physicochemical properties of pristine and used Ac-ZVI were characterized, indicating that the used Ac-ZVI had an increased BET specific surface area due to the formation of Fe₂O₃ nanoparticles during PMS/PDS activation. Nevertheless, both systems displayed good reusability and stability for at least three cycles, indicating that the systems are promising for pollutant removal.     

Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily

AmtB, a member of the Rh/Amt/MEP superfamily, is responsible for ammonia transport in Escherichia coli. The ammonia pathway in AmtB consists of a narrow hydrophobic lumen in between hydrophilic periplasmic and cytoplasmic vestibules. A series of molecular dynamics simulations (greater than 0.4 μs in total) were performed to determine the mechanism of solute recruitments and selectivity by the periplasmic vestibule. The results show that the periplasmic vestibule plays a crucial role in solute selectivity, and its solute preferences follow the order of NH4(+) > NH3 > CO2. Based on our results, NH4(+) recruitment is initiated by its interaction with either E70 or E225, highly conserved residues located at the entrance of the vestibule. Subsequently, the backbone carbonyl groups at the periplasmic vestibule direct NH4(+) to the conserved aromatic cage at the bottom of the vestibule (known as the Am1 site). The umbrella sampling simulations suggest that the conserved residue D160 is not directly involved in the ammonia conduction; rather its main function is to keep the structure of periplasmic vestibule intact. The MD simulations also revealed that two partially stacked phenyl rings of F107 and F215, separating the periplasmic vestibule from the hydrophobic lumen, flip open and closed simultaneously with a frequency of approximately 10(8) flipping events per second. These results show how the periplasmic vestibule selectively recruits NH4(+) to the Am1 site, and also that the synchronized flipping of two phenyl rings potentially facilitates the solute transition from the periplasmic vestibule to the hydrophobic lumen in the Rh/Amt/MEP superfamily.     

Automated, simplified GC/MS data processing system for organic acidemia screening and its application

Gas chromatography mass spectrometry (GC/MS) is widely used in diagnosis of organic acidemias. However, GC/MS has not yet become a routine laboratory test, because of the complexity in interpretation of GC/MS data. We developed a personal computer-based system of automated metabolic profiling and disease detection for the screening of organic acidemias by GC/MS. The data were processed after the GC/MS analysis of urinary organic acids. In this system, 130 kinds of metabolites and 25 disorders of organic acids were enrolled for the search and detection, respectively. Metabolites were identified with methylene unit values (MU). target ions (Q- and C-ions) and their intensity ratios, and semiquantified by peak relative area (%) of the Q-ions to that of an internal standard. Metabolites whose values exceeded the cutoff of the control table were flagged as abnormal. The diseases or pathological condition were automatically evaluated by combination of the abnormal compounds. In this system, index metabolites were categorized into three groups. "AND, "OR" and "NO". The groups, "AND" and "OR" comprised essential and optional compounds, respectively, for the specific diagnosis. The third group, "NO", included compounds which must be absent to reach a diagnosis. We compiled data of MU values and mass spectrum of 130 kinds of index metabolites, and tested the usefulness of this system by analysis of 74 patients with 19 kinds of diseases. In all cases, at least a correct diagnosis could be found among the disease names outputted. We have successfully applied this to a pilot neonatal screening by GC/MS in our regional area, and acylglycine analysis by the stable isotope dilution method with tert-butyldimethylsilyl derivatization. With our system, many people can attend for screening programs using GC/MS.     

