Clsi HS2 A

HS2-A
Vol. 23 No. 5
Replaces HS2-P
Vol. 20 No. 3
Provider-Performed Microscopy Testing; Approved Guideline
This guideline provides information on specimen collection, test methodologies, procedural steps,
reporting of results, and the quality assurance aspects of provider-performed microscopy.
A guideline for global application developed through the NCCLS consensus process.
NCCLS...
Serving the Worlds Medical Science Community Through Voluntary Consensus
NCCLS is an international, interdisciplinary, nonprofit, the need for field evaluation or data collection, documents
standards-developing, and educational organization that may also be made available for review at an intermediate
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consensus standards and guidelines within the healthcare
Proposed An NCCLS consensus document undergoes the
community. It is recognized worldwide for the
first stage of review by the healthcare community as a
application of its unique consensus process in the
proposed standard or guideline. The document should
development of standards and guidelines for patient
receive a wide and thorough technical review, including an
testing and related healthcare issues. NCCLS is based on
overall review of its scope, approach, and utility, and a line-
the principle that consensus is an effective and cost-
by-line review of its technical and editorial content.
effective way to improve patient testing and healthcare
services. Tentative A tentative standard or guideline is made
available for review and comment only when a
In addition to developing and promoting the use of
recommended method has a well-defined need for a field
voluntary consensus standards and guidelines, NCCLS
evaluation or when a recommended protocol requires that
provides an open and unbiased forum to address critical
specific data be collected. It should be reviewed to ensure its
issues affecting the quality of patient testing and health
utility.
care.
Approved An approved standard or guideline has achieved
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HS2-A
ISBN 1-56238-487-2
Volume 23 Number 5 ISSN 0273-3099
Mina L. Harkins, MSB, M.T.(ASCP), Chairholder
Patricia Higgins, D.O.
Gregory D. Metzger, CLS, CLDir(NCA)
C. Anne Pontius, MBA, CLC(AMT), M.T.(ASCP)
Elmer F. Wahl, M.D.
Michael J. Wajda, M.S., J.D.
Abstract
NCCLS document HS2-A Provider-Performed Microscopy Testing; Approved Guideline provides information, instructions,
and performance criteria to assist providers who perform microscopy procedures (provider-performed microscopy [PPM]), with
accurate reporting of diagnostic information from their observations.
These are appropriate procedures for the examining room, emergency room, or clinic environment as an adjunct to traditional
clinical laboratory testing. This testing may also provide for a rapid diagnosis of the patient condition. The guideline relates
information concerning specimen collection, methodologies, procedural steps, reporting of results, and the quality assurance
aspects of PPM.
NCCLS. Provider-Performed Microscopy Testing; Approved Guideline. NCCLS document HS2-A (ISBN 1-56238-487-2).
NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2003.
THE NCCLS 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 NCCLS documents. Current editions are listed in the NCCLS 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 NCCLS Catalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100;
Fax: 610.688.0700; E-Mail: exoffice@nccls.org; Website: www.nccls.org
Number 5 NCCLS
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system,
transmitted, or made available in any form or by any means (electronic, mechanical, photocopying,
recording, or otherwise) without prior written permission from NCCLS, except as stated below.
NCCLS hereby grants permission to reproduce limited portions of this publication for use in laboratory
procedure manuals at a single site, for interlibrary loan, or for use in educational programs provided that
multiple copies of such reproduction shall include the following notice, be distributed without charge,
and, in no event, contain more than 20% of the documents text.
Reproduced with permission, from NCCLS publication HS2-AProvider-Performed

Microscopy Testing; Approved Guideline (ISBN 1-56238-487-2). Copies of the current
edition may be obtained from NCCLS, 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
exemptions granted here or under the Copyright Law must be obtained from NCCLS by written request.
To request such permission, address inquiries to the Executive Director, NCCLS, 940 West Valley Road,
Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Copyright 2003. The National Committee for Clinical Laboratory Standards.
Suggested Citation
(NCCLS. Provider-Performed Microscopy Testing; Approved Guideline. NCCLS document HS2-A

[ISBN 1-56238-487-2]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898
USA, 2003.)
Proposed Guideline
February 2000
Approved Guideline
February 2003
ISBN 1-56238-487-2
ISSN 0273-3099
ii
Volume 23 HS2-A
Committee Membership
Subcommittee on Provider-Performed Microscopy Testing
Mina L. Harkins, MBA, Michael J. Wajda, M.S., J.D. Staff

M.T.(ASCP) State of MD Dept. of Health &
Chairholder Mental Hygiene Jennifer K. McGeary, M.T.(ASCP),
Quest Diagnostics, Inc. Baltimore, Maryland M.S.H.A.
Baltimore, Maryland Staff Liaison
Advisors NCCLS
Patricia Higgins, D.O. Wayne, Pennsylvania
Chicago College of Osteopathic
Jerry Bennington, B.S.M.T.(AMT)
Medicine David E. Sterry, M.T.(ASCP)
Marshfield Clinic
Downers Grove, Illinois Project Manager
Marshfield, Wisconsin
NCCLS
Gregory D. Metzger, CLS, Wayne, Pennsylvania
CLDir(NCA) Richard C. Marsella, M.D., Ph.D.
UW Medical Center Roosevelt LabCorp, Inc.
Patrice E. Polgar
Seattle, Washington Columbia, Maryland
Editor
NCCLS
C. Anne Pontius, MBA, Christine Wright, M.S.,
Wayne, Pennsylvania
CLC(AMT), M.T.(ASCP) M.T.(ASCP)
Laboratory Compliance Consultants American Academy of Family
Donna M. Wilhelm
Raleigh, North Carolina Physicians
Assistant Editor
Kansas City, Missouri
NCCLS
Elmer F. Wahl, M.D. Wayne, Pennsylvania
Medical College of Ohio
Toledo, Ohio
iii
Number 5 NCCLS
iv
Volume 23 HS2-A
Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
The Quality System Approach............................................................................................................ viii
Foreword ................................................................................................................................................ix
1 Introduction................................................................................................................................1
2 Scope..........................................................................................................................................2
3 Safety .........................................................................................................................................2
3.1 Standard Precautions .....................................................................................................2
3.2 Personal Protective Equipment......................................................................................2
3.3 Hand Washing ...............................................................................................................3
3.4 Food, Drink, Cigarettes, and Cosmetics ........................................................................3
3.5 Routine Cleaning ...........................................................................................................3
3.6 Infectious Waste Disposal .............................................................................................4
4 Definitions .................................................................................................................................4
5 Equipment..................................................................................................................................5
5.1 Materials ........................................................................................................................5
5.2 Microscope ....................................................................................................................6
6 Quality Assurance....................................................................................................................10
6.1 Microscopy Provider ...................................................................................................10
6.2 Equipment and Supply Management...........................................................................12
6.3 Patient Test Management ............................................................................................12
6.4 Procedure Manual........................................................................................................13
6.5 Quality Control ............................................................................................................14
6.6 Proficiency Testing......................................................................................................15
6.7 Relationship of Patient Information to Patient Test Results........................................15
6.8 Accreditation ...............................................................................................................16
7 Urine Sediment Examinations .................................................................................................16
7.1 Principle.......................................................................................................................16
7.2 Materials ......................................................................................................................17
7.3 Specimen Collection....................................................................................................18
7.4 Testing Procedure ........................................................................................................19
7.5 Quality Control ............................................................................................................21
7.6 Reporting Results ........................................................................................................22
8 Direct Wet Mount Preparations and Potassium Hydroxide (KOH) Preparations....................22
8.1 Principle.......................................................................................................................22
8.2 Materials ......................................................................................................................24
8.5 Quality Control ............................................................................................................26
v
Number 5 NCCLS
Contents (Continued)
8.7 Limitations of the Procedure .......................................................................................26

