Clsi GP28 A

GP28-A
Vol. 25 No. 7
Replaces GP28-P
Vol. 24 No. 9
Microwave Device Use in the Histology

Laboratory; Approved Guideline
This document provides recommendations for reproducing the performance of

microwave-accelerated procedures to prepare biological specimens in the histology
laboratory.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
Clinical and Laboratory Standards Institute
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GP28-A
ISBN 1-56238-563-1
Volume 25 Number 7 ISSN 0273-3099
Microwave Device Use in the Histology Laboratory; Approved
Guideline
Gary R. Login, DMD, DMSc
Ellyn S. Beary, BS
H. Skip Brown, BA, HT(ASCP)
Cheryl H. Crowder, BA, HTL(ASCP)
Maureen Doran, BA, HTL(ASCP)
Richard T. Giberson, BA, MS
H. M. Skip Kingston, PhD
Anthony Leong, MD
Max Robinowitz, MD
Steven E. Slap, BA, MA, MPhil
Franco Visinoni, PhD
Abstract
This document provides recommendations for quality assurance and safety procedures for microwave equipment use, and
provides a means to understand and troubleshoot conditions that contribute to variability in microwave-accelerated procedures in
human clinical, veterinary, and research histopathology laboratories. Safety issues unique to microwave instrumentation in
histopathology laboratory settings are emphasized. In addition, the document discusses microwave device process control,
procedure validation, and results verification.
Clinical and Laboratory Standards Institute (CLSI). Microwave Device Use in the Histology Laboratory; Approved Guideline.
CLSI document GP28-A (ISBN 1-56238-563-1). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA, 2005.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are
listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is
not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax:
610.688.0700; E-Mail: exoffice@clsi.org; Website: www.clsi.org
Number 7 GP28-A
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system,
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Clinical and Laboratory Standards Institute hereby grants permission to reproduce limited portions of this
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notice, be distributed without charge, and, in no event, contain more than 20% of the document’s text.
Reproduced with permission, from CLSI publication GP28-A—Microwave Device Use

in the Histology Laboratory; Approved Guideline (ISBN 1-56238-563-1). Copies of the
current edition may be obtained from Clinical and Laboratory Standards Institute, 940
West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the
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Standards Institute by written request. To request such permission, address inquiries to the Executive Vice
President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898, USA.
Copyright ©2005. Clinical and Laboratory Standards Institute.
Suggested Citation
(Clinical and Laboratory Standards Institute. Microwave Device Use in the Histology Laboratory;
Approved Guideline. CLSI document GP28-A [ISBN 1-56238-563-1]. Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.)
Proposed Guideline
February 2004
Approved Guideline
February 2005
ISBN 1-56238-563-1
ISSN 0273-3099
ii
Volume 25 GP28-A
Committee Membership
Area Committee on General Laboratory Practices

Sheila M. Woodcock, ART, MBA Margaret M. Grimes, MD Stephen J. Sarewitz, MD
Chairholder Virginia Commonwealth University Valley Medical Center
QSE Consulting Richmond, Virginia Renton, Washington
Rose Bay, Nova Scotia, Canada
Theresa D. Stokeld, MBA, Jennifer Schiffgens, MBA,
Albert Rabinovitch, MD, PhD MT(ASCP)DLM MT(ASCP)
Vice-Chairholder Remel Inc. California Pacific Medical Center
Abbott Laboratories, Hematology Lake Charles, Louisiana San Francisco, California
Business Unit
Santa Clara, California Advisors Daniel W. Tholen, MS
Dan Tholen Statistical Consulting
Eric Arendash, MT(ASCP) Kay M. Creed Traverse City, Michigan
Centers for Medicare & Medicaid St. Mary’s Hospital
Services Richmond, Virginia Marla Thomas
Philadelphia, Pennsylvania Litton Pathology Associates
Steven I. Gutman, MD, MBA Blue Springs, Missouri
Miguel Azar, MD FDA Ctr. for Devices/Rad. Health
Dept. of Veterans Affairs Medical Rockville, Maryland Eleanor M. Travers, MD, MHA
Center State of Connecticut Dept. of Public
Minneapolis, Minnesota Gerald A. Hoeltge, MD Health
The Cleveland Clinic Foundation Hartford, Connecticut
Lucia M. Berte, MA, MT(ASCP), Cleveland, Ohio
SBB, DLM; CQA(ASQ)CQMgr
Quality Systems Consultant
Westminster, Colorado
Subcommittee on Microwave Ovens
Gary R. Login, DMD, DMSc

Chairholder H. M. Skip Kingston, PhD Stacie Kirsch
Harvard School of Dental Duquesne University Electron Microscopy Sciences
Medicine Pittsburgh, Pennsylvania Fort Washington, Pennsylvania
Boston, Massachusetts
Anthony Leong, MD Susan Meloan, BBA, HT(ASCP)
Ellyn S. Beary, BS Hunter Area Pathology Services HTL
NIST Newcastle, Australia Medical College of Georgia
Gaithersburg, Maryland North Augusta, South Carolina
Max Robinowitz, MD
H. Skip Brown, BA, HT(ASCP) FDA Ctr. for Devices/Rad. Health Diane G. Miller, HT(ASCP)
Lab Management Consultants Rockville, Maryland Miller Consultant Services
Maryland Heights, Missouri Beaverbow, Oregon
Franco Visinoni, PhD
Cheryl H. Crowder, BA, Milestone s.r.l. Jan Minshew, HT(ASCP) HTL
HTL(ASCP) Fratelli, Italy Leica Microsystems, Inc.
Louisiana State University Bannockburn, Illinois
Baton Rouge, Louisiana Advisors
Shan-Rong Shi, PhD
Maureen Doran, BA, HTL(ASCP) Charles J. Churukian, BH, USC Keck School of Medicine
SIU Medical School HT(ASCP) HTL Los Angeles, California
Carbondale, Illinois University of Rochester Medical
Center Albert J.H. Suurmeijer, MD, PhD
Richard T. Giberson, BA, MS Rochester, New York University Hospital Groningen
Ted Pella, Inc. Groningen, The Netherlands
Redding, California Alton D. Floyd, PhD
Norfolk Associates, Inc.
Edwardsburg, Michigan
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Staff
Clinical and Laboratory Standards Institute

Wayne, Pennsylvania
Tracy A. Dooley, MLT(ASCP)

Staff Liaison
Donna M. Wilhelm
Editor
Melissa A. Lewis
Assistant Editor
Acknowledgement in Memoriam of our Subcommittee Member and Colleague
The Clinical and Laboratory Standards Institute (CLSI) and the Subcommittee on Microwave Ovens
acknowledge the contributions of Mr. Steven E. Slap, BA, MA, M Phil. Steve was one of the early
promoters of the first commercial laboratory microwave device for histology. His energy and foresight
stimulated many educational seminars, and he introduced many histologists to the benefits of microwave
methods.
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Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope..........................................................................................................................................1
2 Introduction................................................................................................................................1
3 Definitions .................................................................................................................................2
4 Precautions and Safety...............................................................................................................5

4.1 Microwave Radiation Exposure Standards and Regulations ........................................5
4.2 Electrical Precautions ...................................................................................................7
4.3 Biological Precautions ..................................................................................................8
4.4 Chemical Handling Precautions....................................................................................8
4.5 High Temperature Precautions .....................................................................................9
4.6 Recommended Safety Procedures for Microwave Devices and Applications..............9
4.7 Microwave Safety Inspection .....................................................................................11
5 Principles of Microwave-Accelerated Methods.......................................................................12
5.1 Power Level ................................................................................................................13
5.2 Energy Absorption Mechanisms and Reaction Conditions ........................................14
6 Recommendations for Documenting Microwave Methods .....................................................15
7 Critical Descriptors for Microwave-Accelerated Procedures ..................................................15

7.1 Temperature Control and Measurement .....................................................................16
7.2 Microwave Power .......................................................................................................21
7.3 Specimen Handling.....................................................................................................22
7.4 Process Time...............................................................................................................26
8 Special Microwave Procedures................................................................................................27
8.1 Antigen Retrieval Protocol .........................................................................................27
9 Template for Documentation of Microwave Methods.............................................................29
10 Troubleshooting Results ..........................................................................................................30
References.............................................................................................................................................32
Summary of Delegate/Consensus Comments and Subcommittee Responses ......................................35
The Quality System Approach..............................................................................................................40
Related CLSI/NCCLS Publications ......................................................................................................41
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Volume 25 GP28-A
Foreword
The ease and rapid pace with which microwaves have entered the clinical laboratory are raising many
questions for laboratory administrators. Are laboratory personnel aware of and trained in safety issues
unique to microwaves? Do laboratory directors have a quality assurance program for microwave
procedures? Does the leadership of the national societies that represent medical and research communities
have the information they need to respond to local and national regulatory agencies regarding the safe and
efficacious use of microwave technology? Are equipment manufacturers promoting equipment that meets
the highest safety standards?
Several basic science and clinical research laboratories in North America, Europe, Asia, and Australia
working independently during the past 31 years have identified important principles for using microwave
technology reliably in laboratory medicine. This guideline emphasizes the scientific principles and
practices involving the safe and effective use of microwave ovens in the histology laboratory. However,
it is also important to be aware of national and local governmental regulatory requirements before
microwave ovens are selected and used in clinical laboratories. The guideline only provides examples of
the regulatory requirements that are current in the United States. Users in other countries are advised to
consult with their national and local authorities for requirements.
Readers wishing for a quick start are directed to the following sections:
• Table 4, Safe Laboratory Use of Microwave Devices;
• Section 9, Template for Documentation of Microwave Methods; and
• Section 10, Troubleshooting Results.
Key Words
Antigen retrieval, biopsy, electron microscopy, fixation, histology, immunocytochemistry,

immunohistochemistry, light microscopy, microwave, processing, resin curing, staining
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Microwave Device Use in the Histology Laboratory; Approved Guideline
1 Scope
This CLSI guideline is written for multiple audiences, including: laboratory technicians, microwave
device manufacturers, microwave device resellers, compliance and safety officials, and administrators.
The goals of this document are to 1) provide a scientific basis for reproducible sample preparation of
biological specimens for diagnostic purposes; 2) advise laboratory personnel on the best safety guidelines;
and 3) discuss the limitations of domestic microwave ovens in a hospital laboratory. Original sources are
cited in the References section for those individuals seeking additional information.
NOTE: The reader is encouraged to supplement the information in this document with continuing
education courses on microwave device safety and use.
To ensure the success of microwave-accelerated procedures in the histopathology laboratory, this

document provides:
• general definitions of common microwave terminology;
• detailed discussion of safety issues particular to microwave heating of samples;
• guidelines to identify potential sources of variability; and
• a “hands-on” troubleshooting guide to improve microwave-accelerated procedures.
2 Introduction
Microwave-accelerated sample preparation of biological specimens is a field that continues to grow
rapidly as evidenced by the number of innovative articles written each year. The reason for this increase
in the microwave literature is simple. Microwave-accelerated procedures are useful in almost every step
of sample preparation for microscopy. Microwave procedures speed up reaction processes and save time.
Even more important, microwave procedures improve the retention of soluble antigens and often preserve
antigen immunoreactivity better than conventional fixation methods.1,2 In short, microwave-accelerated
techniques can be used to improve the efficiency of a variety of histopathology laboratory procedures,
such as fixation, decalcification, processing of specimens for paraffin wax or resin embedding, and
staining. Hundreds of laboratory procedures using microwave devices for histopathology have been
published.1,3-5 A brief list of these procedures is provided in Table 1.
Table 1. Application of Microwave-Accelerated Methods in Histopathology (see Sections 4.3 through

4.6 for Safety Precautions) 1,3-5
Examples of Microwave-Accelerated Methods
Fixation of human and animal Histoprocessing Immunogold Protein A labeling
specimens
Fixation of marine specimens Decalcification Streptavidin-biotin- In situ hybridization
peroxidase labeling
Fixation of plant specimens Immunoperoxidase Immunofluorescence Rapid drying
Fixation of insect specimens Antigen retrieval Lectin labeling Enzyme-linked
immunosorbent assay
(ELISA)
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Clinical and Laboratory Standards Institute. All rights reserved. 1
Number 7 GP28-A
Three types of microwave devices are being used in a histopathology laboratory setting: 1) microwave
instruments specifically designed and certified as medical devices; 2) commercial grade microwave
devices converted for laboratory/clinical use; and 3) consumer household microwave units modified for
laboratory/clinical use. Laboratory microwave devices are designed with exhaust fans and safety features
to protect the operator, sensors to protect the instrument, and sophisticated temperature monitoring and
intricate electronics that allow improved quality control of the specimen. Household microwave units are
designed for food preparation, and they are not certified for laboratory use unless they meet the
requirements outlined in Section 4.
All three types of microwave devices have a large chamber in which samples are heated. Large-chamber
microwave devices are often described in the microwave literature as “large-cavity” or “multimodal
devices.” For the purposes of this document, we will use the term “large-cavity microwave devices.”
Several features have been added to microwave devices to reduce heating damage to biological specimens
and microwave equipment (see Table 2). Manufacturers of laboratory microwave equipment have
improved temperature control by adding temperature probes with feedback systems for process
automation, specialized power supplies for generation of microwave power,4,6-10 controllable magnetron
duty cycles,3 and variable power output.11 Highly specialized microwave devices have platforms with
very high rotational speed,12 vacuum and pressure cycling,13-15 and hybrid equipment combining
ultrasound and microwave irradiation.16
Table 2. Use of Large-Cavity Microwave Devices With Basic or Advanced Features

Large-Cavity Large-Cavity
Microwave Microwave Devices
Histopathology Laboratory Devices with with Advanced
Procedure Basic Functions Functions Rationale for Equipment Choice
Drying Slides X Minimal control necessary
Melting Agar X Minimal control necessary
Enzyme/Antigen Retrieval X Minimal control necessary
Staining (nontoxic reagents) X Minimal control necessary
Staining (toxic reagents) X Exhaust fume safety
Tissue Processing X Precise temperature control/exhaust
fume safety
In situ Hybridization X Precise temperature control
Decalcification X Process control, safety
Fixation X Exhaust fume safety
Resin Embedding X Exhaust fume safety
To date, there are no regulations or benchmarks specifically for microwave devices in the clinical
laboratory. There are, however, many regulations regarding electrical safety and general laboratory
equipment safety that include microwave devices (see Section 4 for more details). In addition, the
potential to overheat and damage diagnostic biological specimens in microwave procedures is great (e.g.,
tissue shrinkage, denatured connective tissue, pyknotic nuclei). The need for guidelines and quality
assurance for microwave procedures is well recognized.4,17-26
3 Definitions
This section provides a brief list of the most common terms and definitions to facilitate reading the
microwave literature. A detailed report of terminology related to microwave safety has been published.27
Accuracy (of measurement) – Closeness of the agreement between the result of a measurement and a
true value of the measurand (VIM93).28
Anode – A positively charged conductor by which electrons leave an electrical device.

