[Histonet] ki-67 staining
Dear all, I am working on imumunohistichemistry of ki-67 antibody (DAKO MIB-5, a monoclonal mouse anti-rat) on EDTA-decalcifed rat spine and femur. I used DAKO's reagents such as Dual enzyme block, antigen retrieval solution pH6.0, normal goat serum, anti-mouse Envision plus kit and the DAB kit. I washed with TBS (prepared from Invitrogen's 1M Tris-HCl pH7.5 + NaCl to 50mM Tris.HCl pH 7.4, 150mM NaCl without pH adjustment). I used 10% NBF fixed rat liver as a control. However, none in the liver was stained and there were non-specific staining in the samples. Below is my protocol. 1. Dewax and rehydrate sections 2. a. Prepare DAKO antigen retrieval solution by diluting 1:10 with distilled water; b. preheating the solution by microwave (boil); c. heat antigen retrieval solution in a copin jar with microwave; d. put slides to the solution and leave for 20 minutes under low to medium microwave power. e. Cool for 20 minutes 3. Wash with TBS 4. Quench sections in Dual Endogenous Enzyme Block for 10 minutes 5. Rinse with TBS 6. Blocking the slides with 5% goat serum for 20 mins 7. Incubate with 1° antibody (1:25) in 5% goat serum for 1 hours RT 8. wash in TBS @1min x 3 9. Incubate with EnVision HRP-conjugated anti-mouse antibody for 30 mins 10. Wash in TBS @1min x 3 11. Add DAB (20ul into 1ml DAB+substrate buffer, mix immediately) to develop 12. Wash in water 13. Counterstain in haemotoxylin 14. Wash in running tap water 15. Dehydrate, clear and mount Could anyone help me in troubleshooting which steps went wrong or giving suggestion on the staining protocol? Any suggstion in the antigen retrieval procedures are also welcomed. Many thanks in advance. Best Regards, Victor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] workload limits
I am a little concerned about some possible misunderstandings regarding slide limits, so below is a CAP article written on the topic that still applies. Understand that the elevated limits for image related instruments (which now include the ThinPrep Imager and the SurePath GS systems) have specific limits set for specific ways those instruments are used--and they are not the same for both instruments). Note well, liquid based paps do NOT count as 1/2 slides under any circumstances. You can only get to 200 slides per day if you are using the ThinPrep Imager and ONLY looking at fields of view. If you have to do a full manual review of the slide, you must reduce you limit accordingly. CAP Today August 2004 Special Section Q & A Q. We use a combination of manual and location-guided screening in my laboratory. How do I calculate cytotechnologist workload limits in this setting? A. Maximum workload limits should be calculated based on the following factors: Manual versus computer-assisted screening. CLIA regulations specify a 100-slide maximum limit for manual screening in 24 hours over a minimum eight-hour work day. This translates to a maximum screening rate of 12.5 slides per hour for manual screening. The Centers for Medicare and Medicaid Services recently set the maximum workload limit for location-guided screening at 200 slides per day. This limit is equivalent to 25 slides per hour for location-guided screening. All Pap tests count as whole slides. Slides from nongynecologic cases count as whole slides except for concentration and liquid-based techniques that confine the material to less than one-half of the slide surface, which may be counted as one-half slides. Quality control and five-year retrospective rescreens are also included in the workload accounting. Number of hours spent screening. The maximum workload limit should be prorated based on the total number of hours spent screening, using the 12.5 slides per hour maximum rate for manual screening and 25 slides per hour for computer-assisted screening. This time does not include computer data entry, reporting, and other duties. Cytotechnologist ability, experience, QC data, etc. It must be emphasized that the maximum workload limits are the maximum allowable by CLIA/CMS and are not to be used as a productivity goal. Each cytotechnologist's workload limit should be carefully determined and individualized based on expertise, time spent screening, and screening method(s) used. Here are a few scenarios to illustrate maximum workload limit calculations in different settings: Cytotechnologist A works in a high-volume laboratory and screens Paps using location-guided screening. She is a highly skilled cytotechnologist with 10 years of experience and has no other duties (no data entry or reporting, no answering phone). Her maximum workload limit is 200 slides per day over eight hours (25 slides/hour). This case scenario is a rare exception. Most cytotechnologists have other duties or have a lower personal workload limit set by the laboratory supervisor and medical director. Cytotechnologist B's laboratory just brought a computerized imaging system in house and is gradually making a transition to computer-assisted screening. She now screens about two hours per day using the computer-assisted device and three hours per day screening Paps manually. She spends the rest of her day doing clerical and cytopreparatory work. Her maximum workload allowed by CLIA is: (2 hrs x 25 slides/hr) + (3 hrs x 12.5 slides/hr)=87.5 slides Cytotechnologist C screens Paps every morning using location-guided screening and screens nongyns in the afternoon. Today he spent four hours screening Paps. Although the maximum limit by CLIA would be 25 x 4=100, his individual maximum screening limit is set a bit lower (20 slides per hour), so his maximum limit is 80 for four hours of screening. This afternoon is busy with several FNAs, each of about 20 slides. With four hours left of screening time at 12.5 slides per hour, he is allowed by CLIA to screen a maximum of 50 slides this afternoon. However, his individual workload limit is less, and he will be permitted to screen a maximum of 35 of the FNA slides, and other cytotechnologists will screen the rest. Although computer-assisted screening methods afford greater productivity, care should be taken to set reasonable maximum workload limits based on the ability of the individual cytotechnologist, to avoid compromising screening accuracy. Theresa M. Voytek, MD Department of Pathology Bill Tench Palomar Medical Center 555 E Valley Parkway Escondido, Ca 92025 Voice: 760-739-3037 Fax: 760-739-2604 [None] made the following annotations - NOTICE: This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use
