Rapid Quantification of Human Peripheral Blood CD4+ T Lymphocytes

• in a fluorescence microscope after staining with Acridine Orange
• in a conventional light microscope after staining with Sternheimer-Malbin solution or Turck solution The results show the number of cells per microlitre of whole blood.

• 2 ml Dynabeads® CD14 (blue label): for monocyte depletion coated with a mouse monoclonal antibody specific for the CD14 antigens of monocytes
• 2 ml Dynabeads® CD4 (yellow label): for CD4⁺ T cell isolation coated with a mouse monoclonal antibody specific for the CD4 antigens T cell subset 7 ml Lysis solution: designed to viruses and release lymphocyte nuclei. (contains formaldehyde – use with care)
• The kit is sufficient for 80 tests of 125 μl whole blood

• Magnet: Dynal® MPC™-S (Cat. no. 120.20D)
• Mixer allowing both tilting and rotation (e.g Dynal® MX1, Cat. no. 159.07).
• Dynabeads® CD8 for CD8⁺ T lymphocyte isolation (Cat. no. 111.47D).
• Acridine Orange staining solution: for use with fluorescence microscopy (Cat. no. 930.01).
• Sterheimer-Malbin staining solution: for use light microscopy (Cat. no. 930.04)
• Turck staining solution: for use with light microscopy (Cat. no. 930.05).
• Microcentrifuge tubes (Cat. no. 930.02) or Rhesus tubes & caps (Cat. no. 930.06)
• Mallassez Haemocytometer (Cat. no. 995.03).
• PBS-BSA-Sodium azide Washing/dilution buffer (Cat. no. 9408).

• Acridine Orange staining solution

• Stock solution:

• Working solution

• Sternheimer-Malbin staining solution:

• Turck staining solution:

• Washing/dilution buffer

• Blood collecting tubes containing anticoagulant (ACD or EDTA).
• Pipettes (Volume range: 25 μl – 500 μl).
• Parafilm.
• Fluorescence microscope, light microscope.

Cell Isolation Procedure

• Blood samples should be as fresh as possible and preferably not older than 24 hours. Blood samples stored at either 2-8°C or RT for up to 24 hours do not show significant decrease in cell counts.
• When heparin is used as an anticoagulant, fibrin deposits have been observed in the blood samples. Heparin will also activate platelets. Invitrogen Dynal® does not recommend using heparin as an anticoagulant for this procedure.

Monocyte depletion

1. Collect blood in a standard blood collection tube containing ACD or EDTA. Incubate for at least 5 minutes at RT, with gentle tilting and rotation.


2. Resuspend the Dynabeads® CD14 use to obtain homogenous dispersion solution.


3. Add the following in a tube:
   • 350 μl washing/dilution buffer
   • 125 μl blood
   • 25 μl Dynabeads® CD14




4. Cap the tube and carefully mix the sample. Incubate the sample with gentle tilting and rotation for 10 minutes at RT using the sample mixer (e.g. MX1)


5. After incubation, shake down any blood droplets remaining under the cap. Carefully remove and dispose of the cap. Place the tube in the magnet for 2 minutes.


6. Transfer 200 μl of the monocyte-depleted blood (supernatant) to another tube without disturbing the cells held on the tube wall by the magnet. (For CD8⁺ T cell quantification, transfer an additional 200 μl of the sample to a second tube for CD8⁺ T cell isolation). Discard the initial sample tube containing the monocytes.

CD4⁺ T Cell Isolation

For microcentrifuge tubes (Cat. no. 930.02), fold a piece of parafilm three times and place it between the tube and cap to avoid trapping blood in the hole of the cap. If the tube is left in the magnet for more than 5 minutes, sedimentation can occur, affecting the number of T4 cells that are transferred. In this case, resuspend gently with the micropipette the sample well in this tube before collecting 200 μl aliquots for CD4⁺ T lymphocyte isolation.