9 Pinworm Examinations............................................................................................................27
9.1 Principle.......................................................................................................................27
9.2 Detection of Ova: Cellophane Tape Test.....................................................................27
9.3 Detection of Adult Parasites ........................................................................................29
10 Fern Test ..................................................................................................................................31
10.1 Principle.......................................................................................................................31
10.2 Materials ......................................................................................................................32
10.5 Quality Control ............................................................................................................32
11 Postcoital, Direct, Qualitative Examinations of Cervical Mucus ............................................33
11.1 Principle.......................................................................................................................33
11.2 Materials ......................................................................................................................33
11.5 Quality Control ............................................................................................................35
12 Qualitative Semen Analysis.....................................................................................................35
12.1 Principle.......................................................................................................................35
12.2 Materials ......................................................................................................................36
12.5 Quality Control ............................................................................................................37
13 Nasal Smears for Inflammatory Cells......................................................................................38
13.1 Principle.......................................................................................................................38
13.2 Materials ......................................................................................................................38
13.5 Quality Control ............................................................................................................39
14 Fecal Leukocyte Examination..................................................................................................40
14.1 Principle.......................................................................................................................40
14.2 Materials ......................................................................................................................40
14.5 Quality Control ............................................................................................................41
vi
Volume 23 HS2-A
Contents (Continued)
References.............................................................................................................................................43
Additional References...........................................................................................................................44
Appendix A. Microscopic Components in Urine Sediment.................................................................45
Appendix B. Microscopic Components in Vaginal Fluid and KOH Preparations...............................52
Summary of Comments and Subcommittee Responses........................................................................55
Summary of Delegate Comments and Subcommittee Responses.........................................................74
Related NCCLS Publications................................................................................................................75
vii
Number 5 NCCLS
The Quality System Approach

NCCLS 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 through a gap analysis. The approach is based on the model presented in the most current edition of
NCCLS HS1A Quality 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 services path of workflow.
The QSEs provide the framework for delivery of any type of product or service, serving as a managers 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
HS2-A addresses the quality system essentials (QSEs) indicated by an "X." For a description of the other NCCLS
documents listed in the grid, please refer to the Related NCCLS Publications section at the end of the document.
Purchasing &
Improvement
Management
Management
Organization
Information
Assessment
Satisfaction
Facilities &
Occurrence
Documents
Equipment
& Records
Service &
Personnel
Inventory
Control
Process
Process
Safety
X X GP5-A2
GP2-A4 GP21-A X X M29-A2
Adapted from NCCLS document HS1 A Quality System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, GP26-A2 defines a clinical laboratory path of workflow which
consists of three sequential processes: preanalytical, analytical, and postanalytical. All clinical laboratories follow
these processes to deliver the laboratorys services, namely quality laboratory information.
HS2-A addresses the clinical laboratory path of workflow steps indicated by an "X." For a description of the other
NCCLS documents listed in the grid, please refer to the Related NCCLS Publications section at the end of the
document.
Preanalytic Analytic Postanalytic

Interpretation
Management
Test Request
Assessment
Laboratory
Collection
Specimen
Specimen
Specimen
Specimen
Transport
Post-test
Review
Receipt
Testing
Results
Patient
Report
X X X X
GP16-A2 GP16-A2 GP16-A2 GP16-A2 X X
Adapted from NCCLS document HS1 A Quality System Model for Health Care.
viii
Volume 23 HS2-A
Foreword
Provider-performed microscopy (PPM) is a testing modality that requires the use of a microscope and is
performed by a clinical practitioner (referred to as a provider in this guideline) during a patient
encounter. Specimens utilized in PPM testing are considered labile or unstable after a very short period of
time. PPM testing permits providers to render a rapid diagnosis that can, in turn, facilitate the rapid
initiation of treatment.
It has always been recognized that providers use certain microscopic procedures to supplement their
physical examinations in the diagnosis of patients. Unfortunately, many nonpathologist providers were
not afforded adequate training time to fully comprehend good laboratory principles that ensure accurate
results. Accurate results come from following standardized practices for the preanalytical (prior to
testing), analytical (testing), and postanalytical (reporting) phases of testing.
The purpose of this guideline is to present critical aspects of the preanalytical, analytical, and
postanalytical processes that contribute to accurate test results. This includes specific guidance to assist
providers with specimen collection and handling, competency assessment, testing procedures and
interpretation, proficiency testing, quality control, quality assurance, and recommended documentation.
This document is not intended as a recipe for complying with any particular federal laboratory laws, local
laws, or accrediting organization requirements, but is intended to assist clinical practitioners by providing
information that will increase the reliability and utility of microscopic testing done during the course of a
client visit.
For those clinical practitioners who become involved in the testing arena, many will need external
resources to supplement their education. This guideline is not only an excellent resource but, if adhered
to, will reduce the potential liability of performing nonstandardized PPM procedures.
This document addresses certain characteristics of the diagnosis and management of patients in the
clinical office setting. The subcommittee requests reviewers comment on this aspect of the document,
i.e., appropriateness and necessity of clinical points to the utility of the guideline.
Also, it is the subcommittees intention to include, as appropriate, photomicrographs of various

