User talk:Gerd Egger, Md

REQUEST FOR COOPERATION
THE PMN MIGRATION TEST

THE PMN MIGRATION CHAMBER

The problem

Polymorphonuclear leukocytes (PMN; neutrophil granulocytes) represent the foremost line of host defence against bacteria and fungi. A fundamental element of their functional repertoire is their ability to active locomotion in the tissues of the organism, to migration. Migration deficiencies lead to infections, while migratory overreaction can cause self-damage of the organism. Therefore, the measurement of PMN migratory capacity is an essential concern in medical research and clinical medicine.

History

Since about 1988, Egger and coworkers are engaged in the development of a method of an easy, fast and reliable estimation of PMN migration. The method is based on membrane filters as a substrate for PMN migration that simulate well the meshwork of natural tissues. The criterion for an estimation of PMN migration is how many PMNs have migrated how far into a filter under standardized conditions. A “migration chamber” using membrane filters as a tissue substitute was first described by BOYDEN. The “Boyden chamber”, however, has several disadvantages that hamper wide application in the clinical routine: time-consuming filling of the containers with test agents, PMN separation from whole blood, troublesome, arbitrary and inaccurate estimation of the results. Egger and coworkers have tried to eliminate the disadvantages of this type of migration chamber and reduced the elements and the size of the device to a minimum, while care was taken to improve the accuracy and reproducibility of the results. A task in parallel was to develop an automated evaluation of the results. The work was supported by two Austrian government grants. In the later granted study, the companies CLINICO, IMATEC Image Analysis Systems, and OLYMPUS Optical Co were involved.

The method

aims at an easy, fast and reliable estimation of PMN migration. PMN migration is measured in a migration “chamber”. The one-way, ready-to use device consists of a 26 x 76 mm plastic substrate to which chemoattractant depots are attached, followed by a 3 µm pore-size membrane filter topped by silicon rings. A routine unit comprises two verum chambers containing the chemoattractant N-formyl-met-leu-phe (FMLP) in solid form (though any chemoattractant or test substance soluble in water can be used) and two blank control chambers. Duplication increases the accuracy of measurements. Fresh whole blood is diluted with saline solution and pipetted into the silicon rings. The chemoattractant dissolves in the blood suspension and diffuses into the membrane filter. The PMNs follow the chemotactic gradient and penetrate into the filter. The blank controls reflect the non-stimulated, spontaneous migration.

Why using fresh whole blood?

Storage longer than one hour or cell separation activate the PMNs and alter their reactivity to stimuli (the “priming”). Results obtained under such conditions have to be considered as handling artefacts. The use of fresh whole blood preserves the PMNs near their genuine, diagnostically relevant priming state. Moreover, the use of fresh whole blood saves time, working power and money.

Further preparation

After 15 min incubation at 37°C, cell migration is stopped by fixation, the red blood cells adherent to the filter surfaces are lysed and the PMNs in the filters are stained. After drying, the filters are made transparent by soaking them in immersion oil. For an entire test, 24 µL blood are sufficient. The test can be made with venous or capillary blood.

Evaluation of migration

can be done by two ways: ·	The “leading front” of the immigrant PMN bulk and/or the PMN numbers in a certain focal plane (e.g. in 20 µm depth) are determined visually in a normal light microscope with an oil immersion objective. ·	The immigrant PMNs are counted in an automated image analyser in consecutive layers of 7 µm throughout the depth of a filter. The image is recorded by a commercial CCD 768x494 pixel black-and-white camera. The program reproduces the 3-dimensional situation in the filters and registers numbers and distribution of the cells and, on the basis of the white blood count, calculates statistical data. The results are shown numerically and graphically on a printed sheet. The expenditure of work is confined to the fixation of the chambers on the microscope stage, input of the patients’ data and white blood cell counts, and pushing the “start” button. The stage used in the previous analyser prototype had positions for four migration chambers that were evaluated successively. Stages with eight positions are possible and available. The image analyser was developed in cooperation with IMATEC Image Analysis Systems. During the development of the hard- and software until maturity more than 2200 migration tests were evaluated.

Values representative for PMN migration are the reactivity to FMLP estimated by the share of migratory PMNs in their blood total and their depth of penetration into the filters, and the relation: reactivity to the chemoattractant and to blanks.

