[Cytometry] PNH

D. Robert Sutherland rob.sutherland at utoronto.ca
Mon Jul 6 10:10:31 EDT 2009

Hi Kathy,
If I were you, I would just get the method established as we did.
You can use different conjugates in your cocktail if you wish, but  
just titrate and validate each reagent in your chosen cocktail first.  
Then test it on a couple of normal blood samples. You will see the  
CD33, CD14 and CD45 working as expected on the 3 major leukocyte  
subsets present in normal blood (lymphs, monos and grans).
We are a fairly big centre so we find PNH clones in samples  
relatively frequently. However, when we first set up the methodology,  
there was no QC/PT material suitable for our assay.
In this setting, what CAP suggests is that you exchange samples with  
other (nearby) centres that are conducting PNH screening, and this is  
what we did.  So if you or any nearby centre get a PNH clone- 
containing sample, you get it from them, run your assay and compare  
the results you get with theirs. You reciprocate with any PNH samples  
your centre may stumble across. If you do this once or twice a year  
with shared samples and show you are getting the same results, this  
is all that CAP can ask of you for such a rare disease, the absence  
of anything suitable from CAP that can be assessed with this assay.

We have another manuscript coming out in Am J Clin Path soon:  
Included therein is the description of a patient sample that R&D  
systems stabilised for us. This stabilised material was excellent and  
lasted for several months before we used it all up. I have been  
trying to interest CAP in setting something up whereby a couple of  
the big centres could supply R&D with material about twice a year  
(even pooled patient material would probably be OK), have them  
stabilise it and then have CAP distribute this stuff for QC/PT  
purposes. This is still a work in progress (but not much progress so  
Alternatively, you could register with UK NEQAS to obtain their  
stabilised material. It works fine in our assay, as I indicated in  
our 2007 Clinical Cytometry paper you have already seen.

Incidentally, Protox Biotech who supply FLAER, have recently improved  
the stability and staining efficiency of the FLAER reagent. The  
reagent is now available in liquid form as well as in powdered form  
and both exhibit significantly stronger signals than the original  
powder and the stability of both have been improved by the addition  
of BSA (Tom Buckley, personal communication).  Ongoing tests of the  
stock solutions of both liquid and powdered FLAER indicate that they  
retain strong signals for three months (and counting...) in the  
refrigerator. We and a number of other labs have assessed these new  
'formulations' and there seems to be general agreement that they  
represent a significant improvement over the original freeze-dried  
powder. Both are now available worldwide through the Canadian  
company, Cedarlane Laboratories (cedarlanelabs.com).

HTH, but if not, keep in touch I will see if there is anything else I  
can help you with.

rob sutherland
Toronto General Hospital/University Health Network

On 3-Jul-09, at 9:04 AM, Katzke, Kathleen A. wrote:

> I have read the article "Diagnosing PNH with FLAER and Multiparameter
> Flow Cytometry" by R. Sutherland. We are starting a method development
> for PNH. We used to use the RedQuant and CellQuant kits for testing
> years ago, but quit as I couldn't get the support I needed and then  
> the
> results weren't coming out well either after years of testing  
> different
> methods and finally bringing the kits live. So, we quit that testing
> after 5 months.
> My question is, with using the procedure from the article, we had
> thought of doing 20 samples of patients with anemia or those samples
> that are sent out for PNH testing for comparison. We only sent out 16
> samples last year so don't have a lot of reference work for this  
> test. I
> was wondering what is recommended for testing and comparisons for this
> test. I realize we will rarely see a positive PNH patient, esp.  
> with our
> initial testing, but wanted some feedback on how to proceed.       
> Thanks
> for all your help.
> Kathy Katzke, MT (ASCP) QCym
> Flow Cytometry
> OSF System Lab
> 1224 Berkley St
> Peoria, IL 61637
> work 309-624-9036
> fax 309-624-9037
> kathleen.katzke at osfhealthcare.org
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