Women's and Children's Hospital, Adelaide
National Referral Laboratory

National Referral Laboratory

Methods for Sample Preparation

Contents


Preparation of peripheral blood leucocytes for lysosomal enzyme assay

There are 2 methods available for preparation of leucocytes from whole blood - either method is acceptable and merely depends on the availability of expertise, resources and equipment.

Approximately 10 mL (8-12 mL) whole, fresh, EDTA blood required for either method. If the test is for Tay-Sachs disease heterozygote testing, an additional 1 mL serum is also required.

NOTES

1. Date of preparation MUST accompany all specimens. Clinical history should accompany sample or must follow shortly after dispatch of sample. Analysis of samples arriving without these details may be delayed until such correspondence is received

2. The quality of blood collection significantly affects the leucocyte pellet quality. Blood must not contain clots or plastic mixing beads and must be fresh.

3. If a small volume only of blood is obtained (2-4 mL), it is better to prepare only a single leucocyte pellet. In this case, however, only limited enzymological examination may be practicable.


METHOD 1 - Preparation of leucocytes by dextran sedimentation

PRINCIPLE

Leucocytes are separated from erythrocytes by differential sedimentation in a dextran/saline solution. The cells are washed briefly in 0.2% saline to haemolyse the red cells, and then brought back to isotonicity with 1.8% saline. In order to facilitate subsequent analytical operations, two leucocyte pellets are produced from the original single heparinised whole blood sample.

REAGENTS

Keep all reagents refrigerated and ensure that all washing operations are carried out in the cool; 10 degrees Celsius is recommended, certainly no lower than 5 degrees, no higher than 15.

1. Dextran/Saline

    Dissolve 5 g dextran (MW 150,000-200,000) and 0.7 g NaCl in 100 mL deionised water (DIW).

    NOTE: Bulk reagent tends to grow fungi unless used rapidly. 100 mL lots should be autoclaved at 121 degrees Celsius/100 kPa/30 minutes, and kept stored at 4 degrees.

    NOTE: Not all batches of dextran are suitable for use as some cause substantial loss of leucocytes. Each batch must be tested for suitability. We currently use BDH Dextran Grade B, lot# 380153F

2. 0.2% Saline. Dissolve 2 g NaCl/litre DIW

3. 1.8% Saline. Dissolve 18 g NaCl/litre DIW

4. 0.9% Saline. Dissolve 9 g NaCl/litre DIW

METHOD

1. Divide 8-12 mL whole EDTA blood (collected without the use of plastic mixing beads) into two equal volumes in a pair of 10 mL plastic centrifuge tubes; centrifuge both at 3,000 rpm (1550 x g) for 5 min at 10 degrees Celsius

2. Remove all plasma from both tubes, pool and immediately store in freezer (-20 degrees Celsius or below).

3. To each tube of 'packed cells' add 0.9% saline to give final volume of approximately 8 mL.

4. Add 2-3 mL dextran/saline then mix tubes well, but gently, by repeated inversion. Remove the lids or caps of the tubes and carefully pipette off any bubbles on the surface of the blood.

5. Allow the blood to stand at ambient room temperature for approximately 30 - 45 min or until the red cells have settled out sufficiently (red cells should occupy about the lowest one-third of the tube).

6. Carefully remove the leucocyte-containing supernatants of both tubes and transfer to a pair of fresh, clean 10 mL centrifuge tubes. Discard the red cells.

7. Centrifuge all tubes at 3,000 rpm (1500 x g) for 2 min at 10 degrees Celsius. Pour off supernatants and retain the leucocyte pellets.

8. To each pellet add 4 mL 0.2% saline and mix by pipetting gently up and down with wide bore pasteur pipette. Do not leave the cells exposed to the 0.2% saline longer than about 1 min.

9. Add to each tube 3.2 mL 1.8% NaCl and mix gently by inversion. Centrifuge once more at 3,000 rpm (1550 x g) for 2 min. Pour off and discard the supernatant.

10. Repeat steps 8 and 9 once more to obtain a white cell pellet free of red cells.

    NOTE: The pellet at this stage should be white or, at most, just slightly pink in color.

11. After the final centrifugation, suck off all supernatant and transfer the clearly labelled pellets immediately to the freezer and keep frozen at -20 degrees Celsius or below (-80 is preferred).

12. If sending the prepared samples to the ACH Chemical Pathology Department for enzyme assay, transport the clearly labelled leucocyte pellets (from Step 9) and the plasma (from Step 2) frozen in solid CO2 (transportation logistics are described in Information Sheet 3 (from within Australia) or Information Sheet 4 (from overseas)).


METHOD 2 - Preparation of leucocytes by differential lysis of erythrocytes

PRINCIPLE

Leucocytes are isolated by centrifugation after specific lysis of erythrocytes

REAGENTS

1. Lysis Buffer (155 mmol/L ammonium chloride; 10 mmol/L sodium bicarbonate; 0.1 mmol/L EDTA).

    Dissolve 8.3 g ammonium chloride, 0.84 g sodium bicarbonate and 29.3 mg EDTA in about 900 mL reagent-grade water. Titrate to pH 7.4 with HCl, then make the volume to 1 litre. Store at 4oC.

2. Isotonic Saline (0.9%, w/v)

    Dissolve 9 g NaCl in 1 litre reagent-grade water. Store at 4 degrees Celsius.

METHOD

1. Centrifuge 10 mL EDTA blood to pellet all cells (e.g. 1,500 g for 10 minutes at 4 degrees Celsius).

2. Remove plasma into a clean container and freeze.

3. Restore original blood volume with 0.9% saline and transfer the blood suspension into a 50 mL conical centrifuge tube.

4. Add 40 mL cold lysis buffer.

5. Stand on ice, mixing occasionally, until erythrocytes are lysed (the red cell suspension remains red in colour, but becomes transparent). This should take only about 5-10 minutes.

6. Centrifuge 1,500 g for 5 minutes at 4 degrees Celsius.

7. Discard supernatant and resuspend leucocyte pellet in 5 mL cold lysis buffer.

8. Stand on ice for 10 minutes.

9. Dilute cell suspension to 50 mL with cold 0.9% saline. Mix and centrifuge 1,500 g for 5 minutes at 4 degrees Celsius.

10. Discard supernatant, then resuspend leucocyte pellet in 10 mL 0.9% saline. Care should be taken to obtain an even cell suspension without being too vigorous and causing cell disruption.

11. Divide the cell suspension into two equal aliquots, into two 10 mL conical centrifuge tubes.

12. Centrifuge 1,500 g for 10 minutes at 4 degrees Celsius.

13. Remove all supernatant and dry walls of centrifuge tube with a tissue.

14. Store leucocyte pellets and plasma at -20 degrees Celsius (or -80).

15. Send both leucocyte pellets and plasma frozen on dry ice for analysis.

Back to National Referral Laboratory Home Page

last modified: 26 Jul 2018