One key component of immune monitoring of patients on immunotherapeutic trials is the clinical sample collection process. Peripheral blood is typically sampled in order to evaluate systemic immune responses since it contains circulating lymphocytes, monocytes, and mediators that regulate immunity. Examining changes in these peripheral blood components may elucidate biomarkers that correlate with clinical activity.
The blood collection process begins with patient consent for the collection of their samples for research.
The patient arrives at the clinic where a clinician reviews the consent documents with the patient and answers any questions the patient may have. Once patients provide consent, they are enrolled in the study.
The research blood sample is drawn at the clinic by a phlebotomist working with the clinical team of the study.
The phlebotomist records what time the blood was drawn on the requisition form.
The blood sample and the requisition form are then delivered by stat messenger from the clinic to the lab.
The messenger notes the drop off time which allows the lab member receiving the sample to know how much time has elapsed from when the blood was drawn and when it arrived at the lab. This is important because if there is a delay in the sample arriving at the lab, that can impact the integrity of the sample.
Lab members frequently check the bin outside the processing room for new samples so that the samples can be processed quickly.
The sample is then taken into the processing room and documentation begins in order to ensure accurate sample tracking. The lab member adds information to the requisition form such as the time blood processing began, how many samples were received, and which study protocol the samples are for.
While documentation is happening, the samples are run through the centrifuge to separate and remove the red blood cells to isolate the peripheral blood mononuclear cells or PBMC.
The lab member then finds the clinical study binder that is relevant for the study the sample is associated with and notes this is a new addition to that study.
The lab member then signs the sample in to the C-R-D-B clinical research database, which is an MSK in-house software used for tracking clinical specimens.
Once the samples are spun in the centrifuge processing begins. The samples move to the tissue culture hood where the PBMC monolayer is carefully transferred to a separate tube and washed multiple times to remove unwanted materials such as contaminating platelets and residual density gradient solution.
Plasma can also be collected from the separated blood samples and banked as well.
Now the cells are ready to be counted. A portion of the sample is taken for staining with a viability dye and counted using an automated cell counting instrument to distinguish which cells are alive and which are dead to help get an approximate sense of the viability of the samples.
The viable PBMC concentration is then documented on the requisition form and in the clinical research database.
The samples are then resuspended in cell freezing medium and put in controlled cooling containers prior to transfer to the minus 80 degree C freezer.
The samples typically stay in the minus 80 degree freezer for 24-72 hours.
Then they are taken out of the cooling containers and placed into cryo-boxes and are ready to be moved to liquid nitrogen freezers.
A lab member refers to the current freezer map to find the next available slots in the liquid nitrogen freezer for the samples to be placed and documents their location as part of sample inventory management to ensure accurate sample tracking.
Now that the final sample locations are documented, the sample requisition forms go into the appropriate study binder.
The PBMC samples remain in the liquid nitrogen freezer until they are ready to be analyzed. The principle investigator or PI dictates what type of experiment will be conducted on the samples.
There are a number of immune monitoring assay platforms in the lab that can be carried out, including multiparameter flow cytometry, multiplex cytokine immunoassays, serological assays, and protein micro-arrays.
For a typical flow staining experiment, the lab member working on the project retrieves the relevant samples to be analyzed from the liquid nitrogen freezer and brings them to the lab to prepare them for the assay. Samples are typically batch analyzed in order to minimize the inter-assay variability across experimental runs.
First, the PBMC samples need to be thawed, washed again and counted. This is done to determine what the live cell recovery is after thawing the cells and calculate how much sample there is to work with in the assay.
Then the samples are taken to the bench and the lab member will set up the experiment.
This is where the samples are stained with a number of different antibodies conjugated to unique fluorescent molecules. Depending on the flow staining panel used, the results can measure the frequency of specific immune cell subsets in patients and evaluate whether the cells of interest are activated, proliferating, or expressing certain exhaustion or maturation markers.
After the cells have been stained and washed with the relevant antibody panel, they are ready for acquisition and analysis on the flow cytometer.
The results are collected, analyzed using flow analysis software, and data is then exported into Excel. Graphs are produced from the Excel statistics and summarized into PowerPoint slides showing a summary of the cell marker data for every sample that was run.
That data is then shared with the lab head of the immune monitoring core for review and quality control.
After the lab head edits and approves the final results, they then go to the PI where the data can be subsequently transferred for bioinformatics processing and correlation with clinical outcomes.