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2 Clinical and Laboratory Standards Institute. All rights reserved.
Volume 25 GP28-A
Arcing – Electrical conduction through a gas in an applied electric field.
Calorie (C) – 1) The amount of heat it takes to raise the temperature of 1000 grams or 1 kg of water one
degree centigrade; NOTE: A Calorie is actually a kilocalorie or 1000 calories; 2) calorie (c) – The
amount of heat it takes to raise the temperature of 1 gram of water 1 degree centigrade; NOTE: The
cal/min is the unit of heat per minute.
Cathode – Negatively charged conductor that is the source of electrons in an electrical device.
Conduction – The flow of heat by conduction occurs via collisions between atoms and molecules in the
substance and the subsequent transfer of kinetic energy; NOTE: When there exists a temperature
gradient within a body, heat energy will flow from the region of high temperature to the region of low
temperature.
Convection – The flow of heat through a bulk, macroscopic movement of matter from a hot region to a
cool region.
Dielectric constant – The measure of a sample’s ability to obstruct the microwave energy as it passes
through the medium; the loss (dielectric) factor measures the sample’s ability to dissipate that energy;
NOTE: The term “loss” is used to indicate the amount of input microwave energy that is lost to the
sample by being dissipated as heat in the sample.
Diode – A device that conducts electric current run in one direction only.
Dipolar molecules – Molecules that are configured such that electrons favor one region of the molecule,
resulting in an uneven spatial distribution of electrons and charge so that one side is slightly negatively
charged relative to the somewhat more positively charged other side.
Dipole rotation – The net alignment, due to the electric field, of molecules in the sample that have
permanent or induced dipole moments.
Duty cycle (magnetron cycling) – Microwave power can be applied continuously but is usually pulsed.
The power output of the magnetron is controlled by “cycling” the magnetron on and off at full power for
some fraction of time to obtain an average power level. The duty cycle of a magnetron is the time the
magnetron is ‘on’ divided by the total time of the cycling period; NOTE: Domestic microwave devices
have relatively long cycling periods based on intervals of 1/6 min. (10 seconds) and longer as compared
to laboratory analytical grade microwave equipment which has cycling periods of 1/60 min. (1 second),
making heat control more difficult in household microwave devices.
Entropy (thermodynamics) – A thermodynamic quantity representing the amount of energy in a system

that is no longer available for doing mechanical work.
Hertz – The derived Standardized International (SI) Units of inductance, defined as the frequency of one
cycle per second, having units of S-l (reciprocal seconds).
Inductance – A magnetic field produced by the presence of an electric current.
Ionic conduction – The conductive (i.e., electrophoretic) migration of dissolved ions in the applied
electromagnetic field.
Magnetron – A cylindrical diode with an anode and a cathode; NOTES: a) Superimposed on the diode is
a magnetic field that is aligned with the cathode; b) A ring of mutually coupled resonant cavities is in the
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anode so a potential of several thousand volts is reached across the diode; c) The released electrons, under
the influence of the magnetic field, resonate, and the magnetron oscillates.
Microwave field – Electromagnetic, nonionizing radiation that is produced by the magnetron; NOTES:
a) The microwave field refers to the density of microwave energy in a defined space; b) This microwave
energy density in a space is sometimes referred to as “field strength.”
Microwaves – Electromagnetic energy between far infrared (IR) and radio waves that corresponds to
wavelengths of 1 cm to 1 m; NOTE: The frequency range of these sources is between 30 GHz and 300
MHz with a frequency of 2450 MHz being the most common.
Microwave-transparent – Having the property of transmitting microwaves, materials such as glass or

plastics that do not absorb microwave energy.
Mode stirrer – A device that disrupts standing microwave patterns and distributes the microwave energy
more homogeneously throughout a microwave cavity.
Power output of the magnetron – A measure of heat per unit time, i.e., 1 W = 14.33 cal/min; NOTE:
The microwave energy output from the magnetron is generally measured in watts.
Reflected power – The power that occurs when the traveling electromagnetic waves are reflected and the
flow of energy is partly in the reverse direction; NOTES: a) Reflected microwave energy can result in
overheating or a deficiency of microwave energy in localized areas; b) Devices that remove reflected
microwaves have been designed to protect the magnetron from microwave energy reflected back into the
wave-guide.
Reproducibility – Precision under conditions where test results are obtained with the same method on
identical test items in different laboratories with different operators using different equipment (ISO 5725-
1).29
Sample – One or more parts taken from a system and intended to provide information on the system,
often to serve as a basis for decision on the system or its production (ISO 15189)30; NOTE: For example,
a volume of serum taken from a larger volume of serum (ISO 15189).
Specific heat – The heat required to raise the temperature of one gram of a substance one degree
centigrade.
Specimen – The discrete portion of a body fluid or tissue taken for examination, study, or analysis of one
or more quantities or characteristics to determine the character of the whole.
Thermodynamic – The branch of physics concerned with the conversion of different forms of energy; it
is the study of the effects of work, heat, and energy on a system.
Watt – 1) In electrical terms, one watt is the power produced by a current of one ampere flowing through
an electric potential of one volt; 2) The power which in one second gives rise to energy of one joule;
NOTES: a) The SI unit of power; power is the rate at which work is done, or (equivalently) the rate at
which energy is expended; b) One watt is equal to a power rate of one joule (force per distance) of work
per second of time.
Wave-guide – A device that delivers the microwave energy from the magnetron to the microwave cavity.
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4 Precautions and Safety

Microwave techniques introduce unique safety considerations that are not encountered by technicians
using traditional laboratory heating devices. Microwave irradiation has the capability of rapid high-
temperature heating, with the potential for vaporization and pressurization. The majority of unsafe
conditions or practices that can arise during the use of microwave systems in the laboratory are avoidable.
It is essential that microwave units be operated in a manner that ensures maximum safety to the operator
and laboratory personnel. In the U.S., many regulatory bodies (e.g., Food and Drug Administration
[FDA], Occupational Safety and Health Administration [OSHA]) require compliance with provisions of
the Code of Federal Regulations (CFR) and testing laboratories and certification organizations (e.g.,
Underwriters’ Laboratories [UL]). One such provision for laboratory equipment is 29 CFR 1910.399.
This requirement states that an installation of equipment (including household microwave ovens) could
be acceptable (to OSHA) and approved within the meaning of Subpart S requirement if it is accepted,
certified, listed, labeled, or otherwise determined to be safe by a nationally recognized testing laboratory
(NRTL). However, even if a device or equipment is “approved,” 29 CFR 1910.303(b) (2) requires that the
“listed or labeled equipment should be used or installed in accordance with any instructions included in
the listing or labeling.”
NOTE: Current FDA and OSHA regulations do not prevent anyone from purchasing and using a
microwave oven for other than its intended purpose. However, once a household or commercial
microwave unit has been modified for use in a clinical laboratory (i.e., it is no longer considered to have
been “designated to heat, cook, or dry food”), the original oven manufacturer is neither liable nor
responsible for the compliance of the modified ovens. Thus, the person who is modifying the oven is
subject to the provisions of Chapter V, Subchapter A- Drugs and Devices of the Federal Food, Drug, and
Cosmetic (FFD&C) Act.
Microwave-accelerated sample preparation is not exempt from traditional safety considerations, and
references to general laboratory safety are available.31 (Please refer to the most current edition of
CLSI/NCCLS document GP17—Clinical Laboratory Safety for additional information.)
In this section, the unsafe conditions and practices that may occur in the preparation of biological
specimens related to the operation of microwave devices are evaluated. A detailed discussion of relevant
equipment standards, safety code requirements, and general safety guidelines relating to laboratory
microwave systems is presented. A recommendation for periodic updating of safety information is
included.
NOTE: Readers are also encouraged to consult frequently updated resources on general microwave
laboratory safety for additional information.32
Hazards related to microwave use for specimen preparation are organized into five groups: 1) radiation
exposure standards and regulations; 2) electrical precautions; 3) biological precautions; 4) chemical
environment; and 5) high-temperature hazards.
4.1 Microwave Radiation Exposure Standards and Regulations
4.1.1 Microwave Radiation Safety
Studies into the biological effects of microwave radiation exposure have been extensively detailed (~1000
references) in several reviews dealing with scientific, industrial, and medical applications.27 Currently in
the United States, microwave energy exposure from all large-cavity microwave devices operating at 2450
MHz is limited to 5 mW/cm2 at a distance of 5 cm from any surface of a product. 27 The maximum human
exposure to radio frequency (RF) energy for operating in a safe workplace is <10 mW/cm2 averaged over
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a six-minute period.33 Although no microwave leakage limit exists specifically for laboratory and
scientific units, manufacturers of these products are subject to other Federal Drug Administration (FDA)
regulations.34-36
Microwave device performance standards are incorporated in the Radiation Control for Health and Safety
Act, Federal Law enacted in 1968.27 The FDA regulates microwave-heating devices under two different
provisions of the FFD&C Act. Under the FFD&C Act, they are regulated as electronic products in
Chapter V, subchapter C—Electronic Product Radiation Control. Under the Radiation Control
provisions, the FDA regulates the manufacture of electronic products but not their use (written
communication, April 2002). If the microwave-heating device is designed for heating, cooking, or drying
food and “manufactured for use in homes, restaurants, food vending, or service establishments…” then it
is subject to the Federal Performance Standards for Microwave and Radiofrequency Emitting Products,
21 CFR 1030.10 (written communication, April 2002).
4.1.2 CDRH Consumer Information—Microwave Device Radiation
Ovens and Pacemakers: At one time there was concern that leakage from microwave ovens could
interfere with certain electronic cardiac pacemakers. There was similar concern about pacemaker
interference from electric shavers, auto ignition systems, and other electronic products. Because there are
so many other products that also could cause this problem, FDA does not require microwave ovens to
carry warnings for people with pacemakers. The problem has been largely resolved, since pacemakers are
now designed so they are shielded against such electrical interference. However, patients with
pacemakers may wish to consult their physicians about this electrical interference.
Many references to other international standards are available and should be consulted if the user’s
geographical location places the microwave under other jurisdictions.37
4.1.3 Microwave Equipment Classified as Medical Devices
This section provides regulatory information primarily for microwave device manufacturers, microwave
device resellers, compliance and safety officials, and administrators. The information below may also
assist laboratory technicians making purchasing decisions of microwave devices that are in compliance
with FDA and OSHA regulations.
In the U.S., OSHA regulations require that, if microwave equipment is modified or the integrity of a
safety device is violated, the product must be demonstrated to be safe by measuring the microwave
radiation exposure potential.27 As long as damage, wear, or misuse have not lessened the effectiveness of
the instrument, all the exposure limits of various national/international standards are met or exceeded. 27,38
The FFD&C Act defines a medical device as an instrument, apparatus, implement, machine, contrivance,
implant, in vitro reagent, or other similar or related article including a component or accessory that is
intended for use in the diagnosis of disease or other conditions, or in the cure, mitigation, treatment, or
prevention of disease, in man or other animals.39 A summary of FDA regulations of Microwave
Equipment is in Table 3.
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Table 3. Summary of FDA Regulations of Microwave Equipment

