HLA Isolation

A one-step method for In-vitro Isolation of pure lymphocytes.

INTRODUCTION TO THE TECHNIQUE

Early methods for separating lymphocytes from whole blood involved mixing the blood with a compound that causes erythrocytes to clump together. These clumped aggregates then settle to the bottom, leaving the lymphocytes in the upper layer. Granulocytes are also sedimented because they are larger than lymphocytes and tend to slightly aggregate. Boyum (1964) developed a system where the aggregating agent is not mixed directly with the blood. Instead, the aggregating agent is combined with a high-density compound (e.g., Sodium Diatrizoate), and the entire mixture is carefully layered on top. As erythrocytes reach the interface of the two liquid phases, they contact the aggregating agent, form large clumps, and sediment to the bottom. Granulocytes also sediment, resulting in a high concentration of lymphocytes just above the interface.

Using Ficoll® as the aggregating agent, Boyum (1968) created a one-step centrifugal technique for isolating lymphocytes. Thornsby and Brallie (1970) made slight modifications to this technique for lymphocyte isolation before cytotoxicity tests and lymphocyte culturing. Harris and Ukayiofo (1969) and Ting and Morris (1971) highlighted the reliability of this method for fresh blood, stored anti-coagulated blood, and cadaver blood.

ISOLYMP®

Isolymph® is an aqueous solution of Sodium diatrizoate and Ficoll 400®*, having a density of 1-077 ± 0.001 g/ml. (9.0g Sodium diatrizoate and 5.7 g Ficoll 400® per 100 mL). Isolymph® is supplied in a clear, 100 ml bottle with a rubber septum closure for sterile syringe withdrawal. Sodium diatrizoate is the sodium salt of 3, 5.Diacetamido.2.4, 6,-tri-iodobenzoic acid. It forms solutions of low viscosity and high density making it ideal for this application. Since it is light sensitive, Isolymph® must be stored protected from light. Room temperature storage is sufficient, but storage at 4-6°C will increase shelf life.

Materials and Solution
  1. Two siliconized 10 ml glass tubes for each sample (refer to notes for details).
  2. Siliconized Pasteur pipettes (refer to notes for details).
  3. Two siliconized glass centrifuge tubes for each sample. A convenient size is 15 ml with an internal diameter of 1.3 cm for larger sample volumes (refer to notes for details).
  4. A centrifuge.
  5. A sterile syringe and needle for the sterile withdrawal of Isomlymph from its container.
  6. Isolymph®: 3 ml required per sample (refer to notes for details on larger samples).
  7. 20 ml of balanced salt solution for each sample (refer to notes for details).

Fresh blood is recommended to ensure high lymphocyte viability.

1. Put 2 ml of defibrinated or anticoagulant-treated blood into a siliconized 10 ml tube and add an equal amount of balanced salt solution. Mix by drawing in and out of a Pasteur pipette.

2.  Ficoll 400® is a registered trademark of Pharmacia Fine Chemicals AB., Uppsala, Sweden

  1. Transfer the lymphocyte layer to a clean centrifuge tube.
  2. Add at least three volumes of TRIS-balanced salt solution to tube 1 and suspend the lymphocytes by gently drawing them in and out of a Pasteur pipette,
  3. Centrifuge at 60-100 x g for 10 minutes at l8-20°C.
  4. Remove the supernatant.
  5. Add 6-8 ml of TRIS-balanced salt solution and resuspend the cells as above.
  6. Centrifuge at 60-100 x g for 10 minutes at 18-20 °C.
  7. Remove the supernatant.
  8. Resuspend the lymphocytes in a suitable medium for your subsequent work.
  1. Transfer the lymphocyte layer to a clean centrifuge tube.
  2. Add at least three volumes of TRIS-balanced salt solution to tube 1 and suspend the lymphocytes by gently drawing them in and out of a Pasteur pipette,
  3. Centrifuge at 60-100 x g for 10 minutes at l8-20°C
  4. Remove the supernatant.
  5. Add 6-8 ml of TRIS-balanced salt solution and resuspend the cells as above.
  6. Centrifuge at 60-100 x g for 10 minutes at 18-20 °C.
  7. Remove the supernatant.
  8. Resuspend the lymphocytes in a suitable medium for your subsequent work.
  1. All glassware that contacts the blood sample should be siliconized. Immerse it in a 1% silicon solution for 10 seconds, wash with distilled water six times, and dry in an oven.
  2. To ensure reproducibility of the lymphocyte technique, use the same volume of blood and centrifuge tubes of uniform size. This is critical because the taller the blood column, the longer the centrifugation time needed, and the higher the erythrocyte contamination. For larger samples, use a wider diameter tube to keep the height of the blood sample and Isolymph consistent.
  3. The optimal centrifugation temperature is 18-20°C; at higher temperatures (37°C), red cell aggregation increases, but lymphocyte viability decreases. At lower temperatures (4°C), the separation process takes longer, reducing lymphocyte viability.
  4. Defibrinated blood is preferred for this procedure, but anticoagulants such as ACD, CPD, Heparin, EDTA, or Citrate can also be used.
  5. Prepare TRIS-balanced salt solution.
  6. The erythrocyte contamination in the lymphocyte suspension is usually between 1-5% of the total cell count.
Solution 1g/L
D-Glucose (anhydrous)1 .0
CaCl2 • 2HO0.0074
MgCl 20.1992
KCl0.4026
TRIS17.565
Dissolve in 950 ml distilled water. Adjust pH to 7.6 using 1N HCI, q.s. to one liter
Solution IIg/L
NaCl8.19
Working TAIS-balanced salt solution:

Mix 1 volume solution I with 9 volumes of solution II. Prepare fresh weekly.

HLA Isolation

  1. Boyium, A (1964): Separation of White Blood Cells. Nature 204, p. 793.
  2. Boyium. A. (1968): Separation of Leucocytes from Blood end Bone Marrow. Scand. J.Clin. Lab Invest. 21 Suppl. 97
  3. Favour C B. (1964): Antigen-antibody Reaction in Tissue Culture. Immunological Methods, Ed. J. R. Ackroyd, p.195-223. Blackwell Scientific Publ., Oxford.
  4. Harris, R. and Ukaejiofo. E. V. (1969): Rapid Preparation for Lymphocytes for Tissue Typing. Lancet 327. 7615.
  5. Ting. A and Morris. P. J (1971): A Technique for Lymphocyte Preparation from stored Heparinized Blood. Vox Sang, 20. 561.
  6. Thorsby E. and Bratlie, A (1970): A Rapid Method for Preparation of Pure Lymphocytes Suspensions. Histocompatibility testing. 1970 Ed P. I. Terasaki. p. 655 Munksgaard Copenhagen