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Preparation for the HT and HTL (ASCP) Examination Robert A Brunner, BA, HT(ASCP) Senior Field Support Specialist Leica Biosystems 2017 Tri-State Spring Symposium May 3rd to May 5th, 2017 Rochester, Minnesota
Objectives ASCP CAT exam procedures Eligibility Routes for HT and HTL Exam Content Topics
Outline
PART I ◦ General Background ASCP ◦ Eligibility Routes ◦ Application and Required Documents ◦ Computer Assisted Testing (CAT) ◦ Certification Maintenance (CMP) ◦ Study Materials
PART II- Exam Content
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PREPARATION FOR THE HT AND HTL (ASCP) EXAMINATION PART I
ASCP and NAACLS
NAACLS ◦ National Accrediting Agency for Clinical Laboratory Sciences ◦ Nationally recognized agency for assuring quality clinical laboratory training programs
ASCP ◦ American Society for Clinical Pathology ◦ Only agency in the US that has Certification Exams for HT and HTL
Historical Background Pre-2004- HS with OJT could take HT exam January 2004- NAACLS, NSH and ASCP approved changes to eligibility requirements for HT ASCP Exam
◦ Required that applicants successfully complete a NAACLS Accredited HT Program to sit for exam ◦ Allows certificate level programs to train HS Diploma, AA and Baccalaureate for exams
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Historical Background Initiative took over 10 years to get approval (initial proposals were in late 1980’s and early 1990’s) Was important due desire to elevate the field and to address ASCP pass rates and competency 2014- NAACLS proposes to change eligibility to match MLT requirements
ASCP CAT Exam Stats January to December 2004 Exam #of Examinees HT all 1673 HT NAACLS 1st Time 178 HT Non NAACLS 1495
Pass 708(42%) 132(74%) 576 (38.5%)
(HS OJT, AA OJT, etc)
Pass Rates 2000-2008 100 80 60 40 20 0 FY2000 FY2001 FY2002 FY2003 FY2004 FY2005 FY2006 FY2007 FY2008
HT HT =
HTL
45%
48%
49% 46% 40%
64%
69% 59% 64%
HTL = 52%
53%
50% 58%
82%
87% 65%
63%
61%
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ASCP Web Site Helpful!
HT Eligibility Routes Current Successful completion of a NAACLS accredited HTprogram within the last 5 years OR 2. At least -60 semester hours of academic credit (or an Associates Degree) 1.
Must contain at lease of 12 semester hrs. of biology and chemistry (must include credits in both) , AND one (1) year full time experience in the last 5 years (Clinical, Veterinary, Industrial or Research)
-Must have experience in the following areas: - Fixation, Processing, Microtomy, Staining - With in the last 10 years
HTL Eligibility Routes Baccalaureate degree with 30 semester hrs. of biology and chemistry, AND successful completion of a NACCLS HT or HTL program with in the last 5 years 2. Baccalaureate degree with 30 semester hrs. of biology and chemistry, AND one (1) year full time experience in the last five years. 1.
(Clinical, Veterinary, Industrial and Research)
-Must have experience in the following areas: - Fixation, Processing, Microtomy, Staining - With in the last 10 years
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Documentation Needed
Eligibility established by: ◦ Meeting Minimum requirements HT or HTL category of certification ◦ Submission of Appropriate Application form ◦ Payment of Appropriate Application fee ◦ Submission of All necessary documents Experience Documentation form Letter of Authenticity Transcripts
Academic Verification
Official transcript from a regionally accredited college/university ◦ bearing the seal of the college/university ◦ Signature of registrar ◦ Date the degree was completed/granted
•
ASCP will verify if chemistry/biology credits meet requirements
Experience Verification
Download experience documentation form from:
Form must be filled out by immediate supervisor or laboratory director Signed “letter of authenticity” signed by immediate supervisor or laboratory director
◦ www.ascp.org/certification
◦ Original letterhead ◦ Stating that form was completed by employer
Immediate supervisor or laboratory director should be someone who can verify your technical performance of ◦ Fixation, Processing, Microtomy and Staining
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Work Experience
ASCP Defines Full Time Experience as: (1 year) ◦ Minimum of 35 hours worked per week
If working part time then hours are prorated ◦ 20 hours per week then use the following formula 20 divided by 35 multiplied by 52 weeks or an equivalent of 29.7 weeks of work experience
Training Verification NAACLS Accredited Programs Should have completed program with in the last 5 years Printed application form must have:
◦ Program Directors name ◦ School Code number
Official Program completion requires that all final grades are submitted to college/university registrar
Application for BOC
Online ◦ Go to www.ascp.org/certification ◦ Need to use Credit card or Pay Pal to pay fee ◦ All other forms and documents still need to be mailed into ASCP
Mail ◦ Go to www.ascp.org/certification ◦ Need to print off form and pay by check or money order ◦ All forms and documents need to be mailed to ASCP
NOTE: Follow directions carefully to assure all necessary information is provided
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Address for submission
Online ◦ Board of Certification 33 West Monroe Street Suite 1600 Chicago, IL 60603 ◦ NOTE- College transcripts are submitted directly from college/university to the address above
If paying by Check of money Order follow directions with online application Only submit via US Postal Service regular mail.
ASCP Contact Info Address:
ASCP Board of Registry 33 W. Monroe, St-Suite 1600 Chicago, IL 60603-5617
Phone: (312)541-4999 (800)267-2727 Fax: (312)541-4845 Email: Go to Contact Us. Click the link next to “E” for Email and fill in your information. •Office Hours: 8:00 a.m. to 5 p.m. Central Time (Monday - Friday)
All correspondence from Board of Certification is done via Email. It is your responsibility to keep email address current with the BOC
Fees Assigned to Exams Histotechnician, HT(ASCP) Exam $215.00 • Medical Laboratory Technician, MLT(ASCP) $215 • All Technologist Level $240 •
◦ Histotechnology (HTL)
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Application Processing
Application acknowledgement will be done via Email Do not contact ASCP about application until 30 days after submission Online applications usually acknowledged with in 1 day of receipt Required Documents and forms need to be received at ASCP within 45 days of application Applicant will be notified of missing documentation within 6 weeks of submission
Administration of Exam Pearson Vue
http://www.pearsonvue.com/ascp/
More than 202 company-owned and operated test centers throughout US and its territories.