From blood puncture until the printed results, a migration test can be done within one hour.

CLINICAL RELEVANCE OF THE PMN MIGRATION TEST

Some application fields of the method are shown in the publication list. Beyond that, we have an ample quantity of unpublished studies and observations.

The first study applying the migration test was done with juvenile rheumatoid arthritis and was published 1994. Since then, a series of various medical topics was investigated, and the clinical relevance of the test was checked. At the beginning, evaluation of the results was done visually. Later on, the image analyser was used. During the period of development, the assay was progressively improved, whereby the improvement focussed on a simplification of the device and on a reduction in the blood amounts necessary for the test. Moreover, some adaptations facilitating the automated evaluation had to be made. However, the basic principle of the chamber, namely the migration filter and FMLP deposited in solid form, remained the same. The hitherto experience with the PMN migration test comprises an estimated 5000 samples (visual estimation and analyser counting).

The following clinical fields were studied:

Surgical stress, severe multiple trauma, wound healing after surgical trauma, organ transplantation, burns, severe infection and sepsis, rheumatoid arthritis, juvenile rheumatoid arthritis, lupus erythematodes, atherosclerosis, premature preterm rupture of membranes, hormone therapy during in-vitro fertilisation, intensified chemotherapy followed by G-CSF therapy, infections in infants.

Summarising the results of these studies

Despite the differences in the clinical aspects, a common trend can be assessed: Impairment of PMN migration capacity is a risk factor for bacterial and/or fungal infections, whereby the critical parameters are the degree and the duration of PMN migratory impairment. Defective PMN migration occurs several days before clinical manifestation of infection and, therefore, is a predictive marker for subsequent infections. Thus, infections can be “predicted”, or, more accurately expressed, the risk for infections can be defined with the chance to prevent them by early therapeutic measures. The aim must not be treatment, but prevention of infection!

In studies comprehensive enough for statistics (organ transplantation, severe trauma) we found a hit rate of predicting or excluding infections (specificity/sensitivity) between 80 and 90%. Remarkably, patients with markedly impaired PMN migration may show white blood counts in the normal range or even elevated values. On the other hand, patients with autoimmune diseases often exhibit extremely increased PMN migration. Immunosuppressive therapy routinely applied in these patients can dramatically suppress PMN migration and thus may provoke infections.

Clinical outlooks

Fields of application of the method are: Patients and persons at risk of infections by impairment of the immune system: Severe trauma, severe surgical trauma, burns, stress (physical/psychical strain, sport), AIDS. Immunosuppressive therapies: autoimmune diseases, organ transplantation, tumor-chemotherapy, steroid therapy

The method can facilitate medical decisions ( for instance fitness for surgery or when to start or to discontinue antibiotic treatment) and may be an aid for monitoring the course of diseases and the efficacy of therapies.

The standardisation of migration chambers and laboratory handling and the use of calibrated image analysis renders the method independent of individual arbitrariness and allows the comparison of results all over the world. So, the method can be integrated in an evidence-based medicine.

Comparison with other markers of immune activity

In parallel with PMN migration, we have also checked: PMN reactive oxygen species (ROS) release, PMN actin cytoskeleton polymerisation degree, body temperature, white blood count, blood sedimentation rate; blood levels of C-reactive protein, PMN elastase, myeloperoxidase, neopterin, malondialdehyde, fibrinogen, cortisol, lactate, procalcitonin, plasma antioxidative power, IL6, IL8, VCAM1. None of these parameters were found to be predictive for infections; in general, their increase and decrease paralleled the clinical appearance and the intensity of the inflammatory event. In some specific clinical questions indeed a combination of PMN migration with some of these parameters (PMN elastase, ROS release) was favourable and increased the power of evidence.

A more comprehensive description of the theoretical background and the different steps of development of the assay can be found in Egger G, Mitterhammer H, Smolle KH. Current Trends Immunol 2004, Vol.6, 149-176

The problem: The development of a migration device for commercial use, while being in full progress, had to be stopped since the financing company withdrew all supports during to the European financial crisis in 2004. The project could not be restarted, and the team of manufacturers fell apart.

Question:

Which working group/company is interested in continuing and finishing this work that had to be interrupted half on its way to completion? We/I can give free access to the details of how to prepare/produce this migration device, and we place our clinical experiences to your disposal.

Contact me please via E-mail.