[Histonet] Job Opening, Springfield MA
We have an opening for a MOHS tech in our expanding laboratory. Responsibilities include freezing and sectioning of skin excisions . Staining, cover slipping, as well as equipment maintenance and record keeping. Previous experience is a plus, but will train the right candidate. HT(ASCP) or equivalent. Many exciting prospects are becoming available with the expansion of our laboratory service. Get in on the ground floor! The MOHS tech will be working one on one with a micrographic surgeon. Interested candidates should send resume to: Shannon Page, Clinical Manager New England Dermatology & Laser Center 3455 Main St. Springfield MA. 01107 _sp...@nedlc.com_ (mailto:sp...@nedlc.com) ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Cytology 100 slide limit
One other thing that must be considered are any cytotechs that have more than one job. You will need to know how many slides were screened at Job A so that the number is not exceeded at Job B. We had this issue when we were interviewing for a per diem cytotech to cover vacations - many could not guarantee that they would be able to screen the expected number of slides. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu]on Behalf Of Feher, Stephen Sent: Friday, July 16, 2010 5:58 PM To: Rene J Buesa; Histonet; Victor Tobias Subject: RE: [Histonet] Cytology 100 slide limit Victor, Rene is correct in stating that CLIA allows Gyn Liquid Based Paps to be counted as 1/2 slide. It gets tricky when you start mixing 1/2 slide counts and full slide counts in making sure the techs do not exceed 100 slides (or in the case of LBP's 200 slides). If you get a lot of FNA's the counts can go up very quickly. Most LIS systems can prevent Techs from exceeding their limit. I would check again to see why your LIS is not capturing the NON-Gyn slides. Another CAP and CLIA requirement is that each 6 months, the cytotechs are supposed to have a Competency Assessment where the Medical Director signs off on the maximum number of slides each tech is qualified to screen. This is the number that most labs place in the LIS as the max that particular tech can review. What LIS system do you have? Steve -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa Sent: Friday, July 16, 2010 5:21 PM To: Histonet; Victor Tobias Subject: Re: [Histonet] Cytology 100 slide limit Victor: As you wrote, either you have to be diligent in recording your work, or you will have to modify the software of the LIS system to account correctly. On the other hand, if you are dealing with liquid base samples usually using a Thin-Prep Imaging System (TIS) and the sample covers one-half or less of the slide surface, CLIA88 has expanded the limit from 100 to 200 slides/day. René J. --- On Fri, 7/16/10, Victor Tobias wrote: From: Victor Tobias Subject: [Histonet] Cytology 100 slide limit To: "Histonet" Date: Friday, July 16, 2010, 3:33 PM Our Cytology Supervisor was telling me about the 100 slide maximum that they can screen in a day. Our LIS is not capturing the NON-GYN slides being screened, so unless you are very diligent in recording the slides screened, you could go over the 100 limit. Our supervisor also believes the computer system should notify the user when the limit has been reached and prevent them from continuing. Is this a CAP requirement? How are you dealing with this problem or is it a problem for you? Victor -- Victor Tobias Clinical Applications Analyst University of Washington Medical Center Dept of Pathology Room BB220 1959 NE Pacific Seattle, WA 98195 vic...@pathology.washington.edu 206-598-2792 206-598-7659 Fax = Privileged, confidential or patient identifiable information may be contained in this message. This information is meant only for the use of the intended recipients. If you are not the intended recipient, or if the message has been addressed to you in error, do not read, disclose, reproduce, distribute, disseminate or otherwise use this transmission. Instead, please notify the sender by reply e-mail, and then destroy all copies of the message and any attachments. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet CONFIDENTIALITY NOTICE This message and any included attachments are from Somerset Medical Center and are intended only for the addressee. The information contained in this message is confidential and may contain privileged, confidential, proprietary and/or trade secret information entitled to protection and/or exemption from disclosure under applicable law. Unauthorized forwarding, printing, copying, distribution, or use of such information is strictly prohibited and may be unlawful. If you are not the addressee, please promptly delete this message and notify the sender of the delivery error by e-mail or you may call Somerset Medical Center's computer Help Desk at 908-685-2200, ext. 4050. Be sure to visit Somerset Medical Center's Web site - www.somersetmedicalcenter.com - for the most up-to-date news, event listings, health information and more. ___ Histonet mailing l
