Calibrators Standards

Calibration of a flow cytometer is performed by service engineers upon installation and through annual preventative maintenance installation. Calibration is verified by setup beads on each daily shift (see proper manufacturer's instruction).

Suggested Panel of Antibodies for Six-Color Antibody Configuration

Tube no.

FITC

PE

PerCPCy5.5

APC

PE Cy7

APC Cy7

1

IgG1

IgG1

IgG1

IgG1

IgG1

CD45

isotype

isotype

isotype

isotype

isotype

control

control

control

control

control

2

CD2

CD3

CD4

CD8

CD56

CD45

3

CD20

CD19

CD5

CD11c

HLADR

CD45

4

CD38

CD13

CD19

CD23

CD34

CD45

5

CD7

CD64

CD33

CD117

CD34

CD45

6

IgG1

IgG1

IgG1

IgG1

IgG1

CD45

isotype

isotype

isotype

isotype

isotype

control

control

control

control

control

7

Lambda

Kappa

CD5

CD19

CD10

CD45

2.3. Compensation Check, Positive and Negative Control

2.3.1. Compensation Check

The compensation of the six-color leukemia protocol should be verified daily. A normal whole blood sample is stained with six single-color isotypic controls. The matrix of this should be saved.

2.3.2. Positive and Negative Controls

2.3.2.1. Isotypic Negative Controls

1. Check reagent insert for corresponding isotypes of the MAb used for immuno-phenotyping (5,7).

2. For each panel, tubes containing all isotypic controls for all six colors are set up to set the marker dividing positive fluorescence from negative.

2.3.2.2. Normal and Positive Control for Testing Reagents (Antibody Control)

1. A normal blood control is performed to check the working antibody.

2. The purpose of this control is to ensure the integrity of the antibodies used and the accuracy of the staining procedure. Record acceptable/not acceptable results. The staining pattern of the lymphocytes/monocytes/granulocytes should be appropriate for the population (5,7).

a. CD23, CD117, and CD34 are not normally expressed on a normal control. Positive control cells are added only if the positive control cells saved from a known positive patient is available.

b. If any antibody does not perform as expected, make up a fresh working dilution and reset all relevant patient samples for that antibody with a new working dilution.

2.3.3. Parallel Testing of Reagents

1. Prior to putting new lot numbers of MAb in use, parallel testing with the old lot number is performed (5,7).

2. Percent positivity using the new and old lot numbers is compared. The coefficient of variation must fall within the following limits:

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