Located in all 50 states. Search for a center near you.
Pearson Testing Centers
http://www.pearsonvue.com/ascp/
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Determination of Eligibility
If determined eligible ASCP will notify via email ◦ Instructions to scheduling an appointment for examination ◦ Exam must be taken with in 3 month period following the date of the notification
If determined ineligible ASCP will notify you ◦ Application fees are Non-Refundable ◦ Cancelling or not taking exam is non-refundable. ◦ Rescheduling with in the original 3 month period is ok
ASCP COMPUTER ADAPTIVE TESTING (CAT EXAM)
CAT Testing Examinee score not influenced by other examinees scores Criteria referenced Calibrated questions Passing requires answering questions above Minimum competency levels Each correctly answered question will give the examinee a higher level complexity question
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Question Taxonomy Levels
Recall ◦ Measures memory ◦ Recall or recognize previously learned knowledge
Application ◦ Measures Interpretation of Information ◦ Utilize recall knowledge to interpret or apply information
Analysis ◦ Measures Application of Knowledge ◦ Uses all of above to resolve a problem or situation or make appropriate decision
CAT Testing Procedure
Arrive at Testing Center EARLY ◦ At least 30 min early!
Bring the following: ◦ Drivers License or valid State ID Requires them to have photo and signature Valid - NOT expired
◦ Admission Letter ◦ First and Last names MUST EXACTLY match the spelling admission letter
CAT Testing Procedure
May have a Non Programmable Calculator ◦ Writing material for calculations will be provided
NO CELL PHONES allowed Reference Books and notes NOT ALLOWED A photograph and palm vein image will be performed
◦ Assures each candidate has a single record
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CAT Testing Procedures
Taking the test◦ Read and follow directions carefully ◦ Multiple choice 100 questions 2.5 hours to complete
◦ One question on the screen at one time ◦ Graphs, charts and photographs, etc may appear ◦ Answers may be changed but an answer MUST be selected to proceed to the next question ◦ At the end you may review questions
CAT Testing Procedure Preliminary “Pass” or “Fail” will appear on the computer screen Scores will be emailed 4 business days after completion of test
◦ Provided all official transcripts have been verified and appropriate degree received
There will be a “Scaled score” ◦ Scores of other examinees do not impact your score ◦ Minimum passing score is 400 ◦ Maximum achievable score is 999
Certification Benefits
Certificate- suitable for framing Valid for 3 years
Credential Maintenance Program
◦ Valid dates will be on certificate ◦ Renews certification
Assures standard of competence for Histology Professionals Recognized Nationally and Internationally Required by most employers
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CREDENTIAL MAINTENANCE PROGRAM (CMP)
Credential Maintenance Introduced in 2004 for all new ASCP registrants Covers all specialties of certification exams Implemented to help assure laboratory professionals stay current and up to date in their specialties Optional for registrants from prior to 2004
CMP Requirements
HT and HTL Categories 36 CE Points every 3 years ◦ 12 points per year ◦ 1 point = 1 contact hour of training/education
1 Point in Laboratory or Patient safety e.g. – QA and or QC 2 points in specialty of certification Remaining in area of specialty, management, education, related laboratory interests
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CMP Requirements Formal Continuing Education Courses Employer offered courses Competency assessment from Employer
◦ In-service training, Instrument training,Vendor training
Self instructional courses (online) Research Preparation for Workshop Authoring journal articles Poster presentations
Submission of CMP Once every 3 years Declaration of training activities Documentation only submitted when audited by ASCP Obtain Certificates, CE forms etc from trainer/presenter for records and save
BOC RECOMMENDED STUDY MATERIALS
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Suggested Text Books
See ASCP Suggested Reading List ◦ Histotechnology References ◦ Histotechnology Artifacts ◦ Histology (Identification) Reference ◦ Other- Cytoprep etc ◦ Management and Education- HTL
ASCP Suggested Reading List
Histotechnology:A self-instructional Text 4th Edition ◦ 2014 Publication ◦ ASCP Press
3rd Edition
4th Edition
Optional Histotechnology Text
Bancroft’sTheory and Practice of Histological Techniques: Suvarna/Bancroft ◦ 7th Edition ◦ 2012 Publication ◦ Others available
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Histochemical Techniques Histology and Histochemical Techniques 2015 Publication 5th Edition HTL Recommended
Histotechnology Artifacts
Histologic Preparations ◦ Common Problems and Their Solutions ◦ CAP Press- 2009
Chapters are divided by Fixation, Processing ETC Artifacts In Histology and Cytology Preparations, Leica Biosystems, Electronic document
Histology Identification
Atlas of Histology with Functional Correlations ◦ 2017 13th Edition
Wheaters Functional Histology ◦ 2014 ◦ 6th Edt
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Cytology Preparation
Guide to Cytopreparation ◦ ASCT Products
Cytoprep Procedures Common Special Stains used References for
◦ ◦ ◦ ◦
Flow Cytometry Immunohistochemistry Electron Microscopy Microbiologic Studies
ASCP RECOMMENDED STUDY MATERIALS
Study Materials
Histotechnology: A Self-Assessment Workbook ◦ 4nd Edition ◦ 2014 ◦ Pairs with Text Book
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Study Materials
Histotechnology Flash Cards ◦ 2010 ◦ 3rd Edition ◦ Carson and Hladik
Study Materials Dako Education Guide Special Stains and H&E 2009 2nd Edition
Study Materials