Premarket
Notification
21 CFR 21 CFR 21 CFR 1003 and Filing of 21 CFR 820
1002.12 1002.30 Defect of 510(k), GMP/QS
Abbreviated Accidental Failure to Registration Regulation41
Report40 Radiation Comply35 and Listing
Coverage33 MDR, etc.
Microwave Heating
for Nonmedical Use X X X
Microwave Heating
for Medical and/or X X X X
Clinical Use
These regulations can be found in the FDA web pages:
Nonmedical devices: http://www.fda.gov/cdrh/radhealth.html
Medical devices: http://www.fda.gov/cdrh/devadvice
If a manufacturer or user modifies a household microwave oven or commercially promotes histological

use as an indication for use, then the microwave oven would be regarded by the FDA as a medical device
(written communication, April 2002). The medical device would then be subject to provisions under Title
21 of the Code of Federal Regulations CFR 820.180 (records) and 820.198 (complaint files). The final
classification of a microwave device for histological applications appears in 21 CFR 864.3010, Tissue
Processing Equipment. Once a new or modified microwave device is classified, manufacturers should
consult 21 CFR Section 864.9 to determine whether or not subsequent modifications meet limitations of
exemption from Section 510(k) of the Act. The original oven manufacturer is no longer responsible for
the compliance of a modified oven, and the person who is modifying the oven is subject to the provisions
of Chapter V, subchapter A—Drugs and Devices of the FFD&C Act.
Manufacturers subject to the FDA regulations include original manufacturers, importers, and persons who
remanufacture products for distribution to others.27 Remanufacturing includes adapting a product for a
new or intended use, such as converting household cooking devices for laboratory use, and reselling
them.27
4.2 Electrical Precautions
This section provides electrical safety information primarily for microwave device manufacturers,
microwave device resellers, and compliance and safety officials, and administrators.
Laboratory technicians are advised that according to OSHA household microwave ovens do not meet the
more stringent door seal and endurance tests required for applications outside of the home.
Microwave devices, like any appliance used in a laboratory, must be certified as electrically safe for
laboratory use by a recognized testing laboratory (e.g., Underwriters Laboratories [UL], Canadian
Standards Association [CSA], or Conformité Européene [ ], which means European Conformity). The
unit must be grounded in accordance with the manufacturer’s specifications.27 In accordance with the
policy of the end user’s institution, qualified service technicians must do electrical repairs. The use of
metal accessories is not recommended unless they were designed and tested for use with the particular
microwave equipment and are used according to the manufacturer’s specifications.
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UL-listed household and commercial-use microwave ovens are evaluated to the applicable electrical
requirements of the UL 923 standard for safety of microwave cooking appliances. The construction and
type test requirements are substantially similar for household and commercial types except for the more
stringent door seal and endurance tests for commercial types. Laboratory equipment is evaluated to the
applicable requirements of UL 61010A-1, Standard for Safety of Electrical Equipment for Laboratory
Use; Part 1: General Requirements (written communication, April 2002).
The safety instructions in UL 923 state, “Use this appliance only for its intended use as described in the
manual. Do not use corrosive chemicals or vapors in this appliance. This type of oven is specifically
designed to heat, cook, or dry food. It is not designed for industrial or laboratory use” (written
communication, August 2002).
4.3 Biological Precautions
It is often impossible to know what might be infectious; therefore, all specimens are to be treated as
potentially infectious and handled according to “standard precautions.” Standard precautions are
guidelines that combine the major features of “universal precautions” and “body substance isolation”
practices. Standard precautions cover the transmission of any pathogen and thus, are more comprehensive
than universal precautions, which are intended to apply only to transmission of blood-borne pathogens.
Standard precaution and universal precaution guidelines are available from the U.S. Centers for Disease
Control and Prevention (Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital
Epidemiology. CDC. 1996;Vol 17;1:53-80.), (MMWR 1987; 36(suppl 2S):2S-18S), and (MMWR
1988;37:377-382, 387-388). For specific precautions for preventing the laboratory transmission of blood-
borne infection from laboratory instruments and materials, and recommendations for the management of
blood-borne exposure, refer to CLSI/NCCLS document M29—Protection of Laboratory Workers from
Occupationally Acquired Infections.
The utility of using microwave energy to destroy pathogens requires further study. Therefore, until more
data becomes available, all potentially infectious specimens must be handled with standard precautions
during and after the use of microwave irradiation. The inside walls of the microwave device should be
cleaned with a nonabrasive, disinfectant solution. Chemical germicides registered with and approved by
the EPA as “sterilants” can be used for decontamination in accordance with the manufacturer’s
instructions. Microwave devices that are used for any laboratory procedure should not be used to heat
food for human or animal consumption.
4.4 Chemical Handling Precautions
Safety of the technician is the most important consideration in the sample preparation process. Microwave
energy accelerates the rate of sample reactions, but it does not alter the fundamental chemistry involved.
The technician is advised that any reagents that are dangerous to heat by conventional methods are likely
to be more dangerous when heated in a microwave device because of the rapid rate of heating. If there is
any concern about flammability of a reagent do not use it in a microwave oven. Combinations of reagents
that are explosive, or so highly reactive as to be uncontrollable, fall into this category. Please check the
Material Safety Data Sheet (MSDS) or with the manufacturer of the microwave device to determine
safety and test data for a particular reagent or procedure to avoid unreasonable, hazardous misuse of
laboratory microwave systems. 27,32 See http://www.sampleprep.duq.edu/dir/safety.html
Handling chemicals in a microwave environment requires special considerations. Aqueous chemicals that
are dangerous at room temperature (e.g., formaldehyde, osmium tetroxide, lead acetate) pose an added
danger during and immediately after microwave irradiation. Chemicals that are warmed will volatilize in
the microwave cavity. Opening the door of a microwave device that is not properly vented will allow the
volatilized chemical vapors to enter the laboratory environment. Some types of ventilation hoods can
function properly when a microwave device without an exhaust fan is properly placed in the system. End
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users should consult a qualified ventilation specialist to determine if the laboratory hood and ventilation
system are capable of functioning safely with the intended microwave device. Laboratory grade
microwave devices equipped with exhaust fans and connected to an approved ventilation system will
reduce the risk of chemical exposure hazard during microwave heating.
The use of organic solvents and caustic chemicals may accelerate the deterioration of door seals and
safety interlock switches.42 Transport of heated chemicals outside of the microwave cavity or ventilation
hood should be done in a closed container. Chemically resistant gloves and goggles should be used to
handle all containers containing hazardous materials that have been microwaved. All chemical spills
should be handled with appropriate chemical spill precautions and the surfaces cleaned off immediately to
prevent damage to the unit.
NOTE: Do not use corrosive chemicals or vapors in microwave devices unless directed so in the device
manual.
Engineering controls, work practices, and personal protective equipment should be used in accordance
with local and regional regulations (e.g., OSHA) and the end user’s institutional standards. Additional
information about chemical safety according to OSHA standards can be found in documents such as
Occupational Exposure to Hazardous Chemicals in Laboratories (29 CFR 1910.1450), Flammable and
Combustible Liquids standard (29 CFR 1910.106), permissible limits in the Air Contaminants (29 CFR
1910.1000), and specific chemical standards in 29 CFR Subpart Z that apply. For safety concerns within
the hospital, laboratories that must comply with 29 CFR 1910.1450 must designate “personnel
responsible for implementation of the Chemical Hygiene Plan including assignment of a Chemical
Hygiene Officer, and if appropriate, establishment of a Chemical Hygiene Committee” (written
communication, August 2002).
4.5 High Temperature Precautions
Microwave protocols for histology laboratories should specify the use of containers that are vented to
prevent pressurization during microwave heating. Containers must be microwave compatible and not
contain metal parts that may reflect microwave energy or cause arcing of microwave power. Although
special containers are available for pressurization in a microwave, these are generally designed and used
for acid digestion of samples for analytical elemental analyses27 or for high temperature sterilization and
antigen retrieval.
A protective thermal glove should be used when removing samples that are irradiated to high
temperatures in a microwave (i.e., heated solutions may conductively heat the container). Spontaneous
“volcanic” boiling can occur in liquids after microwave irradiation and upon removal of the sample from
the microwave. This is attributed to localized super heating of the liquid which occurs secondary to
bubble nucleation and coalescence of dissolved gases as a result of microwave heating. 27,43 The risk of
spontaneous boil over in histopathology applications is more likely during high temperature heating
procedures, such as microwave staining and antigen retrieval processes.
4.6 Recommended Safety Procedures for Microwave Devices and Applications

Microwave devices designed for the laboratory may include safety features such as isolation of the fume
exhaust from the device electronics, high volume exhaust, safety interlocks, and sensors for flammable
solvents. Table 4 is designed to emphasize safe laboratory practices with microwave devices. Case
histories of microwave accidents have been summarized. 27
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Table 4. Safe Laboratory Use of Microwave Devices

Procedure Safety Equipment Safety
Infectious Materials – Follow standard precautions for Use a nonabrasive disinfectant to keep interior surfaces
blood-borne pathogens with potentially infectious clean. Blot interior surfaces dry after each use to
materials.44 (Please refer to the most current version of prevent local hot spots and corrosion. Clean spills
CLSI/NCCLS document M29—Protection of Labor- immediately.
atory Workers from Occupationally Acquired Infections
for additional information.)
Ventilation – Microwave devices used in the clinical Do yearly inspections of a microwave’s fume exhaust
histology laboratory should be placed in an approved system (check owner’s manual).
ventilation hood to contain airborne contaminants and
potentially infectious agents. Microwave devices used Flammable and/or corrosive reagents must be removed
outside of a fume hood should have an integral fume from the fume hood prior to microwave device operation
extractor that is certified by the manufacturer for use in a to prevent ignition of the reagents or chemical damage to
clinical laboratory (check owner’s manual). Microwave the electronic safety systems in the device.27
devices may be used outside of a hood with
nonhazardous reagents (e.g., water, certain biological Microwave devices without a built-in exhaust should be
stains) determined by the user’s institution. placed six inches from the plane of the hood face and not
block airflow to slots of the baffle.45 Sash height should
be adjusted to the lowest possible position to maximize
containment (see the institutional safety officer for this
determination).
Routine performance tests on ventilation hoods such as

face velocity and smoke visualization are required on
ventilation hoods containing microwave devices and
microwaves with extractor fans connected to a
ventilation hood or other exhaust system.46
Leakage – Have certified personnel do annual Regularly inspect and clean dirty microwave door seals.
monitoring for microwave leakage (<5 mW of Do not operate a microwave with any evidence of
microwave radiation per square centimeter at 5 cm from damaged door frames, hinges, or door latches.
the surface). This safety check should be done to meet
appropriate radiation regulatory standards for the
institution.
Spills – For some applications, a secondary container Pressurization during microwave procedures can only be
may be used to collect spills (e.g., tray or plastic bag done in devices approved by the manufacturer for this
with a vent). Operators must use extra caution when purpose. In particular, containers with screw-type cover
using secondary containers not specifically designed for lids should not be used. A thorough discussion of the
microwave procedures due to the higher risk of safety codes and standards relevant to pressurization
container leakage and contamination. NOTE: Secondary vessels is available.27
containers are very important when transporting samples
to and from the microwave device.
Handling – Use utensils designed to handle containers Use containers made from microwave-compatible
after microwave heating to protect the operator from materials (e.g., polyethylene, polypropylene, PTFE,
burns. Thermal mitts should be used to prevent thermal PFA, borosilicate glass). NOTE: Use caution with
burns from handling containers after microwave heating. laboratory glassware as trace amounts of lead oxide can
Chemically resistant gloves should be used to prevent lead to overheating and breakage. Metal placed alone in
skin exposure to hazardous chemicals or their vapors, a microwave cavity will cause arcing.
which may have contaminated the outside of the
container during microwave heating.
Temperature – Monitor temperature to prevent Temperature measurement devices must be
overheating and specimen damage. manufacturer approved for use in a microwave cavity.
Electrical Safety Microwave devices should have UL, , or CSA
certification for laboratory use.
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4.7 Microwave Safety Inspection
A safety inspection of the microwave device should be done on a regular basis, determined by the
laboratory supervisor and the institution’s safety officer. Table 5 provides a template of a microwave
device survey data sheet for use in microwave safety inspection.
Table 5. Microwave Device Survey Data Sheet
Microwave Device Survey Data Sheet
Date:
Department: Responsible Person:
Location of Unit: Serial/ID#:
Survey Instrument: Control Limit (1): 5 mW/cm2
Inspection Checklist
Yes No N/A
Warning sign for radio frequency radiation posted
Additional information or precautions included on warning label of microwave
Safety interlock prevents the device from operating if the door is opened
Instrument is inspected periodically to ensure proper condition (e.g., corrosion)
Device is maintained in a sanitary condition
A qualified authority certified in using microwave leakage instrumentation should determine microwave
leakage of all microwave devices in the laboratory. Inexpensive microwave leakage detectors typically
result in false-positive leakage readings and are not recommended. Call a qualified service representative
to determine the cause and repair of any leakage. Do not attempt to repair the device yourself. DO NOT
USE THE DEVICE UNTIL IT HAS BEEN REPAIRED.
The institutional radiation safety officer will typically take leakage readings at a distance of 5 cm from the
surface of the unit with a 100 mL water load in place while the device is operating at maximum power
(see Figure 1). In addition to surveying leakage around the door and frame, other locations that must be
checked for leakage include outlets in the device for ventilation tubes and thermocouple lines. Table 6
provides a template for recording microwave leakage readings.
1 2 3
4 5 6
7 8 9
Figure 1. Diagram of Microwave Door and Door Frame
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Table 6. Template for Recording Microwave Leakage Readings
Door closed readings in mW/cm2
Position # Reading
1
2
3
4
5
6
7
8
9
Maximum reading _________ mW/cm2
Surveyed by: ___________________________
5 Principles of Microwave-Accelerated Methods

