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1. The ReaLyse lysing solution contains Diethylene glycol and Formaldehyde.
Formaldehyde is a potential carcinogen and causes irritation to the eyes and
skin. Ingestion of Diethylene glycol can be fatal. Wear suitable protective
ReaLyse Lysing Solution clothing. If swallowed, seek medical attention immediately.
10X Concentrate 2. All blood specimens are considered biohazards. Handle them as if they are
Catalog No. 25237 00 capable of transmitting infection and dispose off with proper precautions in
accordance with governmental regulations. Figure 1. SSC vs. CD45 dot plot; CD3 vs. CD4 dot plot obtained with DryTri
TSTAT CD3/CD4/CD45 reagent.
1. INTENDED USE 3. The addition of the precise volume of deionized water is critical to
obtain correct concentration of the 1X working solution. Use a calibrated
The ReaLyse solution is an erythrolytic reagent intended for lysing red
pipette and operate according to the manufacturer’s instructions.
blood cells to enable a lyseno wash protocol in applications that involve
immunofluorescent staining of human peripheral blood leucocytes. 4. The use of temperatures, incubation and vortexing times other than those
The ReaLyse solution is recommended for use in lysis and fixation of specified in the corresponding product inserts may give erroneous results.
cell preparations stained with ReaMetrix reagents such as Dry Tri TSTAT
Dilution Instructions
CD3/CD4/CD8 and CD3/CD4/CD45, ReaPan 34845, ReaPan B27, and ReaPan
Dilute the 10X concentrate 1:10 with the deionized water at room
S34 which are to be analyzed by flow cytometry. Figure 2. SSC vs. CD3 dot plot; CD8 vs. CD4 dot plot obtained with DryTri
temperature (20° to 25°C) to prepare 1X working lysing solution according to
2. BACKGROUND TSTAT CD3/CD4/CD8 reagent
Table 1. Mix well. /
The quality of immunophenotypic analysis of cell preparations is The dualparameter light scatter plots (SSC versus FSC, Figure 3)
Table 1: Dilution Instructions for Preparation of 1X ReaLyse Lysing (Working)
evaluated on the basis of a high signal to noise ratio. The noise quotient of a illustrates the expected leucocyte spatial distribution of a normal K3EDTA
solution
cell preparation is predominantly caused by the large percentage of red blood anticoagulated blood sample lysed with ReaLyse lysing solution using lyse no
cells (erythrocytes) relative to the fraction of white blood cells (leucocytes) in Required Volume of 1X Working solution (mL) 5 10 15 20 25 wash procedure. Acquisition and analysis are with a BD FACScan flow
Volume of 10X Concentrate (mL) 0.5 1.0 1.5 2.0 2.5 cytometer equipped with Cell Quest Pro software.
the same. The removal of erythrocytes by lysis greatly reduces the background
Volume of Deionized Water (mL) 4.5 9.0 13.5 18.0 22.5
noise, thus allowing for easier analysis of the target cell populations which have
been stained by fluorophore tagged antibodies. Storage and Handling
Use of ReaLyse solution in the processing steps for analysis of the Store the reagent at room temperature in a dry place. Do not use the reagent
sample preparations to be analyzed using the abovementioned reagents after the expiry date on the label.
(Section 1) is highly recommended. This ensures the sufficient lysis of Red Do not freeze the ReaLyse Lysing solution. Follow the instructions given
Blood Cells (RBCs) and fixing of the immuno stained population thus allowing on the container label to ensure good shelf life.
for samples to be analyzed in a lysenowash protocol. The diluted working solution is stable for one month when stored in a glass
3. PRINCIPLE OF LYSIS container at room temperature. Do not use this reagent if discoloration Figure 3. SSC vs. FSC dot plot
occurs or a precipitate forms.
