Microsoft word - a09777en_a.doc

VersaLyse
REF A09777
100 tests; 100 mL 1 mL / test VersaLyse Lysing Solution Ready-for-use Formulation
Number of vials
Volume per test
1 mL (ready-for-use when used with no concomitant fixation) PRECAUTIONS
PREPARATION OF SAMPLES
This reagent is intended for the lysis of red 1. Do not use the reagent beyond the expiry The concentration of leucocytes in the sample blood cells in the preparation of biological must be less than 104 cells / µL (1010/L). If samples for flow cytometry analysis. VersaLyse 2. Do not store in the refrigerator, do not necessary, dilute in PBS to bring the leucocyte is compatible with all types of cytometers and is concentration to 5 x 103 / µL (5 x 109/L). used with or without washing after incubation, 3. Minimize exposure to light during incubation The erythrocytic concentration in the sample as long as properly calibrated fluorescent with fluorescent antibodies. must be less than 6 x 106/µL (6 x 1012/L), it is antibodies are used in the protocol. VersaLyse 4. Avoid microbial contamination of the recommended to dilute sample in PBS in order also makes it possible to treat samples even reagents, or false results may occur. to bring the erythrocytic concentration to when they have been washed earlier, in order 5. Formaldehyde is toxic and allergenic. It is to eliminate serum immunoglobulins for thought to be carcinogenic. Never pipette If both the leucocytic and erythrocytic by mouth and avoid all contact with the concentrations need adjusting, the highest PRINCIPLE
skin, mucosa, eyes and clothing. dilution must be used. The procedures use 6. All blood samples must be considered as 100 µL of a non-prediluted or pre-diluted The sample containing the red blood cells to be potentially infectious and must be handled sample per tube. lysed is exposed to the VersaLyse reagent for with care (in particular: the wearing of at least 10 minutes. The major active ingredient PROCEDURES
protective gloves, gowns and goggles). of VersaLyse is a cyclic amine which, in contact NOTE: The procedures below are valid for
7. Blood tubes and disposable material used with carbonic anhydrase present in red blood standard applications. Sample and/or for handling should be disposed of in ad cells, is transformed into a compound which is VersaLyse volumes for certain Beckman hoc containers intended for incineration. highly lytic for these cells. Coulter applications may be different. If such is This reagent is used with specific conjugated the case, follow the instructions on the antibodies, able to bind to leucocytes due to the Venous blood or bone marrow samples must be technical leaflet of the application. antigenic determinants they express. The taken using sterile tubes containing an EDTA A – Procedure without concomitant fixation
specific staining of leucocytes is achieved by salt as the anticoagulant. Preparation of the reagent
incubating the sample with the antibody or NB: Carefully read in the ANTICOAGULANT
No preparation is necessary. Use VersaLyse antibodies. The red cells are then removed by section, the conditions under which other directly from the vial. lysis and the leucocytes, unaffected by this anticoagulants can be used. PROCEDURE
process, are analyzed by flow cytometry. For each sample analyzed, in addition to the Preparations must be stored between 2 and The samples should be kept at room test tube, one control tube is required in which 8°C and analyzed rapidly using flow cytometry. temperature (18 – 25°C) and not shaken. The the isotypic control corresponding to the In the case of a delay of several hours before sample should be homogenized by gentle specific staining selected is added to the cells. analysis, they may be fixed with PBS containing agitation prior to taking the test sample. 1. In each test tube add the amount of 0.1 % formaldehyde (see the technical leaflet of The samples must be analyzed within 24 hours antibody recommended by the the IOTest®3 Fixative Solution – Ref. A07800 – manufacturer in order to stain 5 x 105 to prepare this PBS with 0.1 % formaldehyde). WASHED SAMPLES
In this case, prior washing is necessary. If you have to wash the samples before staining 2. In each control tube add the amount of the The flow cytometer measures light diffusion and followed by lysis of the red blood cells, it is isotypic control recommended by the the fluorescence of cells. It makes possible the essential to remove all the supernatant after the manufacturer to stain 5 x 105 leucocytes. delimitation of the population of interest within last centrifugation. An excess of washing buffer 3. Add 100 µL of the sample (prediluted or the electronic window defined on a histogram, on the cell pellet can reduce the effectiveness otherwise) to both tubes. Vortex the tubes which correlates the orthogonal diffusion of light (Side Scatter or SS) and the diffusion of 4. Incubate following the conditions set out in narrow-angle light (Forward Scatter or FS). METHODOLOGY
the technical leaflet for the antibodies Other histograms combining two of the different NECESSARY MATERIAL NOT SUPPLIED
