C. Weber¹

¹Millipore SA Dammstr. 19 CH-6301 Zug

New water norms 01.07.2006
CLSI (before NCCLS)
Clinical Lab Reagent Water (CLRW)

1.)Contaminations in water; Organics; Microorganisms; Contamination effects on analysis;
2.)Purification technologies comparison; new technologies;
3.)Effects on the analyzers; Interferences; examples
4.)Effects on other procedures
5.)Water Quality standards; comparison of the different standards
6.)Effects on the Validations of the Laboratory
7.)Solutions

B.K. Ballmer-Weber¹

¹Allergy Unit, Department of Dermatology, University Hospital Zürich, Gloriastr. 31, 8091 Zürich

The primary tools for the diagnosis of food allergy are skin prick testing (SPT) and in vitro determination of food specific IgE. Both diagnostic procedures are mainly based on the use of food extracts. Many commercial food extracts, however, lack appropriate biological standardization or a precise adjustment of the content of individual allergens for instance due to degradation of allergenic proteins during the extraction procedure [1]. This is particularly the case in pollen related food allergy since Bet v 1 homologous food proteins are prone to degradation. Inadequate quality of food derived allergen extracts often results in a low sensitivity of the extract based diagnostic procedure [2-3]. Moreover, even with well prepared food extracts, positive SPT or specific IgE results are frequently observed in the absence of clinical symptoms of allergy due to the phenomenon of subclinical sensitisation or allergen cross-reactivity.
It cannot be expected that the use of recombinant allergens will resolve the dilemma of sensitization not accompanied by clinical allergy, but improved clinical sensitivity is a realistic goal to be achieved.
Recombinant allergens in the diagnosis of food allergy might be used in different ways:
1) Utilizing the traditional extract based diagnostic approach but to determine additionally specific IgE to recombinant food allergens that are underrepresented in the commercial food extract.
This approach proved to be valid in birch pollen mediated soy allergy. When Gly m 4, the Bet v 1-related molecule in soybean, was applied for diagnostic purposes as an ImmunoCAP reagent, determination of specific IgE to Gly m 4 improved the diagnostic sensitivity in patients with combined birch and soy allergy from 45% (soybean extract-based test) to 96% [4].
2) Enhancing the sensitivity of the extract based diagnostic approach by supplementing the extract with the recombinant allergens, that are underrepresented in the extract (spiking of extracts). Spiking of hazelnut extract for instance by r Cor a 1, the Bet v 1 homologue in hazelnut, increased the diagnostic sensitivity from 84% (pure extract) to 100% (spiked extract) [5].
3)Determination of specific IgE to a whole panel of allergens derived from one source, which enables assessment of the sensitization profile in individual patients, a concept that has been defined as “Component Resolved Diagnostics” (CRD).
CRD might detect associations between subpopulations of specific IgE antibodies, measured by the use of individual allergen components, and clinically relevant aspects of the allergic disease such as severity of the allergic response or geographic differences in the sensitisation patterns. A recently published review provides a comprehensive summary on studies regarding CRD in food allergy [3].
4) Determination of specific IgE to mixes of recombinant allergens derived from one source, i.e. to a highly standardized “artificial” extract.
In a recent study in cherry allergy an ImmunoCAP containing a combination of recombinant cherry allergens (rMIX), i.e. rPru av 1 , rPru av 3 and rPru av 4, proved to have a sensitivity of more than 95% whereas the natural extract ImmunoCAP displayed a sensitivity of just 35% [6].
In conclusion: Recombinant allergens are becoming increasingly available and can be used to map, characterize and exploit the clinical significance of individual IgE reactivity profiles.

References
1.Van Ree R, Dorpema JW, Vieths S. Allergy vaccines: a need for standardisation in mass units of major allergen. Pharmeuropa Bio 2005; 2005:27-30.

2.Ballmer-Weber BK, Scheurer S, Fritsche P, Enrique E, Cistero-Bahima A, Haase T, Wüthrich B, Scheurer S. Component-resolved diagnosis with recombinant allergens in patients with cherry allergy. J Allergy Clin Immunol 2002; 110(1): 167-73.

3.Lidholm J, Ballmer-Weber BK, Mari A, Vieths S. Component-resolved diagnostics in food allergy. Curr Opin Allergy Clin Immunol. 2006; 6 :234-40.

4.Mittag D, Vieths S, Vogel L,Becker WM, Rihs HP, Helbling A, Wuthrich B, Ballmer-Weber BK. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004; 113:148-54.

5.Andersson K, Ballmer-Weber BK, Cistero-Bahima A, Östling J, Lauer I, Vieths S, Lidholm J. Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking. Allergy 2007; 62: 897-904.