When whole blood is added to the monoclonal antibody reagent, the The following plots depict peripheral blood sample immuno stained
The ReaLyse lysing solution contains Formaldehyde which is highly
fluorochromelabeled antibodies in the reagent bind specifically to leucocyte with ReaPan 34845 reagent and treated with ReaLyse lysing solution prior to
volatile. Do not leave the lid in a loose or open position. The lid should be
surface antigens. The stained samples are then treated with ReaLyse lysing analysis on a FACS Calibur flow cytometer (Figure 4).
tightly sealed after use.
solution which results in simultaneous lysis of red blood cells under gentle
hypotonic conditions and fixation of the leucocytes resulting in a leucocyte Note Follow appropriate instructions as given in the respective product inserts
suspension substantially free of red blood cells and suitable for flow cytometric for information on instruments, sample collection and sample processing.
analysis.
5. PERFORMANCE CHARACTERISTICS
4. REAGENT
Representative Acquisition Plots of ReaLyse Lysing Solution with
The ReaLyse lysing solution is formulated as a buffered solution and ReaMetrix Reagents
contains Formaldehyde and Diethylene glycol.
Depicted below are acquisition plots of peripheral blood samples incubated
It is provided as a 10X concentrate. A working solution of 1X dilution
with DryTri TSTAT CD3/CD4/CD45 reagent (Figure 1) and DryTri TSTAT
of this concentrate is to be used for the lysis of cell preparations before analysis
CD3/CD4/CD8 reagent (Figure 2); processed with ReaLyse lysing solution and
by flow cytometry. To obtain the working solution, the dilution of the 10X
analyzed on a FACScan flow cytometer.
concentrate is performed in deionized water at room temperature (20°C to
25°C) using reference table1.
Figure 6. Correlation of Absolute Lymphocyte Counts (CD45+) determined 2. Retain samples in VACUTAINER blood collection tubes at room
using the DryTri TSTAT CD3/CD4/CD45 reagent lysed with ReaLyse lysis temperature (20° to 25°C) prior to staining and lysing. Refer to the reagent
solution compared to Absolute Lymphocyte Counts determined using package insert for maximum storage times after collection.
Hematology Analyzer. (n=26) 3. Samples with nucleated red blood cells may show incomplete lysis of red
blood cells. This may also occur when assaying blood samples from patients
with certain hematologic disorders in which red cells are difficult to lyse, as
in sicklecell anaemia, thalassemia, spherocytosis, etc.
Figure 4. SSC vs. CD45 dot plot; SSC vs. CD3 dot plot; CD3 vs. CD4 dot plot;
4. Reagents should be used before the expiration date printed on the reagent
CD3 vs. CD8 dot plot obtained with ReaPan 34845 reagent bottle.
Performance Data 5. Results obtained with flow cytometry may be erroneous if the instrument is
misaligned, compensation spillovers are not correctly compensated or the
The following performance data for lysing solution were generated by
gate is improperly set.
performing clinical validation on DryTri TSTAT CD3/CD4/CD45 reagent.
6. Reproducible results will be obtained as long as the procedure used is in
Accuracy
accordance with this package insert and with good laboratory practice
The absolute counts for CD45+, CD3+ and CD4+CD3+ Tcells were Figure 7. Correlation of Absolute CD3+ cell Counts determined using the Dry guidelines.
determined using DryTri TSTAT CD3/CD4/CD45 reagent. Aliquots of the Tri TSTAT CD3/CD4/CD45 reagent stained and lysed with ReaLyse lysing
same blood samples were stained and lysed with the ReaLyse lysis solution and solution compared to a commercially available lysing solution. (n=30)
a commercially available lysis solution. The samples were analyzed using a BD 7. REFERENCES
FACScan™ flow cytometer. The plots in the figure 5, 6, 7, 8 indicate that the 1. Mandy FF, Nicholson JK, McDougal JS. Guidelines for performing
results from both the lysis solutions are equivalent. singleplatform absolute CD4+ Tcell determinations with CD45 gating
for persons infected with human immunodeficiency virus. Centre for
Disease Control and Prevention. MMWR Recomm Rep 2003 Jan
31;52(RR2):113.
CUSTOMER SUPPORT INFORMATION
ReaMetrix India Pvt. Ltd.
50B, II Phase, Peenya Industrial Area
Peenya, Bangalore560058, India
Ph: 918028378693/5,
Fax: 918041172451
Email: info@reametrix.com
www.reametrix.com
Rev No. 2.0, 15Apr09