Dako IHC Staining Methods ◦ 6th Edition ◦ 2009 ◦ HTL Recommended
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ASCP Board of Certification Study Guide 3rd Edition Published 2013
Freida L Carson, PhD, MASCP, HT(ASCP) Glenda F Hood, MEd, HT(ASCP)
Available in eBooks from ASCP
Additional ASCP Resources Online practice exams- $30.00 90 day access, 5 – 50 question tests ASCP Procedures for Examination and Certification Examination Content Guidelines ASCP Suggested Reading Lists Exam Preparation and Administration Guide
Laboratory Management
HTL Recommended Garcia: Clinical Laboratory Management2nd Edition ◦ 2013 Publication
Harmening: Laboratory Management: Principles and Processes- 3rd Edition ◦ 2012 Publication
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Other Sources
National Society for Histotechnology ◦ Histology Exam Simulator
Centers for Disease Control
College of American Pathologists
◦ Lab safety, precautions etc ◦ Artifact booklet, educational Material
Occupational Safety and Health Administration ◦ Workplace and Laboratory Safety
NSH Self Assessment Series www.nsh.org
On-line Self Assessment Modules Search “Self Assessment” on the NSH website for free PDF versions
NSH Self Assessment Series
Gross Dissection Fixation Processing and Decal Embedding Routine Staining Micro-organisms Connective Tissue Pigments and Minerals
Nervous Tissue Carbohydrates Lab Operations Cyto Preparation Lab Safety *IHC and ISH *Enzyme and Electron Microscopy *HTL only
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PREPARATION FOR THE HT AND HTL (ASCP) EXAMINATION PART II
HT /HTL Content Guidelines
FIXATION
(15 - 25%)
PROCESSING
(10 - 20%)
EMBED/MICRO
(15 - 25%)
STAINING
(30 - 40%)
LAB OPERATIONS (10 - 15%) *Determined by Practice Analysis done by ASCP
Content Outline- Fixation I. FIXATION
(15-25%)
A.Tissues 1. Morphology/anatomy 2. Cell/component preservation 3. Pathology* 4. Biochemistry principles/theories* B. Procedures 1. Light microscopy 2. Electron microscopy 3. Special stains 4. Frozen sections/tissues 5. Enzyme histochemistry 6. Immunohistochemistry 7.Artifacts/precipitates/pigments 8. Quality control 9. Cytologic specimens 10. In-situ Hybridization
C. Parameters 1. Size of specimen 2.Volume of specimen/fixative 3.Time of fixation 4.Temperature of specimen/fixative 5. Other (e.g. , pH) D. Reagents 1.Types/components 2. Properties/functions/actions 3. Quality control 4. Chemistry principles/theories* E. Instrumentation (e.g., microwave) 1. Components 2. Use 3. Maintenance 4.Troubleshooting 5. Quality control
*HTL Only
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Fixation •
Formulas/Functions
•
Actions/Parameters
•
Mechanisms
•
Procedures
Fixation Fixation immersing tissue in a fluid which will: ◦ penetrate, harden and kill in different degrees ◦ preserving all structures and elements as near normal as possible.
Improper fixation cannot be corrected by another step in the processing of tissue, and the finished slide and stain can only be as good as its primary fixation step.
Fixation alters “proteins” in tissue by stabilizing them to resist further changes.
Parameters Affecting Fixation • Volume Ratio • 15-20x the volume of fixative for every volume of tissue
• Penetration • NO fixative will penetrate more than 2-3mm of solid tissue
• Thickness • Tissues no more than 3-4mm thick for proper fixation
• Time • 24 hours in most fixatives is preferred
• Temperature • room temperature is customary; heat increases rate of fixation; cold slows rate of fixation • Increasing Heat also increases rate of Putrefaction and Autolysis
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Methods and Mechanism • Physical: heat or drying
• Chemical: • additive • non-additive
• Coagulation • Non-coagulation
Methods and Mechanism
Additive - links chemically w/tissue
Non- additive - acts on tissue w/o combining with it
Coagulant - forms network or sponge-like meshwork of proteins
Non-Coagulant - forms a gel-like substance of proteins
Non-Coagulant vs Coagulant
Slides from Peggy Wenk
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Coagulant Fixatives
Slides from Peggy Wenk
Fixative Actions/Functions • Acetic acid • softens, swells, fixes nuclei • coag., non-additive
• Formaldehyde • hardens, fixes “protein” • non-coag, additive
• Gluteraldehyde • slow, ultrastructure • non-coag, additive
• Mercuric Chloride • hardens, pigment, toxic • coag, additive
Fixative Actions/Functions • Osmium tetroxide • EM for lipids • non-coag, additive
• Picric acid • slow, washing required • coag, additive
• Potassium dichromate • chromaffin preserved • non-coag, additive
• Zinc salts • slow, morphology improved • coag, additive
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Non-Aqueous Fixatives • Acetone • very rapid, hardens, shrinks • preserves enzymes • coagulant, non-additive
Caution Dehydrants can remove “bound Water”
•
Alcohol
• • • •
(ethyl, methyl) hardens, shrinks penetrates rapidly Recommended for cytology, frozen sections coagulant, non-additive
•
Carnoy Solution
• Abs Alcohol, Chloroform and acetic acid • hardens, shrinks, removes water,
10 % Neutral Buffered Formalin - How it fixes:
Protein
Additive Reaction
Proteins cross link in a meshwork, stabilizing and preserving morphology
it is a strong reducing agent it fixes by an oxidative reaction,
forms methylene bridges between the side amino groups of lysine and glutamine on different protein chains.
Carbohydrate
NO particular chemical reactions with carbohydrates; the protein part of glycoproteins is fixed
The cross-linking does effectively trap glycogen, even though it remains unfixed. Simple carbohydrate is unaffected.
Lipid
Triglycerides remain unfixed; most lipids dissolve during treatment with alcohol and xylene during processing
Histochemistry
Nucleic acids are not fixed and most are removed or reduced by processing. Most enzymes are inactivated, except for a few if used for a short time or at 4°C.