constituents commonly found in microscopic examinations for the tests described herein. Reviewers are
especially encouraged to submit suggestions for photomicrographs (including source information) to be
included in this guideline.
Key Words
Analytic, microscopy, postanalytic, preanalytic, provider
ix
Number 5 NCCLS
x
Volume 23 HS2-A
1 Introduction
Provider-performed microscopy (PPM), as carried out by trained providers, produces rapid, reliable
results intended to be used by the provider to immediately impact patient care decisions.a This type of
testing requires that the performing individual be responsible for all aspects of the testing process,
including:
appropriate use of the procedure (i.e., ordering tests which correspond to patient history and
symptoms, and ensuring correlation between results and clinical picture);
acceptable patient preparation;
proper specimen procurement and handling;
correct selection, use, and maintenance of the microscope;
test methodologies in a procedure manual;
accurate interpretation of observed elements;
quality assurance and competency assessment; and
documentation of results, QA and QC activities.
The appropriate uses of the described procedures may include the following:
urine sediment examination to identify the cause of symptomatic presentations or abnormal chemical
dipstick results;
direct wet mount preparations to detect the presence of bacterial, fungal, or parasitic organisms, and
other cellular elements indicative of pathologic conditions;
potassium hydroxide (KOH) preparations to detect yeast and fungal elements;
pinworm examinations to detect infestation by the Enterobius vermicularis parasite;
fern tests to detect the presence of amniotic fluid in vaginal secretions indicating rupture of the
amniotic sac;
postcoital, direct, qualitative examinations of cervical mucus to investigate infertility;
qualitative semen analysis to test for effective vasectomy or to investigate infertility;
nasal smear for granulocytes to identify an allergic etiology for upper respiratory symptoms; and
fecal leukocyte examinations to suggest or exclude the diagnosis of a number of pathologic

conditions.
a
In the U.S., according to Clinical Laboratory Improvement Amendments (CLIA), it is the designated laboratory directors
overall responsibility to assure the accuracy and reliability of the testing performed.
An NCCLS global consensus guideline. NCCLS. All rights reserved. 1

Number 5 NCCLS
2 Scope
The scope of this guideline is limited to procedures that require the use of microscopic observation with
minimum specimen preparation, typically performed by a provider in near-patient testing environments. It
does not include procedures which require the use of stains to determine the results of the testing.b
It is intended to be used in providers offices, outpatient clinics, public health clinics, health maintenance
organizations, and medical training programs. Providers may include physicians, nurse practitioners,
physicians assistants, or nurse midwives.
3 Safety
Since the anticipated location of PPM testing is the medical office examining room, emergency room, or
clinic environment, there may be written protocols required by regional regulatory bodiesc for worker
safety. A safety manual with specific policies should be available for the facility where testing is located.
Since these locations for direct patient care may be diverse and the provider may be the only one involved
in the testing process, recognition should be made to provide safe work conditions which meet applicable
regional requirements, determine appropriate personal protective equipment, and provide safe disposal of
biohazardous waste.
3.1 Standard Precautions
Because it is often impossible to know what might be infectious, all human body specimens are to be
treated as infectious and handled according to standard precautions. Standard precautions are new
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. 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 the most current edition of NCCLS document M29Protection of Laboratory
Workers from Occupationally Acquired Infections.
The European Union has similarly addressed the precautions of handling human specimens to reduce the
risks associated with exposure to biological agents by passage of a directive (Directive 2000/54/EC on the
protection of workers from risks related to exposure to biological agents at work; 18 September 2000).
3.2 Personal Protective Equipment
Personal protective equipment should be utilized in the following situations:
When the performing providers duties involve occupational exposure, appropriate use of gloves,
gowns, protective coats, face shields or masks, and eye protection is recommended.
b
In the U.S., the CLIA certification for provider-performed microscopy (PPM) described a set of procedures that may be
performed under that designation.
c
In the U.S., for example, the U.S. Occupational Safety and Health Administration (OSHA).
2 An NCCLS global consensus guideline. NCCLS. All rights reserved.
Volume 23 HS2-A
Gloves must be worn whenever collecting, handling, and testing specimens and to provide barrier
protection for cuts, scratches, or breaks in the skin against potential pathogens. Glove use should be
task specific, and be removed to avoid unnecessarily contaminating the microscope.
Protective body clothing (gown, apron, or laboratory coat which is resistive or impervious to liquids)
should be worn whenever collecting, handling, or testing specimens. This clothing should not be
worn into other work areas that are considered free of potentially infectious substances.
3.3 Hand Washing
Hand washing is recognized as the most important deterrent in infection control. Hands should be
thoroughly washed for 20 to 30 seconds under running water either with soap or antiseptic soap as soon
as feasible:
after removing gloves;

