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# System prepended metadata

title: High level questions

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# High level questions

- Does it make sense to try and build a model that tells us which values are important predictors for the initial state of the expansion phase? What would those initial states be? eg "NK Cells" after the last stage or "NK Cell Viability"?

- Which stage is the DHL3-Export dataset about? (working hypothesis: uAPC activation)

- The data we have does not seem to include instrument settings for stages other than the expansion phase and whatever DHL3-Export is about. 


# Datasets

Is there any hierarchy between these datasets? Are some of them compilations of some others, or are they independent?

- `20221219 PRECO Early Process Data.xmlx`: Measurements of the state of samples in the stages pre-expansion (17 donors).
- `CT_Template_wDT`: Data about the expansion stage (16 donors):
    - Initial state of the sample: Z-Variables
    - Continually measured values: X-Variables and Y-Variables
    - Contunually changing instrument settings: W-Variables
- `DHL3-Export-2023-04-14`: Somewhat similar to CT_Template but for a previous stage (3 donors). Which one? (uAPC activation?) Is the sheet name a typo and supposed to be "Stimulation results"? What is "Stim On" / "Stim Off" / "Reseed"? Why never "Stim On"?
- `EAD170 TAK-007 D0-D18 Master Count Sheet` Data about an individual experiment run (just one donor). I guess this is more to see what other data might possibly be available?

# Stages

## D0 Thaw

### Description

First process step, initial cord blood unit is thawed in the plasmatherm thawing device then diluted up to 200mL in thawing media and human serum albumin. This initial dilution step is conducted in order to dilute the DMSO cryoprotectant and prepare the cells for subsequent processing.

### Available Settings

* None (?)

### Output variables of interest

* `20221219 PRECO Early Process Data.xmlx`
    * NC200 Cytometry: Cell viability, cell diameter, number of cells, etc.
    * Flow Cytometer: Immunophenotypic markers

## D0 Wash

### Description

The thawed and diluted cord blood unit is then transferred to the Sepax instrument and a wash operation is conducted. This wash operation transfers the cord blood unit cells into a different cell suspension and washes out the DMSO cryoprotectant, blood plasma, as well as some red blood cells (RBCs) and Platelets (PLTs).

### Available Settings

* ?

### Output variables of interest

* `20221219 PRECO Early Process Data.xmlx`
    * NC200 Cytometry: Cell viability, cell diameter, number of cells, etc.
    * Flow Cytometer: Immunophenotypic markers

## D0 Ficoll

The washed cord blood unit is then processed on a Sepax instrument using a Ficoll operation. This operation serves to debulk the sample of red blood cells (RBCs), platelets (PLTs), and Granulocytes (Grans) to retain only the mononuclear cells, including monocytes (CD14+), NK cells (CD56+), T-cells (CD3+), and B cells (CD19+).


## Prodigy / Isolation

The debulked sample is then processed on the CliniMACs prodigy instrument in order to select out only the
NK cells (the cells of interest). This is conducted by utilizing magnetic beads conjugated with CD14, CD3, and
CD19 antibodies to bind and remove other impurity cell types.

## D0 uAPC Stimulation

The purified NK culture is then stimulated with universal antigen presenting cells (uAPCs), which help to
promote the proliferation of the NK cells. The co-culture is then left for ~4 days in the incubator to allow the
cells to grow.

### Questions

- Sometimes also called "Activation"? Is that the same?

## D5 Pre-Transduction Wash

On D5 the NK cell culture is washed on the sepax in order to remove the cell media supernatant and prepare
the culture for viral transduction.

## D5/D6 Transduction

The NK cell culture is then processed on the sepax instrument utilizing a viral transduction protocol to enable
the transduction of the NK cells with the engineered viral vector. This is conducted in order to promote the
NK cell expression of the chimeric antigen receptor (CAR) to give the NK cells the required drug potency.

### Question

- Very little data. Is the step unimportant, can we ignore it?

## D9 uAPC Stimulation (second round)

## D9/D22 Expansion

## D22 Harvest