Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor ß-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation.

Basic haemoglobinopathy diagnostics in Dutch laboratories; providing an informative test result.

INTRODUCTION: After a first survey in 2001, the Dutch Association of Hematological Laboratory Research (VHL) advised its members to adopt a basic protocol for haemoglobinopathy carrier detection and to provide genetic information with all positive results to allow health-care professionals to inform carriers about potential genetic risks. This article reports on the compliance with these recommendations and their consequences. METHODS: Clinical chemists of all 106 Dutch laboratories were invited to answer a survey on patient population, diagnostic techniques used, (self-reported) knowledge, use and effect of the additional information. RESULTS: The average increase in diagnostic output was over 60% and the recommended basic protocol was applied by 65% of the laboratories. Over 84% of the laboratories reported to be aware of the additional recommendations and 77% to be using them. Most laboratories with limited diagnostic requests were still sending their cases to other laboratories and included the genetic information received from these laboratories in their diagnostic reports. The effect of information on subsequent 'family analysis' was estimated to be between 26 and 50%. CONCLUSIONS: The present study shows an increase in diagnostic potential for haemoglobinopathy over the last decade, especially in the larger cities. Low 'family testing' rates were mostly found in areas with lower carrier prevalence or associated with local reluctance to pass the information to carriers. In spite of a dramatic improvement, too many carriers are still not informed because of lack of awareness among health-care providers and more education is needed.

Iron depletion: an ameliorating factor for sickle cell disease?

We report some observations from our laboratory practice that might be important for the treatment of sickle cell disease (SCD). We describe data from two cases indicating that iron depletion might have a beneficial effect diminishing the formation of HbS in favor of HbF, possibly reducing the severity of the disease. We believe that it would be worthwhile to monitor the course of the disease comparing cases with identical genotypes with and without iron depletion, and we advise to consider chelation therapy to reduce iron overload in patients with SCD.

Fixed versus variable dose of prothrombin complex concentrate for counteracting vitamin K antagonist therapy.

BACKGROUND: Although prothrombin complex concentrate (PCC) is often used to counteract vitamin K antagonist (VKA) therapy, evidence regarding the optimal dose for this indication is lacking. In Dutch hospitals, either a variable dose, based on body weight, target INR (international normalised ratio) and initial INR, or a fixed dose is used. AIM/OBJECTIVES: In this observational, pilot study, the efficacy and feasibility of the fixed dose strategy compared to the variable dosing regimen, is investigated. MATERIALS AND METHODS: Consecutive patients receiving PCC (Cofact®, Sanquin, Amsterdam) for VKA reversal because of a major non-cranial bleed or an invasive procedure were enrolled in two cohorts. Data were collected prospectively in the fixed dose group, cohort 1, and retrospectively in the variable dose regimen, cohort 2. Study endpoints were proportion of patients reaching target INR and successful clinical outcome. RESULTS: Cohort 1 consisted of 35 and cohort 2 of 32 patients. Target INR was reached in 70% of patients in cohort 1 versus 81% in cohort 2 (P = 0·37). Successful clinical outcome was seen in 91% of patients in cohort 1 versus 94% in cohort 2 (P = 1·00). Median INR decreased from 4·7 to 1·8 with a median dosage of 1040 IU factor IX (F IX) in cohort 1 and from 4·7 to 1·6 with a median dosage of 1580 IU F IX in cohort 2. CONCLUSION: This study suggests that a fixed dose of 1040 IU of F IX may be an effective way to rapidly counteract VKA therapy in our patient population and provides a basis for future research.

Vaccine-induced enhancement of viral infections.

Examples of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis have been documented for infections by members of different virus families. Several mechanisms, many of which still are poorly understood, are at the basis of this phenomenon. Vaccine development for lentivirus infections in general, and for HIV/AIDS in particular, has been little successful. Certain experimental lentiviral vaccines even proved to be counterproductive: they rendered vaccinated subjects more susceptible to infection rather than protecting them. For vaccine-induced enhanced susceptibility to infection with certain viruses like feline coronavirus, Dengue virus, and feline immunodeficiency virus, it has been shown that antibody-dependent enhancement (ADE) plays an important role. Other mechanisms may, either in the absence of or in combination with ADE, be involved. Consequently, vaccine-induced enhancement has been a major stumble block in the development of certain flavi-, corona-, paramyxo-, and lentivirus vaccines. Also recent failures in the development of a vaccine against HIV may at least in part be attributed to induction of enhanced susceptibility to infection. There may well be a delicate balance between the induction of protective immunity on the one hand and the induction of enhanced susceptibility on the other. The present paper reviews the currently known mechanisms of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis.