parameters available on the cytometer, can be • Sampling tubes and material necessary for 5. Add 1 mL of VersaLyse and vortex used as an aid in the gating stage depending immediately for 1 second. on the application chosen by the user. • Automatic pipettes with disposable tips for 6. Incubate at least 10 minutes at room The fluorescence of the delimited cells is 10, 20, 100 and 1000 µL. temperature (18 – 25°C), protected from analyzed in order to distinguish the positively- • Plastic haemolysis tubes. stained events from the unstained ones. The • Calibration beads: Flow-Set™ In the case of a procedure without results are expressed as a percentage of Fluorospheres (Ref. 6607007). washing, the tubes are ready for cytometric positive events in relation to all the events acquired by the gating. • Specific fluorescent antibodies. • Isotypic controls. NB: If the preparations need to be washed,
STORAGE AND STABILITY
• Buffer (PBS: 0.01 M sodium phosphate; do not analyze before performing steps 7 VersaLyse is stored between 18 and 25°C. If a 0.145 M sodium chloride; pH 7.2). vial of VersaLyse has been kept inadvertantly in • Fixation reagent: for example IOTest 3 a refrigerator, bring it to room temperature (18 – 7. Centrifuge for 5 minutes at 150 x g at room Fixative Solution (Ref. A07800). 25°C) and wait at least one hour before use. With the vial closed, the reagent is stable up to 8. Remove the supernatant by aspiration. • Automatic agitator (Vortex type). the expiry date shown on the vial. 9. Resuspend the cell pellet in 3 mL of PBS. • Flow cytometer. After opening, the reagent is stable for 90 days. 10. Centrifuge for 5 minutes at 150 x g at room 11. Remove the supernatant by aspiration and resuspend the cell pellet in: A09777EN_A 2003-11-21 − 0.5 mL or 1 mL of PBS plus 0.1% of ACD and heparin give good results with all formaldehyde if the preparations are to procedures except the procedure without be kept for more than 2 hours and less concomitant fixation when it is followed by than 24 hours. (A 0.1% formaldehyde washing. With these anticoagulants, we Cytometer n° 2: PBS can be obtained by diluting 12.5 µL recommend the procedure with concomitant of the IOTest 3 Fixative Solution (Ref. fixation, followed by washing. A07800) at its 10X concentration in 1 mL ANALYSIS
The erythrocyte residues can give rise to a mL of PBS without diffraction signal of the narrow angle light formaldehyde, if the preparations are to LIMITATIONS OF THE TECHNIQUE
("Forward Scatter" or FS) greater than that be analyzed within 2 hours. 1. Flow cytometry may produce false results obtained with other commercial lysis reagents. if the cytometer has not been aligned OTE: In all cases, keep the preparations at
As a result, the usual adjustment (or fixed by between 2 and 8°C and protected from light. perfectly, if fluorescence leaks have not default) of the ("threshold") discriminator on this been correctly compensated for and if the B – Procedure with concomitant fixation
parameter may be too weak for VersaLyse. regions have not been carefully Preparation of the reagent
It is therefore advisable to adjust the cytometer Extemporaneously prepare the "Fix-and-Lyse" during the first moments of the acquisition of a 2. Accurate and reproducible results will be mixture by adding 25 µL of UNDILUTED
typical preparation. In the case of whole lysed obtained as long as the procedures used IOTest 3 10X* Fixative Solution (Ref. A07800) to blood for example, it is advisable first to are in accordance with the technical 1ml of VersaLyse. increase progressively the value of the insert leaflet and compatible with good Vortex the tubes for 3 to 5 seconds. discriminator until the leucocyte populations are laboratory practices. (*) NB: in the context of the "Fix-and-Lyse"
visible on a histogram of the FS type versus 3. In the case of a hyperleucocytosis, dilute mixture, the IOTest 3 Fixative solution (Ref. SS ; then to carefully adjust the amplification of the specimen in PBS so as to obtain 5 x A07800) is used at a forty fold concentrated the FS and SS signals to obtain a distribution of 109 leucocytes/L. solution and not as a 10X solution (nominal the leucocytic populations identical to that of 4. In the case of a polyglobulinaemia, dilute figures 1, 2 and 3. the specimen in PBS so as to obtain 5 x Prepare a sufficient volume of the "Fix-and PERFORMANCE
1012 red blood cells/L. Lyse" mixture depending on the number of 5. VersaLyse must be brought to room samples to be lysed (1 mL of mixture per tube). PURITY AND LYMPHOCYTIC RECOVERY
temperature (18 – 25°C) before use. Procedure
Purity and lymphocytic recovery have been 6. Verify the preparations using the naked For each sample analyzed, in addition to the evaluated according to the recommendations of eye to assess the efficacy of lysis. If they test tube, one control tube is required in which the CDC (1). The blood of 10 healthy donors are cloudy or if the light diffraction the cells are mixed with the isotypic control sampled in K3EDTA was labeled with a mixture histograms are unusual, lysis may be corresponding to the specific staining selected. of monoclonal antibodies CD45-FITC and 1. In each test tube, add the amount of CD14-PE. The mean values of the recovery 7. The erythroblasts may be incompletely antibody recommended by the and purity as well as the range for the different lysed and appear on a light diffraction manufacturer to stain 5 x 105 leucocytes. procedures are given in the following tables: histogram in the same location as the 2. In each control tube, add the amount of isotopic control recommended by the Without washing
