Strategy for cost-effective laboratory testing

Despite the objective and quantitative nature of laboratory information, medical diagnosis still largely rests on clinical information. This paper makes three points to indicate ways of improving the clinical usefulness of laboratory information: (1) Tests should be evaluated from a “diagnostic” rather than from a “technical” viewpoint, since efficacy is defined differently from these two viewpoints. (2) It is proposed to relate the value associated with testing to the combined costs due to “false positives,” “false negatives,” and “late results.” (3) The design of an optimal test strategy requires less emphasis on problems associated with data recognition and more emphasis on problems associated with data interpretation. Multivariate analysis is a particularly powerful method of data interpretation and has facilitated the successful development of clinical medicine despite the limitations of its data base. This analytical procedure can also be successfully applied to data reduction in the laboratory.     

Cytotoxic flow-cytometric crossmatches (flow-tox): a comparison with conventional cytotoxicity crossmatch techniques.

Detection and avoidance of donor-reactive antibodies in the sera of potential organ transplant recipients is key to a successful transplant outcome. Techniques of antibody detection that use flow cytometry are more sensitive than those that rely upon a visual determination of cytotoxicity. However, as conventionally performed, flow-cytometric crossmatches do not distinguish between cytotoxic (complement fixing) and noncytotoxic antibodies because both types of antibodies can bind to a cell and be detected by laser-activated fluorochrome photon emission. In 1989 we described two techniques for detecting cytotoxic antibodies using flow-cytometric techniques [1]. In 1990, we expanded the application of these new techniques that we called flow cytotoxicity assays or "Flow-Tox" [2]. Flow-Tox crossmatches demonstrate an increase in both sensitivity and specificity over conventional cytotoxicity crossmatches.     

Informatics-based,highly accurate,noninvasive prenatal paternity testing

PurposeThe aim of the study was to evaluate the diagnostic accuracy of an informatics-based, noninvasive, prenatal paternity test using array-based single-nucleotide polymorphism measurements of cell-free DNA isolated from maternal plasma.MethodsBlood samples were taken from 21 adult pregnant women (with gestational ages between 6 and 21 weeks), and a genetic sample was taken from the corresponding biological fathers. Paternity was confirmed by genetic testing of the infant, products of conception, control of fertilization, and/or preimplantation genetic diagnosis during in vitro fertilization. Parental DNA samples and maternal plasma cell-free DNA were amplified and analyzed using a HumanCytoSNP-12 array. An informatics-based method measured single-nucleotide polymorphism data, confirming or rejecting paternity. Each plasma sample with a sufficient fetal cell-free DNA fraction was independently tested against the confirmed father and 1,820 random, unrelated males.ResultsOne of the 21 samples had insufficient fetal cell-free DNA. The test correctly confirmed paternity for the remaining 20 samples (100%) when tested against the biological father, with P values of <10⁻⁴. For the 36,400 tests using an unrelated male as the alleged father, 99.95% (36,382) correctly excluded paternity and 0.05% (18) were indeterminate. There were no miscalls.ConclusionA noninvasive paternity test using informatics-based analysis of single-nucleotide polymorphism array measurements accurately determined paternity early in pregnancy.Genet Med 2013:15(6):473–477     

Public interest in predictive genetic testing, including direct-to-consumer testing, for susceptibility to major depression: preliminary findings

The past decade has seen rapid advances in the identification of associations between candidate genes and a range of common multifactorial disorders. This paper evaluates public attitudes towards the complexity of genetic risk prediction in psychiatry involving susceptibility genes, uncertain penetrance and gene–environment interactions on which successful molecular-based mental health interventions will depend. A qualitative approach was taken to enable the exploration of the views of the public. Four structured focus groups were conducted with a total of 36 participants. The majority of participants indicated interest in having a genetic test for susceptibility to major depression, if it was available. Having a family history of mental illness was cited as a major reason. After discussion of perceived positive and negative implications of predictive genetic testing, nine of 24 participants initially interested in having such a test changed their mind. Fear of genetic discrimination and privacy issues predominantly influenced change of attitude. All participants still interested in having a predictive genetic test for risk for depression reported they would only do so through trusted medical professionals. Participants were unanimously against direct-to-consumer genetic testing marketed through the Internet, although some would consider it if there was suitable protection against discrimination. The study highlights the importance of general practitioner and public education about psychiatric genetics, and the availability of appropriate treatment and support services prior to implementation of future predictive genetic testing services.     