This section is intended for readers interested in learning about the principal features and operation of a
microwave device and the mechanisms of microwave heating. This section provides basic background
information for the recommendations presented in Section 6.
The principal features of a microwave unit are illustrated in the cutaway diagram shown in Figure 2. The
primary components include: (A) control pad; (B) power supply; (C) magnetron; (D) wave guide; (E)
distributor; and (F) unit door and walls.47, 48
Figure 2. Principle Features of a Microwave Oven

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5.1 Power Level
Power level in most microwave devices is controlled by switching the magnetron on and off according to
a duty cycle.1,3,48 For example, a typical 600-W unit with a 30-second duty cycle can be made to deliver
an average of 300 W by switching the magnetron on and off every 15 seconds.1,3,48 Long duty cycles (i.e.,
>10 seconds) are undesirable in biological sample preparation where samples may not heat between
switching steps. This is, therefore, one factor that must be considered in adapting certain microwave
devices for tissue sample applications.47 Some microwave devices have a more sophisticated control
system in which the magnetron power is actually variable and not dependent upon switching the
magnetron on and off.4
A wave-guide [see Figure 2 (D)] is a rectangular channel made of sheet metal. Its reflective walls allow
the transmission of microwaves from the magnetron to the microwave cavity or applicator.47,48
The reflective walls of the microwave cavity are necessary to prevent leakage of radiation and to increase
the efficiency of the unit. There is rarely a perfect match between the frequency used and the resonant
frequency of the load. Therefore, if the walls reflect the energy, absorbance is increased because energy
passes through the sample more often and can be partially absorbed on each passage.43 This can be
particularly important if the sample is dimensionally small. If too much energy is reflected back into the
wave-guide, the magnetron may be damaged. Most commercial units are protected by an automatic
cutoff. There may also be protection in the form of a circulator that directs reflected energy into a dummy
load.47,48
NOTE: If working with small loads, poorly absorbing loads, or at high powers, then a dummy load (for
example, a beaker of water) should be placed in the cavity (unless instructed otherwise by the equipment
manufacturer) to absorb the excess energy generated.1,3, 48
In the absence of any smoothing mechanism, the electric field pattern produced by the standing waves set
up in the cavity may be extremely complex. Some areas may receive large amounts of energy while
others may be almost neglected. To ensure that the incoming energy is smoothed out in the cavity, a
distributor (a reflective, fan-shaped paddle) is sometimes used [see Figure 2 (E)]. Some microwave
devices are also supplied with a turntable, which ensures that the samples are moved in and out of areas of
hot and cold spots, as long as the sample is not centrally located on the turntable.1,3, 48
The important features of the magnetron, in which the microwaves are generated, are shown in Figure 3.
A magnetron is a thermionic diode having an anode and a directly heated cathode.47 As the cathode is
heated, electrons are released and are attracted toward the anode (path indicated by the arrow). The anode
(gray area of diagram) is made up of an even number of small cavities, each of which acts as a tuned
circuit.47,48 The gap across the end of each cavity is capable of storing electric charge. The anode is
therefore a series of electric circuits that are tuned to oscillate at a specific frequency or its overtone.47 A
very strong magnetic field (X shown in Figure 3) is induced axially through the anode assembly and has
the effect of bending the path of the electrons as they travel from the cathode to the anode. As the
deflected electrons pass through the cavity gaps, they induce a small charge into the tuned circuit,
resulting in oscillation in the cavity. Once the oscillation has achieved sufficiently high amplitude, it is
taken off the anode via an antenna/aperture. Of the available power used by the magnetron (e.g., between
3000 and 4000 W), only half of this wattage is converted to electromagnetic energy. The remainder is
converted into heat and must be dissipated through air- or water-cooling.47 Microwave units may have
more than one magnetron and/or wave distribution system.
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Figure 3. Magnetron
5.2 Energy Absorption Mechanisms and Reaction Conditions
Absorption of microwave radiation by samples and solutions is a phenomenon controlled by fundamental

thermodynamic properties and is entirely different from conventional heating mechanisms.49,50
In conventional histology procedures, “standard devices” (such as hot plates, heating mantles, heating
blocks, and conventional heat laboratory units) conduct heat to the reaction vessel. The solution is
subsequently heated only through direct contact with the vessel walls.49,50 The heat is distributed via
convection currents throughout the reagent-sample mixture. Because of the nature of this process, these
heating methods are relatively slow. Hot-plate procedures are limited by the boiling points of the
solutions. In addition, there can be variations between laboratories as a result of changes in atmospheric
pressure, colligative properties (convection), and conductive heating properties of conventional
apparatuses.49,50
In contrast, the use of microwave irradiation has a more direct, and thus controllable, heating mechanism
and is less susceptible to the variables mentioned previously. Microwave systems use direct absorption of
microwave radiation through essentially microwave-transparent vessel materials. Atmospheric pressure
microwave systems can generate more stable temperature conditions and are not limited by heating
mechanisms of convection or conduction.49,50
In microwave heating, energy is directly transferred to absorbing molecules in both the sample and
reagents. An examination of the role played by each of the absorption mechanisms will assist the end user
in understanding why unique conditions are achieved in microwave systems. There are two primary
microwave-absorbing mechanisms: ionic conductance and dipole rotation. In simplified terms, ionic
conductance refers to the phenomenon by which ions in solution migrate when an electromagnetic field is
applied. The solution resistance to the free flow of ions results in friction that heats the solution. This
mechanism is much less dependent on microwave frequency than is the dipole rotation mechanism.
Dipole rotation is the alignment of a molecule dipole with the applied field. Molecular “friction” results
from the very rapidly forced molecular movement caused by the oscillation of the applied field. At 2450
MHz, the dipoles align (lose entropy) and then randomize (gain entropy) 4.9 x 109 times per second,
resulting in fast, efficient, and thermodynamically predictable power absorption.49,50
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Water, methanol, and ethanol are examples of molecules likely to be used for tissue preparation.
Microwave absorption by polar reagents (charged molecules) is very high. The importance of this
information will be discussed in greater detail in Section 7.3.2.
6 Recommendations for Documenting Microwave Methods

The reproducibility of microwave methods is often hampered by inadequate documentation. Far too
frequently, microwave methods have not been documented well enough to adequately reproduce methods.
Improved documentation could increase the usefulness of the method in histopathology laboratories. The
following section will discuss what is critical to properly document microwave procedures.
An analogy of microwave method documentation can be drawn from a description of hot-plate

certification. Early procedures for hot plates most likely directed the user to heat the sample until “hot.”
However, probably no information regarding what temperature or how long to run the process was
specified. Similarly early microwave methods often instructed the client to heat the vessel at a designated
power until the desired result was achieved.49
A completely documented microwave method will enhance the likelihood for reproducible results
independent of the specific microwave unit.
7 Critical Descriptors for Microwave-Accelerated Procedures

The most critical descriptors necessary to reproduce a microwave method are organized into four major
headings: temperature, power, specimen handling, and time. Subsequent sections of this guideline
elaborate on the descriptions of each of the categories outlined in Table 7.
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Table 7. Critical Descriptors for Microwave-Accelerated Procedures

Temperature Control
• Temperature Measurement
• Initial Temperature
• Final Temperature (maximum temperature control)
Microwave Power
• Line Voltage
• Magnetron Cycling On and Off (partial and continuous powers)
• Water Load
• Determination of Microwave Power Output
Specimen Handling
• Specimen Container
—Location
—Dimensions
—Composition
—Multiple Samples
—Water Loads and Other Microwave Absorbers
• Specimen Immersion
—Solution Composition
—Multiple Containers
—Solution Mixing
• Samples
—Grouping Samples of Similar Composition
—Position of the Sample Within the Container
—Sample Size
Process Time
• Total Time
• Time at Temperature
7.1 Temperature Control and Measurement
Temperature control is the single most important variable in a microwave procedure. The field of
microwave sample preparation has moved toward using temperature feedback as the most widely
accepted way to gain reproducible and accurate results. However, temperature measurement in a
microwave environment is complicated by the nature of how microwave energy heats samples and by the
type of probes that can be used in a microwave device.
Variation of temperature in microwave-exposed materials depends on exposure level, the dielectric

properties of the load, the thermal properties of the materials, and other physical properties such as
vaporization, size of the object in relation to microwave frequency, the presence of other microwave
absorbers in the vicinity of the sample (i.e., a water load), and the lack of homogeneity of the sample.3
7.1.1 Measuring Microwave Heating of Samples
Insight into the complexity of measuring microwave heating of samples is provided below.
7.1.1.1 Exposure Level
Microwave field intensity is not constant throughout the microwave cavity. Nonuniform microwave field
patterns are present in all multimode microwave cavities. These varying power densities change with
location, time, and the sample placement in the cavity. Since the variations in the field caused by
absorbing samples cannot be modeled or predicted, methods are employed to increase the homogeneity of
the field. Two such devices are the mode stirrer and the turntable or carousel that rotates the sample
through the microwave field to evenly distribute the microwave energy. Field intensity is not uniform
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within samples, because energy is absorbed primarily from the outside boundary of the load. For example,
the half-power penetration depth for a wet tissue is approximately 2 cm at 2450 MHz. At 2 cm into a wet
tissue the power will have been reduced to one half the power available in the bulk of the empty cavity.
This inherent variability of field intensity has been demonstrated in a variety of ways using an array of
neon bulbs48 (see Figure 4), thermo-sensitive paint,3 a sheet of paraffin lining the floor of the microwave
cavity,3 or infrared sensing of samples.3 Variable microwave fields are discussed further in Section
7.3.1.1. To avoid nonhomogeneous energy exposure of the samples placed in multimode microwave
cavity systems, a turntable that passes the sample through the complete microwave field is effective.
These turntables have been found to produce uniform microwave exposure of the samples and specimens,
if the turntable rotates at six revolutions per minute or greater in a nominal 1000-W microwave field. This
method of microwave field energy homogenization has become standard in laboratory chemical reaction
microwave systems.3,10,50
NOTE: Alternatively, without traversing the sample through the microwave field, the microwave heating
of relatively small samples may be controlled by measuring temperature (microwave field density
interactions) of samples placed at specific locations within the microwave device. Reproducibility
requires careful attention to consistent placement of small samples (<50 cc) in specific “charted” areas of
the microwave cavity.4,24,26,51
Figure 4. Measuring Variability of Microwave Field Intensity Using Neon Bulbs Arranged in a
Plastic Base
7.1.1.2 Dielectric Properties of the Material
Materials have very different dielectric constants and hence, penetration depths of microwave energy and
consequently, heating rates (see Table 8).
NOTE: The same solution composition and sample container should be used to obtain results with lower
variability from one application to another.
The following table lists the temperature of 50 mL of several solvents after heating from room
temperature for one minute at 560 W and 2.45 GHz.47
Table 8. Solvent Temperatures
Solvent Temperature Boiling Point

