| | Quality control for the validation of extracorporeal photopheresis process using the Vilbert–Lourmat UV-A irradiation’s systemAbstract In agreement with good practices for therapeutic use of human cells, quality control has to be performed to valid the process of extracorporeal photopheresis (ECP) with the Vilbert–Lourmat system. Since no protocol exists, we evaluated a technique based on the measurement of the inhibition of mitogen (PHA, Con-A, OKT3)-induced proliferation, in 164 procedures from 16 patients. Whatever the pathology, we observed a high proliferation rate in most samples, and we obtained over 90% ECP-induced inhibition in as many as 94% of the cases. Since this approach proved to be relevant regarding our objective, a protocol for the ECP process validation is proposed.
1. Introduction  Extracorporeal photopheresis (ECP), initially developed for the treatment of cutaneous T-cell lymphoma (CTCL) [1], [2], [3], is now currently employed for various hematological disorders with potential expansion of pathological T-lymphocyte clones [4], i.e. graft versus host disease (GVHD) [5], [6], transplant rejection [7], [8] and autoimmune diseases [9], [10], [11]. Two systems are presently used to achieve ECP, the UV-MATIC irradiator from Vilbert–Lourmat system and the Therakos system [12]. In the former technique, which is used in our cellular therapy center, mononuclear cells are collected by cytapheresis, then irradiated with ultraviolet-A (UVA, λ=365 nm) after the addition of a photosensibilizer, the 8-methoxy-psoralen (8-MOP), and next reinfused to the patient within 3 h [12]. Since this technique implies the ex vivo treatment of white blood cells, quality control is needed in order to validate the process. However, there is currently no consensus for any protocol. It is well known that UVA-activated 8-MOP forms covalent cross-links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation and leading to cell death [13]. Therefore, we carried out a study testing whether the measurement of the antiproliferative effect of ECP could be a reliable parameter for such an objective. In this work, we present the results of a systematic analysis of mitogen-induced proliferation inhibition for patients treated with the Vilbert–Lourmat system, and we propose a protocol for the validation of the extracorporeal irradiation process. 2. Material and methods 2.1. Patients A total of 164 processes for 16 patients were analyzed in this study (Table 1). ECP was the only treatment in five cases: four patients with T-cell malignancies, i.e. one case of CTCL, one case of CD8-chronic lymphocytic leukemia (CLL), two cases of CD4-nonHodgkin’s lymphoma (NHL), and one patient with severe autoimme disease (AD). A circulating T-cell clone was present in all patients, including those with NHL (respectively 40%, 95%, 36% and 22% malignant T-lymphocytes) and AD. The remaining 11 patients suffered from severe GVHD, and most of them also received immunosuppressive drugs in association with ECP. |
a
Number of cytapheresis.
b
Number of cases with no significant mitogenic response.
c
Number of cases with residual proliferation over 2000 cpm. |
2.2. ECP ECP cycles were performed using the Vilbert–Lourmat system [12]. Briefly, mononuclear cells were collected in a bag by treating about 1.5–2 whole blood mass by cytapheresis (Spectra cell separator, Cobe, USA). They were next sterilely transferred into an EVA bag specially adapted for irradiation (Maco-Pharma, France), and the volume was adjusted to 300 ml with sodium chloride solution (0.9%). Thereafter the 8-MOP, used as a water-soluble presentation, was added ex vivo at a final concentration of 333 ng/ml. Hematocrit was measured before dilution (STKS, Beckman-Coulter, USA), and recalculated for the bag to be irradiated, by the correcting dilution factor (=corrected hematocrit). The mononuclear cell suspension was exposed to the UVA source at 2 J/cm2 (UV-MATIC irradiator, Vilbert–Lourmat, France) through the EVA-bag. The UVA dose applied to cells was 2.5 J/cm2 when corrected hematocrit was higher than 5%. Irradiated mononuclear cells were then reinfused to the patient as soon as possible (within 3 h). Samples for laboratory control were collected both before (pre-ECP) and after irradiation (post-ECP). They were immediately sent to the laboratory or maintained in the dark at 4 °C until analysis. 