IMMUNOLOGY TEXTBOOK - Mercury Podillia 2013

IMMUNOLOGICAL RESEARCH METHODS

Immunological Tests

Methods based on The Study of lymphocyte surface markers. Isolation of lymphocytes from peripheral Blood by gradient centrifugation.

As a rule, laboratory analysis of lymphocytes involves the Isolation of the mononuclear leukocyte (lymphocyte) fraction from peripheral blood. For this purpose, the Ficoll-Paque density gradient centrifugation method is used. By mixing Ficoll and Paque in a specific proportion, a solution with a density of 1.077 g/cm³ is obtained. Blood is layered onto the Ficoll solution (gradient). As a result, a stepwise density gradient is formed between the plasma and the Ficoll solution. Following centrifugation, erythrocytes and granulocytes pass through the Ficoll and pellet at the bottom, while mononuclear Cells (lymphocytes and monocytes) remain as a ring at the interphase, thus allowing the Separation of cells with lower density (lymphocytes, monocytes) from those with higher density (erythrocytes, granulocytes) than 1.077 g/cm³.

Materials and equipment: 1. Ficoll-400 - polysaccharide. 2. Paque - radiopaque agent (analogues: Hypaque, Isopaque, Omnipaque, Verografin, Urografin, Urotrast, etc.). 3. Medium 199. 4. Sterile distilled Water. 5. 3% acetic acid solution. 6. Centrifuge with a swing-out rotor. 7. Hydrometer with a measuring range from 1.060 g/cm³ to 1.090 g/cm³. 8. Goryaev chamber. 9. Microscope. 10. Laboratory glassware, conical centrifuge tubes, balance for balancing centrifuge tubes, etc.

Method Description

1. Preparation of Ficoll: dissolve 4.32 (8.64) g of Ficoll-400 powder in 48 (96) ml of distilled water.

2. Preparation of the radiopaque agent solution (e.g., Urotrast): bring 10.14 (20.28) ml of a 75% Urotrast solution up to 21 (42) ml with distilled water.

3. Preparation of the density gradient: mix the Ficoll and Urotrast solutions. Measure the density of the resulting solution using a hydrometer; it should be 1.077 g/cm³. If the density is higher than required, add the Ficoll solution; if lower, add the Urotrast solution. The density gradient can be stored for up to 30 days at +4 °C in an amber Glass bottle. If Ficoll is unavailable, the density gradient can be prepared using only the radiopaque agent. For this purpose, mix 10 (20) ml of a 76% Urotrast solution with 43.1 (86.2) ml of distilled water and add 0.45 (0.9) ml of a 0.1% sodium chloride solution. The resulting 14.3% Urotrast solution has a density of 1.077 g/cm³ and can be used as a density gradient.

4. Lymphocyte isolation: collect 10 ml of peripheral blood from the cubital vein into a tube containing heparin at a final concentration of 25 IU per 1 ml of blood (1 ml of heparin solution with a concentration of 200-250 IU/ml). Allow the blood to settle for 40-60 min at room Temperature until a clear separation of erythrocytes and plasma is achieved.

✵ Pipette 2-3 ml of the density gradient into a centrifuge tube, and carefully layer 4-6 ml of the settled plasma and the upper layer of erythrocytes onto it. Maintain the gradient-to-plasma volume ratio within 1:2 to 1:4.

✵ Centrifuge the tubes for 40 min in a swing-out rotor at an acceleration of 200 g (1500-1800 rpm) at 20 °C. The centrifugal acceleration G is calculated using the formula: G = 1.1 × n² × R × 10⁻⁵ (where n is the number of revolutions per minute, and R is the radius from the center of the centrifuge axis to the interface between the Ficoll-Paque mixture and The Cell suspension being separated, in cm). During centrifugation, erythrocytes and granulocytes "fall through" the gradient and pellet at the bottom of the tube. At the upper boundary of the gradient, proper separation results in a loose, whitish ring consisting mainly of lymphocytes with a monocyte admixture. Plasma is located above the lymphocyte layer. Collect the plasma into a separate tube for subsequent immunoglobulin analysis; aspirate the lymphocytes into a dry conical centrifuge tube.