(°C) (°C)
Distilled Water 25 °C 81 100
Methanol 65 65
Ethanol 78 78
Acetic Acid 110 119
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7.1.1.3 Antenna Properties of the Load
This property is determined by the size of the sample relative to the wavelength of the microwave
energy.52 For example, small droplet volumes do not absorb microwave energy as well as larger volumes.
Also, tall samples do not absorb microwave energy as well as flat samples.
NOTE: The same container size and shape and solution volume should be used to obtain results with
lower variability from one application to another.
7.1.1.4 Thermal Properties of the Material
The heat capacity of the object determines how quickly and evenly an object heats. Water has a higher
specific heat than alcohol and therefore, requires more energy and time to heat than alcohol.
NOTE: The same solution composition should be used to obtain results with lower variability from one
application to another.
7.1.1.5 Other Physical Properties
Vaporization is important with respect to small droplets typically used in histochemical procedures. The
combination of large surface area and evaporative cooling determine the rate at which a small sample
heats.
NOTE: The same volume of sample should be used to obtain results with lower variability from one
application to another.
Again, the point must be emphasized that microwave heating is not uniform within the microwave cavity
or within the samples.1,3 A single temperature measurement is rarely useful in process control. It is also
important to note that rotating tables and mode stirrers reduce but do not eliminate variability in heating
between multiple sample containers.
Many laboratory-grade microwave units have a feature in which an aerator or magnetic stirrer is used to
mix fluid during microwave heating. This approach has proven effective in minimizing temperature
variation within a single, large volume. However, the aerator does not equilibrate temperature variation
between containers located in different locations within the microwave cavity.
NOTE: Independent temperature measurement should be performed in each sample container.
7.1.2 Temperature Measurement Devices
Currently, five types of temperature measurement devices are used commercially in laboratory
microwave systems for the direct measurement of the reaction temperature: thermocouples, fiber optics,
infrared (IR), gas-filled bulbs, and liquid crystal temperature strips (LCTS). Each of these temperature
measurement systems ideally overcomes the inherent problems and limitations of working in an
electromagnetic field.
7.1.2.1 Thermocouple Measurement Devices
Thermocouple measurement devices have been used for measuring temperatures for many years. In an
electromagnetic field, this measurement can occasionally lead to problems when sensors are not correctly
engineered. When thermocouples are exposed to microwaves, the microwaves couple with the
thermocouple measurement circuitry.53 Errors are produced when the metal surface interacts with the
electromagnetic field. A combination of shielding, grounding the shielding to the microwave cavity wall,
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and electroplating the thermocouple with gold either eliminates or minimizes these errors.54 The gold-
plated shielding essentially removes the thermocouple from the electromagnetic field. After calibration,
thermocouples are useful from 0 °C to 400 °C in microwave systems, and typically are accurate to
±0.1 °C.49 The cost of these devices is low.
Many microwave units have a basic thermocouple measurement device built into the unit. Inexpensive
probes are usually similar in dimension to meat probes in a domestic microwave unit. Such probes may
only have an accuracy of ±5 °C and may be difficult to place in small sample containers.49 These probes
are more likely to cause arcing and secondary heating (due to electric surface currents) of samples in
contact with the metal probe during microwave irradiation. Built-in temperature probes are designed to
monitor sample heating, but only of one sample container at a time. Stand-alone thermocouple
measurement systems can be purchased at low cost, but they can only be used to measure temperature of
samples after they have been removed from the microwave cavity.
CAUTION: Do not place any wire, such as a thermocouple, into a microwave device not specifically
permitted by the manufacturer. Without grounding the shielding to the cavity wall, microwave energy can
be transmitted along the surface of the shielding and into the laboratory.55
7.1.2.2 Fiber-Optic Systems
Fiber-optic systems are microwave transparent and nonperturbing to the microwave field. Fiber-optics
has been used in phosphorus-based and remote IR temperature sensors. Phosphorus fiber-optic sensors
use a light source outside of the microwave cavity that emits an excitation wavelength that travels through
the fiber-optic cabling to a phosphor tip.49 The measurement system converts the temperature-dependent
fluorescent decay signal into a temperature measurement by comparing the decay rate with calibration
values.56 After calibration, phosphorus fiber-optic thermometers are linear from 0 to 250 °C in microwave
systems (dependent on the specific phosphor and fiber-optic cabling). They typically have an accuracy of
±2 °C, down to ±0.2 °C with calibration near the measurement temperature. These systems can measure
multiple sample vessels at a time. The cost of these systems is very high.
7.1.2.3 Infrared Temperature Sensors
Infrared (IR) temperature sensors have been used for direct and indirect temperature measurements.
Direct IR sensors use fiber optics to directly measure the solution temperature.49 Indirect IR sensors
measure the temperature of the sample vessel, usually the bottom,57 by the IR emission. The temperature
of the sample vessel may lag behind the temperature of the solution.
IR sensors may, in the future, be adapted to measure the temperature of multiple sample vessels. As the
turntable revolves, the IR sensor would measure the temperature of each vessel. If any one vessel surface
reaches the selected target temperature, the microwave power would be shut off until the temperature has
dropped below the programmed level. The cost of IR systems is typically medium to high.
7.1.2.4 Gas-Filled Bulb Thermometers
Gas-filled bulb thermometers have recently been introduced.57 The measurement of temperature with
these gas-filled bulbs is based on the gas law principle; the temperature is proportional to the internal gas
pressure. After calibration, glass-bulb thermometers are linear from 0 to 250 °C and have an accuracy
between ±2 and 5 °C.49 The cost of these devices is low.
7.1.2.5 Liquid Crystal Temperature Strips
Liquid crystal temperature strips (LCTS) can measure temperature of the sample(s) or the sample
vessel(s) (depending on placement of the strip) during microwave irradiation.48 LCTS can be monitored
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during irradiation by direct observation through the microwave unit window or after sample removal from
the unit cavity. LCTS typically appear black, but a bright green color becomes prominent when a
temperature specific to the reaction temperature of the particular crystal on the strip is attained. Accuracy
is within ±5 °C. There is no feedback control to the microwave device. The cost of LCTS is typically very
low, and they are reusable.
7.1.3 A Recommended Temperature Measurement System
An electronic temperature probe should be accurate to ±2 °C and should be used according to the
manufacturer’s recommendations and specifications. The end user should check these instruments
periodically for accuracy.
Many microwave devices have a single shielded microwave probe. This is useful for process control
within a single container, but it is not capable of determining temperature in multiple containers.
An independent, external, electronic temperature probe should be used to determine the temperature of a
solution or sample immediately after the sample is removed from the microwave device. These
temperature measurements can be used to troubleshoot areas within the microwave device in which there
are insufficient heating or overheating of specimens (see Table 9).
NOTE: Repeating the measurement three times, and using the average reading, will provide estimates
with low uncertainty. For example, if the average temperature change were about 20 °C, experimental
data such as 19 °C, 20 °C, and 21 °C would be reasonable just from the measurement aspect (this does not
take into account any other source of uncertainty or variability).
Table 9. Selecting an Electronic Temperature Probe for Microwave Procedures

Parameter Rationale
Probe length ~ 1 cm Small enough to measure temperature changes in discrete areas
of small samples and solution volumes
Probe equivalent to a 25-gauge Minimal mass so that it can quickly respond to temperature
needle diameter or thinner changes before the sample cools
+1.5 °C accuracy Immunohistochemical procedures require the most stringent
temperature measurements
Response within 10 seconds Rapid probe response is essential for measuring temperature
accurately within small sample volumes or multiple sample
containers
Minimum 18” flexible length Facilitates ease of rapid probe placement
between probe and base unit
7.1.4 Initial Irradiation Temperature
If a microwave device is not equipped with an accurate temperature control mechanism, then the
laboratory personnel should adopt the practice of using solutions at the same starting temperatures for
every use.
7.1.5 Final (Maximum) Irradiation Temperature
The precise final temperature parameters supplied by the manufacturer or contained in published
protocols should be followed.
The temperature endpoint for tissue fixation is generally below 65 °C to prevent collagen denaturation
and sample shrinkage. Most special stains in histology are done at a temperature below 60 °C.
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Alternatively, staining and antigen retrieval procedures generally require final sample temperatures above
90 °C.
NOTE: This parameter depends on the specific procedure being followed.
7.2 Microwave Power
The consistency of the power output from a microwave device is directly related to the constancy of the
line voltage to the unit and the performance of the magnetron. A simple calorimetric method can be used
to determine microwave power output by the device (see Section 7.2.3).
7.2.1 Regulated Electrical Line Voltage on a Dedicated Circuit
Microwave equipment should be used on a dedicated electrical circuit. Check with the institution’s
electrical department for more information.
Magnetron output wattage is dependent on input line voltage. Variance (e.g., decreased line voltage from
the electrical utility company [brown outs]) will affect microwave power output.
7.2.2 Water Load
A water load is used as the primary absorber of microwave energy in a microwave.
Recommendations contained in published protocols or in instructions supplied by the manufacturer

should be followed.
Some microwave devices can be damaged if they are operated with a solution volume below their
minimum recommended by the manufacturer. This is especially important when using microwave devices
for heating microliter volumes of reagents for certain immunostaining procedures.
Other methods have been developed to smooth out nonuniform microwave field patterns.58
7.2.3 Power Output Determination
Measure microwave unit power output to determine if the magnetron is functioning properly. These
measurements should be performed at initial instrument set-up and quarterly thereafter.
Magnetrons and power supply ratings vary due to manufacturing tolerances by as much as 10% between
microwave units, after repair, and over the lifetime of the instrument.48
7.2.3.1 Method to Determine Power Output of a Large-Cavity Microwave Device
The following steps describe a method to determine the power output of a large-cavity microwave device:
(1) Place 1 L of distilled water in a 1-L narrow-mouth plastic bottle in the center of the microwave.
NOTE: A narrow-mouth bottle is used to minimize heat loss from the container.
(2) Adjust the initial temperature to 25 °C ± 1 °C.
(3) Irradiate the uncovered bottle for two minutes at 100% power.
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(4) Carefully remove the bottle from the microwave device and place an appropriate cover of plastic or
foil over the mouth of the container.
(5) Invert the container or use a magnetic stirrer to mix water for five seconds immediately after
irradiation to reduce temperature variation in the heated sample.
(6) Measure the final temperature.
NOTE: Perform the procedure three times to obtain sufficient readings to average.
Procedures must be reported in terms of actual microwave power, not percentages set on the microwave
unit. The following formula should be used: (change in temperature) x 35 = power in watts (35 is a
constant for converting between calories and power).
NOTE: If temperature measurement varies by more than 2 °C for a 700-W microwave device, an error of
greater than 10% can result between sample processing batches. (18 °C x 35 = 630 W; 20 °C x 35 = 700
W; 22 °C x 35 = 770 W. Mean and standard deviation of three measurements is 700 W ± 70 W. 70/700 =
10%). Since a temperature difference is measured and linearity of the thermometer within the range is a
reasonable assumption, the absolute accuracy of the thermometer is less important.49
7.2.4 Magnetron Duty Cycle (Magnetron “On-Time”)
Longer cycle times in some microwave devices do not permit smooth transitions of temperature.
Microwave devices with shorter controllable duty cycles or continuous variable power should be selected
for use.
Lack of attention to the duty cycle settings can lead to overheating samples. For example, if the duty cycle
is relatively long, at reduced power, the time that the magnetron is both on and off is longer, resulting in
possible initial overheating followed by significant heat loss causing temperature swings in the sample.3,48
Some microwave devices use continuous variable power and do not have a duty cycle. Users should
follow the manufacturer’s recommendations.
7.3 Specimen Handling
Several factors in specimen handling will determine the variability of a procedure including container
position, quantities of the solutions, and the number and types of microwave vessels.
7.3.1 Specimen Container
7.3.1.1 Container Location
The container location should be marked on the microwave device floor to ensure reproducible container
placement. A useful guide is an alphanumeric grid (see Figure 5) placed on the floor of the microwave
device (see Figure 6).48
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Figure 5. Alphanumeric Grid
Figure 6. Placement of Alphanumeric Grid for Determining Container Location
The alphanumeric grid can be made from a plastic sheet with letters written along the right and left
columns. Numbers are written along the top and bottom rows. The grid spacing is 1 cm. A large dot is
marked in the top left corner to key the alphanumeric grid to the left rear corner of the microwave cavity
(see Figures 5 and 6).
Microwave power can vary significantly within a microwave (see Figure 7). Wickersheim, et al showed a
70 °C difference in temperature within a 2-cm radius in a microwave oven56 (see Figure 8). A turntable
does not eliminate these hot and cold spots; it simply circulates the sample(s) through these areas. Some
laboratory microwave devices are designed for consistent sample placement and do not require an
alphanumeric grid (check with the microwave device manufacturer).
Figure 7. Variance in Microwave Power

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y/cm 40 Back Wall of Oven Cavity 44

