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14. Histograms 1 and 2: Recall file 1 (negative control) and use the position of the negatively stained peaks to delineate positive and negative expression. Record percentages of negative and positive cells.

15. Histogram 3: Recall file 2 (CD235a) and use the marker settings from the negative control histogram to determine the percentage of CD235a-positive red blood cells.

This tube not only checks the efficiency of gating, but also confirms normal expression of a non-GPI linked antigen by red blood cells.

16. Histogram 4: Recall file 3 (CD59). Set three markers on this histogram:

a. Define type III cells (complete deficiency of GPI-antigens) using the marker setting from the negative control.

b. Define type I cells (normal expression) from processing a normal control or from the normal red blood cells present in the sample.

c. Classify type II cells (partial GPI-deficiency) as those cells that fall between type III and type I cells.

Record percentages of type I, II, and III cells. The total PNH clone = percentage type

II cells + percentage type III cells.

Ideally the fluorescence peak from the normal control should cover the third logarithm fluorescence decade.

17. Histogram 5: Recall file 4 (CD55). For this antigen define type I, type II, and type

III cells as established in step 16.

Type III cells will be clearly identified and can be defined using the marker settings from the PE-negative control antibody. The percentage of type III cells from the CD59 and CD55 plots should correspond.

The PNH cell populations may not be as clearly defined with this antibody, particularly type II cells.

18. Report PNH clone size and distribution of type II and type III cells.

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