2.3. Mitogen-induced proliferation, and ECP-related inhibition The most commonly used technique for measuring cellular proliferation is quantification of incorporated 3H-thymidine into newly synthetized DNA. Therefore, cells were washed in PBS by centrifugation at 200 g, for 10 min at 4 °C. On day 1, 105 lymphocytes in 200 μl of culture medium were seeded in a 96-well flat-bottomed plate (Falcon, Becton Dickinson). Culture medium was composed of RPMI-glutamax 1640 (Gibco) supplemented with 10% human serum AB, 100 IU penicillin, 100 μg/ml streptomycin (Roche), 1% (v/v) nonessential amino acids (BioWittaker), 1 mM sodium pyruvate (Gibco) and 50 μM 2-mercaptoethanol (Gibco). Mitogens were added at the beginning of the culture at the following concentrations: 2 μg/ml phytohemaglutinin (PHA) (Murex Diagnostic); 20 μg/ml concanavalin-A (Con-A) (Sigma); 0.1 μg/ml and 0.01 μg/ml CD3 (Orthoclone OKT3, Janssen-Cilag). All assays were performed in triplicate. A background proliferation control was included, consisting in lymphocytes cultured in medium alone. After a 3 day incubation at 37 °C in a humidified atmosphere with 5% C02, 1 μCi of 3H-thymidine (Amersham, France) was added into each well. DNA was then transferred onto filter paper 8 h later, using a multiwell harvester, and 3H-thymidine incorporation was measured in counts per minute (cpm) using a gaz-ionisation counter. Proliferation is calculated as the mean value of the triplicates, and inhibition as follows: { before ECP}. 3. Results The quality control we propose is based on the measurement of the antiproliferative effect of ECP. Since lymphocytes usually do not spontaneously proliferate, it was necessary to stimulate them in vitro. Therefore, we used mitogens that have the property to induce sustained proliferation of most T-lymphocytes. In the present work, we compared three of them: PHA and Con-A, which are very powerful activators leading to a high rate of 3H-thymidine incorporation, and OKT3, which induces more physiologic activation via the TCR-associated CD3 molecule [14]. This analysis was performed before and after ECP, thus allowing us to calculate its antiproliferative effect. 3.2. Post-ECP mitogen-induced proliferation: calculation of inhibition The effect of ECP on mitogen-induced lymphocyte proliferation can be evaluated using two parameters: residual proliferation (cpm) on the post-ECP sample, and % of inhibition. It is first noteworthy that mitogen did not induce any lymphocyte proliferation in most samples after the ECP process (Table 1): 3H-thymidine uptake was less than 2000 cpm in 90% of the post-ECP samples with the four conditions of mitogenic stimulation. It was over 2000 cpm in 12–14 samples from five to nine patients, depending on the mitogen (Table 1). The rate of the remaining proliferation was variable, ranging from 2047 cpm up to 26 050 cpm. Using the calculation of the inhibition of the mitogen-induced proliferation, a powerful antiproliferative effect of ECP could be demonstrated: more than 90% inhibition (mean=97%) was obtained for the four mitogenic conditions in 94% of the cases. Inhibition ranged between 70% and 90% in 4%, and it was less than 70% in 2% of the cases (Fig. 1). In most samples, all the results revealed to be concordant with regard to the four conditions of mitogen stimulation. 3.3. Characteristics of the cases with less than 90% inhibition The presence of at least one sample with less than 90% inhibitory effect of ECP could be observed in 10 patients out of 16. Of note, hematocrit in the irradiated bag (corrected hematocrit) was always over 2% when a partial inhibition was observed (Fig. 2). This observation is in agreement with the notion of UVA absorption by hemoglobin, leading to less efficient irradiation of mononuclear cells [15]. However, hematocrit was also higher than 2% in many post-ECP samples showing total inhibition. In fact, most of the haematological automates do not give exact values for red cells parameters, including hematocrit, when cellularity of the sample is very high (data not shown). So, such results might be interpreted with caution. Thus, we are presently investigating the conditions allowing correct appreciation of the red blood cell contamination. Otherwise, no defect along the process has been observed, such as problems with the UVA-lamp or the 8-MOP. 4. Discussion In agreement with good practices, quality controls have to be performed to valid processes implying mononuclear cells for therapeutic use. In the present work, we propose a protocol validating the ECP process using 8-MOP plus UVA-irradiation with the Vilbert–Lourmat system. This validation is based on the demonstration of an inhibitory effect of ECP on mitogen-induced proliferation [13]. Therefore, the measurement of 3H-thymidine incorporation is particularly interesting for several reasons. First, it is applicable even if ECP treatment is associated to powerful immunosuppressive drugs. Moreover we have never encountered so far mitogen unresponsiveness in patients with T-cell malignancies. Secondly, results are unambiguous: when ECP is performed