after completing work and/or before leaving the work area to eat, drink, smoke, apply cosmetics,
handling contact lenses, and using restroom facilities;
before touching eyes, nose, mouth, glasses;
immediately after contamination with a specimen or reagent; and
after contact with each patient or before contact with another.d
Alternatively, an alcohol-based, waterless, rapid-kill gel can be rubbed thoroughly over the surface of the
hands.
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 NCCLS document M29Protection of Laboratory Workers from Occupationally Acquired
Infections or ISO/DIS 15190 Medical Laboratories Requirements for Safety.
3.4 Food, Drink, Cigarettes, and Cosmetics
Eating, drinking, smoking, and applying cosmetics should not take place within the testing environment,
due to possible contamination of the hands with infectious organisms.
3.5 Routine Cleaning
The work area should be kept clean to prevent contamination from infectious organisms from specimens
to the performing provider or other persons utilizing the area. A 1:10 solution of household bleach and
water (one part bleach plus nine parts water to result in a 0.5% aqueous solution of sodium hypochlorite)
is an effective disinfectant for both bacterial and viral organisms. All work surfaces should be wiped and
allowed to soak a minimum of 15 minutes. To retain potency and effectiveness, the 1:10 solution must be
prepared daily. NOTE: Aluminum and stainless steel are corroded by sodium hypochlorite, and, therefore
other disinfectants may be preferred. (See the most current edition of NCCLS document M29
Protection of Laboratory Workers from Occupationally Acquired Infections or ISO/DIS 15190 Medical
Laboratories Requirements for Safety for additional information.)
If blood or other biohazardous material contaminates equipment such as the microscope, carefully wipe
the area with bleach solution. Care should be taken not to spill solutions onto the working mechanisms of
the microscope, computers, and other equipment.
d
In the U.S., employees must wash their hands with soap and running water as soon as feasible, as recommended above.
Number 5 NCCLS
3.6 Infectious Waste Disposal
Disposal of medical waste in general trash exposes sanitation workers to unknown hazards and may
create a risk of legal liability to the facility or organization. Therefore, handling and disposal of medical
waste should be by trained personnel, utilizing materials and procedures which protect the employees,
patients, and the environment. Infectious waste (biohazard) containers should be conveniently located and
of sufficient volume to accommodate the infectious waste generated at the site. Containers must be
labeled to warn of biohazardous material. The containers should be constructed of materials appropriate
to the type of waste generated and to contain any fluids. Biohazard containers should be handled with
gloved hands. Disposal of medical and infectious waste must comply with all local and regional
regulatory requirements.
4 Definitionse
Accreditation - The process by which a private, peer-level commission or association evaluates and
ensures that a program of professional study or activity, such as laboratory testing, in an institution is
meeting appropriate standards of organizational performance.
Accuracy - Closeness of the agreement between the result of a measurement and a true value of the
measurand/analyte.
Analyte Component indicated in the name of a measurable quantity [ISO FDIS 17511].
Analytical process - The technical process including the operation of equipment and the performance of
the defined steps of a testing procedure designed to produce data.
Biohazard A biological agent or condition that constitutes a hazard to human beings or their
environment.
Competence Demonstrated ability to apply knowledge and skills [ISO 9001] [ISO 9000, 3.9.2].
Exogenous - Developed or originating outside an organism; caused by external factors.
Immersion oil - A liquid medium, occupying the space between the object and microscope objective,
used to optimize the resolution of the image being magnified.
Measurand A particular quantity subject to measurement.
(Microscope) objective - The lens in a microscope that is nearest to the object under examination and
magnifies the specimen in relation to the power of the objective.
Ocular (eyepiece) - The lens used in a microscope for magnification of the primary image; NOTE: The
lens nearest the eye.
Postanalytical//post-examination process - All processes following laboratory examination including

systematic review, formatting and interpretation, authorization for release, reporting of results,
transmission of the results, and storage of the samples of the laboratory examinations [ISO FDIS
15189.2].
e
Some of these definitions are found in NCCLS document NRSCL8Terminology and Definitions for Use in NCCLS
Documents. For complete definitions and detailed source information, please refer to the most current edition of that document.
Volume 23 HS2-A
Preanalytical//pre-examination process - Steps starting in chronological order from the clinicians

request, including the laboratory examination requisition, preparation of the patient, collection of the
primary sample, transportation to and within the laboratory and ending when the analytical examination
procedure starts [ISO FDIS 15189.2].
Precision The closeness of agreement between independent test results obtained under prescribed/
(stipulated) conditions.
Proficiency testing, PT - A program in which multiple specimens are periodically sent to members of a
group of sites for analysis and/or identification; in which each site's results are compared with those of
other participants in the group and/or with an assigned value, and reported to the participating facilities
and others.
Provider - In the medical context, a clinical practitioner.
Qualitative - A characterization applied to laboratory tests that detect the presence or absence of a
particular analyte, constituent, or condition; NOTE: Specific identification may be performed.
Quality assurance - All the planned and systematic activities implemented within the quality system and
demonstrated as needed, to provide adequate confidence that an entity will fulfill requirements for quality
[ISO 8402:94-3.5].
Reagent - A substance that produces a chemical reaction in a sample that allows an analyte to be detected
and measured.
Semiquantitative tests Those that yield results in an approximate range of values (i.e., trace, moderate,
etc.).
Spinnbarkeit A measure of tenacity (from German), i.e., the ability to form a thread, viscosity.
Stability - The capacity for a product to retain its composition, characteristics, and properties during
specified conditions.
5 Equipment
5.1 Materials
Materials vary by procedure. Specific needs and recommended supplies are addressed with each
procedure. Materials include:
collection devices;
centrifuge tubes;
transfer pipettes;
microscope slides and cover slip(s);
cotton-tipped applicators;
immersion oil;
lens paper; and
lens cleaning solution.

Number 5 NCCLS
5.2 Microscope
Microscopes have various attributes and should be carefully maintained. Proficiency testing and control
procedures may not be available for all microscopic examinations. Therefore, analyzing specimens with
the microscope requires training in microscopic techniques, knowledge of standard precautions, and an
understanding of the capabilities, use, and care of the microscope.
A modern, high-quality microscope with the following characteristics is desirable for examinations of
urine sediment, vaginal wet mounts, KOH preparations, pinworm preparations, fern tests, postcoital
examinations, qualitative semen analysis, nasal smears, and fecal leukocyte preparations:
binocular head to allow the use of both eyes when viewing the specimen;
built-in light source with field diaphragm;
mechanical stage to allow the easy and smooth positioning of the slide; and
basic set of objective (10x, 40x, and 100x oil immersion) and ocular (10x or 12.5x) lenses.
Prior to purchase and use, a microscope should be evaluated for the following:
proper illumination and use of the objectives;

