[Histonet] RE: Histonet Digest, Vol 25, Issue 5

Wed Dec 7 04:08:32 CST 2005

I would like to pursue the comment that CLIA requires a positive and
negative control for AFB.

I went looking under CLIA (see below). I'll put my comments/questions in
CAPS after each section I could find that somewhat pertained. (So, no, I'm
not shouting. Just trying to differentiate between CLIA regs and my
comments/questions.)

- - - -
Sec.  493.1256  Standard: Control procedures

(a) For each test system, the laboratory is responsible for having control
procedures that monitor the accuracy and precision of the 
complete analytical process.
(b) The laboratory must establish the number, type, and frequency of testing
control materials using, if applicable, the performance 
specifications verified

d) Unless CMS approves a procedure, specified in Appendix C of the State
Operations Manual (CMS Pub. 7), that provides equivalent quality 
testing, the laboratory must-
    (1) Perform control procedures as defined in this section unless
otherwise specified in the additional specialty and subspecialty 
requirements at Sec. Sec.  493.1261 through 493.1278.
    (2) For each test system, perform control procedures using the number
and frequency specified by the manufacturer or established by 
the laboratory when they meet or exceed the requirements in paragraph (d)(3)
of this section.
    (3) At least once each day patient specimens are assayed or examined
perform the following for--
         (i) Each quantitative procedure, include two control materials of
different concentrations;
         (ii) Each qualitative procedure, include a negative and positive
control material;

- - - -
SO FOR QUALITATIVE PROCEDURES IN ALL LABS, THEY MUST HAVE NEGATIVE AND
POSITIVE CONTROL MATERIAL - DOES THAT MEAN ALL OUR HISTOCHEMICAL SPECIAL
STAINS NEED A POSITIVE AND NEGATIVE CONTROL , NOT JUST AFB?,  

CONTINUING WITH CLIA, UNDER MYCOBACTERIOLOGY:
- - - -

Sec. 493.1262  Standard: Mycobacteriology

(a) Each day of use, the laboratory must check all reagents or test
procedures used for mycobacteria identification with at least one acid-fast
organism that produces a positive reaction and an acid-fast organism that
produces a negative reaction.

- - - - -
SINCE THIS SECTION IS SPECIFIC FOR MYCOBACTERIOLOGY, DOES IT APPLY TO
HISTOLOGY'S AFB HISTOCHEMICAL STAINS?

THE STANDARD FOR THE HISTOLOGY LAB STATES:
- - - - -

Sec. 493.1273  Standard: Histopathology

(a) Fluorescent and immunohistochemical stains must be checked for positive
and negative reactivity each time of use. For all other differential or
special stains, a control slide of known reactivity must be stained with
each patient slide or group of patient slides. Reaction(s) of the control
slide with each special stain must be documented.
(b) The laboratory must retain stained slides, specimen blocks, and tissue
remnants as specified in Sec.  493.1105. The remnants of tissue specimens
must be maintained in a manner that ensures proper preservation of the
tissue specimens until the portions submitted for microscopic examination
have been examined and a diagnosis made by anindividual qualified under Sec.
Sec.  493.1449(b), (l), or (m).
(c) An individual who has successfully completed a training program in
neuromuscular pathology approved by HHS may examine and provide 
reports for neuromuscular pathology.
(d) Tissue pathology reports must be signed by an individual qualified as
specified in paragraph (b) or, as appropriate, paragraph (c) of this
section. If a computer report is generated with an electronic signature, it
must be authorized by the individual who performed the examination and made
the diagnosis.
(e) The laboratory must use acceptable terminology of a recognized system of
disease nomenclature in reporting results.
(f) The laboratory must document all control procedures performed, as
specified in this section.

- - - - 
POSITIVE AND NEGATIVE CONTROLS ARE MENTIONED FOR IHC AND FLUORESCENT STAINS,
BUT FOR SPECIAL STAINS, ONLY A KNOWN POSITIVE CONTROL IS REQUIRED. 

