Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

This video describes The procedure developed by the HIV Vaccine Trials Network at Fred Hutchinson Cancer Research Center for staining fresh whole blood in multicenter clinical trials to be analyzed by a centralized laboratory. The procedure utilizes tru count tubes made by Becton Dickenson biosciences, each of which contains a lyophilized pellet that dissolves during sample preparation to release a known number of fluorescent beads. Fluorescent antibodies are used to stain for surface markers that are specific to different cell populations.

The number of cells in these different populations can be compared to the number of fluorescent beads during flow cytometry analysis and absolute concentrations of different leukocyte populations in whole blood can be tracked over time in relation to intervention. Hello, my name is Erica Anderson Nissan and I'm a staff scientist in the HIV Vaccine Trials Network. The procedure that you're about to watch is one that we've developed for use in our multicenter clinical trials.

In these trials, blood processing laboratory technicians receive fresh hole blood specimens, stain them with fluorescently labeled antibodies, and then ship the specimens to a centralized laboratory that's capable of analyzing them on the flow. Cytometer fresh hole blood is often not analyzed in the context of clinical trial settings due to cost and feasibility issues, but we've developed a single antibody staining panel that can identify the major leukocyte populations in the peripheral blood and staining for these populations takes under 45 minutes making it feasible for use in clinical trials in the HVTN, we're using this panel to understand the early events that occur after vaccination with candidate HIV vaccines, but this PI panel could be further modified to focus in on cell types of interest to the individual investigator. Thank you very much for watching and we hope that this will be of use to you in the future.

Table One shows an 11 color antibody staining panel developed to phenotype and enumerate the major leukocyte populations within the peripheral blood. The antibody cocktail is made by the central assay lab and sent to the processing labs to ensure consistency between sites and to allow for cocktails to be tested on a control blood draw before they're used to stain trial samples. Each fluorescent conjugated antibody is titrated separately and then the antibodies are combined using appropriate titers into a single mixture with flow wash buffer containing delcos PBS with 2%heat and activated fetal bovine serum.

The antibody cocktail is labeled with a batch number and expiration date and shipped from the central assay lab to the processing labs using a packaging system that maintains a temperature of four degrees Celsius during shipping. Once received, the antibody staining panel can be stored at four degrees Celsius in the dark for up to eight weeks. BD fax slicing solution is used after staining to lice the red blood cells and fix the antibody stains while preserving the leukocytes.

The solution is received from the manufacturer at a 10 x concentration and is diluted one to 10 by the central assay lab with deionized water. The diluted solution is then labeled with a lot number and expiration date and shipped at ambient temperature to the processing labs once received. Fax lacing solution is stored at room temperature between 15 and 30 degrees Celsius prior to drawing blood For true count staining.

The processing lab must make sure that all of the appropriate reagents have been obtained from the central assay lab. These include BD TRU count tubes, the combined antibody staining panel, and one x fax licensing solution. The HVTN utilizes a laboratory data management system to record all relevant information for each sample.

A unique identifying code is printed on a label which is placed on the true count tube. To track the specimen throughout the process, label a cryo vial to identify the sample being stained. This will usually include a patient or volunteer identifier, A visit identifier and the date of blood collection aliquot the required amount of blood into the labeled cryo vial using aseptic technique.

The amount of blood used for staining depends on the staining panel being used, but is usually 50 or 100 microliters per panel. The cryo vial can then be set aside for up to four hours at room temperature before staining. One more time sensitive procedures are performed on the remaining blood.

Verify that there is an intact bead pellet in the bottom of the tube and label the tru count tube. With the relevant specimen information record the lot numbers and expiration dates of all reagents. Worksheets are useful for tracking this information throughout a clinical trial.

The tru count tube bead count number and lot number can be found on The front of the bag of tru count tubes. When you are ready to begin the tru Count staining process, make sure that you have all the materials and reagents ready for use in the biological safety cabinet. These include the blood aliquot labeled tru count tube combined antibody staining panel tube, one x fax lacing solution, 200 microliter and 1000 microliter micro pipettes and pipette tips, foil bleach, laboratory film, and a timer after staining.

You'll also need a stained specimen box, but this does not need to be in the hood. The amount of blood and antibody to use varies depending on which cells are distinguished by the panel and how many cells are needed for proper analysis. Each panel is tested on control samples to determine the optimal amount of blood for staining and the amount of antibody used is equal to the amount of blood used for the panel shown in this video.

100 microliters of blood and 100 microliters of antibody will be used before pipetting. Mix the blood by gently inverting the tube several times and gently pipette up and down several times. To further ensure that the blood is well mixed.

Then use reverse pipetting to accurately pipette whole blood from the aliquot into the tru count tube just above the metal retainer. Avoid smearing blood down the side of the tube. Replace the pipette tip between each tru count tube.

Now it is time to stain the blood with the antibody staining panel before pipetting. Mix the staining panel by gently pipetting up and down several times. Add 100 microliters of the antibody mixture to the blood in the true count tube.

Do not reverse pipette in this step as it will waste valuable reagent. Cap the tube and vortex at low speed for approximately 15 seconds to mix. Make sure the bead pellet is dissolved completely.

Cover the tube with foil and set a timer to stain the blood for 15 minutes. The amount of one x fax slicing solution needed is nine times the amount of blood used in the assay, so in this example, 900 microliters of one x fax slicing solution is used. After the 15 minute incubation, add 900 microliters of the one x fax slicing solution to the stain blood to fix the stain and lice the red blood cells.