adequate resolution, i.e., crisp, clear images at all magnifications; and
convenience and comfort with respect to placement of focusing controls and the angle of viewing of
the binocular tube head.
5.2.1 Parts of the Microscope (See Figure 1.)
5.2.1.1 Lenses
The objective lens magnifies the specimen a defined amount. The objective produces the primary image
and the eyepiece magnifies it. The total magnification of the image is the product of the magnification of
the objective multiplied by the magnification of the eyepiece. Magnification is the relationship between
the size of the image and the size of the specimen.
The objectives are important lenses in the image-forming system. They are screwed into a revolving
nosepiece attached to the stand. Only one objective at a time is moved into the illumination path when the
nosepiece is rotated.
The microscope and objectives are constructed so that as each objective is rotated into the light path.
After the first objective has been focused, only a slight adjustment of the fine adjustment knob is required
to focus the specimen. Also, an object seen in the center of the field when objectives are changed should
stay close to the center of the field. The clarity, sharpness, detail, and visibility needed for good
performance in microscopy depends on the quality and care of the objectives.
The oculars (eyepieces) are placed in the top openings of the observation tubes of the microscope and
magnify the primary image projected by the objective.
5.2.1.2 Stand
Characteristics of the stand include the following:
rests on the base of the microscope; and

carries the arm of the microscope and the stage on which the specimen is placed.
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5.2.1.3 Stage
Characteristics of the stage include the following:
horizontal platform upon which the specimen slide is placed;

a slide holder fitted to the top to hold the slide in position; and
knobs to control the movement of the stage and the slide-holder mechanism (slight movement of the
slide can carry the observed area out of view, so movements should be slow and smooth).
5.2.1.4 Condenser
Characteristics of the condenser include the following:
mounted under the stage to concentrate and focus light from the light source;
can be raised or lowered by means of a condenser knob below the stage;
has an aperture iris diaphragm which can be opened or closed to control the amount of light striking
the specimen;
centering screws center the circle of light in the viewing field; and
special purpose condensers, such as phase contrast condensers, can be used with specially constructed
phase contrast objectives to make unstained material visible.
5.2.1.5 Illumination
A built-in light source, usually a tungsten bulb or tungsten-halogen bulb, works by plugging the
microscope cord into an electrical socket and using the on-off switch to turn the bulb on.
Better microscopes have a field diaphragm on the collector lens. The adjustment of this diaphragm
increases or decreases the circle of light in the viewing field.
A switch or dimmer may be used to control the intensity of the light. Lowering the intensity before
turning off the switch will lengthen the life of the bulb.
Most microscopes have a blue filter that is used to alter the quality and/or intensity of the light passing
through the specimen.

Number 5 NCCLS
Stand
Field Diaphragm
Figure 1. Standard Laboratory Microscope
5.2.2 Operation
Before using the microscope, read the manufacturers instructions to become familiarized with the
features of the specific microscope. The optimal conditions for the observations discussed in these
procedures require that steps be followed to focus the image and adjust the illumination.
To focus a specimen and adjust the microscope for bright, even light and good contrast:
(1) Open the diaphragm(s), i.e., the condensers aperture and the field diaphragm; if appropriate, lower
the condenser, and turn on the illuminator to low power.

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(2) Using the low power (10x) objective, focus on a slide by raising the stage with the coarse
adjustment knobs, observing the slide from the side, until the slide comes close to the objective.
Looking through the objectives, focus slowly with the coarse and/or fine focusing knobs until the
image is sharpest.
(3) Close the field diaphragm almost completely and raise the condenser until the edges of the
diaphragm are sharply focused (the condenser is usually at about its highest position). Open the
field diaphragm slowly, stopping just as it disappears from view.
(4) Open and close the aperture diaphragm to optimize contrast. Contrast is increased by closing the
aperture. If more light intensity is needed, increase the illuminator.
The microscope is now optimally adjusted for the objective; other objectives will require some
readjustment.
To use the high (40x) objective:
(1) Focus and center the specimen with the 10x objective, rotate the nosepiece slowly to bring the 40x
objective into the light path.
(2) Move the fine adjustment knob slightly to bring the specimen into focus. If it is not clear, refocus
with the 10x, making sure the specimen is in the center of the field; switch back to the 40x
objective. NOTE: Never raise the stage with the coarse adjustment knobs when using the 40x lens.
This may cause the lens to break by hitting it with the slide.
To use the 100x oil immersion objective:
(1) Focus and center the specimen with the 10x objective, followed by the 40x objective; lower the
stage using the coarse adjustment knobs, with the 40x objective in the light path.
(2) Place a drop of immersion oil on the cover slip of the slide being examined, directly over the light
path, making certain the drop is placed so that the objective will dip into the drop when the stage is
raised. Rotate the nosepiece until the 100x oil immersion objective comes into the light path.
(3) Looking peripherally at stage level and NOT through the eyepieces, raise the stage slowly until the
objective makes contact with the oil drop causing a flash of light.
(4) Raise the stage slowly using the fine adjustment knobs while looking through the eyepieces; at the
slightest resistance, stop, because the cover slip may be too close (at this time the specimen should
come into focus).
(5) After using the oil immersion objective, it should be cleaned with a piece of lens paper dampened
with lens cleaning solution. Avoid getting oil on the 40x objective. Make sure to dry the objective
by either gently blowing with air or using a clean, dry sheet of lens paper.
5.2.3 Care of the Microscope
To ensure long-term quality microscope use, follow the manufacturers guidelines for care and
maintenance of the microscope. At a minimum, maintenance should be performed annually. This
timeframe, however, is dependent on the number of providers and procedures performed on the
microscope. Also:
cover the microscope when not being used and leave the 10x objective in position;
Number 5 NCCLS
do not expose the microscope to extreme heat, cold, or temperature changes;

keep all parts clean; clean external optical surfaces onlybottom of objectives, top of condenser and
illuminator, top eyepiece lenses;
wear smudge-proof make-up when using the microscope;
wipe dust off mechanical surfaces with a soft, lint-free cloth;
remove dust from glass surfaces with a gentle puff of air and a natural bristle brush;
clean oil from objectives, stage, condenser, and illuminator immediately following use or spill with
lens paper moistened with a commercial cleaning solution for lenses or a solution recommended by
the manufacturer;
never use cleaning fluid on the underside lenses of the objective;
use only the bulb and illuminator as specified by the manufacturer to avoid excess heat;
do not mix brands of immersion oil, and use only microscope immersion oil;
never force open diaphragm leaves;
schedule periodic maintenance to include professional servicing (lubrication, fine tuning, and
realignment will be done at this time); and
keep records of cleaning and other maintenance procedures.
6 Quality Assurance
Quality assurance (QA) is an ongoing process established to assure that laboratory test results reliably and
accurately facilitate the diagnosis or treatment of the patients condition for a quality outcome. Major
elements in the QA operation include:
identifying problems and errors;