DOES THIS SENTENCE NEGATE THE TWO STANDARDS ABOVE (ALL LABS AND
MYCOBACTERIOLOGY), WHEN IT PERTAINS TO HISTOLOGY SPECIAL STAINS? I WOULD SAY
YES. BUT
I WOULD APPRECIATE VERY MUCH, IF SOMEONE WITH MORE EXPERTISE IN CLIA REGS
WOULD COMMENT ON POSITIVE/NEGATIVE CONTROLS IN HISTOCHEMICAL SPECIAL STAINS.

THANK YOU. 

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gus
Mondragon
Sent: Tuesday, December 06, 2005 3:30 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Histonet Digest, Vol 25, Issue 5

A positive and a negative control is required by CLIA in all AFB stains
(Leprosy and TB). A known positive control validates your staining
procedure; a negative control eliminates the presence of artifact in your
sections Gus Mondragon gmondragon <@t> gsopath.com

-----Original Message-----
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histonet-request <@t> lists.utsouthwestern.edu
Sent: Saturday, December 03, 2005 1:09 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 25, Issue 5

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Today's Topics:

   1. Re: RE: Paraffin sections (Maria Mejia)
   2. Extracellular matrix proteins fixation. Details
      (Diego J. Rodr?guez Gil)
   3. RE: Specail stains Negative controls (Smith, Jeffery D.  (HSC))
   4. Re: Specail stains Negative controls (Bryan Hewlett)
   5. RE: Goat polymer (pruegg <@t> ihctech.net)
   6. Knife holder (arunams)
   7. RE: Question: Inventory (Favara, Cynthia (NIH/NIAID))
   8. AW: [Histonet] Question: Inventory (Gudrun Lang)

----------------------------------------------------------------------

Message: 1
Date: Fri, 02 Dec 2005 10:16:05 -0800
From: Maria Mejia <maria <@t> ski.org>
Subject: Re: [Histonet] RE: Paraffin sections
To: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>
Cc: histonet <@t> lists.utsouthwestern.edu,	"C.M. van der Loos"
	<c.m.vanderloos <@t> amc.uva.nl>,	BMolinari <@t> heart.thi.tmc.edu
Message-ID: <43908F65.6010808 <@t> ski.org>
Content-Type: text/plain; charset=us-ascii; format=flowed

This type of experimenting would be interesting to check out especially with
lower percentages of BSA. Andrea, I would agree that this overnight storage
step probably is antigen dependent (Chris, that's what I meant by too much)
and it would require a whole gamut testing of antibodies to see which
respond positively and which would not...if one is inclined to do so.

Yours

Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185

Andrea T. Hooper wrote:

> Very interesting Chris! I have found sometimes this works and 
> sometimes it doesn't and you get complete loss of staining - 
> frustrating when one was just trying to save time to begin with!! I 
> imagine it's antigen dependent but I have not had time to check.
>
> In my opinion Betsy, if you can avoid it (because you are just doing 
> it to save time instead of setting up a workshop) then avoid it ... it 
> simply isn't worth the risk IMHO.
>
>
>
> At 9:52 AM +0100 12/2/05, C.M. van der Loos wrote:
>
>>    Betsy,
>>
>>    For my NSH wet-workshops I tested the option of going up to endogenous
>>    PO blocking, HIER and then overnight storage at 4C in PBS with 1% BSA.
>>    Next  day  we  started at the protein blocking step and then the rest.
>>    Staining was as good as running the whole procedure in one day.
>>
>>    Hope this info helps.
>>
>>    Chris van der Loos, PhD
>>    Dept. of Pathology
>>    Academic Medical Center M2-230
>>    Meibergdreef 9
>>    NL-1105 AZ Amsterdam
>>    The Netherlands
>
>

------------------------------

Message: 2
Date: Fri, 02 Dec 2005 13:55:04 -0500
From: "Diego J. Rodr?guez Gil"	<Diego.RodriguezGil <@t> yale.edu>
Subject: [Histonet] Extracellular matrix proteins fixation. Details
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1133549704.43909888f401e <@t> webmail.med.yale.edu>
Content-Type: text/plain; charset=ISO-8859-1

Hi:

     Thanks a lot to all of you who answer!. Since some of you asked, I am
sending some more details.
     I am working on mice (CD-1), and the molecules I am trying to stain are
named Wnt (Wingless-Int). Antibodies are from R&D systems (most of them are
goats), and there are no previous works reporting the use of these
antibodies. I am only making the assumption that they work, based on the
datasheet the company provides.  I could see some very faint positive stain
for some of them, but I am sure I losing some because of fixation. Besides
that, I know they are expressed based on RT-PCR and in situ hybridization
experiments.
     Brief protocol is:
         Perfusion with 4% PFA, and then overnight deep in 4% PFA at 4 C.
Transfer to PBS, then 30% sucrose til they sink, freeze on OCT and then
section on cryostat at -18C.
         The antigen retrieval protocols that I tried were steaming (2, 5
and 10 min with sodium citrate) or pepsin in HCl. None of them gave me any
better stainings.
         Staining protocol: Wash in TBS and then add 2% BSA in TBS-T, for 30
min. Primary antibody (on 2% BSA) Over night .
	Wash 3 x TBS-T.
	Add secondary antibody on 2% BSA for an hour.
	Wash 2 x TBS-T.
	Wash TBS and mount with Crystal Mount.

      Thanks again!
      Best regards,
      Diego.

--
Diego J. Rodríguez Gil
Postdoctoral Associate
Department of Neurosurgery
Yale University School of Medicine
PO Box 208082
333 Cedar St, FMB-430
New Haven CT, 06520-8082
U.S.A.

------------------------------

Message: 3
Date: Fri, 2 Dec 2005 13:23:19 -0600
From: "Smith, Jeffery D.  \(HSC\)" <Jeffery-Smith <@t> ouhsc.edu>
Subject: RE: [Histonet] Specail stains Negative controls
To: "Grant, Debra, R" <drgrant <@t> cmh.edu>,
	<Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<EA80A7E0A540C44A8A339A3561E35C60063E262B <@t> PERCHERON.hsc.net.ou.edu>
Content-Type: text/plain;	charset="iso-8859-1"

Debby, 
   Routine special stains only require a positive control as you are looking
to see if the stain worked or not.  Specimen is either positive or negative.
No need for negative control.  IHC uses both pos. and neg. controls because
you are looking for varying intensities of staining as well as pos. or neg.

Best of Luck,
 Jeff

________________________________

Hi Histonetters,

Does anyone run negative control slides with their GMS, AFB, and Gram
special stains, and if so why?

Kind Regards,

Debby R. Grant HT(ASCP)
Histology Coordinator
The Children's Mercy Hospital & Clinics
2401 Gillham Rd.
Kansas City, Missouri 64108
lab (816)234-3827
fax (816) 802-1492

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------------------------------

Message: 4
Date: Fri, 2 Dec 2005 15:23:28 -0500
From: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>
Subject: Re: [Histonet] Specail stains Negative controls
To: "Smith, Jeffery D.  (HSC)" <Jeffery-Smith <@t> ouhsc.edu>,	"Grant,
	Debra, R" <drgrant <@t> cmh.edu>, <Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <002001c5f77e$786f0990$6400a8c0 <@t> mainbox>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
	reply-type=original

Debby and Jeff,

Negative control slides are useful for GMS, Gram and AFB in order to 
eliminate false positives due to contamination from the reagents, washing, 
carry-over etc.

Regards,

Bryan

----- Original Message ----- 
From: "Smith, Jeffery D. (HSC)" <Jeffery-Smith <@t> ouhsc.edu>
To: "Grant, Debra, R" <drgrant <@t> cmh.edu>; <Histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, December 02, 2005 2:23 PM
Subject: RE: [Histonet] Specail stains Negative controls

Debby,
   Routine special stains only require a positive control as you are looking

to see if the stain worked or not.  Specimen is either positive or negative.

No need for negative control.  IHC uses both pos. and neg. controls because 
you are looking for varying intensities of staining as well as pos. or neg.

Best of Luck,
 Jeff

________________________________

Hi Histonetters,

Does anyone run negative control slides with their GMS, AFB, and Gram
special stains, and if so why?

