Normal Cut-off values for FISH

[parkermw79]

Hi All-

I am working on our CAP checklist and was wondering if anyone has
ideas on how to organize the cut-off values for each probe. I was also
wondering if anyone would share their written protocol for the
establishment of these cut-off values. We currently run the assay on
twenty patients before putting a probe into use. I was just curious
what other labs do to establish normal cut-off values. In particular,
single fusions in a dual fusion probe set.

Thanks in advance,

Melissa

[Gwenevere Carter]

I need help with that too. My issue is valadating a protocol that is different from what it was designed for and setting those cutoff limits. Help. Gwen

---

From: parkermw79 <parke...@yahoo.com>;
To: Cytogenetics methods and trouble-shooting <cytogenetics-methods...@googlegroups.com>;
Subject: Normal Cut-off values for FISH
Sent: Thu, Oct 28, 2010 3:11:44 PM

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[Andrea]

For hematological FISH, we identify 20 known normal male patients
(46,XY[20], no known hematologic maliganacy, clean hematopathology
report) and, after the required CAP retention period, use their
pellets to make FISH slides for normal limit testing. We score a
minimum of 500 nuclei (split evenly between 2 scorers) and record each
and every signal pattern. By recording all the signal patterns that
we see, we can determine if an unexpected (varient) signal pattern
seen in a patient sample is statistically significant. We also rotate
the scoring so that all techs with FISH responsibility contribute to
the normal limit data. We compile this data on an Excel spreadsheet,
each tab being a different probe and the different probe signals
across the top of the columns. With this data, we use a two sample
test to calculate the 95% and 99% confidence intervals.

For interphase FISH testing on patient samples, we use slides with a
Control Box etched on. In this control box, we apply a known 46,XY
blood metaphases (from PHA stimulated cultures). The patient sample
is applied to the rest of the slide. We apply the FISH probe across
both sections. Before scoring the patient sample, to validate that
the correct probe was applied, we photograph control metaphases.
Then, using reverse DAPI to get pseudo-Gbands, we verify the probe
location matches the probe ordered.

[Iqbal, Anwar]

Could you send your Excel spreadsheet template.

Thanks

AI

________________________________________
From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] On Behalf Of Andrea [andrearh...@gmail.com]
Sent: Friday, October 29, 2010 1:32 PM

To: Cytogenetics methods and trouble-shooting

Subject: Re: Normal Cut-off values for FISH

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[Robyn Lukeis]

A good reference for validation of FISH testing in haematological malignancy is:
Wolff DJ et al. (2007) J Mol Diagn 9:134-143.

Cheers
Robyn

>>> parke...@yahoo.com 10/29/10 2:11 AM >>>
Hi All-

I am working on our CAP checklist and was wondering if anyone has
ideas on how to organize the cut-off values for each probe. I was also
wondering if anyone would share their written protocol for the
establishment of these cut-off values. We currently run the assay on
twenty patients before putting a probe into use. I was just curious
what other labs do to establish normal cut-off values. In particular,
single fusions in a dual fusion probe set.

Thanks in advance,

Melissa

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[dollmtz]

Hello group,

I was wondering if I could get a copy of the spread sheet in this thread? We are hoping to bring on more probes and we need to know where to begin.

Are the patient samples for the 20 normal males pooled? Can anyone share thier validation protocal for validating a single probe?