Introduction

This protocol is designed for quick and easy quantification of human CD4+ T cells after monocyte depletion using the Dynal® T4 Quant Kit . The same procedure can also be used to quantify CD8+ T cells with Dynabeads® CD8 (Cat. no. 111.47D).

Principle of Isolation

Immunomagnetic cell separation using Dynal® T4 Quant Kit enables a rapid and direct quantification of CD4+ T cells directly from samples of ACD or EDTA anti-coagulated blood. Cell isolation takes place at room temperature (RT) in 30 minutes with three steps:

1. Depletion of Monocytes

A significant fraction of human monocytes express low CD4 antigen levels (1, 2). Consequently, Dynabeads® CD4 could bind CD4+ monocytes, thus producing artificially high CD4+ T cells counts. To prevent this, efficiently deplete monocytes in 10 minutes from the blood sample using Dynabeads® CD14.

2. Isolation of CD4+ T Cells

Isolate CD4+ T cells from monocyte-depleted blood within 10 minutes using Dynabeads® CD4. Wash beadbound cells to remove contaminating cells.

3. Counting of CD4+ T Cells

After isolation, lyse the CD4+ T cells and count the liberated nuclei with one of these 2 options:

  • 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.

Description of Materials

Dynabeads® are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with mouse monoclonal antibodies specific for the CD14 antigens of monocytes, or the CD4 antigens of the CD4+ T cell subset respectively. The products are supplied in a suspension of phosphate buffered saline (PBS) containing 0.1 % bovine serum albumin (BSA) and 0.02 % sodium azide line.

The human monoclonal secondary antibody coated onto the Dynabeads® is produced in a hybridoma cell line.
The human donor was free of Human Immunodeficiency Virus (HIV), Hepatitis-B Virus (HBV) and Hepatitis-C Virus (HCV).

Materials Supplied

Dynal® T4 Quant Kit contains:

  • 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

Additional Products Required

Materials available from Invitrogen Dynal®
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  • 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).

Solutions:

  • Acridine Orange staining solution
  • Stock solution:

Acridine Orange                       15 mg
Ethanol 96%                                1 ml
Distilled water                           49 ml
Mix well
Divide into 1 ml aliquot and stock at -20°C

  • Working solution

Thaw 1 ml stock solution and add 9 ml PBS (pH 7.4). Mix well. Store in an amber bottle at 2-8°C.

  • Sternheimer-Malbin staining solution:

Crystal Violet                             100 mg
Safranin                                     200 mg
Ethanol                                          10 ml
Ammonium Oxalate                      30 mg
Distilled water to                         100 ml
Store in an amber bottle at 2–8°C


  • Turck staining solution:
Glacial acetic acid                         2 ml
Gentian Violet                        0.0128 g
Deionised water to                   100 ml
Store in an amber bottle at 2–8°C

  • Washing/dilution buffer

Isotonic phosphate buffered saline (PBS) pH 7.4.

Invitrogen Dynal® recommends the following phosphate buffered saline (PBS) pH 7.4:

NaH2PO4 × H2O                      0.16 g
Na2HPO4 × 2H2O                    0.98 g
NaCl                                         8.10 g
Distilled water to 1 litre

Add 0.1% (w/v) bovine serum albumin to PBS. All reagents should be analytical grade. For long-term storage, 0.02% sodium azide (final concentration) may be added as a preservative.

Materials not available from Invitrogen Dynal®

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

Sku Name Size Price Qty
11321D Dynabeads® T4 Quant Kit 2 mL USD 493.00

Protocols

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.

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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.
                                                                                                                                                                                 
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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 visualise 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:
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Perform three blind tests using the same sample within a series of tests. The reproduction of results in a series
of tests using the same sample is fundamental as the aim of the counting is to detect increasing and decreasing variations in the number of CD4+ T lymphocytes.

External quality control:

Transfix® can be sent to the reference laboratory for Flow Cytometer measurements, then compared with the local measurements using Dynabeads® technique.

Performance and Limit

Specificity:
The correlation coefficient, for the CD4+ T lymphocyte quantification between the Dynal® T4 Quant kit and flow cytometry reference standard, is in a range of 0.89-0.96 (3, 4, 5, 6, 8).

Sensitivity: CD4+ T lymphocytes were counted on 32 points by a series of 3 sample dilutions. The detection limit is 6 CD4+ T lymphocytes/μl of whole blood with a background of +2 standard Reproducibility: The coefficient of variation is 8.4%

Limit: The result can be underestimated above 800 CD4+ T lymphocytes counted in microscopy. Blood 2–a decrease in CD4+ T lymphocytes count. The pH of the washing/dilution buffer is fundamental, a pH lower than 7.2 makes capture of the T CD4+ lymphocytes difficult.

Reference Intervals

From 2 studies intended to validate the Dynal® T4 Quant kit regarding flow cytometry (FCM), 43 individuals were selected to determine the reference intervals. These people had no known sickness and were not taking medication. The 2.5% to 97.5% confidence limit of ranked data is the reference range defined by this product (see Table 1) and flow cytometry, which has been taken as the reference method for comparison.

Table 1 – Reference intervals of 95% for CD4+ T lymphocyte quantification


Technique Mean Lower limit Upper limit No. of cases within 95% confidence
Dynal® T4 Quant 948438157041
FCM 964421159241
Difference between T4 Quant-FCM -1617-22N/A

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


Dynal® T4 Quant FCM
Dynal® T4 Quant 1
0.90
FCM 0.901

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

Class Lower limit Upper limit Mean No. of cases
Female 540157099817
Male 438150491224

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

Class Upper limit Lower limit Mean
No. of cases
Less than
15 years old
734
1570123010
16 to 25 years 656124292711
26 to 45 years 556
13748349
Older than
46 years

438
1504
809
5

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

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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

  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.


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113.21D_4_sprak.indd    Rev 005     5-May-2008