European Communities Confederation of Clinical Chemistry Working Group on Accreditation: past, present and future.

During the past 10 years, many activities have taken place in the field of quality systems and accreditation in medical laboratories. Each country in Europe has a slightly different approach. The Working Group on Accreditation of the European Communities Confederation of Clinical Chemistry (EC4) tries to support harmonisation of these efforts. For this purpose, they edited the Essential Criteria for quality systems of medical laboratories and supported the forthcoming International ISO standard "Quality management for the medical laboratory". At this moment, a Model Quality Manual is nearly ready for publication. The next items are setting up criteria for auditing the quality system and criteria for the accreditation process.

Quantitative and qualitative analysis of haemoglobin variants using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) has been introduced into the clinical chemistry laboratory because of its range of potential applications. In this paper, we evaluate an alkaline CZE method for the quantification of HbA2 and HbF and also assess the combination of the alkaline CZE method with an acid CZE method for the determination of haemoglobin variants in an automated fashion. Correlation of the HbA2% determined between the HbA2-CZE method (alkaline conditions) and the Helena Sickle Thal Quick Column method was good (r = 0.91). The correlation between the HbF% determined by the HbA2-CZE method and by the alkaline denaturation method was acceptable (r = 0.81). The HbA2-CZE method was able to identify a large number of haemoglobin variants. The variants HbC and HbE or HbS and HbD, however, had the same characteristics under alkaline conditions and could therefore not be discriminated from each other. The identification of these overlapping variants could be accomplished by the analysis of the blood specimens in combination with the HbA1c-CZE method (acid conditions). We conclude that the presented applications for capillary zone electrophoresis can be used for quantitative and qualitative analysis of haemoglobin variants.

Fecal continence after rectocele repair: a prospective study.

Combined transvaginal/transanal rectocele repair was performed in series of 89 consecutive women (mean age 55, range 35-81 years) with obstructed defecation due to a rectocele with a depth of more than 3 cm. The impact of this procedure on anal sphincter pressure and continence status was evaluated prospectively. Anorectal manometry was carried out before and after surgery (at 3, 6, 12, and 24 months). The following measurements were performed: maximal anal resting pressure (MARP), maximal anal squeeze pressure (MASP), and rectal sensory perception including first initial sensation, urge to defecate, and maximum tolerable volumes (MTV). The outcome was successful in 71% of patients with respect to symptoms such as the need for straining at defecation, manual assistance, feelings of incomplete evacuation, sense of rectal fullness, constipation, abdominal pain, and the use of laxatives. However, after rectocele repair seven patients experienced deterioration in fecal continence, and dyspareunia developed in 41% of the sexually active patients. Manometric studies revealed a significant decline in mean of 18% of MARP and 16% of MASP. In contrast to MASP, MARP gradually improved during the follow-up period. Distending volumes required for initial sensation and urge to defecate did not change after the procedure. MTV values were significantly lower 3 and 6 months after rectocele repair than those before and 24 months after surgery. MARP and MASP values after surgery did not differ between patients with impaired and those with normal continence. In conclusion, transvaginal/transanal rectocele repair is beneficial for patients with obstructed defecation; however, care should be taken in sexually active patients, and patients at risk of developing fecal incontinence.

Usefulness of EC4 essential criteria for quality systems of medical laboratories as guideline to the ISO 15189 and ISO 17025 documents. European Community Confederation of Clinical Chemistry (EC4) Working Group on Harmonisation of Quality Systems and Accreditation.

Many medical laboratories have made a start with the introduction of quality management systems. However, it is still not clear against which standards such systems should be measured. The existing ISO and CEN standards do not cover essential aspects of medical laboratories. The publication of the EC4 Essential Criteria has stimulated the development of the ISO/Draft International Standard 15189. This standard seems adequate for our type of laboratories. However, it is not easy to read. The EC4 Essential Criteria could well serve as a guide, covering additional aspects, e.g. on total quality management and budget management as required in the EFQM model, that are not (yet) included in the ISO standard. In the present article the EC4 Essential Criteria are cross-referenced with two new international ISO standards, ISO/FDIS 15189 and ISO/FDIS 17025, the latter being the successor of ISO guide 25 and EN 45000. Both new ISO documents are in compliance with the new ISO 9000:2000 standard.