assessment of the causes;
designing corrective actions; and
monitoring to assure correction.
QA is an important part of a comprehensive quality system consisting of policies, procedures, and

practices that regularly evaluate all aspects of laboratory testing that can affect test quality and patient
outcomes. Examples include: specimen collection and handling (preanalytical); performance of the test or
examination (analytical); and test reporting and documentation (postanalytical). Collectively the QA
system involves review and evaluation of provider training and competency, safety, equipment function,
supply selection, patient test management (ordering and reporting), procedures for test performance,
mechanisms for quality control of the test, proficiency testing, test results, error detection, investigation
and implementation of corrective actions, and appropriate certification and/or accreditation. This broad
QA approach assures that 1) test procedures and protocols are established and followed; 2) test or
examination systems function properly when patient results are produced; 3) providers perform the test or
examination competently; 4) patient test results are consistent with the patient's clinical presentation; and
5) written records are available to demonstrate procedures and protocols are uniformly followed.
6.1 Microscopy Provider
6.1.1 Training
The person responsible for oversight of the testing should first establish the necessary qualifications
(education and experience) required for someone performing microscopic examinations, and then train
qualified individuals. Training should be developed and provided to all providers who conduct the PPM

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testing. The scope of the position in the workplace should be documented by a position description or
contractual documents to comply with any regulatory agencies or standards of practice in the medical
community.
It is helpful to use a training checklist for each procedure to be performed, so that each critical step in the
process is understood and can be performed correctly before testing patient specimens. The same
checklist may also be used for subsequent competency testing. The training protocols must include
preanalytical aspects such as patient preparation and specimen collection, analytical steps in the
procedure, and postanalytical aspects such as standardized recording of results and correlation with
presenting symptoms. The training activity and results should be documented in provider personnel
records. As part of the training, it is important that providers read and sign all procedure manuals, safety
procedures, policies, and equipment manuals that are associated with microscopic examinations.
6.1.2 Competency Assessment
All providers performing microscopic examinations should be assessed periodically (such as twice during
the first year of testing and annually thereafter) to assure that they maintain competency to perform test
procedures and report test results promptly, accurately, and proficiently.f
Procedures for evaluating the competency of the testing provider may include a documented system of:
direct observation of patient test performance by another experienced provider;

monitoring of the recorded results of testing by a consultant or experienced provider, with
consideration for the correlation of patient information to patient test results;
assessment through review of testing of previously analyzed specimens or proficiency test samples by
the provider responsible for the testing location;
assessment examinations using microscopy images in atlases or textbooks, photographs, or computer
software; and
self-assessment through optional proficiency testing surveys and split samples.
The occurrences of errors detected through the QA system offer another opportunity to assess
competency. Such detected errors should be investigated, analyzed, and corrected according to QA
protocols, with appropriate documentation and follow up to prevent recurrence.
6.1.3 Continuing Education
Continuing education for microscopy providers is essential for maintaining the skills/knowledge required
for performing PPM tests. The individual performing tests should receive regular in-service training and
education appropriate for the type of services offered. Continuing education can take the form of
seminars, journal clubs, and clinical rounds where current developments can be presented and discussed.
Microscopy providers should be encouraged to attend meetings and seminars, and to read current
literature, including textbooks and journals, and commercially available educational packages. Reference
materials such as this document and others referenced here should be made readily available to
microscopy providers.
Because of the unique nature of proficiency testing (PT) samples (discussed later), past PT slides and
pictures can be compiled into an atlas or album and used for training purposes or used as a reference to
help with identifications on patient samples.
f
In the U.S., CLIA requires that competency testing is performed twice in the first year of testing and once per year thereafter as
stated in 493.1413(b)9.

Number 5 NCCLS
6.2 Equipment and Supply Management
6.2.1 Equipment
Accurate and reliable patient test results are achieved only when the equipment used in the testing process
is properly operated and maintained. It is important to ensure that all providers follow the manufacturers
instructions in the owner/operators manual for the operation and maintenance. Training or orientation
sessions must be provided to all intended users and include the proper use and care of the equipment, as
well as any function checks or adjustments that affect the accuracy of the examination.
If not provided by the equipment manufacturer, the development of preventive maintenance protocols is
necessary to ensure that the equipment is operating and functioning properly. Microscopes should be
inspected, cleaned, and checked on a daily basis. This assures that eyepieces, objectives, and condensers
are free of fingerprints and immersion oil, and that the fine and course adjustments are smooth and
accurate. Centrifuges should be inspected and cleaned daily, and require periodic routine rotor rpm and
timer checks, because speed and time have a direct effect on test quality.
A qualified service technician should perform major repairs and an annual equipment performance check,
preferably one recommended by the manufacturer. All maintenance activities should be documented and
maintained as part of the testing sites QA system.
6.2.2 Supplies
Products to be purchased for use in testing should undergo the same evaluation as equipment for needs
assessment and quality evaluation. All reagents must be labeled with the identity, storage requirements,
date of receipt or preparation, the date placed in service, and the expiration date. The characteristics of the
product will determine the time period in which the product can be used after opening. Many products,
such as stains, may have data supplied by the manufacturer that provide details on recommended storage
and useful life. Storage conditions such as temperature, light, humidity, and length of time exposed to an
open environment may affect the performance of test solutions and even microscope slides. Some
products may become more concentrated over time until their functional characteristics have been
materially altered. It is also important to check a new reagent lot against an old reagent lot when possible
with the same sample or control material to compare performance. All glassware used for PPM
procedures should be single-use.
6.3 Patient Test Management
Patient test management involves all phases in the testing process, including specimen collection, test
records, and the system for reporting patient results, as well as ordering, recording, and reporting
activities. It is important to establish procedures that describe the process for patient test management,
from the determination of the need for the test and handling of the patients specimens, through the actual
testing and reporting of the test results. Even in the environment where PPM is an infrequent occurrence,
a description that shows consideration of the necessary elements is needed. The established standard of
practice is that there be a written description of whatever system is used for test requests, criteria and
recording of unacceptable specimens, recording of test results, and reporting patient results which meet all
applicable local and regional requirements.
The significant elements of patient test management include:
criteria for patient preparation and specimen collection, labeling, preservation and transportation,
ensuring optimum patient specimen integrity and identification throughout the testing process;
if a requisition is used, the information necessary and relevant to complete the testing;