Carson 4th Edition, Page 18
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Good Fixation
Delayed Fixation
Fixation Incomplete
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Formalin Pigment
Formalin Pigment Light Microscopy
Polarized Light
Nuclear Bubbling
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Nuclear Bubbling Theoretical
Causes
Incomplete/inadequate formalin fixation ◦ Formalin- at least 12-24 hours to fix
Excessive heat while drying slides
Microwave Fixation •
Microwave Stabilization of Unfixed Tissue
•
Microwave-Stimulated Fixation with Fixatives
•
Hybrid Procedure ◦ Combination of Microwave and Routine Fixation methods
Microwave Fixation Brings on own set of Artifacts Used in some Rapid tissue processing equipment Can see under fixation as fixation is a Chemical reaction Uses microwave energy to heat fixative reagents Can cause uneven fixation
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Example Fixative Study Sheet
Fixative Study Information
Ingredients
Color
Artifacts
◦ Not amounts of each ingredient ◦ What ingredient gives the color ◦ Preventable/removable ◦ How to Prevent and or remove
Specific Uses
Reasons to Avoid
◦ What fixative is good for ◦ If there are negatives of the use
Example Question Fixatives are classified as additive because of the: a. addition of several chemicals of the solution b. addition, or binding of the fixative, to tissue proteins c. additional reactions occurring with longer fixation d. additional reactive tissue sites available for binding
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Example Question Fixatives are classified as additive because of the: a. addition of several chemicals of the solution b. addition, or binding of the fixative, to tissue proteins c. additional reactions occurring with longer fixation d. additional reactive tissue sites available for binding
Example Question A fresh surgical resection specimen is received in the laboratory from a patient with a history of malignant melanoma. The choice of fixative for this specimen should be compatible with the use of which of the following special stains or diagnostic procedures? a. b. c. d.
colloidal iron stain immunohistochemistry immunofluorescence digestion procedures
Example Question A fresh surgical resection specimen is received in the laboratory from a patient with a history of malignant melanoma. The choice of fixative for this specimen should be compatible with the use of which of the following special stains or diagnostic procedures? a. b. c. d.
colloidal iron stain immunohistochemistry immunofluorescence digestion procedures
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Content Outline- Processing II. PROCESSING (10-20%)
A.Tissues
C. Instrumentation
1. Morphology/anatomy 1. Components 2. Cell/component preservation 2. Use B. Procedures 3. Maintenance 1. Light microscopy 4. Troubleshooting 2. Frozen sections/tissues 5. Quality control 3. Enzyme histochemistry* D. Reagents 4. Calcified/decalcified tissue 1. Types/components 5. Immunohistochemistry 2. Properties/function/action 6. Quality control 3. Quality control 7. Cytologic specimens 4. Chemistry principles/theories* 8. In-situ Hybridization *HTL Only
Processing Decalcification Processing Instrumentation Reagents
Processing The purpose of processing is to get the tissue from the natural aqueous state to paraffin (or other supporting medium). Includes:
◦ Dehydration ◦ Clearing ◦ Infiltration
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Processing
Dehydration ◦ removal of water, which is replaced by alcohol or other dehydrating fluid which is miscible with water and the clearing agent.
Clearing ◦ intermediate step between dehydrant and wax; refractive index should be close to that of tissues to render them “clear.”
Infiltration (impregnation) ◦ introduces the supportive medium into the tissue; removes the clearing agent.
Dehydration Reagents
Must be miscible with? ◦ Fixative and Clearing Agent
Examples ◦ Ethanol ◦ Methanol ◦ Isopropanol ◦ Denatured Alcohol ◦ Acetone
Excessive Dehydration
Over Dehydration ◦ Removal of Bound Water
Unbound (free)water should be the only water removed from the tissue. Bound water keeps the tissue from overdrying and helps prevent distortion from the chemicals used.
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Clearing Reagents
Must be miscible with?
Examples (Aromatic Hydrocarbons)
◦ Dehydrating agent and Paraffin (Infiltration) ◦ ◦ ◦ ◦ ◦ ◦
Benzene Toluene Xylene Chloroform Carbon Tetrachloride Essential Oils
Xylene Substitutes (Aliphatic Hydrocarbons)
Aromatic Hydrocarbons
Xylene
Benzene
Colorless, water-insoluble, flammable, toxic, isomeric liquids, C8H10, of the benzene series, obtained mostly from coal tar: used chiefly in the manufacture of dyes.
Aliphatic Hydrocarbons Xylene Substitutes Some are Limonene based Straight Chained Hyrdrocarbons Very Intolerant of Water
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Universal Solvents Must be miscible with? Fixative and Paraffin (infiltration) Examples
◦ Dioxane ◦ Tertiary Butyl Alcohol ◦ Tetrahyrdrofuran ◦ Isopropanol ◦ Acetone
Infiltration Media Paraffin wax Water soluble wax Celloidin Plastic Resins
◦ Glycol Methacrylate ◦ Epoxy Resins
Agar and Gelatin Sucrose (30%)
Paraffin Wax
Most Commonly Used ◦ Varying Melting points ◦ Additives to adjust properties
Knowledge of: ◦ Range of Temperature of melting points ◦ Melting point and its affect on tissue ◦ Additives and benefits
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Tissue Processors
Types of Tissue Processors
Manual Open Tissue processor
Enclosed Tissue Processor
◦ Tissues are transferred to reagent ◦ Reagents are moved in and out of a common chamber
Microwave Tissue Processor ◦ Enclosed tissue processor with Microwave heat and proprietary reagents
Rapid Tissue processor ◦ Efficiencies in reagent transfer and Heating
Processing Protocols Reagents Used Reagent Sequence Protocol times Evaluate protocols and select appropriate protocols for given scenarios Heat and Vacuum and affects Temperature range of reagents
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Troubleshooting Processing Over Dehydration Poor Processing Desiccated tissue Ability to:
◦ Recognize tissue characteristics of above issues ◦ How to correct the issue ◦ Effects of above on tissue microtomy, staining and morphology
Processing Troubleshooting
Issue ◦ When Facing block the tissue smells of Clearing agent
Cause ◦ Clearing agent not completely removed- poor infiltration
Remedy ◦ Melt wax and place in infiltration media ◦ Re-embed
Processing Troubleshooting
Issue ◦ Sections expand or disintegrate on water bath
Cause
Remedy
◦ Poor Processing or water bath temp to high ◦ Reprocess tissue ◦ Cool water bath
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Processing Troubleshooting
Poor Fixation, Processing and Microtomy
Poor Fixation, dehydration and Clearing
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Extreme Parched Earth
Decalcification The process of dissolving or removing the calcium salts from tissue to facilitate sectioning. Calcium may appear in sections as a complete bone, or as clumps or granules in various tissues. • Before decalcification, bone must be fixed properly • After decalcification, bone should be washed thoroughly to stop action of decal solution.
