Novel Automated Blood Separations Validate Whole Cell Biomarkers

Background Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. Methods and Findings To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. Conclusions Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

P6: Span_8_200uL named TB1. Discard the tips to "<Tipbox>". When done, move the box to "<Home>". Use these tips "1" times. tipboxlid_lowgrip with an unknown amount of an unknown liquid.
Pelt1: RM4_Magnet named Magnet with an unknown amount of an unknown liquid.
R1: rm4_tubeholder named Cryo1 with an unknown amount of an unknown liquid.tipboxlid_lowgrip with an unknown amount of an unknown liquid.
Wash only the probes that have been used.

Span-8 Discard Tips
Discard tips from all probes on Pod2.
Wash all probes of Pod2 at the closest wash station; do a passive wash by dispensing 1 mL while in the wells and 1 mL to waste. Delay for 300 ms after each dispense.
Wash only the probes that have been used.

Span-8 New Tips
Get new Span_8_1000uL_LLS tips for probes 1-6 on Pod2.
- - Wash all probes of Pod2 at the closest wash station; do a passive wash by dispensing 1 mL while in the wells and 2 mL to waste. Delay for 300 ms after each dispense.
Wash only the probes that have been used.     ' start dispensing at 2 mm below liqlevel Extend "ZDispBeads", (DispHeightCM * 10) -2 Prompt: [IDispatch] Tooltip: Scripted Let Using Pod2, execute the following transfer: From: Cryo2, with the following pattern: Proceed down first, then left to right.
Start from the beginning of the selection.
Set the mark at the last well transferred.
Use the following custom technique: Use the following pipetting template: Span-8 P1000 2 Calibration Offset: 0 Probes 1-6 will be used.
Start from the beginning of the selection.
Do not set the mark.
Start from the beginning of the selection.
Do not set the mark.
- Start from the beginning of the selection.
Set the mark at the last well transferred.
Use the following custom technique: Use the following pipetting template: OS Span-8 P1000 SlowRetract 2 To: Cryo1, =VolTripWBµL, sections specified by "=Wells", Water Proceed down first, then left to right.
Start from the beginning of the selection.
Set the mark at the last well transferred.
Use the following custom technique: Use the following pipetting template: Span-8 P1000 2 Calibration Offset: 0 Probes specified by "=TwoTips()(MFX.NumSamples)" will be used. Start from the beginning of the selection.
- Start from the beginning of the selection.
Do not set the mark.
- Start from the beginning of the selection.
Set the mark at the last well transferred.
Use the following custom technique: Use the following pipetting template: OS Span-8 MultiDispense Slow Retract 2 Aspirate at most =MaxVolInTips µL per transfer for repeated dispensing.
Create 1 replicate(s) of each source well.
Load Span_8_1000uL_LLS onto the pod.
Keep the tips when finished.
Stop when finished with Destinations.
- Start from the beginning of the selection.
Set the mark at the last well transferred.
- Start from the beginning of the selection.
Set the mark at the last well transferred.
- Start from the beginning of the selection.
Set the mark at the last well transferred.