A rapid and accurate assay for assessing the cytotoxicity of viral proteins

A fluorescence-based assay is presented for measuring the cytoxicity of viral proteins added exogenously to cells. The assay is based on the use of two fluorescent dyes, calcein-AM and ethidium homodimer (EtD-1) to specifically stain living and dead cells respectively and employs fluorescence activated cells sorting (FACS) to achieve a rapid and accurate measurement of the cytotoxic capacity of a potential viral toxin. The assay has been developed using the group B homologue (ADRV-NSP4) of the NSP4 enterotoxin encoded by Group A rotaviruses but should be applicable to assaying any viral protein exhibiting cytotoxic activity.     

Comparison of implantation and cytotoxicity testing for initially toxic biomaterials

To evaluate the predictive value of cytotoxicity testing, the present study compares the in vivo tissue responses to in vitro cytotoxicity before and after implantation. Material toxicity was caused by addition of the toxic substance Zincdiethyldithiocarbamate (ZDEC) that is used as a standard for in vitro cytotoxicity testing. Polyurethane discs with the addition of 0.5% or 1% ZDEC as well as nontoxic discs were inserted in the abdominal wall of rats for 1 day up to 6 weeks. After explantation the foreign body response was analyzed immunohistochemically. An in vitro reanalysis of the explanted reference materials (RMs) revealed remaining high concentrations of toxic compounds after 1-week implantation, whereas no toxicity was seen after 6 weeks implantation. This was reflected in the foreign body response where a significantly thicker capsule and more inflammatory cells were seen at 1 week for the toxic implants. Over time, with decreasing toxicity, these differences disappeared. Test samples that only were subjected to in vitro extraction with water did not elute toxic compounds to the same extent as the in vivo conditions. It is concluded that many clinically useful implant materials may be unnecessarily rejected due to the results of in vitro tests.     

Bloodless testing for microporous membrane oxygenator failure: a preliminary study

The use of a bloodless solution and high pressure to accelerate microporous membrane oxygenator (MMO) failure was investigated. It was hypothesized that albumin acts as a wetting agent, contributing to plasma leakage through the membrane, and that high MMO outlet pressure accelerates the process. Three MMO, B-Bentley BCM-40 (n = 7), M-Medtronic Maxima (n = 4), and S-Sarns 16310 (n = 7) were tested at 37 +/- 2 degrees C using three identical closed recirculating circuits and four conditions: 1) Lactated Ringer solution (LR) with MMO outlet pressure (Pmo) 750 mmHg; 2) LR + albumin (4 g/100 ml), Pmo 150 mmHg; 3) LR + albumin, Pmo 300 mmHg; and 4) LR + albumin, Pmo 750 mmHg. "Blood" flow and gas flow were maintained at 2 l/min. Failure was indicated when Na+ was detected in the effluent of the MMO exhaust gas. There were no failures without albumin in the solution. B and M showed no signs of failure under any of the test conditions at 78 hours. S failed at (mean +/- SEM) 4.9 +/- 1.0, 12.1 +/- 0.2, and 19 hours for conditions 4, 3, and 2 respectively. Preceding failure, inlet gas pressure increased more than eightfold (27 +/- 1 to 224 +/- 34 mmH2O). These preliminary results are similar to previous findings with blood and suggest that high MMO outlet pressure and the presence of albumin may promote plasma breakthrough for S. The combination may provide a basis for an accelerated bloodless test for MMO compatibility with long-term respiratory support.     

A method for freezing sheep lymphocytes prior to cytotoxicity testing

The two stage freezing technique was adapted for use with sheep lymphocytes Parameters investigated were lymphocyte concentration, composition of thawing medium and DMSO concentration. The main difference between this and earlier published techniques is the use of a high DMSO concentration (17%). The technique adopted was both simple and reliable. It consistently gave lymphocyte viabilities of 95% or more in thawed cell suspensions. The procedure is apparently without effect on lymphocyte antigen expression and thus appears very suitable for use in microlymphocytotoxicity tests.     

Frequency of biochemical hypothyroidism in sera referred for autoantibody testing.