8. Acetazolamide, an inhibitor of carbonic manufacturer to stain 5 x 105 leucocytes. Without concomitant
With concomitant fixation
fixation
anhydrase can completely inhibit the 3. Add 100 µL of the sample (prediluted or Recovery
Recovery
action of VersaLyse. otherwise) to both tubes. Vortex the tubes 96.4 92.3 96.6 91.6 9. In certain disease states, such as severe renal failure or haemoglobinopathies, 4. Incubate following the conditions set out in 90.1 / 94.3 95.4 / 98.1 90.1 / 92.6 lysis of red cells may be slow, incomplete the technical leaflet for the antibodies used. With washing
or even impossible. In this case, it is 5. Add 1 mL of the "Fix-and-Lyse" mixture and Without concomitant
With concomitant fixation
recommended to isolate mononucleated vortex immediately for 1 second. fixation
cells using a density gradient (Ficoll, for 6. Incubate at least 10 minutes at room Recovery
Recovery
example) prior to staining. temperature (18 – 25°C), protected from 95.6 94.0 96.2 97.2 90.3 / 97.3 95.4 / 97.5 92.7 / 99.4 In the case of a procedure without washing, the tubes are ready for cytometric analysis. INTRA-LABORATORY REPRODUCIBILITY
See the Appendix for examples and references. NB: If the preparations need to be washed,
On the same day and on the same cytometer, do not analyze before performing steps 7 to 31 measurements of the percentage of CD3+ lymphocytes were carried out on the same TRADEMARKS
The Beckman Coulter logo, COULTER,
7. Centrifuge for 5 minutes at 150 x g at room sample (whole blood from a healthy donor). The EPICS, EXPO, Flow-Set, IOTest, System II, lysis procedure used was concomitant fixation XL, Cytomics are registered trademarks of 8. Remove the supernatant by aspiration. in the absence of washing. The results obtained Beckman Coulter Inc. 9. Resuspend the cell pellet in 3 mL of PBS. are summarized in the following table: FACSCalibur is a registered trademark of BD 10. Centrifuge for 5 minutes at 150 x g at room Biosciences and Company. 11. Remove the supernatant by aspiration and resuspend the cell pellet in 0.5 mL or 1 mL MANUFACTURED BY:
of PBS plus 0.1% of formaldehyde if the preparations are to be kept for more than 2 INTER-LABORATORY REPRODUCIBILITY
a Beckman Coulter Company hours and less than 24 hours. (A 0.1% On the same day and on the same sample 130 avenue de Lattre de Tassigny formaldehyde PBS can be obtained by (whole blood from a healthy donor), 31 B.P. 177 – 13276 Marseille Cedex 9 diluting 12.5 µL of the IOTest 3 Fixative measurements of the percentage of CD3+ Solution (Ref. A07800) at its 10X lymphocytes were carried out by two Customer Services: (33) 4 91 17 27 27 concentration in 1 mL of PBS). technicians and the preparations analyzed www.beckmancoulter.com These preparations may be kept 24 hours using two different cytometers. The lysis between 2 and 8°C and protected from light. procedure used was concomitant fixation in the absence of washing. The results obtained are The tripotassium salt of EDTA (K summarized in the following tables: the best results, irrespective of the methodology Cytometer n° 1: used, with or without washing after lysis. A09777EN_A 2003-11-21 APPENDIX TO REF A09777
EXAMPLES
REFERENCES
The graphs below are biparametric representations (Forward Scatter 1. Nicholson, J.K.A., Hearn, T.L., Cross, G.D., White, M.D., "1997 vs. Side Scatter) of a normal whole blood sample lyzed with VersaLyse revised guidelines for performing CD4+ T-cell determinations in (Ref. A09777) using the no fixation concomitant to lysis and no wash persons infected with human immunodeficiency virus (HIV)", 1997, MMWR 46, RR-21-29. 2. Ashmore, L.M., Shopp, G.M., Edwards, B.S., "Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage", 1989, J. Immunol. Methods, 118, 209-215. 3. Macey, M.G., McCarthy, D.A., Milne, T., Cavenagh, J.D., Newland, A.C., "Comparative study of five commercial reagents for preparing normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry", 1999, Communications Clinical Cytometry, 38, 153-160. 4. Macey, M.G., McCarthy, D.A., van Agthoven, A., Newland, A.C., "How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures", J. Immunol. Methods, 204, 175-188. 5. Vuorte, J., Jansson, S-E., Repo, H., "Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay", 2001, Cytometry, 43, 290-296. Figure 1: Acquisition is with a BECKMAN COULTER® CYTOMICS™ FC 500 flow cytometer. Analysis is with CYTOMICS™ RXP Analysis software. Figure 2: Acquisition and analysis are with a COULTER® EPICS® XL™ flow cytometer equipped with System II™ software. Analysis is with EXPO™ Cytometer software (Ref. 6605434). Figure 3: Acquisition is with a BD Biosciences FACSCaliburTM flow cytometer. Analysis is with EXPO™ Cytometer software (Ref. 6605434) A09777EN_A 2003-11-21

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