A multi-step centrifuge method for extraction of leucocytes and preparation of Tc-HMPAO labeled leucocytes

Labeled leucocytes could be used for localization of infection foci after surgeries or in inflammatory diseases including inflammatory bowel diseases. Extraction of leucocytes needs 10% Hetastarch which is not available in Iran. We provide a method employing multiple centrifuges to extract and label leucocytes with Tc-HMPAO.

The study was conducted from April to June 2018 in the Nuclear Medicine Unit of Valiasr Hospital. Leucocytes were extracted from a 60 ml blood sample anticoagulated with Acid-citrate-dextrose through four-step centrifugation as below: 1-whole blood was centrifuged at 1k cycle per minute (CPM) for eight minutes to precipitate red blood cells (RBC). Supernatant including RBC free plasma, WBC, and platelet was extracted for the next step. 2-WBC was precipitated at 1.8k CPM for five minutes and platelet-rich plasma (PRP) as supernatant. 3- PRP was centrifuged at 3k for five minutes and cell-free plasma (CFP) was extracted as supernatant, and 4- precipitate WBS at step two was washed with saline and centrifuged at 0.5k CPM to achieve washed WBC. Then the leucocytes were labeled with 40 mCi Tc-HMPAO through 15 minute incubation at 37-38 degrees centigrade. The extra free pertechnetate was eliminated using two additional centrifugation steps as follows: 1-0.5k CPM for five minutes to dispense free pertechnetate, and 2-0.5 for five minutes to achieve high radiochemical purity labeled WBC. Finally, the labeled WBC was re-suspended in CFP and reinjected to the patient. Imaging at 2-4 hours was done. The pathology and imaging of labeled WBC distribution are reported

No RBC was detected in microscopy. The majority of the leucocytes were lymphocytes with rare accompanying platelets. The radiolabeling efficiency of the procedure was higher than 40%. The viability test indicated more than 80% of viable cells. The radiochemical purity of the final product was more than 95%. Two to four hours after injection, low background images were acquired. The liver and spleen were target organs with low-grade urinary, thyroid, and GI activity.

Employing multi-stage centrifugation, Tc-HMPAO labeled leucocyte scan could be efficiently performed.