We examined sera submitted for autoantibody testing for thyroid microsome antibodies (TMA), elevated thyroid-stimulating hormone (TSH), and free thyroxine concentrations. The frequency of TMA in antinuclear antibody-positive sera was higher (19%) than that in antinuclear antibody-negative sera (12%). Elevated TSH concentrations in serum and subnormal thyroxine concentrations in serum were associated with the presence of TMA; TMA titer and the frequency of elevated TSH concentrations were also associated with the presence of TMA.     

Polymers with tunable toxicity: a reference scale for cytotoxicity testing of biomaterial surfaces

A series of copolymers, with varying ratio di-methylamino-ethylmethacrylate (DMAEMA) and methyl-methacrylate (MMA), was designed as a potential scale for cytotoxicity. These copolymers were characterized for toxicity of their surface. The surfaces of washed copolymers display increasing toxicity with increasing DMAEMA content. The toxicity was observed for three different cell-types, namely mouse fibroblasts, human endothelial cells and human osteoblast-like cells. With an increasing toxic surface, cell growth was inhibited as was indicated by the proliferation marker Ki-67. Staining for F-actin revealed that with increasing DMAEMA, cells adopted a more and more round morphology, resulting in decreased surface-contact area. Immuno-staining for phospho-tyrosine or vinculin demonstrated gradual loss of focal adhesions on increasingly toxic surfaces. Surprisingly loss of focal adhesions coincided with an increase in paxillin and vinculin protein, indicating cells try compensating for loss of adhesion. This series of copolymers may have potential as a cytotoxicity scale. They provoke cellular responses ranging from highly toxic to completely non-toxic, with some showing intermediate toxicity.     

Improving the performance of virtual crossmatch results by correlating with nationally-performed physical crossmatches: Obtaining additional value from proficiency testing activities

Purpose

When donor specific HLA antibodies (DSA) are identified, the predictive value of whether a certain strength of reactivity (mean fluorescence intensity, MFI) leads to a positive crossmatch is uncertain. To determine this, we compared the DSA MFI results we generated locally for nationally distributed proficiency samples against the percentage of other laboratories reporting a positive crossmatch.

Attitudes towards future testing for bipolar disorder susceptibility genes: a preliminary investigation

The discovery of susceptibility genes for the major psychiatric illnesses may lead to the development of presymptomatic and prenatal tests. In a preliminary study we assessed the attitudes of 147 bipolar patients, 90 attendees at their family doctor (GP) and 32 psychiatrists to the possible development of genetic tests for bipolar disorder susceptibility genes. Our results suggest that patients and the public will look favourably on the development of presymptomatic (but not prenatal) testing for bipolar disorder susceptibility genes. Psychiatrists, who will have to administer such tests, appear significantly more cautious.     

Inhibition of antibody-dependent cellular cytotoxicity by artificial immune complexes and pathological sera.

Detection of immune complexes by inhibition of antibody-dependent cellular cytotoxicity (ADCC) is based on the principle that soluble complexes can compete with target cell-bound antibody for receptors (FcR) on cytotoxic lymphocytes. The objective of this study was to define a cytotoxicity system for the determination of soluble immune complexes in the sera of patients with inflammatory bowel disease (IBD). For this purpose, the conditions under which soluble complexes of rat serum albumin (RSA) and rabbit anti-RSA inhibited human K-cell mediated lysis of sensitized Chang cells were examined, on the assumption that the behaviour in the system of circulating immune complexes putatively present in inflammatory bowel disease, is similar to that of artificial immune complexes. Inhibition of ADCC by a standard amount of artificial complex in different normal human sera was relatively uniform provided that the final concentration of the latter did not exceed 10% of the culture medium. In the absence of extraneous complexes, the effect of both normal and IBD sera on ADCC varied widely. Differential inhibition of ADCC by sera from patients with IBD and normal subjects was thus expressed as a function of ADCC in a standard batch of foetal bovine serum (FBS). Under these conditions differences between pathological (n = 51) and normal (n = 52) sera were highly significant (P less than 0.001), which could not be explained by the presence in the patients' sera of HL-A antibodies reactive with the effector cells, nor by a deficit in nutritional support of ADCC. The absence of a correlation between inhibition of ADCC and total serum IgG or IgM inferred that inhibition was attributable to immune complexes in the IBD sera. The limitations of this assay for assessment of the incidence of immune complexes in pathological sera are discussed.     

A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing

Background

Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column.

Methods

Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test.

Results

The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods.

Conclusions

Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.