Preparation of Control Materials
In this part, methods are given for preparation of control materials. All laboratories should be able to make a lysate as well as a preserve and stabilize blood specimens for internal quality control of their blood counts. The staff of a central laboratory should be able to make all the preparations for a national external quality assessment scheme.
Accordingly, at least some laboratory workers will be expected to accept this responsibility and must be familiar with the preparation of quality control materials.
Some of these materials are required for the exercises. Organizers of training courses must be able to prepare small batches of the various materials for use by the course participation.
2) General Notes:
How to prepare CPD and ACD (NIH A)? CLICK HERE
Human blood for use as calibration and control material for blood counts should be HBsAg and HIV and hepatitis C virus (HCV) antibody negative. Anitcoagulated blood is usually available from Blood Transfusion Services; the anticoagulant is either citrate-phosphate-dextrose (CPD) or acid citrate dextrose (ACD-NIH A). Lysates blood in other anticoagulants (e.g. EDTA or heparin) can also be used.
In the following procedures care should be taken at all stages to avoid contamination. Where possible sterile glassware and reagents should be used and aseptic handling procedures observed.
Broad spectrum antibiotics should be added to aid sterility, e.g. 25-50 mg of pencillin and 25-50 mg of gentamycin per 500 ml material has been found to be satisfactory.
3) Preparation of some Control materials:
A) Preparation of hemolysate:
1) Centrifuge anticoagulated blood in bottles of appropriate size (e.g. 30ml screw-cap “universal” containers). Remove the plasma and buffy coat aseptically.
2) Add to each red cell deposit an excess of physiological saline (9 g/l NaCl), mix well, and re-centrifuge. Discard the supernatant and any remaining “buffy coat”.
3) Repeat saline wash two times to ensure complete removal of plasma, white cells and platelets, each time removing the top layer of packed red cells.
E.g. If the washed cells is 2 mL then add 2 mL of water and 1 ml of Carbon tetrachloride (i.e. 0.5 volume) or 800 μl of toluene (i.e. 0.4 volume)
4) Add to the washed cells an equal volume of water and 0.5 volumes (i.e. 50% of the volume used) of carbon tetrachloride or chloroform (may not be available in the lab, an alternative is 0.4 volumes of toluene), cap the containers and then shake vigorously on a mechanical shaker or vibrator for one hour. Refrigerate overnight to allow the lipid/cell debris to form a semi-solid interface between carbon tetrachloride and lysate.
5) On the following day centrifuge at about 2500 g for 20 minutes. Transfer the upper lysate layers into universal containers and centrifuge at about 3000 g for an hour. Collect the upper 95% and pool into a clean bottle.
6) To each 70 ml of lysate add 30 ml of glycerol. Add the antibiotics (mentioned in the introduction) , then store stock material at 4˚C for short periods or frozen at -20˚C for a longer period until required for dispensing.
7) If a lower concentration is required add an appropriate volume of 30% (v/v) glycerol in 9 g/l NaCl to the stock then mix well
8) With continuous mixing, dispense aseptically into sterile containers. Then cap and seal.
9) Assign the Hb conc. Of the hemolysate by the Hemiglobincyanide method
The CV should be less than 2% (What is the CV and how to calculate it will be prepared soon).
Stored frozen product should maintain its assigned value for several years