First, assume a patient sample contains 10 IU/ml plasma. When the blood has a hematocrit of about 50%, the plasma volume will constitute about 50% in the sample. All tPA is present in the plasma fraction. The initial collection of blood in citrate reduces the tPA plasma concentration about 10%, resulting in a concentration of about 9 IU/ml. Mixing 1 volume of citrated blood with 1 volume of acetate buffer corresponds, with a hematocrit of 50%, to mixing 0.5 volumes of plasma with 1 volume of acetate buffer. The plasma is diluted 1:2, and at this stage has a tPA concentration of 3 IU/ml. The addition of 10 ml HCl to 150 ml acidified plasma results in a tPA concentration of 2.8 IU/ml. Thus, a plasma sample containing 10 IU/ml will actually contain 2.8 IU/ml, after the prescribed procedure. The 10 IU/ml tPA standard is diluted from 20 ml to 340 ml (100 ml tPA/PAI depleted plasma + 200 ml acetate buffer + 20 ml HCl + 20 ml tPA standard) and its actual concentration is therefore 50 x 20/340 = 2.9 IU/ml.