6.Reuter A, Lidholm J, Ostling J, Scheurer S, Enrique E, Cistero-Bahima A, San Miguel-Moncin M, Ballmer-Weber BK, Vieths S. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. Clin Exp Allergy 2006; 36: 815-823.

M. Balerna¹

¹Ente Ospedaliero Cantonale, Clinical Chemistry Division, Department of Laboratory Medicine, 6500 Bellinzona, Switzerland

On principle, scientists active in Laboratory Medicine have a special status: coming from scientific or technological education, they are given the possibility to apply their knowledges to the human context and to diagnostic and therapeutic problems in particular. In our Country, their role is perhaps even more relevant than that of the Collegues working elsewhere in Europe: today, Switzerland has many private or hospital laboratories led by people with sound backgrounds in chemistry, biochemistry, pharmaceutics or biology who have passed in addition a federal, usually pluriannual mono- or pluri-disciplinary competence diplome (the so-called F.A.M.H. title).
From another point of view, it is not unusual that clinicians still look at scientists as a sort of “strange” (if not “dangerous”) people, too much prone to show their scientific skill - or cleverness ! - but unable in practice to understand what Medicine really is and needs. From a pluri-annual perspective it is however my opinion that MDs and PhDs do often not realize that they can be seen as “the two banks of a same river”, the patients constituting the water flowing between them. In such a context, the will of the patients has to come into consideration: patients simply expect that the clinicians and their collaborators (and therefore also the scientists) work together with one objective: that to improve their health.
This view of the things has strong ethical, cultural and practical consequences: it asks for a continuous improvement of the paradigm that clinicians and scientists have not only to do well their respective jobs, but also that they continuously have to meet in a sort of “hot pot” allowing the exchange of the mutual competences nourished by mutual respect as well as - if possible - of human warm-heartedness. In other words, “the river of patients” asks for a continuos and untiring “crossing the bridge” between Medicine and Science.
This issue assumes a particular relevance in the context of the education of the future generations of clinicians and scientists. The Author will discuss about the various possibilities to create “bridges” between Clinic and Science.

J. Bille¹

¹Clinical Microbiology and Infectious Diseases, University Hospital, Lausanne, Switzerland

Bacteremia and/or septicemia constitute a frequent and severe infection, particularly in intensive care units, with a high mortality (30%). Early treatment, in particular early antibiotherapy, is essential for prognosis.
Microbiological investigations are important to determine the etiology and establish the antibiotic susceptibility profile. Blood culture are still the cornerstone of this diagnosis, even if they have some limitations due to the patient (variable bacterial loads), to microbial determinants (various doubling time), or related to technical determinants (volume of blood , delay of incubation).
Molecular detection of microorganisms in blood at time 0, when blood is drawn, is very appealing but quite challenging. Molecular methods have been used mainly with blood cultures at time of positivity, to speed-up (4-6 h.) the identification, or to detect some molecular determinants of resistance such as the gene mecA for methicillin resistance among staphylococci or Van A/B for glycopeptide resistance among enterococci.
One of the most clinically relevant indications to use molecular methods in positive blood culture bottles at detection time is the rapid differentiation between S.aureus and coagulase neg. staphylococci, as well as the search for mecA gene. Other approaches than PCR involve in situ hybridization methods.
Detection of bacteria or fungi in the blood at time 0 has been tried for over 10 years, often as narrow spectrum (Neisseria, mycobacteria, Aspergillus), or more recently as broad-range PCR. The method is still confronted with many problems linked to the limited amount of bacteria, the potential presence of dead organisms and of contaminants during the venipuncture, and the presence of inhibitors.
The first commercially available PCR system to be used as broad-range PCR directly in blood specimens (Septifast Roche) has been evaluated in a few clinical settings so far (oncohematologic neutropenic febrile patients, ICU patients). Septifast detects in a shorter time (5-6 h.) as many or often more bacteria and fungi than blood cultures, some being potentially pathogens, other probable contaminants. The gold standard of positive blood culture should be challenged, of course. The system seems particularly promising when patients have previously received antibiotics, for the detection of fungi, and in patients persistently febrile while on antibiotics. Before we can really evaluate the benefits of this new system additional studies in different clinical settings are needed. The current version is very cumbersome. Many developments using chips micro arrays, nanotechnology, or calorimetry, are currently in development.
Today, classic automated blood cultures are still largely used, with many post-detection improvements aimed to speed-up the results both of identification and susceptibility testing. The ultimate goal is to establish in less than one hour the microbial etiology and susceptibility pattern of a sepsis episode using a simple POCT at the bedside.