CD4% easy count kit – dry

Product Name CD4% easy count kit – dry IVD

Code No. 05-8405-d

Content Packing contains reagents for 100 tests:

• 100 sample tubes (yellow lid) with lyophilized CD4 mAb PE
(MEM-241, PE-conjugated monoclonal antibody to human
CD4) and lyophilized CD45 mAb PE-Dy647 (MEM-28, PE-
Dy647-conjugated monoclonal antibody to human CD45)
• 100 small sample tubes (orange lid) with each 410 µl Buffer 1
• 100 small sample tubes (black lid) with each 410 µl Buffer 2

General Information: Patients with AIDS exhibit T-cell
lymphopenia, a loss of CD4+ lymphocytes and a relative
increase in the CD8+ subtype and in the CD3+CD4-CD8-
subtype. The generally used threshold of 200 CD4+
T-cells/µl blood to start antiretroviral therapy (ART) for adult
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
pediatric patients for reliable and controlled ART and patient
monitoring.
Specificity and Antigen Distribution: The mouse
monoclonal antibody MEM-241 recognizes the human CD4
antigen, a transmembrane glycoprotein (55 kDa) of the
immunoglobulin supergene family, present on a subset of
T-lymphocytes (“helper/inducer” T-cells) and also expressed
at a lower level on monocytes, tissue macrophages and
granulocytes. Approximately 20-60% of human peripheral
blood mononuclear cells as well as a subpopulation of
monocytes but with a weaker signal are stained. The
antibody has been studied at the 8th International Workshop
on Human Cell Differentiation Molecules HCDM (former
HLDA VIII), May 2006, Québec, Canada. CD4 is the primary
cellular receptor for the human immunodeficiency virus
(HIV).
The mouse monoclonal antibody HI-28 reacts with all
alternative forms of the human CD45 antigen (Leukocyte
Common Antigen), a 180-220 kDa single chain type I
transmembrane protein expressed at high level on all cells of
hematopoietic origin, except erythrocytes and platelets. 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).
Clone CD4 mAb PE MEM-241
CD45 mAb PE-Dy647 MEM-28
Isotype CD4 mAb PE Mouse monoclonal IgG1
CD45 mAb PE-Dy647 Mouse monoclonal IgG1
Applications:
• Identification and enumeration of the CD4+ helper/inducer
T-lymphocytes subset as well as CD45+ cells of
haemopoietic lineage
• Determination of percentage of CD4+ T-cells in relation to
all lymphocytes
• Studies of HIV infection. Patients infected with HIV loose
CD4+ helper/inducer lymphocytes and contemporarily
increase CD8+ T-cells
• Characterization of subtypes of T-cell leukemias and
lymphomas
• For in vitro diagnostic use
Method:
• Add 20 µl whole blood (EDTA as anticoagulant) to a
Partec sample tube (yellow lid) with lyophilized CD4 mAb
PE and lyophilized CD45 mAb PE-Dy647. Place blood on
top of the lyophilized antibodies.
• Mix gently and incubate for 15 minutes at room temperature
protected from light. After 5 minutes incubation time mix
again gently.
• Pour 410 µl of Buffer 1 out of a small sample tube (orange
lid) to the stained blood sample and shake gently
• Directly prior to the measurement pour 410 µl of Buffer 2
out of a sample tube (black lid) and analyze immediately
(within 10 minutes)
• Analyze blood sample on a Partec CyFlow device

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. 001 Date 2010-01-12 05-8405-d CD4% easy count kit – dry
 CD4% easy count kit – dry – Page 2
Example of a direct CD4% counting result using the CD4% easy
count kit – dry – display of a Partec CyFlow® Counter. 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, LYM and CD4; they are used in order to calculate the
CD4 % T-cell count.
Recommended and Required Materials:
• Partec Flow Cytometry instrument (e.g. CyFlow® Counter
or CyFlow® SL_3)
• 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 are supplied as lyophilisate. The amount of lyophilized
CD4 mAb PE and CD45 mAb PE-Dy647 are sufficient for
labelling 1x106 cells. When stored at 2-35°C in the dark, the
CD4% easy count kit is stable until the expiration date printed
on the kit label. Do not freeze or expose to elevated
temperatures.

Warnings and Precautions: Reagents contain 0.09%

Example of a direct CD4% counting result using the CD4% easy
count kit – dry – 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 below.
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)
Cell Comment Concentration Concentration Göhde, W. et al.: A New Protocol to Follow-up Immune Impairment in HIV/AIDS-
Population (Diluted blood (Whole blood Children, Scientific Presentation - Abstract No. MoPeLB151C02 at the 3rd IAS
sample) sample) Conference on HIV Pathogenesis and Treatment in Rio de Janeiro, July 2005
CD4+ T-cells Polygon R1 15.010 cells/ml 630 cells/µl 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),
Lymphocytes Polygon R2 34.890 cells/ml 1.465 cells/µl
30: 8-13
Leukocytes Polygon R3 91.165 cells/ml 3.829 cells/µl Imade, G. et al.: “Comparison of a New, Affordable Flow Cytometric Method and
CD4 percent R1/R2 43.0% 43.0% 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
Flow Cytometric Analysis: CD4-PE and CD45-PE-Dy647 Instruments, Journal of Acquired Immune Deficiency Syndromes, Volume 39,
fluorescence can be analysed on a Partec Flow Cytometer Number 1, May 1 2005
with an excitation light source of 532 nm (green solid state Fryland, M. et al.: The PARTEC CyFlow counter for CD4+ T-cell counting
laser). To count CD4+ T-cells and CD45+ cells connect the produces high quality results and is robust when evaluated under routine field
test tube with 840 µl of the ready prepared blood sample (see conditions in Malawi, Scientific Oral Presentation - Abstract No. TuOrB1149 at the
XV. International AIDS Conference in Bangkok, July 2004
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® Counter and CyFlow® SL_3 this
dilution factor is already set in the instrument software
(CyView™ or FloMax™/Word-Report respectively). 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.

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. 001 Date 2010-01-12 05-8405-d CD4% easy count kit – dry