CD4% easy count kit

Product Name CD4% easy count kit IVD

Code No. 05-8405

Content Packing contains reagents for 100 tests:

• 100 ml Buffer 1
• 100 ml Buffer 2
• 1000 µl CD4 mAb PE (MEM-241, PE-conjugated
monoclonal antibody to human CD4)
• 1000 µl CD45 mAb PE-Dy647 (MEM-28, PE-Dy647-
conjugated monoclonal antibody to human CD45)

General Information: Patients with AIDS exhibit T-cell Clone CD4 mAb PE MEM-241
lymphopenia, a loss of CD4+ lymphocytes and a relative CD45 mAb PE-Dy647 MEM-28
increase in the CD8+ subtype and in the CD3+CD4-CD8-
subtype. The generally used threshold of 200 CD4+ Isotype CD4 mAb PE Mouse monoclonal IgG1
T-cells/µl blood to start antiretroviral therapy (ART) for adult CD45 mAb PE-Dy647 Mouse monoclonal IgG1
patients may not be appropriate for children since the CD4
counts in infants are significantly higher than for adults.
Therefore a CD4% measurement should be performed in Applications:
pediatric patients for reliable and controlled ART and • Identification and enumeration of the CD4+ helper/inducer
patient monitoring. T-lymphocytes subset as well as CD45+ cells of
haemopoietic lineage
Specificity and Antigen Distribution: The mouse • Determination of percentage of CD4+ T-cells in relation to
monoclonal antibody MEM-241 recognizes the human CD4 all lymphocytes
antigen, a transmembrane glycoprotein (55 kDa) of the • Studies of HIV infection. Patients infected with HIV loose
immunoglobulin supergene family, present on a subset of CD4+ helper/inducer lymphocytes and contemporarily
T-lymphocytes (“helper/inducer” T-cells) and also increase CD8+ T-cells
expressed at a lower level on monocytes, tissue • Characterization of subtypes of T-cell leukemias and
macrophages and granulocytes. Approximately 20-60% of lymphomas
human peripheral blood mononuclear cells as well as a • For in vitro diagnostic use
subpopulation of monocytes but with a weaker signal are
stained. The antibody has been studied at the 8th Method:
International Workshop on Human Cell Differentiation • Add 20 µl whole blood (EDTA as anticoagulant) to a
Molecules HCDM (former HLDA VIII), May 2006, Québec, Partec test tube
Canada. CD4 is the primary cellular receptor for the human • Add 10 µl of CD4 mAb PE and 10 µl CD45 mAb PE-Dy647
immunodeficiency virus (HIV). Mix gently and incubate for 15 minutes at room temperature
The mouse monoclonal antibody HI-28 reacts with all protected from light
alternative forms of the human CD45 antigen (Leukocyte • Add 400 µl of Buffer 1 and shake gently
Common Antigen), a 180-220 kDa single chain type I • Directly prior to the measurement add 400 µl of Buffer 2
transmembrane protein expressed at high level on all cells and analyze immediately (within 10 minutes)
of hematopoietic origin, except erythrocytes and platelets. • Analyze blood sample on a Partec CyFlow device
The antibody has been clustered to CD45 at the 3rd
International Workshop on Human Leukocyte
Differentiation Antigens (Ref.: Leucocyte Typing III,
McMichael A. J. et al. (Eds.), Oxford University Press,
Oxford 1987).

For in vitro diagnostic use with Partec IVD Flow Cytometers.

Contact Information:

Partec GmbH • Am Flugplatz 13 • D-02828 Görlitz • Germany

Tel +49 3581 8746 0 • Fax +49 3581 8746 70 • E-mail: mail@partec.com
Rev. 016 Date 2009-06-18 05-8405 CD4% easy count kit
 CD4% easy count kit – Page 2

Cell Population Comment Concentration Concentration

(diluted blood sample) (Whole blood sample)
CD4+ T-cells Polygon R1 15010 cells/ml 630 cells/µl
Lymphocytes Polygon R2 34890 cells/ml 1465 cells/µl
Leukocytes Polygon R3 91165 cells/ml 3829 cells/µl
CD4+ T-cell percent R1/R2 43.0% 43.0%
Example of a direct CD4% counting result using the CD4% easy count kit – display of a Partec CyFlow® SL_3. With the
2-colour analysis of CD4/CD45, the absolute number of leukocytes, lymphocytes and the CD4+ T-lymphocytes are displayed in
the polygons R3, R2 and R1; they are used in order to calculate the CD4 % T-cell count as shown above.

