這裡將向您展示如何在Kaluza 分析軟體中 修改參數名稱和描述 , * 如何載入和解除安裝參數 以及如何 將這些更改傳輸到更多數據集。將提供範例以示範如何 使用編輯參數名稱將 Kaluza 分析Protocol傳輸到使用不同儀器配置獲取的數據集 以及 以及如何使用 Kaluza Cytobank 外掛程式從數據集中刪除參數。

流式細胞術中的參數名稱

在細胞術中，參數是從流式細胞儀偵測器收集的數據，包括光散射、螢光、面積、高度和寬度的測量值。有關獲取參數的訊息存儲在符合FCS3.0或FCS3.1標準檔的TEXT段中（Spidlen 等人，2009）.  FCS 關鍵字 $PnN 用於存儲參數 n 的短名稱。Kaluza 將此資訊顯示為參數名稱。給定參數的短名稱可以來自細胞儀獲取的參數序列 （例如，CytoFLEX平臺上符合FCS3.0標準（Seamer等人，n.d.）的FLn） ，或者可以反映通常在這裡檢測到的螢光染劑的名稱。

Figure 1. Kaluza Parameters Pane showing Parameter Names ($PnN) and Description ($PnS) for data acquired on a CytoFLEX LX instrument. The Information Plot shows the Description, FCS keyword and value for the cytometer type, short name for parameter 4 and name used for parameter 4 as an example. 

Editing Basic Parameter Data

To update basic parameter data such as Parameter Name and Description:

1. Highlight the text in the field that you wish to change.


2. Press the Delete key on your keyboard.


3. Type the new content into the field and press Enter.

TIPS FOR SUCCESS:

• When parameters are updated from the Parameters pane, all references to that parameter in the Protocol are also updated.


• The described procedure changes FCS Keywords $PnN (Name) and $PnS (Description) in files saved in Kaluza Analysis only. The raw data file keywords remain unchanged. A new FCS file showing the updated values for $PnN (Name) and $PnS (Description) may be exported using the Kaluza Cyotbank Plugin. 


• There is a limit of:
  • 5,000 characters for parameter name.
  
  
  • 5,000 characters for parameter description.
  
  
  • 5,000 characters for parameter detector.

Editing Loaded Parameters

Kaluza can load up to 64 parameters. Files with more parameters can be opened, but additional parameters are disabled and cannot be used for analysis. The parameters that are loaded into Kaluza Analysis from the Data Set can be edited to select specific parameters for analysis. Editing available parameters is done through the Edit loaded parameters window .

Figure 2. Edit loaded parameters window

1. From the Parameters pane, select the (Edit Loaded Parameters) icon to open the Edit loaded parameters configuration window.


2. Make all necessary configurations by making the necessary entries or clicking the desired buttons or check boxes.


3. When finished with all modifications, click OK. The parameters listed in the Parameters pane (and available in the Protocol) will now reflect the changes.

Copying Parameters to Other Data Sets

This function allows you to copy parameter details (Name, Description, and corresponding meta data) to other Data Sets.

1. Right click on the Analysis List entry from which to copy the parameter data, and then select Copy.


2. On the Analysis List, select the Data Sets to receive the updated parameters.


3. Right click on any of the selected rows, and then select Paste Special > Parameters.


4. Verify the updated Analysis List row(s) to ensure the parameters were updated.

TIPS FOR SUCCESS:

Copying Parameter information using the Paste special functionality will not apply the selection of loaded parameters. The Batch Processing functionality may be used to load the same parameters in a large number of data sets. See Kaluza Analysis Flow Cytometry Software IFU C10986 for detailed instructions on using Batch Processing. 

• Load data sets to be processed.


• In the first data set, load the desired parameters and edit Parameters Names and Descriptions as needed.


• Ensure the Cascade parameters values from previous analysis check box is selected.


• Ensure Save batched files as .analysis files check box is selected.


• Choose Process all to automatically process all files without further review, choose Next to manually advance file by file. Kaluza Analysis will prompt you by displaying a Browse for Folder window to specify a folder for file outputs. Kaluza Analysis will prompt you by displaying a Browse for Folder window to specify a folder for file outputs. 

Use Case: Editing Basic Parameter Data to Apply Protocols Across Different Instrument Configurations

Figure 4. List of Parameters showing Names and Descriptions and Analysis Protocol established on CytoFLEX LX data.

Figure 5. Three Parameters that are used in the protocol are not available in the data set. Non-available parameters are marked in red. 

When acquiring the same panels on instruments with different configurations, the Parameter Names ($PnN) assigned for specific markers may depend on the instrument used for acquisition. When applying a Kaluza Protocol from one data set (e.g. for CytoFLEX LX) to another data set with a Parameter Name mismatch (e.g. from a V5-B5-R3 CytoFLEX), an error message will indicate parameters used in the Protocol that are not present in the second data set (Figure 5).
To prevent the Parameter Name mismatch, update the Parameter Names in the original data set that was used to establish the Kaluza Analysis Protocol to match the Parameter Names of the data set from the second instrument. 