78 120
60 78
30
77 50
100 82
62 92
63 68
109 115
20
92 103
100
110 84
99 90
90 80
79 55
10
65 85
94 62
89 64
66 130
0
98 Front Door of Oven 58
0 10 20 30
x/cm
Figure 8. Microwave Temperature Differences Within a 2-cm Radius. Numeric readings are
temperature, °C. (From Wickersheim K., M. Sun, and A. Kamal, 1990. A small microwave E-field probe utilizing fiberoptic
thermometry. Journal of Microwave Power and Electromagnetic Energy. 25(3):141-148. Reprinted courtesy of the International
Microwave Power Institute (Wickersheim, 1990).
7.3.1.2 Container Dimensions
The smallest container possible that the procedure will permit should be used. Do not interchange
containers with different dimensions for a given procedure. In addition, the aspect ratio of the container
used to hold the solution is important, i.e., for some given volume of solution, short, wide containers
absorb microwave energy more efficiently than tall, narrow containers.48,59
Fifty to eighty percent of microwave energy is absorbed within the first 2 cm of a solution.48,59 Therefore,
microwave heating may not be uniform throughout the volume of a solution. Some experts recommend
using rectangular containers, because cylindrical containers may focus microwave energy (e.g., similar to
a lens), resulting in sample overheating.59,60
7.3.1.3 Container Composition
Use microwave-transparent materials such as ceramics, sodium borosilicate glass, unleaded quartz,
fluoropolymers, and nonpolar plastics like polypropylene, polyethylene, and PTFE compounds.
Microwave-transparent materials do not absorb microwave energy and permit more uniform transmission
of power to the sample.
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7.3.1.4 Number of Containers
When more than one container is heated in a microwave device, it is likely that the temperature will vary
from container to container.
Many experts recommend microwave units with a rotating table when heating multiple sample containers.
The rotating table should rotate at six revolutions per minute.27
Another method for determining the best placement of multiple containers for uniform heating in the
microwave cavity is by trial and error. Reproducible heating results depend on using consistent container
placement, consistent load (with respect to container size, container shape, solution volume, and solution
chemistry), and consistent bubble agitation within each container. Final irradiation temperature of each
container is measured immediately after the microwave heating cycle ends. The locations of each
container are marked on the floor of the microwave cavity with an indelible pen. This calibration should
be checked periodically or immediately after a change is made to the microwave device.
Uneven distribution of microwave fields within the microwave cavity is the primary cause for uneven
heating. Secondary causes include variation in airflow within the cavity and inconsistent agitation in the
containers.
7.3.2 Specimen Immersion
7.3.2.1 Solution Composition in a Single Container
Salt concentration, ionic conductivity, and polarity affect microwave absorption. An analytical-grade
balance should be used to measure solution components. The balance should be calibrated to at least 1%
accuracy.
Table 10 shows microwave (frequency 3 GHz) penetration depth (distance in which the intensity is
reduced in half) in various solutions typically used in clinical laboratories.
Table 10. Microwave (2.45 GHz) Penetration Depth in Various Materials (From Kok LP, Boon ME.
Microwaves for the Art of Microscopy. Fourth revised edition. Leiden: Coulomb Press Leyden; 2003. Reprinted with permission).
Medium Depth
Distilled water 25 °C 3.4 cm
0.1 M sodium chloride 2.0 cm
Ethanol 7.0 cm
Ethylene glycol 1.3 cm
Epon resin 120 cm
Paraffin wax (molten 55 °C) 150 m
7.3.2.2 Solution Composition in Procedures Using Multiple Containers
The same solution should be used in all containers during a single procedure. Preparing the solution in
bulk ensures a diluent of constant composition. Solutions with different compositions absorb microwave
power differently (see Table 10).
7.3.2.3 Solution Agitation
Solution agitation during microwave irradiation improves uniformity of sample heating. Examples of
commonly used mixing methods are manual mixing by inverting the sample after it is removed from the
microwave device or bubble agitation and magnetic stirring while the sample is in the microwave device
(manufacturer dependent).
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Microwave energy is introduced through a wave-guide, thereby preferentially heating one surface of the
container. Agitation prior to temperature measurement is necessary.
7.3.3 Samples
7.3.3.1 Grouping Samples of Similar Composition
Samples with similar composition should be batch processed together. Fatty tissues (i.e., breast) and
calcified tissues do not absorb microwave energy as readily as solid organ tissues such as liver (see Table
11).3
Table 11. 2.45 GHz Microwave Penetration Depth in Biological Tissues (From Kok LP, Boon ME.
Microwaves for the Art of Microscopy. Fourth revised edition. Leiden: Coulomb Press Leyden; 2003. Reprinted with permission).
Medium Depth
Muscle and skin 2.0 cm
Fat and bone 9.0 cm
7.3.3.2 Position of the Sample Within the Sample Container
Specific procedures may require optimal positioning of the sample within its container.
Due to differences of absorption of microwave energy and shielding by some samples and reagents,
position should follow guidelines for specific applications. For example, slides placed near other slides in
a staining jar may receive less microwave energy from the field. Also, tissue samples in a fixative will
receive more microwave exposure the closer they are to the outside wall of the container.
7.3.3.3 Sample Size
The smallest and thinnest sample size possible should be used. Thicker specimens require longer
processing times.
All sample preparation procedures are dependent on solution penetration and chemical reactivity with the
biological sample.1,3,59 In addition, 80% of the microwave energy incident on a sample is absorbed within
the first 1 cm of the sample depth.
7.4 Process Time
Many published microwave protocols will distinguish between “total time” and “time at temperature.”
7.4.1 Total Time
Total time defines the length of time that the specimen is exposed to microwave energy. This process
includes the time it takes for the set temperature to be reached and the time the sample remains at that
temperature. Increasing the power will decrease the total processing time. Heating time may be
programmable on microwave devices by controlling power output or magnetron cycle time.
Recommendations supplied by the manufacturer or contained in published protocols should be followed.
7.4.2 Time at Temperature
Time at temperature is defined as the amount of time the sample remains in solution that has reached a
preset incubation temperature. The time at temperature definition does not include the time needed to
heat the sample to the preselected temperature. A temperature sensor is necessary for this function.
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Multiple containers may not be maintained at the same temperature, because they are exposed to different
microwave fields in different positions within the microwave cavity. This procedure is independent of the
power setting.
Recommendations supplied by the manufacturer or contained in published protocols should be followed.
8 Special Microwave Procedures

Procedures that are often done in microwave devices and require additional explanation regarding control
of parameters for reproducible results are described below.
8.1 Antigen Retrieval Protocol
The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of
tissues, is used as a nonenzymatic pretreatment for immunohistochemical staining of formalin-fixed,
paraffin-embedded tissue sections. It has been widely applied in pathology and analytical
morphology.18,22,23,60-63 (Please refer to the most current version of CLSI/NCCLS document MM4—
Quality Assurance for Immunocytochemistry for additional information.)
The use of a standardized AR protocol is recommended for all tissues that may have been over-fixed by
exposure to formalin. Variables in the AR process include the choice of retrieval solution, pH,
temperature, time, and heating method. Conventional heating in a laboratory dry-heat oven, microwave
heating, pressure-cooking, steaming, and autoclaving all may give equivalent results if temperature and
time are adjusted to optimal values. The microwave, pressure cooker, and steamer are easiest to use, but
the final choice for each laboratory may be based upon availability of equipment and convenience. Once a
heating method has been selected, each antibody procedure should be titrated to determine the optimal
staining and retrieval conditions of the corresponding antigen, for tissues processed in that laboratory. A
simple test panel (consisting of identical tissue samples) may be employed for this purpose, varying the
pH and heating time to achieve accurate, true retrieval that is traceable to optimally fixed and processed
tissue; one of the more commonly used retrieval solutions such as citrate buffer or Tris-HCl buffer with or
without 5% (w/v) urea should be used. An example of this test battery approach is shown in Table 12.23
Table 12. Test Battery for Optimal pH and Heating Using Choice Antigen Retrieval (AR) Solution
and Selected Heating Methods (Shi SR, Cote RJ, Yang C, et al. Development of an optimal protocol for antigen
retrieval: A ‘test battery’ approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed
paraffin sections. J Pathol. Copyright© 1996. Pathological Society of Great Britain and Ireland. Reproduced with permission.
Permission is granted by John Wiley & Sons Ltd. on behalf of the Pathological Society).
Heating Condition (Temperature x Time)
pH Value of the Antigen Mid-high High Super-high
Retrieval (AR) Solution (90 °C x 10 min) (100 °C x 10 min) (>100 °C x 10 min)
Low (pH 1-2) Slide #1 Slide #4 Slide #7
Middle (pH 7-8) Slide #2 Slide #5 Slide #8
High (pH 9-10) Slide #3 Slide #6 Slide #9
8.1.1 Test Battery for Optimal pH and Heating
The temperature of super-high at 120 °C may be reached by either autoclaving or microwave heating at a
longer time or with a microwave-safe pressure cooker.64 In contrast, the low temperature of mid-high at
90 °C may be obtained by either a water bath or a microwave monitored with an electronic temperature
probe or with LCTS. In addition to the nine slides used for this test battery, one more slide may be used
for control without AR treatment. Tris-HCl buffer solution is recommended for this test; citrate buffer of
pH 6.0 may be used to replace Tris-HCl buffer of pH 7-8, as the results are the same (see Table 12).64
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Good immunohistochemical staining practice requires the use of valid positive and negative control
tissues. For a discussion on the use of controls in IHC tests, please refer to the most current edition of
CLSI/NCCLS document MM4—Quality Assurance for Immunocytochemistry.
The antigen retrieval (AR) procedure:
• is a controlled process that restores (recovers) antigenicity of proteins modified (or masked) by
fixation in formalin; and
• can be used to determine that the IHC result truly detects the presence or absence of target antigen
(analyte) in the sample. If the IHC test is semiquantitative (i.e., 1, 2, 3+, or quantitative [in actual
units of measure]) the IHC result after AR should be traceable to known samples that contain the
target antigen (positive controls) and samples that lack the target antigen (negative controls).
– Examples of positive and negative tissue samples include human tumor samples that have been
analyzed with quantitative biochemical methods for the concentration of the target analyte, e.g.,
human breast cancer with biochemical analysis of estrogen and progesterone receptors; and human
tumor cell lines with quantitative biochemical, immunological, and/or nucleic acid-based analyses
(e.g., fluorescent in-situ hybridization [FISH]) of the target analyte (antigen).
– Normal human samples are excellent sources for positive and negative control samples. They may
be made into multiple tissue blocks and used as controls for all steps of the IHC testing process,
including AR procedures.
The same “test battery” approach may be used for selected, preferred heating methods or preferred AR
solutions by holding other variables (such as pH and time of heating) constant. The use of slide adhesives
is recommended. In addition, do not allow slides to dry out during microwave staining; this can lead to
artifacts in the background.
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9 Template for Documentation of Microwave Methods
The following template may be used for documentation of microwave methods.
Documentation of Microwave Methods
Procedure Name (e.g., fixation, staining, antigen retrieval [AR])
Microwave Device
Manufacturer: Model: Year:

Rated Wattage: Actual Measured Wattage:
Magnetron Cycle Time (if unit selection is available):
Power Used for Procedure:
Process Time
Time at Temperature or Total Process Time:
Sample
Sample Type (e.g., bone, fat, organ, plant, bacteria):

Sample Dimensions:
Typical Number of Samples Used:
Sample Container
Dimensions: Composition:
Number of Containers Heated at One Time:
Position of the Container Within the Cavity (alphanumeric coordinates):
Use of a Water Load and Its Volume:
Sample Immersion
Solution Type (e.g., water, ethanol, salt concentration):

Volume:
Time in Solution:
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10 Troubleshooting Results
The troubleshooting guide summarizes key points in this document. The information presented here can
be useful when introducing microwave procedures in the laboratory or customizing protocols for the
microwave device. The guide summarizes results, possible causes of a particular problem, and trouble-
shooting recommendations for microwave leakage, microwave fixation, microwave staining, microwave
AR, and microwave curing of embedments.
Table 13. Troubleshooting Guide48

Microwave Leakage:
Results Possible Causes Recommendations
• Qualified inspection reveals • Damage to door or frame • Do not use microwave
microwave leakage from device • Faulty interlock switch • Call for service
• Wire leads from an external
thermometer are placed in
door frame
• Corrosion/rust resulting in
holes in the interior walls
• Sparks occur within microwave • Metal in microwave • Use plastic or glass in
microwave
• Temperature probe is too close • Submerge tip of probe into
to wall water or specimen
• Insufficient load • Add water load
Microwave Fixation:
• Nonuniform fixation of sample • Sample is too large • Check power settings of
• Only edges of sample are fixed microwave based on protocol
and device power output
• Solution volume is too large • Position sample based on
around sample published protocol
• Excess heat around edges from • Tissue dimensions ideally <0.3
conductive heating cm thick x 1 cm2
• Sample placed in wrong • Minimize solution depth
location of microwave around sample to <1 cm
• Microwave power is too low • Increase power or increase
exposure time
• Improper infiltration • Bisect capsular specimens
(lymph nodes, polyps)
• Bread loaf large specimens
• Samples fragment during • Built-in temperature probe • Place sample in a plastic tissue
irradiation near samples results in cassette to avoid contact with
localized overheating temperature probe
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Table 13. (Continued)