Flow Cytometric Analysis: CD4-PE and CD45-PE-Dy647
fluorescence can be analysed on a Partec Flow Cytometer
with an excitation light source of 532 nm (green solid state
laser). To count CD4+ T-cells and CD45+ cells connect the
test tube with 840 µl of the ready prepared blood sample (see
Method) to your Partec Flow Cytometer and carry out a count
analysis. For detailed information on the handling and
calibration of the instrument please refer to the operating
manuals and application notes.
The blood preparation allows one count procedure on the
same sample. Following the above procedure the dilution
factor is 42. In the CyFlow® SL_3 this dilution factor is
already set or can be directly set by the instrument operator in
the instrument software. Counting results will be displayed
automatically as CD4+ T-cells per µl whole blood and as
percentage of CD4+ cells in relation to all lymphocytes.
Recommended and Required Materials:
• Partec Flow Cytometry instrument (e.g. CyFlow® SL_3)
• Partec test tubes (Code No. 04-2000)
• Micropipettes and pipette tips (e.g. Eppendorf, Code No.
3112 000.029 and 3111 000.0165)
• Powder-free latex gloves (e.g. Safeskin, Code No. 545-950-
06)
• Venous Blood Collection System with EDTA as
anticoagulant (e.g. Greiner Bio-One: Vacuette® EDTA
Tubes, K3E/EDTA K3, 3 ml, Code No. 454217, Vacuette®
Blood Collection Needles 38 x 0.8 mm, Code No. 450076,
Vacuette® Tube Holders, Code No. 450201)
Handling and Storage: CD4 mAb PE and CD45 mAb PE-
Dy647 each are supplied in 1 ml of phosphate-buffered saline
(PBS) pH 7.4, containing 0.2% BSA and 0.09% sodium azide,
sufficient for 100 tests. 10 µl of CD4 mAb PE and 10 µl of
CD45 mAb PE-Dy647 are sufficient for labelling 1x106 cells.
Warnings and Precautions: Reagents contain 0.09%
sodium azide that under acidic conditions yields hydrazoic
acid, an explosive compound. Sodium azide may react with
lead and copper plumbing to form potentially explosive metal
azides. When disposing of sodium azide, flush plumbing with
a large volume of water to prevent build-up. Very toxic if
swallowed. After contact with skin, wash immediately with
plenty of water.
Harmful if swallowed (R22). Contact with acids liberates very
toxic gas (R32). Keep out of reach of children (S2). Keep
away from food, drink and animal feeding stuffs (S13). Wear
suitable protective clothing (S36). If swallowed, seek medical
advice immediately and show this container or label (S46).
Handle all specimens and materials as potentially infectious
materials. Decontaminate with 0.5% sodium hypochlorite. The
product should be handled by trained staff only. Reagents are
not sterile. Not for in vivo use. For in vitro use only.
Literature:
Cassens, U. et al.: Simplified volumetric flow cytometry allows feasible and
accurate determination of CD4 T lymphocytes in immunodeficient patients
worldwide”, Antiviral Therapy 9: 395-405 (2004)
Göhde, W. et al.: A New Protocol to Follow-up Immune Impairment in HIV/AIDS-
Children, Scientific Presentation - Abstract No. MoPeLB151C02 at the 3rd IAS
Conference on HIV Pathogenesis and Treatment in Rio de Janeiro, July 2005
Greve, B. et al.: ”A New No-Lyse, No-Wash Flow-Cytometric Method for the
Determination of CD4 T Cells in Blood Samples”, Transfus Med Hemoth (2003),
30: 8-13
Imade, G. et al.: “Comparison of a New, Affordable Flow Cytometric Method and
the Manual Magnetic Bead Technique for CD4 T-Lymphocyte Counting in a
Northern Nigerian Setting”, Clinical and Diagnostic Laboratory Immunology, Jan.
2005, p. 224-227
Dieye, T.N. et al.: Absolute CD4 T-Cell Counting in Resource-Poor Settings: Direct
Volumetric Measurements Versus Bead-Based Clinical Flow Cytometry
Instruments, Journal of Acquired Immune Deficiency Syndromes, Volume 39,
Number 1, May 1 2005

When stored at 2-8°C in the dark, the CD4% easy count kit is Fryland, M. et al.: The PARTEC CyFlow counter for CD4+ T-cell counting
produces high quality results and is robust when evaluated under routine field
stable until the expiration date printed on the kit label. Do not conditions in Malawi, Scientific Oral Presentation - Abstract No. TuOrB1149 at the
freeze or expose to elevated temperatures. XV. International AIDS Conference in Bangkok, July 2004

For in vitro diagnostic use with Partec IVD Flow Cytometers.

Contact Information:

Partec GmbH • Am Flugplatz 13 • D-02828 Görlitz • Germany

Tel +49 3581 8746 0 • Fax +49 3581 8746 70 • E-mail: mail@partec.com
Rev. 016 Date 2009-06-18 05-8405 CD4% easy count kit