Kind Regards,

Debby R. Grant HT(ASCP)
Histology Coordinator
The Children's Mercy Hospital & Clinics
2401 Gillham Rd.
Kansas City, Missouri 64108
lab (816)234-3827
fax (816) 802-1492

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http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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------------------------------

Message: 5
Date: Fri, 2 Dec 2005 13:39:43 -0700
From: pruegg <@t> ihctech.net
Subject: RE: [Histonet] Goat polymer
To: "'Orr, Rebecca'" <ROrr <@t> enh.org>,
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200512022039.jB2KdkXX005415 <@t> pro12.abac.com>
Content-Type: text/plain;	charset="us-ascii"

Becky,
Invitrogen is the company who makes the goat labeled polymer.  As for
blocking, I use serum free protein block before the primary and then before
the rab. Anti-goat secondary and then again before the rabbit labeled
polymer.  If I still encounter non-specific staining I put some serum (from
the host of the secondary, about 10%) in with the protein block.  This
method is very sensitive and you can bring up lots of non-specific BG so be
careful to optimize your dilutions and times for the antibody and for the
detection reagents. Patsy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Orr, Rebecca
Sent: Friday, December 02, 2005 6:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Goat polymer

Hello everyone,

Just recently there was a posting on how to run a rabbit polymer with goat
antibodies...or something to that nature, using an anti rabbit secondary and
then the Dako polymer...could that person send me an email please?  I had a
quick question about blocking...

Also, does anyone have the name of the company that makes a goat polymer?  I
know there's a company out there...I've seen the ads but can't locate it...

Many thanks and have a great weekend!

Becky

 Becky Orr CLA,HT(ASCP)

IHC Lead 

Evanston Northwestern Healthcare

847-570-2771

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------------------------------

Message: 6
Date: Fri, 02 Dec 2005 14:04:38 -0800 (PST)
From: arunams <arunams <@t> interchange.ubc.ca>
Subject: [Histonet] Knife holder
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<5641228.1133561078869.JavaMail.myubc2 <@t> portal9.itservices.ubc.ca>
Content-Type: text/plain; charset=us-ascii

Hi All
Does eny one know where to find a Tungston Carbide Knive holder for a
Sorvall JB-4 Microtome. I really need one urgently. Thanks for your help
Aruna Somasiri

------------------------------

Message: 7
Date: Fri, 2 Dec 2005 17:35:26 -0500
From: "Favara, Cynthia \(NIH/NIAID\)" <cfavara <@t> niaid.nih.gov>
Subject: RE: [Histonet] Question: Inventory
To: "Luis Chiriboga" <Luis.Chiriboga <@t> med.nyu.edu>,	"Histonet"
	<Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<A985B7D45D355D4EB363288E717394375B8090 <@t> NIHCESMLBX5.nih.gov>
Content-Type: text/plain;	charset="us-ascii"

I do not do high volume consistently but have some weeks where I am doing
200-300 stains per week. I do not register my reagents as the come into the
lab. c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Luis Chiriboga [mailto:Luis.Chiriboga <@t> med.nyu.edu] 
Sent: Thursday, December 01, 2005 11:14 AM
To: Histonet
Subject: [Histonet] Question: Inventory

Hi All
For those of you who are in high volume labs using ventana stainers, do you
register all your reagents when they arrive? or do you register as you use?
Thanks in advance Luis _______________________________________________
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------------------------------

Message: 8
Date: Sat, 3 Dec 2005 08:52:40 +0100
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] Question: Inventory
To: "Histonetliste \(Histonetliste\)"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<mailman.0.1133632800.18983.histonet <@t> lists.utsouthwestern.edu>
Content-Type: text/plain;	charset="us-ascii"

We register the bulk-reagents and prep-kits in the Ventana System just
before use. The antibodies are registered, when they arrive, to have them on
the list.

Gudrun Lang

Hi All
For those of you who are in high volume labs using ventana stainers, do you
register all your reagents when they arrive? or do you register as you use?
Thanks in advance Luis _______________________________________________
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End of Histonet Digest, Vol 25, Issue 5
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