1. Add 200 μl washing/dilution buffer to a tube containing 200 μl monocyte-depleted blood.


2. Resuspend the Dynabeads® CD4 (yellow label) beforeuse to obtain a homogenous dispersion of beads in suspension.


3. Add 25 μl Dynabeads® CD4 to the tube.


4. Cap the tube and carefully mix the sample. Incubate with gentle tilting and rotation for 10 minutes at RT.


5. After incubation, shake down any blood droplets remaining under the cap. Carefully discard the cap. Place the tube in the magnet for 2 minutes to isolate the bead-bound CD4⁺ T cells.


6. Discard the supernatant while the cells remain attached to the tube wall by the magnet. Avoid disturbing the bead-bound cells.


7. Wash the isolated CD4⁺ T cells by removing the magnet slide from the magnet and adding 500 μl washing/dilution buffer to the tube. Place a pre-cut sheet of parafilm over the tube. Rotate the tube a few times unti  the bead-bound cells are completely resuspended


8. Carefully discard the parafilm sheet. Refit the magnet slide in the magnet for 2 minutes to isolate the bead-bound CD4+ T cells. We recommend that inexperienced users of this technique perform a second washing procedure.

CD4⁺ T Lymphocyte Counting

2 different alternatives are proposed for CD4⁺ T cell counting:

Alternative A: Fluorescence microscopy haemocytometer reading

Alternative B: Light microscopy haemocytometer reading

• In a few cases (<2%), contamination of isolated CD4⁺ T cell lymphocytes with polymorphonuclear granulocytes has been observed, where cell counts exceeded the normal range of CD4⁺ T cell counts by several hundred percent. Such heavy contamination of granulocytes is readily visible in the microscope due to the characteristic shape of their nuclei.

Alternative A: Fluorescence

Microscopy Haemocytometer Reading

1. Completely remove the last washing buffer. Avoid disturbing the isolated beads on the tube wall. Remove th  magnet slide from the magnet and flush all the bead-bound cells from the tube wall with 50 μl Lysis Solution. Resuspend thoroughly by vortexing.


2. Leave the samples for 5 minutes at RT.


3. Add 50 μl acridine orange staining solution. Store the samples in the dark at 2–8°C until counting. The sample  should be counted within one hour after adding the Lysis Solution.


4. Resuspend the samples thoroughly by vortexing for 5 seconds. Transfer about 20 μl of the suspension to the haemocytometer and count the number of cells in a known volume using a fluorescent microscope with both blue (450/ 490 nm) excitation and white light to visualize the grid. If less than 100 cells are present in the haemocytometer chamber, repeat counting and take an average of two counts.

The concentration of lysed CD4⁺ T cells in the sample corresponds to half the concentration of CD4⁺ T cells in whole blood.

Convert the number of cells counted in the haemocytometer to the concentration of CD4⁺ T cells in whole blood with the formula:

Cells/μl = n × 2
                   v
n = number of cells counted
V = volume counted in the haemocytometer (in microlitres).

Alternative B: Light Microscopy

Haemocytometer Reading

Staining solutions are not included with this kit. Use Sternheimer-Malbin or Turck staining solutions in the following procedure.

Staining with Sternheimer-Malbin solution                                                                                                                        TOP

1. Completely remove the last washing buffer. Remove the magnet slide from the magnet and flush all the bead-bound cells from the tube wall using 80 μl Lysis Solution. Resuspend thoroughly by vortexing.


2. Leave the samples for 5 minutes at RT.


3. Add 20 μl Sternheimer-Malbin staining solution. Store the samples in the dark at 2–8°C until counting. Count the samples within one hour of adding the Lysis Solution.


4. Resuspend thoroughly by vortexing for 5 seconds. Transfer about 20 μl of the suspension to the haemocytometer and count the cells in a known volume using a conventional white light microscope. If less than 100 cells are present in the haemocytometer chamber, repeat counting and take an average of two counts.

The concentration of lysed CD4⁺ T cells in the sample corresponds to half the concentration of CD4⁺ T cells
in whole blood.

Convert the number of cells counted in the haemocytometer to the concentration of CD4⁺⁺ T cells in whole blood with the formula:

Cells/μl = n × 2
                   v
n = number of cells counted
V = volume counted in the haemocytometer (in microlitres).