Decalcification
Properties to consider complete removal of calcium absence of damage to tissue cells or fibers non-impairment of subsequent staining methods reasonable speed of decalcification process
Volume of decal solution should be 75100 times that of the tissue Change Decal solution as it can become saturated with Ca Ions
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Decalcification
Methods ◦ Acids (includes ion exchange and electrolytic methods) ◦ Chelating agents
Know advantages of each, which preferred, any specific uses for each method) End Point Testing ◦ Physical ◦ Chemical ◦ X-ray
Staining Alterations ◦ Usually an increase in time in hematoxylin is necessary due to the decal solution’s action on nuclei.
Poor/Incomplete Fixation
Decalcified Bone
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Over Decalcified Bone
Under Decalcified Bone
Decalcification
End Point Testing ◦ Physical Probing Bending Floating
◦ Chemical Testing Decal solution for presence of Ca ions
◦ X-ray
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Sample Question Processed tissue that was fixed in zinc formalin is very hard and brittle. Stained sections show artifacts similar to the figure on the right.This problem may be corrected in the future by: a) b) c) d)
leaving the tissue in the fixative for less than 4 hours. treating the tissue for removal of pigment placing the tissue in a buffer solution after fixation selecting a shorter schedule for processing
Sample Question Processed tissue that was fixed in zinc formalin is very hard and brittle. Stained sections show artifacts similar to the figure on the right. This problem may be corrected in the future by: leaving the tissue in the fixative for less than 4 hours. treating the tissue for removal of pigment c) placing the tissue in a buffer solution after fixation d) selecting a shorter schedule for processing Carson text states min 4-6 hours for fixation. a)
b)
Sample Question What would be the optimal paraffin infiltration protocol for complete infiltration of paraffin into 3 mm thick piece of Colon resection tissue with the use of vacuum? a. 3 changes of paraffin 10 min each b. 3 changes of paraffin 20 min each c. 3 changes of paraffin 60 min each d. 3 changes of paraffin 90 min each
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Sample Question What would be the optimal paraffin infiltration protocol for complete infiltration of paraffin into 3 mm thick piece of Colon resection tissue with the use of vacuum? a. 3 changes of paraffin 10 min each b. 3 changes of paraffin 20 min each c. 3 changes of paraffin 60 min each d. 3 changes of paraffin 90 min each Carson, Chapter 2, Processing, example protocols
END OF PART 1
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Preparation for the HT and HTL (ASCP) Examination Robert A Brunner, BA, HT(ASCP) Senior Field Support Specialist Leica Biosystems 2017Tri-State Spring Symposium May 3rd to May 5th, 2017 Rochester, Minnesota
MICROTOMY, STAINING AND LABORATORY OPERATIONS
Content Outline- Embedding/ Microtomy III. MICROTOMY (15-25%) A.Tissues 1. Morphology/anatomy 2. Cell/component demonstration B. Procedures 1. Paraffin 2. Frozen section 3. Agar/gelatin 4. Quality control 5. Plastic/resin*
C. Instrumentation 1. Components 2. Use 3. Maintenance 4. Troubleshooting 5. Quality control
*HTL Only
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Microtomy Routine -
paraffin
Cryotomy -
frozens
Agar/Gelatin
cell blocks
QC / Artifacts
Instrumentation
Microtomy
Types of Microtomes
Rotary ◦ Most Commonly used for Paraffin and frozen sectioning
Sliding ◦ Used in large plastic embedded brain tissue sectioning
Ultra-Microtome ◦ Ultra thin sections ◦ Used for Electron Microscopy sectioning
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Microtomes
Have three basic parts ◦ Block holder and adjustment screws ◦ Knife holder ◦ Ratchet mechanism for advancement of the block or knife
Use knives ◦ Disposable, Steel, etc
Knife Angles
Knife Angle Adjustments
Diagram from Carson Text, 2nd edition pp 49
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Microtome Knives
Steel Knife Profiles ◦ Wedge plane (Flat) surface on both sides; used in paraffin and frozen sectioning; used in any rotary microtome
◦ Planoconcave hollow ground (concave) on one site and plane (flat) on the other side; used in celloidin sectioning
◦ Biconcave hollow ground (concave) on both sides; used for paraffin sectioning on rocking or rotary microtomes
Microtome Knives
Other Microtome Knives ◦ Diamond EM preparation of plastic (resin) sections; expensive and must be professionally sharpened or reconditioned
◦ Glass Knives Sectioning of plastic sections for light microscopy; made by breaking glass at correct angles
◦ Disposable Widely used; less space needed; no expense of knife sharpeners
Microtomy Error
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Moth Eaten
Venetian Blind
Scratches
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Tissue Contaminate
Thick Section
Extreme Separation
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Microtomy Troubleshooting
Alternate sections coming off microtome thick and thin? ◦ Block loose ◦ Knife loose ◦ Insufficient clearance angle ◦ Possible microtome problem
Microtomy Troubleshooting
Sections lift from microtome blade on the upstroke? ◦ Wax/debris on knife edge ◦ Debris on block edge ◦ Insufficient clearance angle ◦ Static Electrical charge
Microtomy Troubleshooting
Excessive compression of sections? ◦ Dull knife ◦ Knife angle to wide ◦ Wax too soft ◦ Block not cool
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Microtomy Troubleshooting
Sections show venetian blind (chatter) effect? ◦ Loose Knife ◦ Loose Block ◦ Knife angle too wide ◦ Tissue is very dense/hard
Frozen Sections-Cryotomy Purpose
•
• rapid diagnosis for surgical patients • demonstrate fats, lipids, or enzymes that would be lost in routine fixation/processing
Advantages
• •
rapid preservation of cells for enzyme and fluorescent studies
Miscellaneous
• • • •
cryostat temperature is approximately -15 to -25°C. knife angles are approximately 30° Sections cut at 6-8-10 microns for routine sections
Cryostat
• • •
maintained at -20°C for routine frozen sectioning. brain, -15°C is better.
