[Histonet] Fading of H&E controls -long response with portions of SOP

Elizabeth Chlipala liz at premierlab.com
Thu Jul 21 09:57:41 CDT 2016


We use recycled xylene in our H&E stainer and have never experienced any fading of the eosin.  When you recycle your clearing agent do you check for purity and contamination, you have to also take into consideration that xylene substitutes may behave differently than xylene.   We have an SOP that governs how we recycle, we keep tract of runs and assign lot numbers to recycled products, and we also record what lot numbers were used when we change the stainer or tissue processer, so if we do have problems (we normally do not) we could trace back to the lot of recycled reagent. 

This is how we test for xylene purity - see below.  For our recycled alcohol we do merge multiple runs since we will use this to make up solutions but we also have a defined procedure for that and it includes determining  the  percentage of alcohol via hydrometer and adjusting for temperature prior to making up a reagent with it.

Excess alcohol in your xylene can also cause reagents to fade, especially ones that are susceptible to fading in alcohol such as some of the red chromogens for IHC.  This could be due to the process of recycling or your overall QC and how often you change your stainer.

8.6	Testing the Purity of Recycled Xylene or Propar

8.6.1	Pour 85ml of recycled xylene or propar into a clean, dry 100ml graduated cylinder, then add 15ml deionized water.
8.6.2	Seal the top of the graduated cylinder and invert the mixture a couple of times.
8.6.3	Allow the mixture to settle. Make sure all the water settles to the bottom of the graduated cylinder and does not cling to the sides above the xylene/ or propar/water separation point (approximately the 15ml level of the graduated cylinder).
8.6.4	Record the water/solvent separation point as indicated by the bottom of the meniscus on Form 1: Solvent Recycler Log - EQ-0014.1.
8.6.5	Use the following equations to calculate the percent impurities and percent purity of the recycled xylene:                                                                            
(measured separation point) - 14.9 = (% impurities)     
 100 - (% impurities) = (% pure xylene)
8.6.6	Enter the percent pure xylene on Form 1: Solvent Recycler Log - EQ-0014.1.

8.7	Assigning Control Numbers for Xylene and Propar

8.7.1	Each recycled lot will be kept in a separate carboy, and assigned a unique control number.
8.7.2	Control numbers for xylene have the format: two digit year (YY)-RXYL-lot #, and control numbers for propar have the format: two digit year (YY)-RPRO-lot #. 
8.7.3	Write the assigned control number and the expiration date (one year from recycling) on Form 1: Solvent Recycler Log - EQ-0014.1.
8.7.4	Fill out a reagent control label (see Appendix 1) to label the carboy.
8.7.5	Recycled alcohol will not be assigned control numbers since multiple lots are combined and it is only used in making other reagents. Instead, control numbers are assigned to the solutions made from recycled alcohol (50%, 70%, 80%, 95% or other).

8.8	Testing Recycled Alcohol for Xylene Contamination

8.8.1	Every batch of recycled alcohol should be tested for xylene contamination.
8.8.2	Mix a small volume of recycled alcohol with an equal volume of deionized water. 
8.8.3	If the recycled alcohol is contaminated with xylene, the mixture will become cloudy or milky.
8.8.4	If contamination is discovered, dispose of the recycled product, clean all containers thoroughly (see 8.1.1), and flush the recycler (see 7.3.7) before proceeding with the next batch. 

8.9	Measuring the Percent Alcohol of Recycled Alcohol Using a Hydrometer 

8.9.1	A hydrometer measures specific gravity and is used to determine the percent alcohol of recycled alcohol produced by the CBG Biotech Solvent Recycler System.
8.9.2	When using recycled alcohol to make alcohol solutions (50%, 70%, 80%, 95% or other) it is necessary to determine the percent alcohol of the current stock of recycled alcohol.
8.9.3	Since water and alcohol have different densities, and therefore different specific gravities, the specific gravity of a mixture of these two liquids indicates the percent alcohol of the solution.
8.9.4	The temperature of a liquid affects its density, and therefore its specific gravity. Because of this, hydrometer reading must be adjusted for temperature. 
8.9.5	Since the alcohol collected from the recycler is slightly warm, it is best to let it stand at room temperature overnight before using it to make alcohol solutions. 
8.9.6	Fill a transparent 500ml graduated cylinder with a sample of the recycled alcohol and gently place the hydrometer into the solution, such that it is floating with the weighted end down. Allow the hydrometer to stop moving, then record the reading (Tralles, not Proof) at the bottom of the meniscus on the reagent control form.
8.9.7	Measure the temperature of the alcohol in the graduated cylinder and record it on the reagent control form.
8.9.8	Like most standard hydrometers, the hydrometer used in Premier Laboratory is calibrated at 60°F. 
Form 2: Temperature Correction Chart for Hydrometer Readings - EQ-0014.2 shows the correction factors for hydrometer readings over a range of temperatures.
8.9.9	Calculate the actual percent alcohol from the measured percent alcohol using the following equation: (measured % alcohol) - (measured % alcohol x correction factor) = (corrected % alcohol)
8.9.10	Record the corrected percent alcohol on the reagent control form, and use it to calculate the correct volumes of recycled alcohol and deionized water or reagent alcohol for the desired solution.

Good Luck


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: Vickroy, James via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Thursday, July 21, 2016 8:09 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Fading of H&E controls

We are getting ready for our first CAP inspection in the new laboratory.    I was checking over the H&E controls that we do daily and made a discovery.   Several of the first slides we stained, over a year ago, are now washed out.  The eosin staining is probably worse than the hematoxylin however both have faded.    The H&E slides are in a file box that sits on the counter.   I next checked our surgical slide files and found that there were a very few slides from the same time period that have faded but most were still very good.

One of the weird things I have found in the controls is that some slides are as vibrant and brilliant as the day we stained them and some are almost completely faded with little color.   I am trouble shooting to figure out why the slides are fading.  I am trying to think of the variables  between the H&E control slides and the routine surgical slides.   The only thing that is different is that the routine surgical slides are filed in a rack and not exposed to the fluorescent light all day.  Is this possible?

Other thoughts:

If it is not the exposure to light than I am wondering if there is a problem with the staining protocol.   Another variable is that we use recycled clearing reagents.   On the stainer we use Fisher's Citrus Clearing agent and we recycle.  We process with Clearite III.  I know xylene is the best but do not want to use Xylene because of health issues.   I also wonder if there may still be some alcohol in the sections since some frozen sections fade when the clearing is not complete.  I do think the fading problem would be more evident in the routine filed slides if the problem was a staining protocol problem, but the recycled clearing agent is still on the table of suspects.
In the beginning we did not change the reagents as often as we do now but of course our volumes have increased also.

Please share with me your thoughts.

Jim Vickroy
Histology Manager
Springfield Clinic, Main Campus, East Building
1025 South 6th Street
Springfield, Illinois  62703
Office:  217-528-7541, Ext. 15121
Email:  jvickroy at SpringfieldClinic.com<mailto:jvickroy at SpringfieldClinic.com>

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