✵ Add 3-4 ml of Medium 199 containing 10% calf serum to the lymphocyte suspension and mix the contents thoroughly.

Next, centrifuge the tubes at an acceleration of 200-300 g at 20 °C, discard the supernatant, and repeat the washing procedure once more. Instead of Medium 199, any buffer solution with pH 7.0-7.2 lacking Ca²⁺ ions can be used (the presence of Ca²⁺ ions promotes cell aggregation and coagulation), along with human blood group IV (AB) serum. When using the latter, inactivation at 56 °C for 30 min and erythrocyte absorption are required. To do this, add 0.5 ml of washed erythrocyte pellet to 1 ml of serum and incubate for 1 h at 37 °C with periodic shaking. Serum absorption with a leukocyte pool is desirable, given the potential presence of antileukocyte Antibodies. When using human serum, The Effect of autocytolymphotoxins, which exhibit maximum activity at 4 °C, must be taken into account.

✵ After washing, prepare a working lymphocyte concentration containing 2×10⁶ cells in 1 ml (20 cells in a large square of the Goryaev chamber). Before preparing the working concentration, calculate the initial number of lymphocytes:

- add 0.9 ml of Medium 199 to 0.1 ml of the washed cell pellet and mix the suspension thoroughly;

- add 0.38 ml of a 3% acetic acid solution and 0.02 ml of the lymphocyte suspension to a clean tube;

- count the lymphocytes in 100 large squares using the Goryaev chamber and multiply the obtained number by 50,000. The result corresponds to the number of lymphocytes in 1 ml of suspension. To prepare the working lymphocyte concentration, divide the obtained number by 2×10⁶, subtract 1 from the result, and the remainder represents the volume of nutrient Medium 199 in ml that must be added to the tube with lymphocytes.

The lymphocyte suspension prepared according to this method can be stored at +4 °C for up to 2 hours.

Currently, three main groups of methods are used to identify Surface structures of lymphocytes and A number of other cells: 1) Methods for determining T- and B-lymphocyte subpopulations using erythrocyte diagnostic Reagents; 2) laser flow cytometry; 3) methods for determining T-lymphocytes via spontaneous rosette formation with sheep erythrocytes (E-RFC) and B-lymphocytes via rosette formation with sheep erythrocytes in the EAC system.

METHOD FOR DETERMINING T- and B-lymphocyte subpopulations using "Anti-CD3", "Anti-CD4", "Anti-CD8", "Anti-CD22", and "Anti-CD16" erythrocyte diagnostic reagents

The Principle of the method is based on the determination of T- and B-lymphocyte subpopulations using a rosette formation assay with erythrocytes onto which Monoclonal Antibodies against CD3 (T-lymphocytes), CD4 (T-helper cells), CD8 (T-suppressor cells), CD19 or CD22 (B-lymphocytes), and CD16 (natural killer cells) receptors are adsorbed.

Test results are evaluated using a Light Microscope with an immersion system.

List of required equipment: Centrifuge. Tubes (10 ml). Incubator. Refrigerator. Automatic micropipettes (20 - 200 µl). Microscope with an immersion system. Glass slides.

Additional reagents: density gradient solution d-1.077. Physiological saline or phosphate-buffered saline, pH 7.2-7.4. 0.12% glutaraldehyde solution. May-Grunwald-Giemsa stain.

Preparation of leukocyte suspension. Blood is drawn from a vein into a heparinized tube. 3 ml of blood is sufficient for these assays. The mononuclear cell suspension (lymphocytes) is obtained using a d-1.077 density gradient. Wash the cells 2-3 times with physiological saline or phosphate-buffered saline, pH 7.2-7.4. The desired cell concentration in the suspension is 2x106/ml (20 cells in the large square of the Goryaev chamber).

