2

Appendix

Flow chart and timing of study procedures in the FIRSTLINE-AMI protocol

Calculation of mobilized MNCCD34+

Calculation of the quantity of MNCCD34+ likely to exposed to reperfused myocardium exemplified in one representative case subjected to 10µg G-CSF/kg body weight given subcutaneously over 6 consecutive days after acute myocardial infarction. At baseline 4 MNCCD34+ were detected per microliter (µl) whole blood, with a rapid increase to 54 MNCCD34+ per µl at peak stimulation on day 6. Based on daily MNCCD34+ counts post-ischemic myocardium subtended by one major coronary artery was exposed to 2.8x1010 MNCCD34+ over 8 days with 6 days of G-CSF stimulation.

Day / MNCCD34+ count/µl / MNCCD34+/day
baseline / 4 / 4.6x108
1 / + G-CSF / 8 / 9.2x108
2 / + G-CSF / 18 / 2.1x109
3 / + G-CSF / 27 / 3.1x109
4 / + G-CSF / 38 / 4.4x109
5 / + G-CSF / 47 / 5.4x109
6 / + G-CSF / 54 / 6.2x109
7 / - / 30 / 3.5x109
8 / - / 18 / 2.1x109
∑ 2.8x1010

For the final number of MNCCD34+ the following factors were applied:

x 1000 to convert to ml; x 80 assuming that post-ischemic myocardium is perfused by approximately 80 ml whole blood per minute (or LAD or RCA flow/minute at rest); x 60 to convert to MNCCD34+ offered per hour; and x 24 to convert to MNCCD34+ offered with myocardial perfusion per day. ∑ equals the sum of MNCCD34+ postischemic myocardium is exposed to over 8 days.


Calculation of mobilized leukocytes

Calculation of the quantity of leukocytes likely to expose to reperfused myocardium exemplified in one representative case subjected to 10µg G-CSF/kg body weight given subcutaneously over 6 consecutive days after acute myocardial infarction. At baseline 12 leukocytes were detected per nanoliter (nl) whole blood, with a rapid increase to 56 leukocytes per nl at peak stimulation on day 6. Based on daily leukocyte counts post-ischemic myocardium subtended by one major coronary artery was exposed to 3.1x1013 leukocytes over 8 days with 6 days of G-CSF stimulation.

Day / WBC count/nl / Leukocytes/day
baseline / 12 / 1.4x1012
1 / + G-CSF / 15 / 1.7x1012
2 / + G-CSF / 25 / 2.9x1012
3 / + G-CSF / 33 / 3.8x1012
4 / + G-CSF / 40 / 4.6x1012
5 / + G-CSF / 48 / 5.5x1012
6 / + G-CSF / 56 / 6.5x1012
7 / - / 25 / 2.9x1012
8 / - / 15 / 1.7x1012
∑ 3.1x1013

For the final number of leukocytes the following factors were applied:

x 1,000 000 to convert to ml; x 80 assuming that post-ischemic myocardium is perfused by approximately 80 ml whole blood per minute (or LAD or RCA flow/minute at rest); x 60 to convert to leukocytes offered per hour; and x 24 to convert to leukocytes offered with myocardial perfusion per day. ∑ equals the number of leukocytes the postischemic tissue is exposed to over 8 days.