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Cryotomy- The Cryostat Clinical or research Protect against biohazards Set temperature for tissue type before cryotomy Equilibrate -80°C blocks in chamber ~30 min Know chamber’s cold and warm areas High vs low profile Brush or Anti-roll?
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Cryotomy Cutting temperatures • Colder for fatty tissues • Warmer for friable tissues Blade & block @ same temperature Orient block • For least cutting resistance • Slow, steady stroke
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Sample Question Special stains for which of the following require that sections be cut at 8 – 10 um? a. amyloid, phospholipids, central nervous system b. bacteria, reticulin, adrenal, hepatitis B antigen c. calcium, bile, blood, cholesterol d. elastic fibers, connective tissue, carbohydrates
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Sample Question While the block is being rough trimmed, the knife digs into the tissue and gouges out a chunk. The most likely cause is that the : a. block is too cool b. knife is dull c. block is loose in the holder d. cutting stroke is too rapid
Sample Question Which of the following should be used to sterilize the interior chamber of the cryostat? a. 70% alcohol b. 10% Bleach c. xylene d. formalin
Embedding Refers to the orientation of tissue in a mold and allowing the embedding medium to harden Place cut side down. Press (tamp) the tissue down to the bottom of the mold as the wax hardens. Place the tissue in the mold before the paraffin forms a solidified layer
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Embedding
Methods of marking tissue for orientation are tattoo dyes, India ink, nicks cut into the tissue.
Each lab has its own protocols
Embedding/Paraffin
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Embedding
Make sure that the top of the cassette is filled with paraffin since it will shrink
Embed tissue at an angle so that the knife hits the tissue a little at a time.
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Embedding Error
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Improper Orientation
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Paraffin Embedded blocks
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Content Outline- Staining IV. STAINING (30-40%)
B. Procedures- Continued 7. Nerve
A. Tissues 1. Morphology/anatomy 2. Cell/component demonstration 3. Function 4. Pathology* 5. Biochemistry principles/theories* B. Procedures 1. Nucleus/cytoplasm (e.g., H&E) 2. Bone marrow 3. Carbohydrates 4. Connective/supporting tissue 5. Lipids 6. Microorganisms *HTL Only
8. Pigments/minerals/granules 9.Tissues/cells/components (e.g., fibrin, mast cells) 10. Enzymes* 11. Immunohistochemistry (e.g., basic staining theory, retrieval techniques, selection of controls*, antibody preparation*) 12. Quality Control 13. Cytological stains (e.g., Papanicolaou)
14. In Situ Hybridization*
Content Outline- Staining(Cont) C. Instrumentation E. Mounting Procedures 1. Components 1. Media 2. Use 2. Coverslip 3. Maintenance 3. Refractive index* 4. Troubleshooting 5. Quality control D. Reagents/Dyes 1. Types/components 2. Properties/functions/actions *HTL Only 3. Quality control 4. Chemistry principles/theories*
Staining “Biological stains” is the term used to denote the purified dyes adapted for microscopic work.
The three broad categories of biologic staining are:
• general staining • differentiation of cytoplasmic and nuclear elements
• special staining • demonstration specific tissue elements or disease processes
• heavy metal impregnation • visualization of tissues or tissue elements with silver impregnation and reduction
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Staining Terminology • Dye • a benzene ring with chromophores and auxochrome
• Chromophore • atomic groups associated with color (“color bearing”) •
Auxochrome • • • •
gives the dye affinity for attachment to tissue a common acid auxochrome is -COOH (carboxyl group) a common basic auxochrome is -NH2 (amino group) auxochromes may alter the shade of the dye but does not give the color to the dye compound
Staining Terminology • Chromogen • a benzene compound containing chromophore radicals
• Mordant • salts of metals, used to link dye to tissue elements • may be applied before, with Or after the stain • most common hematoxylin mordants are aluminum or iron
Staining Terminology • Metachromasia • staining different tissue elements different colors by a single dye • it is an adsorption reaction; (reds and blues) • Example- Toludine Blue
• Polychromatic • differential staining of tissue elements by a combination of dyes in one solution • Example- Giemsa stain.
• Orthochromatic • staining of tissue the same color as the dye in solution • Example- Biebrich Scarlet
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Staining Terminology • Basophilia • “basic loving”; acid tissue elements • stained by basic dyes such as hematoxylin
• Acidophilia • “acid loving”; basic tissue elements • stained by acid dyes such as eosin
Staining Terminology • Acid dye • Chromophore group is located in the anionic (acid++) portion of the dye • stains basic tissue elements such as cytoplasm (Eosin)
• Basic dye • Chromophore group is located in the cationic (basic--) portion of the dye • stains acidic tissue elements such as nuclei (Hematoxylin)
Sample Question A benzene compound that containing atomic groupings associated with color is called a/an: a. auxochrome b. chromogen c. chromophore d. dye compound
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Info to Know!!!! Stain Name Demonstrates what Reagents used Functions and Mechanisms Results Pitfalls
Routine Staining
Routine General staining ◦ Theory ◦ H&E ◦ Dyes and Reagents
Types of Routine Staining
Hematoxylin and Eosin ◦ Regressive Differentiation ◦ Progressive
Know Components of all the dyes and reagents and their functions
NUCLEAR STAIN Hematoxylin | DIFFERENTIATION of NUCLEAR STAIN Acid alcohol | BLUING of NUCLEAR STAIN Ammonia water, lithium carbonate, Scott's tap water | CYTOPLASMIC STAIN (COUNTERSTAIN) Eosin, Eosin-phloxine | DIFFERENTIATION of CYTOPLASMIC STAIN Alcohol-water
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Evaluation of Hematoxylin Stain In open epithelial cells, a good H&E stain will show: 1- A well-defined nuclear membrane 2- Clear, open karyoplasm (cytoplasm of the nucleus)
3- Crisp, fine-spiculed chromatin patterns 4- Prominent “eosinophilic” nucleoli. (if present)
*Dense “closed” patterns usually present* The hematoxylin should appear blue to blueblack.