Microwave Staining:
• Variability of stain from slide • Too many staining jars in • Reduce staining jar number or
to slide device at one time reposition staining jar(s) in
microwave
• Slides placed too close • Check agitation device
together
• Sections dislodge from slide • Use tissue adhesive
• Nonspecific staining increased • Overheated sample • Monitor temperature around
slide
• Dried sample • Monitor solution volume
• No improvement of specific • Underheated sample • Monitor temperature around
staining of tissue section slide
• Inadequate incubation time • Increase process time by
reducing microwave power or
increasing volume of stain or
water load
Antigen Retrieval (AR):
• Incomplete retrieval • Inadequate heating • Higher temperature, shorter
temperature heating time (100 °C, 20 min;
70 °C, 10 hours)
• Inadequate heating time • Longer duration heating at
higher temperature generally
increases retrieval; measure
time from start of boiling
• pH • Generally higher pH >6 yields
most intense staining;
alternatively very low pH
effective with some antigens
• Molarity • Minor effect
• Metal ions • Minor effect
• Solution • Minor effect
Microwave Curing of Plastic Resins:
• Embedment discolors • Formulation contains nadic • Use dodecenylsuccinic
methyl anhydride (NMA) or anhydride (DDSA)65,66
nonenyl succinic anhydride
(NSA)
• Embedment burns • Nonuniform power distribution • Use lower power level
• Nonuniform curing • Nonuniform power distribution • Use longer duration heating
• Blocks are fragile • Inadequate cooling time after • Allow blocks to cool at room
curing temperature before removing
blocks from mold
• Embedding mold deforms • Wrong mold used in microwave • Use flat silicon embedding
molds
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www.sampleprep.duq.edu/sampleprep.
33
OSHA. Nonionizing Radiation. 29 CFR §1919.97. U.S. Government Printing Office: Washington, D.C.; 2002.
34
CDRH. Food and Drugs: Records and Reports: Records to be Maintained by Manufacturers. 21 CFR §1002.30. U.S. Food and Drug
Administration.
35
CDRH. Food and Drugs: Notification of Defects or Failure to Comply. 21 CFR §1003. U.S. Food and Drug Administration; 2002.
36
CDRH. Food and Drugs: Repurchase, Repairs, or Replacement of Electronic Products. 21 CFR §1004. U.S. Food and Drug
Administration; 2002.
37
Thuery J. Microwave: Industrial, Scientific and Medical Applications. Norwood, MA: Artech House; 1992.
38
FDA. Performance Standards for Microwave and Radio Frequency Emitting Products. Microwave Ovens. 21 CFR §1030. U.S.
Government Printing Office: Washington, D.C.; 2002.
39
Federal Food, Drug, and Cosmetic Act: Section 201(h). 21 USC 321.
40
CDRH. Food and Drugs: Abbreviated Reports. 21 CFR §1002.12. U.S. Food and Drug Administration; 2002.
41
CDRH. Good Manufacturing Practice for Medical Devices. 21 CFR §820. U.S. Food and Drug Administration; 2002.
42
Kingston HM, Jassie LB. Safety guidelines for microwave systems in the analytical laboratory period. In: Kingston HM, Jassie LB, eds.
Introduction to Microwave Sample Preparation: Theory and Practice. Washington, D.C.: American Chemical Society; 1988.
43
Mudgett RE. Developments in microwave food processing. In: Schwartzberg HG, Rao M, eds. Biotechnology and Food Process
Engineering. New York, NY: Marcel Dekker, Inc.; 1990.
44
CDRH. Performance Standards for Microwave and Radio Frequency Emitting Products. 21 CFR §1030.12. U.S. Food and Drug
Administration; 2002.
45
American Industrial Hygiene Association. American National Standard for Laboratory Ventilation. ANSI/AIHAZ9.5-1992.
46
American Industrial Hygiene Association. American National Standard for the Recirculation of Air from Industrial Process Exhaust
Systems. ANSI/AIHA Z9.7-1998.
47
Mingos DMP, Baghurst DR. Applications of microwave dielectric heating effects to synthetic problems in chemistry. In: Kingston HMS,
Haswell SJ, eds. Microwave-Enhanced Chemistry. Fundamentals, Sample Preparation, and Applications. Washington, D.C.: American
Chemical Society; 1997.
48
Login GR, Dvorak AM. The Microwave Toolbook. A Practical Guide for Microscopists. Boston, MA: Beth Israel Hospital; 1994.
49
Kingston HMS, et al. Environmental microwave sample preparation: Fundamentals, methods, and applications. In: Kingston HMS, Haswell
SJ, eds. Microwave-Enhanced Chemistry. Fundamentals, Sample Preparation, and Applications. Washington, D.C.: American Chemical
Society; 1997:223-349.
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Neas ED, Collins MJ. Microwave heating: Theoretical concepts and equipment. In: Kingston HM, Jassie LB, eds. Introduction to
Microwave Sample Preparation: Theory and Practice. Washington, D.C.: American Chemical Society; 1988:7-32.
51
Jensen FE, Harris KM. Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J Neurosci
Methods. 1989;29:217-230.
52
Choi TS, et al. Advances in temperature control of microwave immunohistochemistry. Cell Vision. 1995;2(2):151-164.
53
Chakraborty DP, Brezovich IA. Error sources affecting thermocouple thermometry in RF electromagnetic fields. J Microw Power.
1982;17(1):17-28.
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54
Kingston HM, Jassie LB, eds. Introduction to Microwave Sample Preparation Theory and Practice. American Chemical Society:
Washington, D.C.; 1988:(6)93-154.
55
Kingston HM, Jassie LB. Microwave energy for acid decomposition at elevated temperatures and pressures using biological and botanical
samples. Anal Chem. 1986;58:2534-2541.
56
Wickersheim KA, Sun MH, Kamal A. A small microwave E-field probe utilizing fiberoptic thermometry. J Microwave Power.
1990;25(3):141-148.
57
Lorentzen EM, Kingston HM. The advantage feedback controlled microwave assisted leaching under atmospheric pressure. In: Total
Microwave Processing Using Microwave Technologies. Federation of Analytical Chemistry and Spectroscopy Societies: Cincinnati, OH;
1995.
58
Giberson RT, Austin RL, Charlesworth J, Adamson G, Herrera GA. Microwave and digital imaging technology reduce turnaround times for
diagnostic electron microscopy. Ultrastruct Pathol. 2003;27(3):187-196.
59
Kok LP, Boon ME. Physics of microwave technology in histochemistry. Histochem J. 1990;22:381-388.
60
Cattoretti G, Suurmeijer A. Antigen unmasking on formalin-fixed paraffin-embedded tissues using microwaves: A review. Adv Anat Path.
1995;2:2-9.
61
Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for
immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39(6):741-748.
62
Shi SR, Cote RJ, Taylor RC. Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem
Molecul Morphol. 2001;9(2):107-116.
63
Leong AS, Milios J. An assessment of the efficacy of the microwave antigen-retrieval procedure on a range of tissue antigens. Appl
Immunohistochem. 1993;1(4):267-274.
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Shi SR, Cote RJ, Yang C, et al. Development of an optimal protocol for antigen retrieval: A ‘test battery’ approach exemplified with
reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections. J Pathol. 1996;179(3):347-352.
65
Giammara BL, Birch DJ, Harper DO. Microwave polymerization of embedding resins for biological/biomedical electron microscopy. In:
Snyder WB, et al, eds. Microwave Processing of Materials II. Pittsburgh, PA: Materials Research Society; 1991:347-353.
66
Giammara B. Microwave embedment for light and electron microscopy using epoxy resins, LR White, and other polymers. Scanning.
1993;15:82-87.
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Clinical and Laboratory Standards Institute consensus procedures include an appeals process that
is described in detail in Section 8 of the Administrative Procedures. For further information,
contact the Executive Offices or visit our website at www.clsi.org.
Summary of Delegate/Consensus Comments and Subcommittee Responses
GP28-P: Microwave Device Use in the Clinical Laboratory; Proposed Guideline
General
1. The document should include microwave technology utilization in the blood bank.
• This technology is outside the scope of this document.
2. Include comprehensive discussion(s) specific to the domestic microwave user. It should identify which procedures may be
suitable for domestic units (outside of regulatory limitations) in more detail than simply listing them in Table 2 and
specifically how those methodologies may be carried out. Expound on the benefits of laboratory-grade microwave devices
for certain applications, explaining why they are advantageous to the user in those specific instances.
• The document is a guideline regarding microwave device safety and reproducibility. The guideline reports well-
documented general principles that laboratory technicians can apply to any published protocol in any microwave
device with an oven-like interior with the goal of improving reproducibility. This information is contained in Section
7, Critical Descriptors for Microwave-Accelerated Procedures and Section 10, Troubleshooting Results.
The document purposely does not describe specific methods, because these are already published in textbooks such
as Kok LP, Boon ME. Microwaves for the Art of Microscopy. Leyden: Coulomb Press; 2003 and Giberson RT,
Demaree RS. Microwave Techniques and Protocols. Totowa, NJ: Humana Press; 2001.
Table 2 in the guideline refers to basic and advanced functionality of FDA- and OSHA-compliant microwave devices.
The subcommittee thought it was important for laboratory technicians to also know this information, so they can
make informed purchasing decisions depending on their intended applications.
3. Include a discussion of the limitations of domestic microwave devices and which methodologies are unsuitable for this type
of device and why. This information will allow those considering the purchase of microwave technology to make informed
decisions appropriate for the procedures they intend to perform.
• This guideline is the first publication to collate, document, and discuss the limitations of domestic microwave ovens in
a hospital laboratory. Domestic microwave appliances do not meet FDA and OSHA guidelines for use in a hospital
laboratory environment. Please refer to Section 4.2, Electrical Precautions.
4. Offer domestic microwave device users strategies to enable them to achieve reproducible results.
• This information is outlined in Section 7, Critical Descriptors for Microwave-Accelerated Procedures and Section 10,
Troubleshooting Results.
5. This guideline calls it “immunohistochemistry” and uses the abbreviation IHC. The NCCLS document MM4-A uses the
word “immunocytochemistry” throughout. Though both are interchangeable, and though most histotechs seem to use the
term “immunohistochemistry” or “IHC,” NCCLS should consider sticking with one of the terminologies for consistency.
• “Histo” refers to tissue seen at the light microscopy magnification, and “cyto” refers to cells seen at electron
microscope magnification. The term “immunohistochemistry” is more accurate in this document.
6. When written after a number, either use the word for a measurement (i.e., 400 Watts), or use the symbol (400 W) throughout
the document. This is varied throughout the document.
• The symbol for a watts measurement (W) has been made consistent throughout the document.
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Section 2, Introduction
7. On page 1, last sentence, I would like to see this sentence rewritten to state, “Household microwave units are designed for
food preparation, and they are not certified for laboratory use; however, many laboratories have used them (effectively) for
staining, heating agar, heat activated epitope retrieval, and drying slides, (as long as proper safety precautions are
followed.)”
I think the average laboratorian trying to read this document will not understand what “Subpart S” or “Chapter V Subchapter
A-Drug & Devices”; or 29CFR 1910 means. I think for the purposes of a user-friendly document, this sentence should be
added in the text.
• The subcommittee acknowledges that domestic microwave ovens have been a cost-effective and successful piece of
equipment for many procedures in the laboratory. Their continued use is subject to the discretion of the individual
laboratory and hospital administrators. However, since domestic microwave ovens unequivocally do not meet the
electrical safety requirements of the FDA and OSHA for hospital laboratories, mention of domestic microwave oven
use has been eliminated from this guideline.
This guideline is written for multiple audiences including laboratory technicians, microwave device manufacturers,
microwave device resellers, compliance and safety officials, and administrators. Section 4.2, Electrical Precautions,
is relevant to technicians who want to know what is necessary to make a domestic appliance compliant with FDA
regulations for hospital laboratory use.
Section 3, Definitions
8. “Arcing” referred to relatively frequently in the text. Define “arc” and any implications with fire hazard.
• This definition has been added as suggested.
9. The majority of histology professionals in the United States are histologic technicians (HT) with a high school diploma
and trained on the job. For most of these individuals, a background in the theory of chemistry and physics is minimal to
nonexistent. Most HT with associate degrees, and the histotechnologists (HTL) with baccalaureate degrees, have not taken
physics in high school or college. Therefore, their background knowledge of microwave radiation and electricity is
minimal, at best.
It would be to the advantage of these readers to include more definitions in the front of the booklet, including units of
measure.
Suggestions of words that need definitions under Section 3 include:
Hertz - include the symbol Hz. Include a brief definition of MHz and GHz. (page 3)
Inductance (page 3)
Diode (page 3)
Anode (page 3)
Cathode (page 3)
Watt — including the symbol W. (page 3) Include W. page 4)
Cal/min (page 3) = calorie/minute
Arcing (page 7)
Thermodynamic (page 12)
Microwave-transparent (page 12)
Convection (page 12)
Conduction (page 12)
Entropy (page 12)
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Incident wave (page 14)
Specific heat (page 15)
• The definitions for the above terms have been added as suggested with the exception of “incident wave.” The term
“incident wave” has been replaced with “microwave frequency” (page 14).
Section 4, Precautions and Safety
10. I believe that the safety sections of the document should be modified and enhanced to increase their value to the reader.
There seems to be an overwhelming emphasis in some of these sections on how domestic units violate various regulatory
standards which are in some cases difficult to follow. I believe that this document should provide useful information
pointing the reader to standards that may apply rather than attempting to interpret the standards in a lengthy discussion that
may lose the reader. Ultimately compliance is up to the user. My suggestions are listed below:
a. It is puzzling that Section 4.1.1 (Microwave Radiation Safety) appears more concerned with FDA regulations than
microwave radiation safety. It never identifies the most basic hazard posed to individuals with pacemakers. I do not believe
that the paragraph at the top of page 5 will be useful to the reader. This section is supposed to be about microwave radiation
safety yet seems to be focused on the issue of “heating, cooking or drying food….and is manufactured for use in homes,
restaurants, food vending or service establishments.” I see no relevance of these remarks to radiation safety concerns unless
it can be demonstrated that domestic microwave devices pose a greater radiation hazard than other types of devices.
• Section 4.1.1 states that all microwave devices must comply with FDA regulations of microwave leakage. The
subcommittee did not find documentation about radiation hazards specific to domestic microwave devices, so this
was not raised as a concern in the guideline. The FDA DOES require that a microwave device be used solely for the
purpose for which it was designed, and this statement is reported in the guideline.
At one time there was concern that leakage from microwave ovens could interfere with certain electronic cardiac
pacemakers. There was similar concern about pacemaker interference from electric shavers, auto ignition systems,
and other electronic products. Because there are so many other products that also could cause this problem, FDA
does not require microwave ovens to carry warnings for people with pacemakers. The problem has been largely
resolved, since pacemakers are now designed so they are shielded against such electrical interference. However,
patients with pacemakers may wish to consult their physicians about this (refer to Section 4 for further details).
b. In Section 4.1.2, the first paragraph appears to have no relevance to the title of this section and will be confusing to the
reader as it discusses “if the equipment is modified or integrity of the safety device is violated...” I have no idea what this
has to do with classifying microwave equipment as medical devices. It appears to me that the content of Table 3 contains
more useful information for the reader than the attempts in the text to interpret the regulations which are confusing and
unclear. Likewise, the last paragraph on page 5 is equally confusing to the reader. Few practitioners will find the statement
“If a manufacturer or user modifies a household microwave oven or commercially promotes histological use as an indication
for use…” relevant to their circumstances. They may utilize a domestic device but are they “commercially” promoting
histological use? In my opinion this section needs a major overhaul. It would seem to me that its intent could be summed
up with a simple statement such as the following:
“Those who use microwave devices for other than that intended by the manufacturer must comply with standards of the
Occupational Safety and Health Administration and the FDA.” The section can then point the reader to the specific
regulations. I can't stress enough that clarity is key to reaching the reader.
• This guideline is written for multiple audiences including laboratory technicians, microwave device manufacturers,
microwave device resellers, compliance and safety officials, and administrators.
Section 4.2 is relevant to technicians who want to know what is necessary to make a domestic appliance compliant
with FDA regulations for hospital laboratory use. This section is also necessary for microwave resellers to be aware
that they cannot put their private label on a domestic microwave oven and promote it as a laboratory device. This
type of device violates FDA regulations as noted in Table 3. The subcommittee thought it was important for
laboratory technicians to also know this information so that they can make informed purchasing decisions.
Additional text has been added as introductory sentences in Section 4.1.3 to clarify this section as follows: “This
section provides regulatory information primarily for microwave device manufacturers, microwave device resellers,
compliance and safety officials, and administrators. The information below may also assist laboratory technicians
making purchasing decisions of microwave devices that are in compliance FDA and OSHA regulations.”
c. In Section 4.2, I find the second and third paragraphs of this section confusing. It appears to be saying that domestic
microwave devices and laboratory-grade devices are covered by different UL standards and that the UL standard for
domestic devices does not cover laboratory applications. Couldn't this section’s message be stated more clearly and
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succinctly as I’ve stated it here? My interpretation of this section is that domestic ovens are not tested according to UL
61010A-1, not that they were tested and failed to pass this electrical certification. It seems to me that the first paragraph
covers the relevant points. The other commentary is difficult to follow and will likely lose the reader.
• The three paragraphs in Section 4.2 actually describe three independent criteria for electrical safety. Paragraph one
outlines that specific electrical safety codes can only be tested by recognized national testing agencies; e.g., recognized
by OSHA (USA), CSA (Canada); or authorized to affix the mark (European States). Paragraph two states
domestic microwave devices are not designed or tested to meet the more stringent door seal and endurance tests
required in 61010A-1. Paragraph three informs the reader that the electrical safety code UL 923 is for domestic
microwave ovens, and because their electrical system is not shielded from the microwave cavity they must not be
used with caustic or corrosive vapors. Manufacturers are required to state in their instructions that household ovens
are only for heating food.
Additional text has been added as introductory sentences in Section 4.2 to clarify this section as follows: “This section
provides electrical safety information primarily for microwave device manufacturers, microwave device resellers,
compliance and safety officials, and administrators. Laboratory technicians are advised that according to OSHA
household microwave ovens do not meet the more stringent door seal and endurance tests required for applications
outside the home.”
Section 4.3, Biological Precautions
11. The first sentence of the second paragraph in Section 4.3, Biological Precautions, suggests that some pathogens are
destroyed by microwave heating, but it never tells the reader which ones are and which ones are not. This statement refers to
a publication that is ten years old. If more current studies do not exist or if the committee is not comfortable identifying
which pathogens are neutralized by microwave heating, why not simply state “the utility of using microwave energy to
destroy pathogens requires further study” followed by “all potentially infectious specimens must be handled with standard
precautions during and after the use of microwave irradiation.” In the absence of definitive information, the first sentence
becomes misleading as if to say, “Microwave irradiation may destroy your pathogen, or it may not!”
• The text in this section has been revised as suggested.
Section 4.4, Chemical Handling Precautions
12. Section 4.4., Chemical Handling Precautions, is a good start but I feel that it should be substantially expanded. What
chemicals should absolutely not be used in a microwave device? Are there any that might explode or ignite? I feel strongly
that this section needs to be much more informative and should even contain a comprehensive listing of chemicals
commonly utilized in histology laboratories and any special precautions for microwave applications.
• This section has been expanded as suggested.
Section 4.5, High Temperature Precautions
13. The last sentence in Section 4.5, High Temperature Precautions, states: “The risk of spontaneous boil over in histopathology
applications is more likely during microwave staining and heat-induced epitope retrieval.” More likely than what? If this
statement is correct, why is this so? Is this possible result limited to a particular type of microwave device and if not, what
can be done to reduce the likelihood of such an outcome? I believe that the reader will want to know if this can be explained
in greater detail.
• The sentence has been modified to state, “…more likely during high temperature heating procedures, such as...”
The explanations provided in the literature for microwave-induced boil over are super heating and bubble nucleation
(refer to cited references 27 and 43). Since there is no method provided in the literature to prevent spontaneous boil
over, technicians should use protective thermal gloves when removing solutions heated to near boiling.
Section 7, Critical Descriptors for Microwave-Accelerated Procedures
14. With the word accuracy, can we add the symbol “±.” Such as,
- page 16, 7.1.2.1, second paragraph, “. . . only have an accuracy of ±5 °C.”

