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CD34/CD133 Enumeration Kit

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CD34/CD133 Enumeration Kit CD34/CD133 Enumeration Kit For in vitro diagnostic use. REF 170-070-709 7 50 Sufficient for 50 tests with 10 cells Miltenyi Biotec B.V. & Co. KG Friedrich-Ebert-Str. 68 51429 Bergisch Gladbach Germany +49 2204 8306-8484 www.miltenyibiotec.com Contents 1. General information 2 1.1 Intended use 2 1.2 Reagents and contents 2 1.3 Materials required but not provided 3 1.4 Background information 4 1.5 Principle of the CD34/CD133 Enumeration Kit 5 2. Warnings and precautions 6 3. Protocol 7 3.1 Sample requirements 7 3.2 Important notes 8 3.3 Preparation of solutions 9 3.4 Staining of CD34+/CD133+ cells 9 4. Flow cytometric data acquisition and analysis 11 4.1 General description 11 4.2 Description of the detailed gating strategy 13 4.3 Data acquisition and analysis 21 4.4 Analysis of results using counting particles 22 5. Quality control 27 6. Reverse pipetting technique 27 7. Performance data 28 8. Limitations 31 9. References 31 10. Glossary of symbols 34 33513v03 | 2020-05 1 General information 1. General information 1.1 Intended use Determination of the absolute cell number of CD34+ and CD34+/CD133+ hematopoietic progenitor cells by flow cytometry in various human blood products, such as whole blood, leukapheresis harvest, bone marrow, cord + + blood and all fractions of CD34 and CD133 cells separated using a CliniMACS System. 1.2 Reagents and contents Components 1 mL CD34/CD133 Staining Cocktail: cocktail consisting of anti-CD45-FITC (clone: 5B1; isotype: mouse IgG2a), anti-CD34-APC (clone: AC136; isotype: mouse IgG2a), and anti-CD133/2-PE (clone: 293C3; isotype: mouse IgG2b). 1 mL CD133 Control: cocktail consisting of anti-CD45-FITC (clone: 5B1; isotype: mouse IgG2a), anti-CD34-APC (clone: AC136; isotype: mouse IgG2a), anti-CD133/2-PE, and anti-CD133/2 pure (clone: 293C3; isotype: mouse IgG2b). 2×10 mL 10× Red Blood Cell Lysis Solution ammonium chloride based lysis solution 2 mL 7-AAD Solution (52.5 µg/mL) 50 Sufficient for 50 tests with 710 cells Product format All components, except 10× Red Blood Cell Lysis Solution, are supplied in buffer containing stabilizer and 0.05% sodium azide. 2 33513v03 | 2020-05 General information Store protected from light at +2 °C to +8 °C. Do not freeze. The use-by date is indicated on the vial label. For in-use stability at +2 °C to +8 °C storage temperature refer to the use-by date indicated on the vial label. Do not use the reagent after the use-by date. Disposal Chemical residues and remains should be routinely handled as special waste. This must be disposed in compliance with anti-polution and other laws of the country concerned. To ensure compliance contact relevant authorities or an approved waste-disposal company. 1.3 Materials required but not provided • Micropipettes with disposable tips: variable micropipettes with volume ranges of 10–100 µL and 100–1000 µL. • Flow cytometer able to detect 4-color fluorescence (488 nm argon laser, 635 nm red diode laser) and equipped with appropriate computer hardware and software. • Vortex mixer • Double-distilled water • BD Trucount™ tubes (BD cat. no. 340334) • Buffer for optional dilution steps: Prepare a PEB (PBS/EDTA/BSA) buffer containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA) or human serum albumin (HSA), and 2 mM EDTA, e.g., by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold +2( °C to +8 °C). Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced 33513v03 | 2020-05 3 General information by other proteins, such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use. 1.4 Background information The CD34 antigen is a single-chain transmembrane glycoprotein, expressed on human hematopoietic stem and progenitor cells constituting a small subpopulation of bone marrow cells and peripheral blood cells. The antigen is absent on fully differentiated hematopoietic cells, such as normal peripheral blood lymphocytes, monocytes, granulocytes, erythrocytes, and platelets. After severe damage, for example, after myeloablative conditioning, the hematopoietic system can be reconstituted by transplantation of allogenic or autologous CD34+ hematopoietic progenitor cells. Clone AC136 recognizes a class III epitope of the CD34 antigen. The CD133 antigen is a 5-transmembrane cell surface antigen with a molecular weight of 117 kDa. It is expressed on a subset of CD34 bright stem and progenitor cells in human fetal liver, bone marrow, cord blood, and peripheral blood but is not found on mature blood cells1. In contrast to the CD34 antigen, CD133 is not expressed by late progenitors, such as pre-B-cells, CFU-E, and CFU-G2,3. CD133 has also been found to be expressed on circulating endothelial progenitor cells and fetal neural stem cells as well as on other tissue-specific stem cells, such as renal, prostate, and corneal stem cells4-6. Clone 239C3 recognizes epitope 2 of the CD133 antigen. CD34+ and CD34+/CD133+ cells may have various therapeutic uses. CD34 and CD133 enriched stem cell grafts have been employed in autologous7,8 and allogenic transplantation both in the haploidentical9-11 as well as the HLA- matched setting12. The high potential for hematopoietic engraftment of isolated CD133+ cells has been shown in NOD/SCID repopulation assays13,14. CD34 and CD133 enriched cell fractions have also been used as starting fraction for ex vivo expansion of hematopoietic progenitor cells from cord blood15-18. 