Reverse pipetting is not necessary in this step. Cap the tube and vortex at low speed for approximately 15 seconds to mix. Push the cap down firmly into locking position and seal.

With laboratory film. Store the tubes in a dark box or completely wrapped in foil at minus 65 to minus 95 degrees Celsius until they're shipped to the central assay.Lab. To date testing has shown Samples to be stable for up to four weeks at this temperature, stain samples must be shipped As category B biological substances following International Air and Transport Association or I ATA.

Regulations at the HVTN samples are shipped weekly from the processing labs to the central assay lab using an insulated shipping system from safety pack ink that is specifically designed to meet these requirements. When using this system, place the inner brown box inside the styrofoam chest, securing it to the indentation to prevent shifting. Fill the chest completely with dry ice and wait five to 10 minutes to allow the box to cool down.

To prepare specimens for shipping, completely wrap each tube and foil and place in the stained specimen box. Place absorbent material in a leakproof poly bag with the stained specimen box. Remove excess air from the bag and tightly seal.

Place the leakproof poly bag inside of a Tyvek bag. Again, removing all of the air from the bag before ceiling. Place the specimen package inside the inner brown box and place the lid tightly on the styrofoam chest.

Securely tape the shipping box and ship as biological substance category B with the proper dry ice markings. Follow I ATA PI six 50 instructions in multicenter clinical trials. It is important to track specimens throughout the short-term storage, shipping, and long-term storage stages.

The data management system used by the HVTN allows for the creation of a shipping manifest that can be imported into the specimen database at the central assay lab where the samples will be stored and analyzed. This creates a record of when the samples were shipped and received, where they were terminally stored And when they were removed. For analysis, remove the stain sample from the Freezer to thaw room temperature in a dark box.

No more than one hour before collecting on the flow cytometer. If collecting data on more than one sample stagger thawing so that the tubes are not sitting at room temperature for more than one hour each samples should be acquired using a four laser flow cytometer equipped with appropriate filters such as the BD LSR two, use standard cytometer calibration and fluorescence compensation methods for data collection if required by the instrument. To set a threshold, set the lowest possible am cyan channel threshold vortex is sample tube for five seconds before loading on the flow.

Cytometer tru count beads fluoresce highly in many channels. Gate the beads during collection by finding the population that is highly double positive. For PE SCI five and a PC, note that the two colors that most easily distinguish the beads from the cells may vary depending on instrumentation.

Select the tru count bead gate as your stopping gate and record data until at least 20, 000 beads are acquired. Analyze the data using appropriate software such as FlowJo. Now we will go through the gating scheme utilized for analysis of major leukocyte populations using data from a representative control blood draw.

More specific cell subsets can also be distinguished using the panel such as NKT cells or neutrophils, but these will not be shown in this example. First, an inclusion gate and exclusion gate are drawn around the tru count beads and placed on top of one another one to get the beads for counting and one to exclude the beads from the cellular analysis. Next, the cellular data excluding the beads is gated to include only single cells using forward scatter height versus forward scatter area.

This gate also excludes small debris. The CD 45 positive cells are then gated, separating the granulocytes from the lymphocytes and monocytes using side scatter. The lymphocytes and monocytes are then separated into three distinct groups using CD 14 and side scatter.

These include CD 14 negative lymphocytes, all CD 14 negative cells and non lymphocytes from the CD 14 Negative lymphocyte gate cells are first divided into three groups, CD three positive CD 19 negative CD three negative CD 19 positive and CD three negative CD 19 negative T cells are gated from the CD three positive CD 19 negative gait. After excluding CD 56 positive and CD 16 positive cells and CD four positive and CD eight positive T cells can be distinguished. B cells are gated from the CD three negative CD 19 positive gait after excluding CD 56 positive and CD 16 positive cells and NK cells are gated from the CD three negative CD 19 negative gait.

After excluding possible contaminating monocytes using CD 11 C and CD four, the NK cells are divided into CD 56 brite, CD 56 DM and CD 56 negative populations. To distinguish dendritic cell population, all CD 14 negative cells are gated to exclude CD three positive, CD 19 positive, CD 56 positive and CD 16 positive cells. From there, the HL A DR positive cells are gated and myeloid dendritic cells and plasmacytoid dendritic cells can be distinguished by their CD 11 C and CD 1 23 expression.

Lastly, from the non lymphocyte gait, monocyte subsets are gated after excluding possible contaminating CD 56 positive CD three positive and CD 19 positive cells. Non-classical, intermediate and classical monocytes are distinguished based on their CD 16 and CD 14 expression. Once the populations of interest are gated concentrations can be calculated.

Comparing the number of events in the bead gate to the total number of beads originally in the tube will allow you to determine the ratio of sample collected, which can then be used to determine the absolute concentration. Our cells per microliter for each population. The equation shown here can be used for this Purpose.

In this video, We demonstrated the procedure used by blood processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stain samples at a central assay lab. In the context of a multicenter clinical trial, we have demonstrated that the assay is simple and relatively shortened duration, making it feasible for blood processing laboratory technicians to perform it and to freeze and shift the samples to essential assay lab for analysis. The key to obtaining accurate and consistent results is to carefully standardize critical steps in the protocol and to utilize a documentation system that keeps track of all of the steps so that any anomalies that are discovered can be traced to the source of the air.

The data generated from this staining panel can be used to track changes in leukocyte concentrations over time and could easily be further developed to assess activation states of specific cell types of interest.