Volume 23 HS2-A
how confidentiality is maintained;

complete, useful, and accurate information produced for the interpretation or utilization of test results;
the timeliness of reporting test results;
a reliable reporting system and maintenance of records, including a specified time period that results
will be kept on file; and
demonstrable retrieval of results showing the date of testing and who performed the test.
The results should be retained for a minimum of two years; however, retention time may vary depending
on local and regional regulatory requirements and standards of practice for the medical community. The
forms used for patient test management will differ from site to site, but the essential elements remain the
same. Where appropriate, good laboratory practice specifies that a record should be made of the patients
name and unique identifier; ordering provider; documentation of the test request; date and time sample
was collected and by whom; date and time sample was tested and by whom; and the test results. In the
PPM environment, the ordering provider and person performing the microscopic examination are often
the same, as are the date collected and the date tested, simplifying the elements required. All of this
information can be included on one form such as the patient chart, a testing log, or a laboratory
request/result form.
6.4 Procedure Manual
Of all the elements in a quality assurance program, the preparation of a comprehensive procedure manual
is extremely critical.g The procedure manual is the compilation of individual procedures. The procedure
manual is essential to provide direction with instructions for the microscopy provider. Each individual
procedure should provide a standardized description with detailed instructions for performing the test. A
procedure manual is also a valuable resource for training new providers, verifying provider competency
(see Section 6.1.2), or troubleshooting testing problems when they arise.
As an adjunct to written procedures, these guidelines (as well as access to visual examples) are useful for
learning and establishing provider competency. It is recommended that a provider review references and
acquire those that can be used to develop confidence in visual recognition of the microscopic elements, as
well as for future reference for less common observations.
The procedure manual should include the following information:
Test procedures Every test performed at the testing site should have its own unique set of
instructions, including collecting and handling the specimen, testing the specimen, and reporting the
test results. Information on reporting abnormal as well as normal results should be included.
Quality assurance plan Addresses overall quality issues such as patient test management, PT, QC,
safety, and personnel policies.
Procedural updates as necessary Any changes to the original procedure should be documented in
writing, dated, and circulated for review by testing providers.
Provider procedure review process All microscopy providers must review the procedure manual,
including an initial approval of the procedure by the provider responsible for the testing site, who
should sign each procedure. It is helpful to include a log sheet in the front of the manual where
microscopy providers can sign off with date and signature that they have completed their reviews of
current procedures, as well as for future revisions as they occur.
g
In the U.S., as required by CLIA.

Number 5 NCCLS
For more information, please refer to the most current edition of NCCLS document GP2 Clinical
Laboratory Technical Procedure Manuals.
6.5 Quality Control
The purpose of quality control (QC) is to monitor the analytical process to determine that the test system
is operating within pre-established limits, thus ensuring the accuracy of each examination or measurement
performed on a patient specimen. The function of a quality control protocol is to give testing personnel,
such as microscopy providers, this guidance. Testing sites that are subject to the control of regulatory
agencies must obtain the specific requirements from those groups.h
There is a limited selection of quality control materials for this particular group of microscopic tests.
Check with suppliers for information on purchasing manufactured control material. In-house control
material can also be used to verify accuracy for these procedures. A patient specimen that contains
multiple cell types can be used as a positive control. For example, a positive urine control could be a urine
specimen with squamous epithelial cells, white blood cells, and/or red blood cells. A negative urine
control could be obtained by using a patient sample that is virtually clear of any cellular material.
Because the survival of positive morphological structures is limited, testing sites that choose to use fresh
patient specimens for control material need to implement a program that is constantly on the look-out
for positive materials.
This process applies to any PPM procedure. For example, additional slides can be made from patients
with positive and negative nasal smears, pinworm preps, and fecal leukocyte exams for future use as
controls. The slide preparations can be kept at room temperature.
The testing site must establish and follow written quality control procedures for each PPM procedure. For
PPM tests there are few available manufacturers' instructions except those which may be provided with
purchased reagents or control materials. The following are steps to be taken when the expected results of
control materials are not achieved:
Review all steps of the procedure to determine if any technical error has occurred. Take corrective
action if necessary.
Repeat the analysis even if no cause of error has been determined. If the new analysis produces the
expected result, the test is considered satisfactory and the patient test can proceed. If on repeat, the
results are still incorrect, no patient testing should be reported until the problem is identified and
resolved.
The following is a list of the most common causes of unexpected control results and their corrective
actions:
error in technique, including anything from improper handling to using the wrong reagent. Review
every step of the testing process;
improper reconstitution or mixing of control materials. Prepare new control material;
incorrect, deteriorated, or improperly made reagents or diluents. Acquire or prepare new reagent;
cross-contamination by other samples, reagents, or water source. Observe when problem occurred.
Change testing protocol; test one sample at a time; and
QC documentation. Verify lot number, date in use, acceptability of results, tests performed, and QC
acceptability limits.
h
In the U.S., all PPM testing sites are subject to CLIA regulations and must comply with all applicable requirements.
Volume 23 HS2-A
A monthly assessment of recorded quality control information should include a written review of the
proper performance of designated QC procedures and documentation of corrective actions. A QC
checklist can be incorporated as part of a monthly QA review which includes a check off of each aspect
of the quality assurance plan.
6.6 Proficiency Testing
Proficiency testing (PT) programs were developed to investigate the variation in results of testing among
different testing sites, assess the ongoing accuracy of test results, and facilitate education through the
resolution of problems causing proficiency testing failures. PT programs are administered by PT
providers. A testing site voluntarily enrolls with a PT provider. A PT program consists of stable
specimens sent to subscribing testing sites, which submit their independently derived results to the
program provider for assessment. Examination of the peer group data gives a guide to accuracy and
methodology comparison. PT should be performed on all PPM tests when available as part of the QA
program to verify and assess accuracy, measure precision, and detect errors. PT samples should be tested
in the same manner as patient specimens; therefore, the handling, preparation processing, examination,
and documentation of results should be performed by those providers who routinely perform the patient
testing. The PT provider grades the results as pass or fail and then sends the grades back to the PPM
provider. The grades should be reviewed and shared with all providers as part of the testing sites QA,
continuing education, and competency evaluation systems.
If proficiency testing is performed on PPM tests, there are several documentation requirements that
should be followed to assure good performance:
Results must be recorded accurately on the PT program's result form. Transcription errors are causes
for PT failure. The significance of this type of error is apparent if one considers the consequences of
transcribing patient results incorrectly.
The entire result form should be copied and retained. When a failure occurs, it is a good idea to
verify that results were entered correctly by the PT program by comparing the evaluation report to
those results documented on the copy of the result form that had been sent to the PT program.
Record the remedial actions taken for PT results that are not graded acceptable, and use the
information to improve performance.
If no PT program is available, the testing site may choose to monitor providers proficiency by
exchanging blind samples with other test sites twice each year. For more information, please refer to the
most current edition of NCCLS document GP29Assessment of Laboratory Tests When Proficiency
Testing is Not Available.
6.7 Relationship of Patient Information to Patient Test Results
It is a vital part of the quality assurance postanalytical process to consider the relationship of known
patient information to the produced patient test results. In addition to the assessment of the accuracy and
reliability of the testing process, a review of test results must include assessing the logic of the
relationship of test results to the patient's age, sex, diagnosis, and to other test results. Since it is intended
that the practitioner performing the test is the same as that assessing the patient's condition, it may appear
obvious that the results would be evaluated for consistency with relevant clinical information. When
results appear inconsistent or absurd, the preanalytical phase of testing must also be examined (as well as
the analytical phase) to determine if the patient sample was correctly collected, identified, and tested
properly.
The consideration of the expected results with the actual results may also include later comparisons with
data obtained through alternate testing methods (i.e., correlation of urine microscopic bacteria results with