Evaluation of Eosin Staining When applied correctly eosin will produce at least a tri-tonal (three-color) effect within the section. 1. Muscle cells (smooth, skeletal, cardiac)/epithelial cell cytoplasm will stain deep red-pink.
2. Collagen will stain a distinct lighter pink. 3. Red blood cells (RBC) will stain a bright orangered. In addition, the nucleoli (if present) should exhibit a reddish-purple color due to their high RNA content.
Hematoxylin and Eosin
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Three Shades of Eosin
Good H & E
Poor Staining Results
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Poor Nuclear Staining
Poor Nuclear Staining
Sample Question Acidophilic tissue components that can be distinguished by light microscopy following H&E staining are: a. muscle, reticulin, and elastic b. collagen, basement membranes, and nucleic acids c. collagen, muscle, and erythrocytes d. Muscle, collagen, and reticulin
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Sample Question Following H&E staining, slides are dehydrated through ascending strengths of alcohol and cleared in xylene; however, the first xylene in the series is milky-white. The best course of action is to: a. Change all of the solutions in the series b. Change the xylenes at the end of the series c. Change the alcohols and xylenes at the end of the series d. Air dry the slides and start them in alcohol at the end of the series
Special Staining Bone Marrow / Nucleic acids Carbohydrates Connective tissues
Bone Marrow Stains
Giemsa Wrights
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Carbohydrate Stains • Glycogen: • PAS (with and without digestion)
• Neutral mucins: • Mucicarmine • PAS-H
• Amyloid: • Benhold’s congo red • Puchtler’s congo red • Crystal violet (metachromatic) • Thioflavine T
• Acid mucopolysaccharides: • Alcian blue • Colloidal iron • Alcian Blue PAS Differentiate Neutral and acid mucins
Carbohydrate Stains
PAS ◦ With and Without Diastase
Parallel sections are ran thru procedure one with diastase digestion and one with out Demonstrates????
OXIDATION periodic acid | CHEMICAL REACTION Schiff's reagent | RECOLORIZATION water | COUNTERSTAIN
PAS With and WO diastase
Diastase Digestion
No Diastase Digestion
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Neutral Mucins Mucicarmine and PAS-H
What stains are used in this procedure?
Mucicarmine
Acid Mucopolysaccarides
Alcian Blue
Amyloid Demonstration
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Connective Tissue Stains Collagen ◦ Masson Trichrome ◦ Gomori Trichrome ◦ ◦ ◦ ◦
Reticulin
◦ Masson Trichrome ◦ Gomori Trichrome
Gomori’s Wilder’s Snook’s Gordon and Sweet
Muscle
Basement Membranes ◦ Periodic Acid Schiffs ◦ Periodic acid Methenamine Silver
Elastic ◦ Verhoff-VanGieson ◦ Aldehyde Fuchsin
Trichrome Staining Mechanism NUCLEAR STAIN Iron hematoxylin | PLASMA STAIN (SECONDARY) Biebrich scarlet-Acid fuchsin | DIFFERENTIATION Phosphotungstic-Phosphomolybdic acid | COLLAGEN STAIN (PRIMARY) Aniline blue, Light green
Collagen/ Muscle stains
Masson Trichrome
Gomori Trichrome
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Elastic Stain Mechanism FORMATION OF DYE LAKE Hematoxylin - Ferric chloride * - Iodine | DIFFERENTIATION Ferric chloride * | REMOVAL of EXCESS IODINE Sodium thiosulfate | COUNTERSTAIN Acid Fuchsin, Picric acid
Elastic Stain
Basement Membrane Stains
Periodic Acid Schiff’s
Periodic Acid Methenamine Silver
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Reticulin Stain
Ammoniacal vs Methenamine
Ammoniacal Silver ◦ ◦ ◦ ◦ ◦ ◦
Oxidation Sensitization Impregnation Reduction Toning Removal of unreduced silver
Methenamine Silver ◦ ◦ ◦ ◦
Oxidation Impregnation Toning Removal of un-reduced silver
Know reagents used to perform the above functions!!