- page 17, 7.1.2.4, “... have an accuracy between ±2 and 5 °C.”
- page 17, 7.1.2.5, " . . . is within ±5 °C.”
• The symbol “±” has been added as suggested.
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Section 7.1.2.1, Thermocouple Measurement Devices
15. ‘One hundred dollars’ – Should a dollar amount be quoted?
• The text has been changed to read “low cost.”
Section 7.1.2.3, IR Temperature Sensors
16. This section starts out with “IR Temperature Sensors,” and the reader has to go to the top of the page to find the definition of
“IR.” Page 17 — 7.1.2.5 starts out with “Liquid Crystal Temperature Strips,” and then abbreviated it in the first sentence to
LCTS. The mode on page 17 appears for the LCTS seems clearer.
• The abbreviation “IR” has been spelled out in the title of this section as suggested.
Section 7.1.5, Final (Maximum) Irradiation Temperature
17. Second paragraph: Does staining really require the final sample temperature to be above 90 °C?
• Most special stains in histology are done at a temperature range of 22 to 60 °C. Text has been added to reflect this.
Section 7.2.3.1, Method to Determine Power Output of a Large-Cavity Microwave Device
18. #4 — "... place an appropriate cover of plastic or foil." The question is where? Over the mouth of the bottle? Around the
outside of the container?
• Text has been added to clarify this.
Section 7.3.1.2, Container Dimensions
19. Is the final recommendation to use rectangular containers?
• There is no consensus about container shape within the subcommittee; however, the recommendation for rectangular
containers is based on cited literature (see cited references 59 and 60).
Section 8, Special Microwave Procedures
20. This section alternates between heat-induced epitope retrieval (without indicating the HIER abbreviation) and AR (without
defining AR as antigen recovery or antigen retrieval. Suggest using HIER consistently, with the abbreviation.
• The term “heat-induced epitope retrieval” has been replaced with “antigen retrieval” (AR) throughout this section.
Table 13
21. “Microwave Curing — Results — Mold Deforms” — Before the word “mold,” insert “Embedding.” Mold, in histology,
can also mean “fungus.”
• This text has been added as suggested.
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The Quality System Approach

Clinical and Laboratory Standards Institute subscribes to a quality system approach in the development of standards
and guidelines, which facilitates project management; defines a document structure via a template; and provides a
process to identify needed documents. The approach is based on the model presented in the most current edition of
CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The quality system approach
applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any healthcare
service’s path of workflow (i.e., operational aspects that define how a particular product or service is provided). The
QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The
quality system essentials (QSEs) are:
Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Service & Satisfaction
Personnel Process Control Assessment Facilities & Safety
GP28-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other
CLSI/NCCLS documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the
following page.
Purchasing &
Improvement
Organization
Management
Management
Information
Satisfaction
Assessment
Facilities &
Occurrence
Documents
Equipment
& Records
Service &
Personnel
Inventory
Control
Process
Process
Safety
X
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
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Related CLSI/NCCLS Publications*

GP17-A2 Clinical Laboratory Safety; Approved Guideline—Second Edition (2004). This document contains general
guidelines for implementing a high-quality laboratory safety program, which are provided in a framework that
is adaptable within any laboratory.
MM4-A Quality Assurance for Immunocytochemistry; Approved Guideline (1999). This document provides
recommendations for the performance of immunocytochemical assays on cytologic and surgical pathology
specimens. It is intended to promote a better understanding of the requirements, capabilities, and limitations of
these diagnostic methods; to improve their intra- and interlaboratory reproducibility; and to improve their
positive and negative predictive values in the diagnosis of disease.
*
Proposed- and tentative-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus
process; therefore, readers should refer to the most recent editions.
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Number 7 GP28-A
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NOTES
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GP28-A

Microwave Device Use in the Histology

This document provides recommendations for reproducing the performance of

Reproduced with permission, from CLSI publication GP28-A—Microwave Device Use

Copyright ©2005. Clinical and Laboratory Standards Institute.

Area Committee on General Laboratory Practices

Subcommittee on Microwave Ovens

Gary R. Login, DMD, DMSc

Clinical and Laboratory Standards Institute

Tracy A. Dooley, MLT(ASCP)

Acknowledgement in Memoriam of our Subcommittee Member and Colleague

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

4 Precautions and Safety...............................................................................................................5

7 Critical Descriptors for Microwave-Accelerated Procedures ..................................................15

10 Troubleshooting Results ..........................................................................................................30

Summary of Delegate/Consensus Comments and Subcommittee Responses ......................................35

The Quality System Approach..............................................................................................................40

Related CLSI/NCCLS Publications ......................................................................................................41

• Table 4, Safe Laboratory Use of Microwave Devices;

• Section 9, Template for Documentation of Microwave Methods; and

• Section 10, Troubleshooting Results.

Antigen retrieval, biopsy, electron microscopy, fixation, histology, immunocytochemistry,

Microwave Device Use in the Histology Laboratory; Approved Guideline

To ensure the success of microwave-accelerated procedures in the histopathology laboratory, this

• general definitions of common microwave terminology;

• detailed discussion of safety issues particular to microwave heating of samples;

• guidelines to identify potential sources of variability; and

• a “hands-on” troubleshooting guide to improve microwave-accelerated procedures.

Table 1. Application of Microwave-Accelerated Methods in Histopathology (see Sections 4.3 through

Table 2. Use of Large-Cavity Microwave Devices With Basic or Advanced Features

Anode – A positively charged conductor by which electrons leave an electrical device.

Arcing – Electrical conduction through a gas in an applied electric field.

Entropy (thermodynamics) – A thermodynamic quantity representing the amount of energy in a system

Microwave-transparent – Having the property of transmitting microwaves, materials such as glass or

4 Precautions and Safety

4.1 Microwave Radiation Exposure Standards and Regulations

4.1.1 Microwave Radiation Safety

4.1.2 CDRH Consumer Information—Microwave Device Radiation

4.1.3 Microwave Equipment Classified as Medical Devices

Table 3. Summary of FDA Regulations of Microwave Equipment

These regulations can be found in the FDA web pages:

Nonmedical devices: http://www.fda.gov/cdrh/radhealth.html

Medical devices: http://www.fda.gov/cdrh/devadvice

If a manufacturer or user modifies a household microwave oven or commercially promotes histological

4.2 Electrical Precautions

4.3 Biological Precautions

4.4 Chemical Handling Precautions

4.5 High Temperature Precautions

4.6 Recommended Safety Procedures for Microwave Devices and Applications

Table 4. Safe Laboratory Use of Microwave Devices

Routine performance tests on ventilation hoods such as

4.7 Microwave Safety Inspection

Table 5. Microwave Device Survey Data Sheet

Microwave Device Survey Data Sheet

Figure 1. Diagram of Microwave Door and Door Frame

Table 6. Template for Recording Microwave Leakage Readings

Door closed readings in mW/cm2

Maximum reading _________ mW/cm2