4 33513v03 | 2020-05 General information CD133+ stem cells can also be utilized in non-hematological applications, such as regenerative medicine. CD133 is expressed on stem/progenitor cells of different tissues including bone marrow, peripheral blood, cord blood, and liver. CD133+ stem cells have been shown to harbor the capability to differentiate into cell types of various tissues, for example, endothelial cells, neural cells, and hepatocytes. CD133+ stem cells have come into focus in non- hematological applications especially in ischemic heart diseases19-26. 1.5 Principle of the CD34/CD133 Enumeration Kit The gating strategy of the CD34/CD133 Enumeration Kit is based on the ISHAGE guidelines27 for simple and standardized enumeration of CD34+ cells. By employing anti-CD45-FITC, anti-CD34-APC, and anti-CD133/2-PE the kit allows the identification of CD45+ leucocytes, of CD34+ hematopoietic progenitor cells, which are dim for CD45+ fluorescence and show a low side scatter and of CD34+/CD133+ hematopoietic progenitor cells, which are also CD45dim with a low side scatter. Additionally, for each sample a control sample is stained with the CD133 control containing anti-CD45-FITC, anti-CD34-APC, anti-CD133/2-PE and anti-CD133/2 pure antibody to block any CD133 staining. This is required for the proper setting of the relevant gate during analysis. The kit is suitable for single platform technique²⁸, enabling the absolute cell count determination by analysis of commercially available counting particles present in BD Trucount™ tubes. The CD34/CD133 Enumeration Kit has been designed for use with flow cytometers able to detect 4-color fluorescence (488 nm argon laser, 635 nm red diode laser) and equipped with appropriate computer hardware and software. Optionally, dead cells can be excluded from the analysis by addition of the DNA stain 7-aminoactinomycin D (7-AAD), which is also included in the kit. 7-AAD diffuses through the cell membrane of dead cells and intercalates with their DNA. 33513v03 | 2020-05 5 Warnings and precautions 2. Warnings and precautions • Analysis results obtained by the use of the CD34/CD133 Enumeration Kit shall never be the sole basis for diagnosis and/or therapy of patients with hematological malignancies. • The interpretation of the results is under the full responsibility of the user. • To verify the analysis results every determination should be repeated. • In case of unexpected results repeat the measurement or contact Miltenyi Technical Support +49 2204 8306-8484. • For all handling, consideration of good laboratory practice (GLP) regulations is recommended. • The use of the CD34/CD133 Enumeration Kit is restricted to trained and qualified personnel only. • The reagent should not be used if signs of leakage are observed. • All biological specimens and all materials that come into contact with blood and blood products must be treated as infectious material. Regulations for the treatment and disposal of infectious material must be followed. • The staining cocktails of the CD34/CD133 Enumeration Kit contain sodium azide (NaN3), a chemical highly toxic in pure form. However, at product concentrations, it is not classified as hazardous. Sodium azide may react with lead and copper plumbing to form highly explosive buildups of metal azides. Upon disposal, flush with large volumes of water to prevent metal azide build-up in plumbing. Safety guidelines must be observed. • The CD34/CD133 Enumeration Kit contains a vial of 7-AAD solution, a chemical highly toxic in pure form. However, at product concentrations, it is not classified as hazardous. Pure 7-AAD is potential carcinogen. Although this component is highly deluted, we recommend to avoid contact with skin and eyes, to wear suitable protective clothing and gloves and appropriate eye/face protection. 6 33513v03 | 2020-05 Protocol • Do not inhale vapors or dusts. Avoid contact with substance. Ensure adequate supply of fresh air. • For material required but not provided the manufacturers recommendations and safety regulations must be followed. • The NH4Cl based Red Blood Cell Lysis Solution is 10× concentrated. Although this mixture is not classified as dangerous we recommend to avoid contact with skin and eyes, to wear suitable protective clothing and gloves and appropriate eye/face protection.
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