Experimental Method for Simultaneous Determination of the Lamb Wave A0 Modes Group and Phase Velocities

Determining Lamb wave dispersion curves when measuring phase and group velocity values at a fixed frequency is now a common and relevant task. In most cases, in order to solve such a problem, it is necessary to know the exact properties of the material, particularly its thickness. In experimental methods, Lamb wave parameters are evaluated directly from the test materials. This paper proposes a new and simple experimental algorithm for A₀ mode group and phase velocity determination based on signal filtering and zero-crossing estimating. The main idea is to capture the zero-crossing instances of the signals closest to the signal envelope peaks and use these time instances to determine the phase and group velocities. The reliability of the proposed method was evaluated using simulated and experimental signals propagating in an aluminum plate. Theoretical modeling has shown that the proposed method enables the calculation of the A₀ mode group and phase velocities with a mean relative error of less than 0.7%. An accuracy of 0.8% was observed during the experimental measurements.     

Cost and Threshold Analysis of an HIV/STI/Hepatitis Prevention Intervention for Young Men Leaving Prison: Project START

The objectives of this study were to: (a) estimate the costs of providing a single-session HIV prevention intervention and a multi-session intervention, and (b) estimate the number of HIV transmissions that would need to be prevented for the intervention to be cost-saving or cost-effective (threshold analysis). Project START was evaluated with 522 young men aged 18–29 years released from eight prisons located in California, Mississippi, Rhode Island, and Wisconsin. Cost data were collected prospectively. Costs per participant were $689 for the single-session comparison intervention, and ranged from $1,823 to 1,836 for the Project START multi-session intervention. From the incremental threshold analysis, the multi-session intervention would be cost-effective if it prevented one HIV transmission for every 753 participants compared to the single-session intervention. Costs are comparable with other HIV prevention programs. Program managers can use these data to gauge costs of initiating these HIV prevention programs in correctional facilities.     

LCT-13910C > T polymorphism-associated lactose malabsorption and risk for colorectal cancer in Italy

Background

The activity of epithelial lactase (LCT) associates with a polymorphism 13910 bp upstream the LCT-encoding gene (LCT-13910C > T). The relationship between LCT-13910C > T polymorphism and risk for colorectal cancer is unclear.

Aims

We examined the relationship between the LCT-13910C > T polymorphism causing lactose intolerance and risk for colorectal cancer/polyps onset in the Italian population.

Patients and methods

793 subjects (306 with colorectal cancer, 176 with polyps and 311 controls) were genotyped for the LCT-13910C > T variant by TaqMan real time-PCR.

Results

Lactose malabsorption linked to the CC genotype did not associate with an increased risk for either colorectal cancer (OR = 1.041; 95% CI = 0.751–1.442; p = 0.868) or polyps (OR = 0.927; 95% CI = 0.630–1.363; p = 0.769). There was no association with colorectal cancer/polyps site. 60% of the subjects overall bore the CC genotype.

Conclusion

In the Italian population the LCT-13910C > T polymorphism is not associated to the risk for colorectal cancer or polyps.     

An Automated Strategy for Bedside aPTT Determination and Unfractionated Heparin Infusion Adjustment in Acute Coronary Syndromes: Insights from PARAGON A

