Physical Address
304 North Cardinal St.
Dorchester Center, MA 02124
-
Chapter Outline
-
Preparation of commonly used reagents 561
-
Preparation of glassware 564
-
Sizes of tubes 565
-
Speed of centrifugation 565
-
Statistical procedures 565
-
Automated (mechanical) pipettes 567
-
Autodiluters 567
Preparation of commonly used reagents
Water
For most purposes, still-prepared distilled water or deionised water is equally suitable. Throughout this text, this is implied when ‘water’ is referred to. When doubly distilled or glass-distilled water is required, this has been specially indicated, and when tap water is satisfactory or indicated, this, too, has been stated.
Anticoagulants and preservative solutions
Acid–citrate–dextrose (ACD) solution – NIH-a
| Trisodium citrate, dihydrate (75 mmol/l) | 22 g |
| Citric acid, monohydrate (42 mmol/l) | 8 g |
| Dextrose (139 mmol/l) | 25 g |
| Water to 1 litre |
Sterilise the solution by autoclaving at 121 °C for 15 min. Its pH is 5.4. For use, add 10 volumes of blood to 1.5 volumes of solution. For use in red cell survival studies, see p. 358.
Acid–citrate–dextrose (Alsever) solution
| Dextrose (114 mmol/l) | 20.5 g |
| Trisodium citrate, dihydrate (27 mmol/l) | 8.0 g |
| Sodium chloride (72 mmol/l) | 4.2 g |
| Water to 1 litre |
Adjust the pH to 6.1 with citric acid ( c . 0.5 g) and then sterilise the solution by micropore filtration (0.22 μm) or by autoclaving at 121 °C for 15 min.
For use, add 4 volumes of blood to 1 volume of solution.
Citrate–phosphate–dextrose (CPD) solution, pH 6.9
| Trisodium citrate, dihydrate (102 mmol/l) | 30 g |
| Sodium dihydrogen phosphate, monohydrate (1.08 mmol/l) | 0.15 g |
| Dextrose (11 mmol/l) | 2 g |
| Water to 1 litre |
Sterilise the solution by autoclaving at 121 °C for 15 min. After cooling to c . 20 °C, it should have a brown tinge and its pH should be 6.9.
Citrate–phosphate–dextrose (CPD) solution, pH 5.6–5.8
| Trisodium citrate, dihydrate (89 mmol/l) | 26.30 g |
| Citric acid, monohydrate (17 mmol/l) | 3.27 g |
| Sodium dihydrogen phosphate, monohydrate (16 mmol/l) | 2.22 g |
| Dextrose (142 mmol/l) | 25.50 g |
| Water to 1 litre |
Sterilise the solution by autoclaving at 121 °C for 15 min. For use as an anticoagulant preservative, add 7 volumes of blood to 1 volume of solution.
Citrate–phosphate–dextrose–adenine (CPD-a) solution, pH 5.6–5.8
| Trisodium citrate, dihydrate (89 mmol/l) | 26.30 g |
| Citric acid, monohydrate (17 mmol/l) | 3.27 g |
| Sodium dihydrogen phosphate, monohydrate (16 mmol/l) | 2.22 g |
| Dextrose (177 mmol/l) | 31.8 g |
| Adenine (2.04 mmol/l) | 0.275 g |
| Water to 1 litre |
Sterilise the solution by autoclaving at 121 °C for 15 min. For use as an anticoagulant preservative, add 7 volumes of blood to 1 volume of solution.
Low ionic strength saline (LISS)
| Sodium chloride (NaCl) (30.8 mmol/l) | 1.8 g |
| Disodium hydrogen phosphate (Na 2 HPO 4 ) (1.5 mmol/l) | 0.21 g |
| Sodium dihydrogen phosphate (NaH 2 PO 4 ) (1.5 mmol/l) | 0.18 g |
| Glycine (NH 2 CH 2 COOH) (240 mmol/l) | 18.0 g |
| Water to 1 litre |
Dissolve the sodium chloride and the two phosphate salts in c . 400 ml of water; dissolve the glycine separately in c . 400 ml of water; adjust the pH of each solution to 6.7 with 1 mol/l NaOH. Add the two solutions together and make up to 1 litre. Sterilise by Seitz filtration or autoclaving. The pH should be within the range of 6.65–6.85, the osmolality 270–285 mmol and conductivity 3.5–3.8 mS/cm at 23 °C.
K 2 EDTA
| Ethylenediaminetetra-acetic acid (EDTA), dipotassium salt | 100 g |
| Water to 1 litre |
Allow appropriate volumes to dry in bottles at c . 20 °C so as to give a concentration of 1.5 ± 0.25 mg/ml of blood.
Neutral EDTA, pH 7.0, 110 mmol/l
| EDTA, dipotassium salt | 44.5 g |
| (or disodium salt | 41.0 g) |
| 1 mmol/l NaOH | 75 ml |
| Water to 1 litre |
Neutral buffered Na 2 EDTA, pH 7.0
| EDTA, disodium salt (9 mmol/l) | 3.35 g |
| Disodium hydrogen phosphate (Na 2 HPO 4 ) (26.4 mmol/l) | 3.75 g |
| Sodium chloride (NaCl) (140 mmol/l) | 8.18 g |
| Water to 1 litre |
Saline (normal ionic strength)
| Sodium chloride (NaCl) (154 mmol/l) | 9.0 g |
| Water to 1 litre |
Trisodium citrate (Na 3 C 6 H 5 O 7 .2H 2 O), 109 mmol/l
Dissolve 32 g ( or 38 g of 2Na 3 C 6 H 5 O 7 .11 H 2 O) in 1 litre of water. Distribute convenient volumes (e.g. 10 ml) into small bottles and sterilise by autoclaving at 121 °C for 15 min.
Heparin
Powdered heparin (lithium salt) is available with an activity of c . 160 iu/mg. Dissolve it in water at a concentration of 4 mg/ml. Sodium heparin is available in 5 ml ampoules with an activity of 1000 iu/ml. Add appropriate volumes of either solution to a series of containers and allow to dry at c . 20 °C so as to give a concentration not exceeding 15–20 iu/ml of blood.
Buffers
Barbitone buffer, pH 7.4
| Sodium diethyl barbiturate (C 8 H 11 O 3 N 2 Na) (57 mmol/l) | 11.74 g |
| Hydrochloric acid (HCl) (100 mmol/l) | 430 ml |
Barbitone buffered saline, pH 7.4
| NaCl | 5.67 g |
| Barbitone buffer, pH 7.4 | 1 litre |
Before use, dilute with an equal volume of 9 g/l NaCl.
You're Reading a Preview
Become a Clinical Tree membership for Full access and enjoy Unlimited articles
If you are a member. Log in here