Preparation of the diagnosticum. Resuspend the erythrocyte pellet by gently swirling the vial without foaming. Alternatively, resuspension can be performed using a sterile 2 ml syringe: pierce the stopper with the needle, draw up the mixture, and expel it back into the vial several times (without foaming). Draw the required volume for the assay (0.05 ml per test) into the syringe and transfer it into sterile tubes.

Performing the Assay

1. Add 0.05 ml (50 µl) of the CD-diagnosticum to the tubes and add 0.05 ml of the lymphocyte suspension.

2. Incubate the mixture for 40 min at 370 °С.

3. Centrifuge at 1000 rpm for 5 minutes.

4. Leave for 1 hour in a refrigerator at +40 °С.

5. Remove the supernatant.

6. Add 0.05 ml of 0.12% glutaraldehyde solution to the pellet and gently resuspend (avoiding foam formation!). Incubate for 5-7 minutes, then resuspend gently once again.

7. Prepare a smear covering approximately 1 cm² of a degreased glass microscope slide.

8. Air-dry, fix with alcohol, and stain with Romanowsky stain.

9. Using a light microscope with an immersion system, count the percentage of rosette-forming lymphocytes binding at least 3 erythrocytes with CD-diagnostica per 200 cells.

Do not count! Granulocytes, cell aggregates, as well as lymphocytes trapped within aggregates.

Rosette-forming lymphocytes can also be counted in a native preparation using a Goryaev chamber.

Evaluation of Results

1. The percentage of T-lymphocytes equals the percentage of rosette-forming lymphocytes with the СD3-diagnosticum.

Normal values for adults are 50-80% (mean 60+5%); for children - 47-76% (mean 55+4.8%).

Absolute count per 1 µl of blood = А х У х С : 10000,

where: A is the leukocyte count per 1 µl of blood, Y is the percentage of lymphocytes in the differential blood count, C is the percentage of rosette-forming T-lymphocytes.

2. The percentage of T-helper cells (Th) equals the percentage of rosette-forming lymphocytes with the СD4-diagnosticum.

Normal range 33-46% (mean 40+3.0%).

3. The percentage of T-suppressor cells (Ts) equals the percentage of rosette-forming lymphocytes with the СD8-diagnosticum.

Normal range 17-30% (mean 22+1%).

IRI - immunoregulatory index. IRI: Th/Ts = 1.4 - 2.0.

4. The percentage of B lymphocytes corresponds to the percentage of rosette-forming lymphocytes with the CD22 diagnostic reagent. The absolute count of B lymphocytes is determined in the same manner as for T lymphocytes.

Normal range: 17–31% (mean 23±3.6%).

5. The percentage of natural killer cells corresponds to the percentage of lymphocytes forming rosettes with the CD16 diagnostic reagent.

Normal range: 12–23% (mean 16±4.5%).

It should be noted that blood smears are recommended to be prepared as a monolayer on glass slides thoroughly degreased with an alcohol-ether mixture. Avoid cell overlapping and multilayer formation. Diagnostic reagents must be stored at temperatures ranging from G2 to G10 °C. Freezing is strictly prohibited!

Immunoregulatory index (CD4/CD8 ratio). In addition to determining the counts of populations and subpopulations, great importance is attached to calculating the CD4/CD8 ratio, known as the immunoregulatory index or helper-to-suppressor ratio (normal range: 1.8±0.4). A decrease in the immunoregulatory index is observed in viral infections, in newborns, and following Bone Marrow transplantation, whereas an increase occurs in autoimmune diseases and allergies (Table 11).

Class="center">Table 11. Clinical Examples of alterations in the immunoregulatory index (helper-to-suppressor ratio, CD4/CD8)

At the same time, it should be noted that some CD8+ T cells function as killers, while some CD4+ cells act as effectors; therefore, there is currently no single, flawless method for assessing the number of lymphocytes with suppressor or helper activity. Furthermore, the antigenic Structure of type 1 and type 2 T-helper cells is virtually identical. For this reason, the evaluation of lymphocyte subpopulation counts should preferably be complemented by functional assays and the Determination of the cytokine profile.