Nuclei stained with Sternheimer-Malbin staining solution appear purple under white light microscopy. These samples are less readable compared to acridine orange staining. Consequently, a high visual acuteness is needed and a decrease in number of samples performed is expected using light microscopy counting.

Staining with Turck solution

1. Completely remove the last washing buffer. Remove the magnet slide from the magnet and flush all the bead-bound cells down tube wall using 50 μl Lysis Solution. Resuspend thoroughly by vortexing.


2. Leave the samples for 5 minutes at RT.


3. Add 50 μl Turck staining solution. Store the samples in the dark at 2–8°C until counting. Count the samples within one hour of adding the Lysis Solution.


4. Resuspend the samples thoroughly by vortexing for 5 seconds. Transfer about 20 μl of the suspension to the haemocytometer and count the number of cells in a known volume using a conventional white light microscope. If less than 100 cells are present in the haemocytometer chamber, repeat the count and take an average. The concentration of lysed CD4⁺ T cells in the sample corresponds to half the concentration of CD4⁺ T cells in whole blood.

Convert the number of cells counted in the haemocytometer to the concentration of CD4⁺ T cells in whole blood by the formula:

Cells/μl = n × 2
                   v
n = number of cells counted
V = volume counted in the haemocytometer (in microlitres).

Nuclei stained with Turck solution appear violet under white light microscopy. These samples are less readable compared to acridine orange staining. Consequently, a high visual acuteness is needed and a decrease in number of samples performed is expected using light microscopy counting.

Interpretation of Results

CD4⁺ T lymphocyte quantification is one of the best indicators of immunodeficiency disease progression such as agammaglobulinemia, Acquired Immunodeficiency Syndrome (AIDS) and thymic aplasia. The result of this CD4⁺ T lymphocyte decrease is an important risk of opportunistic diseases that occur when the CD4⁺ T cell number is less than 200 per μl blood.

Quality Assurance

For all routine laboratory work, a procedure for quality assurance is recommended.

Internal quality control:

Table 2 – Correlation between flow cytometry and Dynal® T4 Quant kit for healthy patients.

Of the 43 individuals, one gave a value above the 97.5 percentile and one below the 2.5 percentile. Tables 3 and 4 provide reference intervals for the Dynal® T4 Quant kit for male, female and age groups.

Table 3 – Reference intervals of 95% for CD4⁺ T lymphocyte quantification by sex

Table 4 – Reference intervals of 95% for CD4⁺ T lymphocyte quantification by age

Each laboratory should establish its own normal reference interval from the local population

1. Wood GS. et al. (1983) Anti Leu-3/T4 antibodies react with cells or monocyte/macrophage and langerhans


2. lineage. J. Immunol. 131, 212-216.


3. Stewart SJ. et al. (1986) Human T lymphocytes and monocytes bear the same Leu-3 (T4) antigen. J. Immunol. 136 3773-3778.


4. Lyamuya, E.F. et al. (1996) Evaluation of the FACS count, TRAx CD4 and Dynabeads® methods for CD4 lymphocytes determination. J. Imm. Methods 195: 103-112.


5. Didier, J.M. et al. (2001) Comparative assessment of alternative methods for CD4⁺ T lymphocytes enumeration in developing countries. JAIDS 26 (2).


6. Crowe, S. et al. (2003) Monitoring of human immunodeficiency virus infection in resource-constrained countries. Clin. Infect. Dis. 37 (Suppl1) : 25-35.


7. Diagbouga, S. et al. (2003) Successful implementation for a low-cost method for enumerating CD4⁺


8. Besson-Faure I. et al. (1993) Numération des lymphocytes CD4 et CD8: Etude d’une nouvelle technique par immunoséparation magnétique.


9. Poncelet P. et al. (1993) Numération des lymphocytes CD4⁺ et CD8⁺ après immunoséparation magnétique. Conférence de Gonsensus l’immunophénotypage lymphocytaire au cours de l’infection à VIHAvril 1993.