Treatment of enterocele by obliteration of the pelvic inlet.

PURPOSE: Enterocele is defined as a herniation of the peritoneal sac between the vagina and the rectum. This hernial sac contains either sigmoid colon or small bowel. It is well known that enteroceles are associated with symptoms of pelvic discomfort. It is unclear whether enteroceles contribute to evacuation difficulties. Controversies also exist regarding their treatment of choice. The aim of the present prospective study was to evaluate the impact of obliteration of the pelvic inlet on evacuation difficulties and on symptoms of pelvic discomfort. METHODS: From October 1994 to August 1996 20 females (median age, 53; range, 41-73 years) with symptomatic enterocele diagnosed on evacuation proctography underwent obliteration of the pelvic inlet with a nonabsorbable Mersilene mesh. All patients presented with pelvic discomfort, characterized by feelings of prolapse (n=20), pelvic pressure (n=16), lower abdominal pain (n=13), and false urge to defecate (n=15). Symptoms of obstructed defecation were noted in 15 patients. Six months after repair, evacuation proctography with opacification of the small bowel and the vagina was repeated. RESULTS: The median duration of follow-up was 25 (range, 10-34) months. A persistent or recurrent enterocele was observed in none of the patients. All symptoms of pelvic discomfort disappeared except feelings of a false urge to defecate, which persisted in 27 percent of cases. Symptoms of obstructed defecation persisted in all patients with evacuation difficulties. CONCLUSIONS: In patients with pelvic discomfort enterocele should be considered as a possible causative factor. It is unlikely that this abnormality contributes to the problem of obstructed defecation. In patients with a symptomatic enterocele, obliteration of the pelvic inlet with a Mersilene mesh is an adequate treatment.

Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection.

Cats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizing antibodies were detected in a Crandell feline kidney cell-based neutralization assay, but not in a neutralization assay based on primary peripheral blood mononuclear cells. Despite the induction of these FIV-specific responses, vaccinated cats were not protected. Instead, accelerated virus replication was found, an observation similar to what previous experiments using other vaccine candidates have shown. Here, the results of the present study are discussed in the light of enhancement of lentivirus infections as a complicating factor in lentivirus vaccine development.

Accelerated viraemia in cats vaccinated with fixed autologous FIV-infected cells.

We have vaccinated cats with fixed autologous FIV infected PBMC to determine whether autologous presentation of antigen is capable of inducing a protective immune response against homologous challenge. To this end autologous PBMC were infected with a FIV molecular clone (19k1). When infection was established, cells were inactivated by dialysis against paraformaldehyde. Upon vaccination, cats developed a virus specific immune response as measured by ELISA against the Gag protein of FIV. No antibodies against the envelope protein were detected with a peptide ELISA. Virus neutralizing antibodies however could be detected with a neutralization assay based on infection of CrFK cells, but not in an assay based on infection of primary T-cells. Although vaccination led to the induction of these virus-specific immune responses, vaccinated cats were not protected against homologous challenge but showed an accelerated viraemia upon infection. This was shown both by PCR and cell-associated viral load. The possible mechanisms underlying this observation are discussed in this paper.

Additional Essential Criteria for Quality Systems of Medical Laboratories. European Community Confederation of Clinical Chemistry (EC4) Working Group on Harmonisation of Quality Systems and Accreditation.

Essential Criteria for Quality Systems of Medical Laboratories have been published recently by the European Community Confederation of Clinical Chemistry (EC4) Working Group on Harmonisation of Quality Systems and Accreditation. The Essential Criteria address the majority of critical aspects of quality management in the medical laboratory. They have been accepted by the EC4 General Assembly and are endorsed by the Forum of European Societies for Clinical Chemistry (FESCC). However, a supplement to the Essential Criteria was necessary, addressing two aspects, which are only partly covered by the Essential Criteria: the management of resources and point of care testing. Thus, the EC4 Working Group on Harmonisation of Quality Systems and Accreditation has decided to formulate Additional Essential Criteria for Quality Systems of Medical Laboratories, directed at the issues of management of resources and point of care testing. Criteria on management of resources address financial aspects, information logistics and acceptance by clients. Criteria on point of care testing address responsibilities, education of non-laboratory staff and operational aspects. The Additional Criteria are supplementary to the previously published Essential Criteria and should be read as an integral part of these.

Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection.

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms + vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms + FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms + vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms + FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.

Salmonella typhimurium aroA recombinants and immune-stimulating complexes as vaccine candidates for feline immunodeficiency virus.

Two experimental feline immunodeficiency virus (FIV) vaccines were tested, either alone or in combination, in four groups of cats (A-D). One vaccine (SL3261-FIV) was composed of live attenuated Salmonella typhimurium aroA (SL3261) strains expressing the capsid (Gag) and part of the envelope (Env) proteins of FIV. The other was composed of FIV Gag and Env proteins incorporated into immune-stimulating complexes (iscom-FIV). Cats of group A were immunized four times with SL3261-FIV. Cats of group B were immunized twice with SL3261-FIV and then twice with iscom-FIV. Cats of group C were immunized twice with SL3261 expressing the B subunit of cholera toxin (SL3261-CtxB) and then twice with iscom-FIV. Cats of group D, which served as negative controls, were immunized twice with SL3261-CtxB and then twice with iscom into which the Gag and Env proteins of simian immunodeficiency virus (SIV) had been incorporated (iscom-SIV). Two weeks after the last immunization, all cats were challenged with FIV. At this time, cats immunized with iscom-FIV (groups B and C) showed strong plasma antibody responses to Gag and Env, whilst these responses were weak or undetectable in the cats immunized four times with SL3261-FIV (group A). Seven weeks after FIV challenge, Env-specific antibody responses had increased considerably in cats of all groups except group A. The mean virus loads in the cats of this group proved to be lower than those of the other groups at all time points, indicating partial protection.

Role of defecography in predicting clinical outcome of rectocele repair.

PURPOSE: The aim of this study was to evaluate the role of defecography in predicting clinical outcome of rectocele repair. METHODS: Between January 1988 and July 1994, 74 consecutive patients (median age, 54 (range, 35-81) years) with a rectocele and symptoms of obstructed defecation were studied prospectively. After preoperative evaluation by a standardized questionnaire, physical examination, and defecography, a combined transvaginal/transanal rectocele repair was performed. At follow-up, all patients had defecography. Long-term results were qualified by an independent observer after a median follow-up of 58 (range, 14-89) months as "excellent," "good," or "poor." RESULTS: Rectocele repair was considered excellent in 37 patients and good in 13 patients. Defecography six months after surgery did not show persistent or recurrent rectocele in any of the patients. Size of the rectocele, barium-trapping in the rectocele, internal intussusception, rectal evacuation, and perineal descent did not appear to influence clinical outcome. Radiologic evidence of anismus did not correlate with long-term results of rectocele repair. CONCLUSIONS: Combined transanal/transvaginal repair of rectocele is an efficient therapy in patients with obstructed defecation. Various defecographic parameters (size of rectocele, internal intussusception, rectal evacuation, perineal descent, radiologic signs of anismus) do not appear to influence clinical outcome of surgery. The main value of defecography is the objective demonstration of rectocele and any associated abnormalities such as an enterocele preoperatively and again in objective assessment of the postoperative results.

Essential criteria for quality systems in medical laboratories.

The introduction of total quality systems in medical and clinical laboratories and accreditation of these laboratories is gaining more and more interest. In several countries laboratories have set up quality systems, and accrediation schemes are also operating. The standards of these schemes have much in common although several differences exist. There exists uncertainty in several countries on the choice of a system. Laboratory specialists are confronted with a new way of thinking concerning the management and daily practice of their laboratories. It is not clear, which standards should be used as a basis, and certainly not how to interpret such standards. Particulary in the European Union, harmonisation of criteria for quality systems is desirable. In the present paper, the document entitled "Essential Criteria for Quality Systems in Medical Laboratories" is presented. The document has been accepted in the general Assembly of the European Communities Confederation of Clinical Chemistry (EC4) and by the working group on Good Laboratory Services of the European Council on Laboratory Medicine (ECLM). The criteria in the document are focussed on the particular situation of medical laboratories, including pre- and post-analytical aspects. Reference is made, where applicable, to EN 45001, ISO 9001 and ISO guide 25 draft 3.