1. Unload all Parameters that are not used for the Analysis.


2. Re-name Parameter Names to match the data set the protocol should be applied to.


3. Apply Protocol to second data set.

Figure 6. Parameter Names A) before and B) after manual adjustment.

When all Parameter Names for the Parameters used in the gating strategy match between data sets, the Kaluza Analysis Protocol can be applied by Drag&Drop in the Analysis list. If gains (or voltages) have been standardized between the instruments, only minor gate adjustments will be required. If gains (or voltages) have not been standardized or data was acquired on instruments from different families, adjustments to the scale of different Parameters may be required.

Figure 8. Kaluza Analysis Protocol established on CytoFLEX LX data applied to data acquired on 3L 13C CytoFLEX after scale and gate adjustments.

TIP FOR SUCCESS:

Instruments belonging to the CytoFLEX family save height and area information as individual parameters for all acquired parameters. If the analysis of only area or only height for fluorescence channels is desired, unloading channels that are not needed can help make the Parameter list more manageable.

範例：使用 Kaluza Cytobank 外掛程式刪除參數

Cytobank外掛程式為Kaluza添加了新功能，可以將FCS檔案從Kaluza上傳到Cytobank雲端平臺以進行進一步分析。Cytobank外掛程式允許Kaluza用戶創建新的FCS檔案，並對參數名稱和FCS關鍵字以及Kaluza中生成的螢光補償和邏輯變換進行編輯。此外，在使用 Kaluza Cytobank 外掛程式匯出新的 FCS 檔案之前，可以使用 Kaluza 中的 合併功能函數將來自多達 48 個數據集的數據連接起來。

為了分析Cytobank平臺上同一Panel內的數據，通道數量需要在所有檔中保持一致。如果您正在處理包含不同通道數的檔案，並且您最終希望這些文件位於 Cytobank 平臺上的同一Panel中，則 Kaluza 和 Kaluza Cytobank 外掛程式可用於生成新的派生 FCS 檔案，其中僅包含使用者定義的通道數。要上傳到 Cytobank 平台的參數是通過在 Kaluza 中載入和解除安裝參數來選擇的。

有關使用 Kaluza 細胞庫外掛程式的詳細說明，請參閱用於 Kaluza 分析流式細胞術軟體的細胞庫外掛程式 PN C56576。Kaluza Cytobank 外掛程式可以從 Kaluza 分析軟體下載頁面進行下載： https://www.beckman.com/flow-cytometry/software/kaluza/downloads。使用外掛程式需要有效的 Cytobank Premium 或 Enterprise 帳戶。

1. Load the data set in Kaluza Analysis.


2. Use the Edit loaded parameters window to load only the parameters desired to be present in the new FCS file


3. Open the Kaluza Cytobank Plugin


4. Login to your Cytobank account through the Kaluza Cytobank Plugin


5. Make adjustments to update the compensation, data transformation or FCS keywords


6. Select “Next“ to select the location where you want to save the FCS file.


7. Select the location where the FCS files will be saved. This step allows you to store the newly generated FCS files locally and to manually add several files to the Cytobank platform in one upload. Define the location and select “Save“.


8. Select an existing experiment or create a new experiment on the Cytobank platform that you want to add the newly generated FCS file to.


9. Select Upload to upload your data to the Cytobank Platform. Confirm the upload request when prompted. 

Figure 9. Generate FCS file window in Kaluza Cytobank Plugin workflow.

TIPS FOR SUCCESS:

• The Kaluza Cytobank Plugin will use the Parameter names from Kaluza and apply them to the FCS file that will be created.


• By default, the compensation matrix from Kaluza Analysis Software, including any adjustments you have made, will be included in the FCS file or applied to the data when exporting logicle transformed data. Uncheck the checkbox for „Including the compensation matrix“ if you wish to export uncompensated data.


• In order to apply the logicle transformation setting as defined in Kaluza, check the „Apply logicle transformation“ box. If this box is checked both compensation and logicle transformation will be applied to the data. You will not be able to adjust the compensation or transformation in the Cytobank software.


• To add or modify FCS keywords click the “+“ icon in the “New and Modified Keywords“ window to select keywords. Keywords that can be edited are displayed in green. Select a keyword to edit it and add the desired value or text to the “New Value“ column.

參考文獻

1. Seamer, L. C., Bagwell, C. B., Barden, L., Redelman, D., Salzman, G. C., Wood, J. C. S., & Murphy, R. F. (n.d.). Proposed new data file standard for flow cytometry, version FCS 3.0. 5.


2. Spidlen, J., Moore, W., Parks, D., Goldberg, M., Bray, C., Bierre, P., Gorombey, P., Hyun, B., Hubbard, M., Lange, S., Lefebvre, R., Leif, R., Novo, D., Ostruszka, L., Treister, A., Wood, J., Murphy, R. F., Roederer, M., Sudar, D., … Brinkman, R. R. (2009). Data File Standard for Flow Cytometry, version FCS 3.1. Cytometry Part A, 9999A, NA-NA. https://doi.org/10.1002/cyto.a.20825

* For Research Use Only. Not for use in diagnostic procedures.