Collection of peripheral blood stem cells (PBSC) must be performed in a safe and effective manner. Issues like automation, collection efficiency (CE), and adverse events must be considered. Auto-PBSC (COBE Spectra) is a fully automated program for PBSC collection. Changes in the protocol were made to achieve high CE, low product volume, and resulted in three groups of patients. Standard operating procedures (SOPs) were developed to reduce citrate toxicity and patients with central venous catheter. Twenty patients and 27 collections (Group 1), 88 patients and 112 collections (Group 2), and 158 patients and 194 collections (Group 3) were recorded. The protocol changes increased CE significantly from 31% (Group 1) to 57 and 59% (Group 2 and 3). Adjusting endpoint according to the preapheresis number of CD34+ cells reduced the collection time and the volume of the product significantly (median 227 min and 56 mL) without affecting CE. Mean level of ionized calcium before collection was 1.22 mmol/L, measured in 31 patients. This declined to a mean of 1.07 mmol/L after 1 h of collection and remained unchanged despite continuous calcium infusion. The number of patients with mild symptoms of citrate toxicity was reduced from 20 to 6%. A central venous catheter was used in 15%. Compared to peripheral access no differences in blood flow rate or time to perform the collection were found. Changes in the Auto-PBSC protocol resulted in an improved CE and a small product. SOPs reduced the number of patients with citrate toxicity and with central venous catheter.