Background: Intravenous unfractionated heparin remains a cornerstone of anticoagulation therapy for patients with acute coronary syndromes, but regulation to a target aPTT is challenging. We assessed unfractionated heparin infusion regulation by bedside, whole-blood aPTT testing and computerized, algorithmic infusion adjustment, and further evaluated the relationship of achieving the target aPTT with clinical outcomes. Methods and Results: We studied 1,275 patients randomized to unfractionated heparin in PARAGON-A, which tested lamifiban with or without unfractionated heparin versus unfractionated heparin. All patients had baseline and 6-hour blinded, bedside aPTTs, then aPTTs per algorithm. A central computer translated encrypted values to algorithmic dose-adjustment commands. We assessed the ability to achieve and maintain aPTTs of 50–70 seconds and associations of 6- and 12-hour aPTTs and time-to-target with 30-day outcomes.Overall, the median 6-hour aPTT was 50–70 seconds and remained so throughout infusion. Individually, only 33.6% of patients achieved 6-hour target-range aPTTs, and only 40% of all aPTTs were in-range. After achieving target, only 42% of subsequent measures were in-range. Thirty-day death or myocardial infarction (death/MI) increased non-significantly as time-to-target increased (p = 0.08). Thirty-day mortality was similar if target aPTT was reached, regardless of timing. Death/MI trended lower if target aPTT was reached by 8 hours (p = 0.10). The best clinical outcomes were associated with in-range aPTTs. Conclusions: This study represents the most systematic monitoring and regulation of unfractionated heparin anticoagulation to date. Although average anticoagulation achieved target range, wide inter- and intra-patient variability may have important implications for clinical outcomes. Abbreviated abstract: Using systematic aPTT testing and computer-directed, algorithmic unfractionated heparin infusion adjustment in 1,275 acute coronary syndrome patients, the overall median aPTT was 50–70 seconds. However, only 33.6% of patients achieved this target 6-hour aPTT range. Only 40% of all aPTTs, and after achieving target, only 42% of subsequent measures, were in this range. Thirty-day death or myocardial infarction increased with increasing time to target aPTT (for trend, p = 0.08). The best outcomes were associated with 6- and 12-hour aPTTs in the target range. Wide inter- and intra-patient variability despite highly systematic, controlled unfractionated heparin infusion regulation has important implications for unfractionated heparin use.     

Threshold of soluble fms-like tyrosine kinase 1/placental growth factor ratio for the imminent onset of preeclampsia

It has not been clarified whether thresholds of soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), soluble endoglin, and the sFlt-1/PlGF ratio for the imminent onset of preeclampsia (PE) exist. We hypothesized that onset thresholds for the imminent onset of PE could be determined by the distributions of these 4 markers just after the onset of PE. Study subjects were 51 PE after the onset of PE; 36 of PE, 20 of gestational hypertension, 142 of a small-for-gestational-age infant, and 400 of normal pregnant controls at 19 to 25 and 27 to 31 weeks of gestation in a prospective cohort study. The current data supported our hypothesis that onset thresholds of sFlt-1 and the sFlt-1/PlGF ratio exist. The onset thresholds of the sFlt-1/PlGF at 26 to 31 weeks of gestation were useful for detecting imminent PE with the onset at <36 weeks of gestation, showing sensitivity of 0.36 and a positive likelihood ratio and 95th percent CIs of 38 (11-132); when positive, PE occurred at 2.2±0.6 weeks (range: 1.4-3.0 weeks) after the measurement of the sFlt-1/PlGF ratio. The combination of sFlt-1 at 26 to 31 weeks of gestation, past history of gestational hypertension or PE, prepregnancy body mass index, and mean blood pressure at 16 to 23 weeks of gestation was useful for detecting PE with onset of <36 weeks of gestation, showing sensitivity of 0.82, and a positive likelihood ratio (95% CI) of 42 (20-88). In conclusion, the onset threshold of sFlt-1/PlGF existed and might be useful for detecting the imminent onset of PE.     

Distinct Advantages of Circumferential Notch Tensile (CNT) Testing in the Determination of a Threshold for Stress Corrosion Cracking (KISCC)

Stress corrosion cracking (SCC) is a vexing problem for load-bearing equipment operating in a corrosive environment in various industries, such as aerospace, chemical and mineral processing, civil structures, bioimplants, energy generation etc. For safe operation, effective maintenance and life prediction of such equipment, reliable design data on SCC (such as threshold stress intensity for SCC, i.e., K_ISCC) are invaluable. Generating reliable K_ISCC data invariably requires a large number of tests. Traditional techniques can be prohibitively expensive. This article reviews the determination of K_ISCC using the circumferential notch tensile (CNT) technique, the validation of the technique and its application to a few industrially relevant scenarios. The CNT technique is a relatively recent and considerably inexpensive approach for the determination of K_ISCC when compared to traditional techniques, viz., double-cantilever beam (DCB) and compact tension (CT) that may be fraught with prohibitive complexities. As established through this article, the CNT technique circumvents some critical limitations of the traditional techniques.