TECHNICAL UPDATE

Miami Technical Support

Number: TU2000M061 Date: August 03, 2000 Page 1 of 4

Subject: How to Handle GEN·S Differential False Negative Flagging Issues

Differential false negative flagging issues, if not handled properly, can escalate rapidly into
adverse situations for both the customer and Beckman Coulter. As there are many factors that
affect flagging – the instrument, the flagging algorithm and user-defined setup, and the
limitations of the technology - this Technical Update will provide guidance for bringing false
flagging situations to a quick resolution.

The Instrument
Many of the differential algorithm criteria for both contouring (gating) and flagging are based on
the DC, opacity, and RLS mean and standard deviation (SD) of the individual white cell
subpopulations. Abnormal cell types will typically exhibit a population mean and/or SD outside
of expected ranges and generate an appropriate Suspect flag.

NERLS SD

NEDC SD NEDC Mean

NEOP SD
NEDC Mean = Neutrophil Volume Mean
NEOP Mean = Neutrophil Opacity Mean
NERLS Mean = Neutrophil Light Scatter Mean NEOP Mean
NEDC SD = Neutrophil Volume Standard Deviation
NEOP SD = Neutrophil Opacity Standard Deviation
NERLS SD = Neutrophil Light Scatter Standard Deviation
NERLS Mean

When white cell populations shift or become diffuse because of instrument related problems, the
means and SDs of populations representing normal cell types may be outside the expected
ranges resulting in an increase in false positive differential flagging and/or incorrect differential
results. The customer complaint will be “too much flagging”. This scenario will typically result in
a service visit to optimize the instrument – bringing the means and SDs back to where they
should be.

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A common misconception is that false positive differential flagging can be minimized by

intentionally shifting the DC mean at the low end of the range. This may contribute to the
opposite problem of false negatives.

False negatives occur when the customer observes a significant number of abnormal cell types
on the manual differential and the instrument generated no Suspect flags. Be aware that a
number of false negatives can be expected as there is a balance between flagging too much
and flagging too little. Our obligation is to minimize the occurrence of false negatives by
ensuring correct placement of all differential populations.

Example 1: If the DC Mean is low, the second population of lymphocytes commonly seen with
CLL and lymphoma patients will be shifted low and may be gated as non-WBC debris instead of
lymphocytes.

Example 2: If the DC Mean is low, the neutrophil population mean may be too low to trigger the
Imm Ne1 flag as needed.

Example 3: If the RLS Mean is high, the neutrophil population may be falsely gated as
eosinophils.

The Flagging
The GEN·S offers three separate levels of flagging along with the ability to disable the Imm NE
1 flag. Appropriate flagging, specific to the needs of the laboratory, at the onset of instrument
implementation is crucial. The laboratory’s patient population and their sensitivity to abnormal
cell types must be taken into consideration when assisting with flagging limits. In addition,
sufficient sample size and type must be used to test the flagging of the system.

Example: If the laboratory is band sensitive, disabling the Imm NE 1 flag without validation may
result in an unexpected increase of false negatives.

The GEN·S flagging algorithm is unique to the GEN·S. There have been many changes in
regards to contouring and flagging when compared to the STKS/MAXM algorithms. Pre-existing
differential review criteria should be validated prior to use with the GEN·S.

The Limitations of the Technology

There are limitations to all technologies employed for counting and sizing cells. This applies to
all instruments and all manufacturers. There are known limitations – such as cold agglutinins
and platelet clumps. And there are unknown limitations – such as new drugs and drug
combinations with some disease states. These limitations can produce either false positive
flagging or false negatives.

It is extremely important to understand that false positives and false negatives are a
normal outcome of screening tests such as the automated differential. Our obligation is
to minimize these occurrences by instrument optimization and appropriate flagging.

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Guidelines for Resolving GEN·S False Negative Flagging Issues

Originator

1. Generate a Service Order within Vantive to document the customer complaint.

2. Explain to the customer that their complaint is being documented, that a Service
Representative will validate the performance of their instrument, optimize the instrument if
needed, and that an Applications Specialists will review their flagging criteria with them to
ensure they are optimally set for their laboratory needs and their patient population.

Field Service Engineer

1. Upon receipt of the Service Order, download the listmode data (both .dat and .inf files) from
the sample(s) in question to a diskette. Retain diskette for future reference.

2. Review Field Trends for proper postional parameters (NEDCMean, NEOPMean, and
NERLSMean). Contact your Hardware Specialist for assistance if necessary.

3. Review patient scatterplots for the following:

a) good separation between populations

b) well defined populations
c) minimal non-WBC debris
d) single population lymphocytes

4. Optimize the instrument as needed.

5. Advise the customer that the instrument has been validated to be within specifications
specific to differential contouring and flagging or optimized to minimize the occurrence of
false negative flagging.

6. Notify Applications Specialist that instrument validation has been completed.

Applications Specialist

Discuss the following with the customer.

1. Were sensitivity and specificty studies performed prior to implementation of the GEN·S?

§ Discuss the need to establish criteria that match the flagging performance of the
GEN·S.
§ Recommend that a study be performed to determine appropriate flagging.

2. Did the orginal studies include a sufficient number of abnormal samples to test the system?

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§ Discuss the need to have a significant number of abnormal samples to determine

flagging capabilities on a wide variety of abnormalities typically seen by the
laboratory.
§ Recommend that a new study be performed or additional samples be added to the
existing study.

3. Did the original studies include samples representative of the laboratory’s typical workload?

§ Discuss the need to meet the laboratory’s flagging needs for pediatrics, oncology,
outpatients, emergency room, etc.
§ Recommend that a new study be performed or additional samples be added to the
existing study.

4. Did the original studies indicate a false positive or false negative rate markedly different than
what is currently being observed?

§ Discuss the balance between false positives and false negatives.

§ Discuss the limitations of 5-part differential technology.

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