Assays for quality control of platelets for transfusion

Quality control of platelets relies on the determination of the extent by which platelets are still able to react with known agonists, and here knowledge of the biochemistry of platelet activation may guide to decide which tests are useful. In vivo, platelets will adhere to collagen exposed upon vessel wall damage, and especially under high shear conditions through von Willebrand factor (VWF) that forms a bridge between collagen and platelet glycoprotein (GP) Ib. Binding of GPVI to collagen next results in the activation of a tyrosine kinase cascade, finally resulting in phosphorylation and activation of phospholipase C (PLC) γ2, which leads to an increase in cytosolic Ca²⁺ levels. High Ca²⁺ levels trigger the production of thromboxane A2 (TXA2) and release of platelet granules containing among others, adenosine diphosphate (ADP). TXA2 and ADP now will activate their receptors on platelets which are, among others, linked to a G-protein that activates PLCβ2 with more Ca²⁺ increase. This ultimately provokes activation of the integrin α_IIbβ₃ (or GPIIbIIIa), which now can bind the symmetrical fibrinogen, thus allowing cross-linking of platelets or platelet aggregation. From the above it is clear that testing whether platelets are fully reactive ideally should be looking at as many of the above parameters as possible, while at the same time being simple and fast. Systems in which blood is flown over collagen surfaces mimic best the physiological situation; however, these tests are not fit for stored platelet testing as haematocrit is a critical factor in these experiments. Thromboelastography at best provides a test for G-protein dependent activation (through thrombin) and integrin α_IIbβ₃ involvement. Platelet agglutination induced by ristocetin (surrogate for the adhesion phase) next to aggregation (involvement of integrin α_IIbβ₃) induced by collagen (Tyr kinase pathway) and ADP (G-protein mediated pathway) can give a comprehensive view on the platelet quality. However, flow cytometry is also an excellent technique to detect (i) bound VWF to platelets in the presence of ristocetin, (ii) collagen- or ADP-induced activation of integrin α_IIbβ₃ (by determining the binding of fibrinogen or of the activation-dependent PAC-1 antibody) and (iii) secretion (by detecting surface expression of P-selectin).     

Criteria for aspiration cytology for the diagnosis of seminoma

Fine-needle aspiration cytology was performed in two cases of intra-abdominal tumors, one from retroperitoneal lymph nodes and one from a cryptorchid testis. The cytological diagnosis was consistent with seminoma in both cases. Cytological features included uniform neoplastic malignant cells with round nuclei and nucleoli. The cytoplasm, easily observed on May-Grünwald-Giemsa-stained smears, contained large vacuoles or lacunes filled with glycogen. Alkaline phosphatase activity was strictly located to one cytoplasmic area of the cells. This cytological and cytoenzymatic pattern is different from that observed in other intra-abdominal tumors, including adenocarcinoma, large-cell lymphoma, and embryonal carcinoma.     

Comparison of Blocking Agents for an Elisa for Lps

ELISA is a sensitive, specific, reproducible and fast method for detection of antigen-antibody reactions. in case of non-protein antigens as LPS, problems exist, such as poor proportion of coating to microplates, non-specific binding of antibodies to the plastic wells. These problems were resolved partially by Takahashi and co-workers using poly-L-lysine for coating of LPS antigens. to reduce non-specific binding, blocking agent, such as bovine serum albumin (BSA) or casein is commonly used. We have to choose the blocking agent carefully because LPS can bind proteins non-specifically. This process can inhibit binding of LPS-specific antibody to LPS and decrease the sensitivity of method. in this paper we describe an ELISA test for LPS in which normal goat serum is used for blocking. This modification increases the sensitivity of ELISA. This method is useful for detection of LPS (S, R form) and anti-LPS antibody reaction in serological cross-reaction studies.     