Lipid Stains Oil Red O Performed on frozen sections Avoid all Solvents Coverslip with Aqueous Media Other Fat Stains
◦ Sudan Black ◦ Osmium Tetroxide
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Microorganism Stains
Acid Fast Bacilli
◦ Kinyouns ◦ Ziehl Neelson ◦ Fite
Fungi ◦ Grocott Methenamine Silver ◦ Periodic Acid Schiffs
Gram Positive/Neg ◦ Brown and Hopps ◦ Brown and Brenn
Spirochete ◦ Steiner,Warthin-Starry
Acid Fast Staining Mechanism PENETRATION OF DYE SOLUTION ”HEATED” Carbol fuchsin in Phenol | DECOLORIZATION 1% HCl in 70% Alcohol | COUNTERSTAIN Methylene blue, Light green
AFB Stain
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AFB Stain
Fite Stain For Leprosy Bacilli
Auromine Rhodamine- (not on exam)
Gram Staining Mechanism "GRAM POSITIVE" STAIN Crystal violet | MORDANT Iodine | DECOLORIZATION Acetone/Alcohol | "GRAM NEGATIVE" STAIN Basic fuchsin, Neutral red | DECOLORIZE BACKGROUND Picric acid-Acetone, Gallego's solution, Alcohol
Gram Stain
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Fungus Stains
Grocotts Methenamine
Periodic Acid Schiffs
Pneumocystis Stain
Spirochete Stain
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Additional Stains
Nervous tissue ◦ Bielschowski ◦ Luxol Fast Blue LFB PAS LFB Cresyl Echt Violet
Pigments and Minerals ◦ Iron Prussian Blue
◦ Melanin Fontana Masson
◦ Bile Halls
◦ Calcium Von Kossa
Pigments and Minerals
Aftifactual
◦ Mercury ◦ Formalin
◦ ◦ ◦ ◦
Exogenous ◦ Carbon ◦ Asbestos
Endogenous
Hemoglobin Bile Melanin Lipofuchsin
Minerals ◦ Iron ◦ Copper ◦ Calcium
Additional Stains
Tissue/Cells/Components
Immunohistochemistry
◦ Toludine Blue (Mast Cells)
For HT and HTL
Enzymes* (HTL only) ◦ APTase ◦ NADH ◦ etc
◦ Fixation ◦ Basic Staining Theory ◦ Retrieval Techniques
For HTL only ◦ Selection of Controls ◦ Antibody Preparation
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Immunohistochemistry Define, understand, and the use of the following IHC terms:
• Fixation for IHC • Antigen • Antibody • Primary • Secondary • Isotype • Controls • Positive • Negative • Serum • Conjugated • Titer
• Antigen Retrieval • Blocking • Detection Systems • Horseradish peroxidase • Alkaline phosphatase • ABC • Chromagen • DAB • AEC
*HTL ONLY
Melan A IHC with DAB and Red
Sample Question- HTL In the bloody areas of a tissue section stained with the immunoperoxidase technique, there is a marked reaction of the red blood cells. This is most likely the result of: A. Selecting the wrong chromogen B. Forgetting to apply the non-immune blocking serum C. Failure to block using hydrogen peroxide D. Prolonging the incubation with the primary antibody
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Additional Stains
Cytology Stains ◦ Papanicolaou ◦ Diff Quick ◦ Wrights ◦ Giemsa
Content Outline- Laboratory Operations V. LABORATORY OPERATIONS (10-15%)
(e.g., microwave, computers, pH meter, solvent recovery)
A. Safety 1. 2. 3. 4. 5. 6.
C. Ancillary Equipment/Instruments 1. 2. 3. 4. 5.
Storage Disposal Hazards Regulations Procedures Quality control
Components Use Maintenance Troubleshooting Quality control
D. Management*
B. Laboratory Mathematics 1. Metric system 2. Percent solutions/dilutions 3. Molar solutions
1. Theories* 2. Procedures*
E. Education* 1. Theories* 2. Procedures*
F. Regulations* * HTL ONLY
1. Federal government* 2. Accrediting agencies
The Safety Chapter in Carson’s book (Chapter 4) is a fairly comprehensive reference for safety information.
MSDS (Material Safety Data Sheet) “Right to Know” law
A.
Flammable
B.
Radioactive
C.
Biohazard
D.
Poison
9 3
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Laboratory Math
(Chapter 5) in Carson’s book is a fairly comprehensive review.
Units of Measure: meter (m) for length liter (L) for volume gram (g) for weight
Temperature Conversion
Solution Dilutions V1 x C1 = V2 x C2
Molar Solutions
Normal Solutions
Laboratory Microscope A lab without a microscope is a lab without quality control.
Types of Microscopes •
LIGHT (brightfield) light passes unaltered up through its lens system to produce a brightly lighted field; commonly used in routine light microscopy.
•
FLUORESCENT: uses a source of intense light together with a filter that removes all except the ultraviolet (LIV) and near ultraviolet rays; an object fluoresces when it absorbs UV light reflected on it or transmitted through it, and then emits the energy in visible lights of specific colors.
•
PHASE: utilizes refraction that occurs when light passes from one medium into another of different density; preferred for studying unstained cells and living material.
•
POLARIZING: can be a special scope or the effect produced by using sheets of film, turning one of them to produce the polarized effect.
discs or
• ELECTRON: light source is a beam of electrons, not visible light; images are captured by ring-shaped electromagnets which function as lenses. Electron microscopy is useful in ultrastructure studies and diagnoses.
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Miscellaneous Equipment • Lab refrigerator • • • • • • • • •
Lab freezer Long-term freezer Microwave Oven Embedding Center Slide drying oven Paraffin bath pH Meter Flotation water bath Adhesives for slides/water bath
• Automatic slide stainer • Automatic tissue processor • Cryostat • Microscope • Microtome • Microtome knife • Balances and Scales • Incubators • Knife sharpening • Slides • Coverslips
Chapter 3 In Carson Text Covers Well
Test Taking Tips!
“There is NO substitute for knowing the subject matter, nor is there a substitute for a good understanding of the process of taking an exam. Both content knowledge and the ability to take examinations successfully are improved by study and practice.”
Test Taking Tips
Get a good night’s sleep Know the test format Organize your test material Read and understand the directions Read the questions carefully Develop a timetable
• Keep your cool • Use the process of elimination • Draw a blank? DON’T PANIC • Have realistic expectations • Celebrate after it’s over !!!
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And Most of All! Do not second guess yourself !! Be thorough in your preparation! Study hard ! GOOD LUCK! ☺☺☺☺☺☺
References 1. 2. 3. 4. 5. 6. 7. 8.
American Society for Clinical Pathology, Board Of Registration website www.ascp.org National Society for Histotechnology, www.nsh.org Peggy Wenk, for photos Indiana University School of Medicine, Histotechnology Program for photos Carson,F.L., Histotechnology: A Self-Instructional Text, 3rd edition, Chicago; ASCP Press, 2009 Bancroft, J.D., Gamble, M., Theory and Practice of Histological Techniques, 6th edition, New York; Churchill-Livingstone, 2007. Sheehan, D.C., Hrapchak, B.B., Theory and Practice of Histotechnology, 2nd edition, St. Louis, MO; C.V. Mosby, 1980 Brown, R.W., Histologic Preparations: Common Problems and Their Solutions, CAP Press, 2009
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