in optimal conditions (hematocrit below 2% in the irradiated bag), as much as 99%±1% inhibition was obtained (min=94%, max=100%). In our hands, values below 90% were observed only when hematocrit in the irradiated bag was above 2% (eight samples). These results outline the importance of a correct appreciation of hematocrit. It is noteworthy that even the lowest values have not led to the identification of other technical problems. Lastly, there is no difficulty with this protocol: special shielding is not required for 3H-thymidine manipulation, as it emits low energy β particles; devices are usual in a cell-culture laboratory (laminar flow hood, CO2 incubator, cell harvester and radioactive counter); the technique is not expensive nor time consuming. It should be noticed that results are obtained only after 3 days, so they cannot represent an authorization to accredit the product to be transfused. They are only useful for a quality control of the process. In the present work, we tested three mitogens. Since all the results were concordant whatever the conditions in most samples, it is no longer necessary to study all of them. For the future, we suggest to systematically use PHA, because it gave the most important 3H-thymidine incorporation in the largest number of samples. It is also possible to go on testing Con-A, to confort the results with another mitogen. For the samples with no mitogen-induced proliferation, no other test is carried out, because this represents very rare event (1.2% with PHA). A further question is to define which threshold should let us consider a process as “abnormal”, and should lead to complementary investigations. We arbitrarily decided to set this “alerting” threshold at 70%. We postulate that, an inhibition comprised between 90% and 70% associated to an hematocrit higher than 2% are two related events, and no further investigations are necessary. When inhibition is below 70%, we cannot exclude other interfering problems, which have to be resolved. Of note, it is not known whether it corresponds to biologically inactive or less active products for the patient. Using the ECP-related inhibition, we consider that as few as 2% of the products have been prepared in “suspicious” conditions. Thus, we think that sampling for quality control must not be systematic, but must be included in a defined protocol. Therefore we propose the following sequence of analyses: (1) for the first cytapheresis collection of each patient, (2) when corrected hematocrit is over 2%, (3) every time a new lot of 8-MOP is used, and (4) according to a sampling protocol for a systematic control, with a frequency depending on the number of treated patients. For less than one cytapheresis a week, the control might be systematic. If procedures are more frequent, analyses could be done on the basis of one control for four procedures. Whenever the rate of inhibition of mitogen-induced proliferation is below 70%, extra samplings must be added for the two following procedures, and for the next procedure for the same patient; if those new results are correct, the usual protocol can be resumed. If two consecutive “abnormal” values are obtained for two different patients, each step of the process must be analyzed to detect the problem, and a reinforced control involving analysis of a sample from each procedure must take place, until the problem is resolved. The present work let us conclude that the evaluation of the inhibition of mitogen-induced proliferation is a reliable test to valid the process of irradiation in ECP. Acknowledgements We thank Agnès Colomer, Sylvie Glaizal, Martine Chauvet, Marie-Claire Legal and Danièle Riebel for their technical assistance. References [1].
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Biol. Med. 1989;62:621–638. a Laboratory of Immunology, UMTCT, Rhône-Alpes French Blood Establishment, site of Grenoble, 29 avenue du maquis du Grésivaudan, 38701 La-Tronche, France b R&D Laboratory, Rhône-Alpes French Blood Establishment, 38701 La-Tronche, France c Apheresis Department, UMTCT, Rhône-Alpes French Blood Establishment, 38701 La-Tronche, France d Quality Control Laboratory, UMTCT, CHU, 38700 Grenoble, France e R&D Laboratory, UMTCT, Rhône-Alpes French Blood Establishment, 38701 La-Tronche, France f Preperation Department, UMTCT, Rhône-Alpes French Blood Establishment, 38330 St Ismier, France g Department of Hematology, CHU, 38700 Grenoble, France h UMTCT, CHU, 38700 Grenoble, France i Laboratory of Immunology, Rhône-Alpes French Blood Establishment, 38701 La-Tronche, France j Laboratory of Immunology, CHU, 38700 Grenoble, France  Corresponding author. Tel.: +33-4-76-42-43-44; fax: +33-4-76-42-94-49
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