Criteria for the Selection of Materials for Implanted Electrodes

There are four criteria that must be considered when choosing material for an implanted electrode: (1) tissue response, (2) allergic response, (3) electrode-tissue impedance, and (4) radiographic visibility. This paper discusses these four criteria and identifies the materials that are the best candidates for such electrodes. For electrodes that make ohmic contact with tissues: gold, platinum, platinum–iridium, tungsten, and tantalum are good candidates. The preferred insulating materials are polyimide and glass. The characteristics of stimulator output circuits and the importance of the bidirectional wave- form in relation to electrode decomposition are discussed. The paper concludes with an analysis, the design criteria, and the special properties and materials for capacitive recording and stimulating electrodes. © 2003 Biomedical Engineering Society. PAC2003: 8754Dt, 8780Fe, 8768+z, 8719Nn     

Comprehensive assessment for serum treatment for single antigen test for detection of HLA antibodies

The single antigen test is widely used in the field of transplantation to determine the specificity of HLA antibodies. It will be beneficial to standardize the procedure of the single antigen test among HLA laboratories. It is not uncommon that single antigen testing on native sera fails to detect antibodies with very high concentrations. It has been shown that cleavage products of activated complement components may mask strongly binding antibodies in single antigen testing. To overcome inhibition by the activated complement products, sera are pretreated with ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), or heat inactivation before single antigen testing. However, no studies have been published to systemically compare the impact of these treatments on single antigen testing. The aim of this study is to understand the different effects these treatments may have on single antigen test results. We found that mean fluorescence intensity (MFI) obtained from sera treated with EDTA and heat inactivation were nearly identical, while DTT treatment was less potent to remove the inhibition. In addition, sera dilution did not further increase MFI of antibodies after EDTA treatment. Our results provide guidance to choose a pretreatment reagent for single antigen testing, and to compare studies obtained from laboratories using different treatments.     

Comparison of blocking agents for an ELISA for LPS

ELISA is a sensitive, specific, reproducible and fast method for detection of antigen-antibody reactions. In case of non-protein antigens as LPS, problems exist, such as poor proportion of coating to microplates, non-specific binding of antibodies to the plastic wells. These problems were resolved partially by Takahashi and co-workers using poly-L-lysine for coating of LPS antigens. To reduce non-specific binding, blocking agent, such as bovine serum albumin (BSA) or casein is commonly used. We have to choose the blocking agent carefully because LPS can bind proteins non-specifically. This process can inhibit binding of LPS-specific antibody to LPS and decrease the sensitivity of method. In this paper we describe an ELISA test for LPS in which normal goat serum is used for blocking. This modification increases the sensitivity of ELISA. This method is useful for detection of LPS (S, R form) and anti-LPS antibody reaction in serological cross-reaction studies.     

A Novel,Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination

Although malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 μl of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three false-negative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/μl. The assay''s simple work flow, high throughput, and sensitivity make it suitable for active malaria screening.     

Importance of object relations theories for development of capacity for mature love

We discuss Klein's, Winnicott's, and Mahler's object relational theories relevant for creating and maintaining the mature love relationship. The concept of love refers to the basic human relationship. The capacity for adult love involves the attainment of the relation towards the object as whole, satisfying the emotional needs of the self, including simultaneous tolerance of the specific needs of the object. It also involves the optimum resolution of anxiety related to schizo-paranoid and depressive positions and phases of separation and individuation. Primitive defense mechanisms, such as splitting, are replaced by more mature defense mechanisms, and primitive idealization is replaced by more mature idealization. The fusion with the object is reversible and helps in creating the experience of closeness with the partner, while the possibility of separation provides the possibility of recognizing and respecting the differences. Obstacles in the development of object relationships from pre-object to object phase, from symbiotic to separation and individuation phase can impair the capacity to love.     

Optimization of a method for purifying antibodies for immunofluorescent diagnosis of influenza

Efficiency and diagnostic adequacy of immunoglobulin isolation from rabbit immune serum by reiterated salting out with ammonium sulfate and subsequent separation of DEAE sephadex A-50 was evaluated. Electrophoresis and immunoelectrophoresis in agar gel demonstrated that the resultant immunoglobulin fraction is much more pure than after one salting out. It contains nothing more than antibodies to influenza virus taken for immunization, and after binding to the stain shows high fluorescent activity and induces but a moderate background nonspecific fluorescence in the subsequent preparations. In clinical trials of conjugates based on the new combined method of purification, influenza A or B could be correctly diagnosed in 97-98% cases.