Number 5 NCCLS
urine culture results). Such comparisons provide additional opportunities to examine concerns with all
phases of provider-performed microscopy testing. Quality assurance reviews should include assessments
as to whether or not the inconsistencies have been resolved after implementing solutions. Unresolved
problems should prompt retesting, if possible, or alternate diagnostic procedures to confirm or rule out the
suggested diagnosis.
6.8 Accreditation
Accreditation is a voluntary process by which a testing site chooses to be inspected and guided by a
professional peer group. It is not a mandated, regulated requirement, but is intended to improve quality
and verify that a defined standard of practice exists when accreditation is achieved. An accreditation body
will provide and require adherence to standards of performance including specific requirements for the
testing sites quality assurance plan. Accreditation also offers opportunities for continuing education,
operational assessment, and performance improvement. i
7 Urine Sediment Examinations
7.1 Principle
The study of urine is one of the oldest testing procedures utilized. It yields a great deal of information
quickly and economically. Urine tests need to be carefully performed and properly controlled.
Examination of the urine may be considered from two general standpoints: 1) diagnosis and management
of renal or urinary tract disease; and 2) the detection of metabolic or systemic diseases not directly related
to the kidney.1
7.1.1 The Urine Sediment Microscopic Examination
The microscopic examination of urine is the most common testing procedure utilized for the detection of
renal and/or urinary tract disease. Interpretation of urine sediment requires time, skill, training, and
experience acquired through the use of various microscopic methods and pathophysiologic correlation of
the sediment findings with the macroscopic results and clinical status of the patient. In order to practice
with competency, microscopy providers should be knowledgeable of numerous morphologic entities, e.g.,
organisms, hematopoietic and epithelial cells, and casts. Also, providers should be alert regarding the
clinical relevance of urine findings, as well as the chemical abnormalities associated with microscopic
interpretations.1 A review of available information, including physicochemical results, is essential before
reporting the microscopic examination, if the information is available. The data contained in these reports
should substantiate the microscopic results and vice-versa; e.g., calcium oxalate crystals are associated
with a neutral to acidic pH, not an alkaline pH. Any discrepancies should be resolved before the final
results are reported.
i
Please note that in the U.S., accreditation is a voluntary process. However, in the U.S., laboratory certification is mandated by
CLIA. U.S. laboratories may choose to obtain a certificate for PPM procedures if only PPM and CLIA-waived testing is
performed. A certificate of compliance or a certificate of accreditation is required for the performance of moderate and/or high
complexity testing. If a certificate of accreditation is obtained, the site must be accredited by an accrediting organization
approved by the Centers for Medicare and Medicaid Services (CMS). Other accreditation may be voluntarily obtained outside of
the CLIA requirements. Laboratories seeking accreditation by a CMS-approved organization to meet CLIA requirements are
mandated to meet the organization's standards which have been determined to be equivalent to or more stringent than CLIA
requirements.

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7.1.2 Formed Elements of Urine
Centrifuged urine sediment contains all the insoluble materials (commonly referred to as formed
elements) that have accumulated in the urine in the process of glomerular filtration and during passage of
fluid through the tubules of the kidney and lower urinary tract. Cells found in urine come from two
sources: 1) desquamation or spontaneous exfoliation of epithelial cells lining the upper (kidney) and
lower urinary tract and adjacent structures; and 2) cells from the circulating blood (leukocytes and
erythrocytes). Casts formed in the renal tubules and collecting ducts are the other formed elements
frequently seen.
Organisms (bacteria, fungi, viral inclusion cells, parasites) and neoplastic cells represent elements foreign
to the urinary system, and proper identification of these elements may provide important diagnostic clues
as to the etiology of certain urinary system disorders.
7.2 Materials
7.2.1 Supplies
The primary collection container and transport container, tubes, and slides should be labeled in a way that
ensures proper patient identification. All materials used to perform a urine microscopic examination
should be particle-free. All glassware should be specified as single-use.
7.2.1.1 Collection Devices
Please see the current edition of NCCLS document GP16Routine Urinalysis and Collection,
Transportation, and Preservation of Urine Specimens for a description of proper containers for urine
collection and transportation, and other related information.
7.2.1.2 Centrifugation Tubes
A proper tube for urine centrifugation has the following features:
clear plastic or single-use glass to permit enough strength to avoid breakage during centrifugation;
volume graduations to ensure a standardized volume of urine;
closures to reduce hazards from spillage and centrifuge aerosols;
conical or constricted bottom to concentrate sediment;
freedom from interfering chemicals; and
labels to ensure proper identification.
NOTE: Do not reuse centrifugation tubes.
7.2.1.3 Transfer Pipettes
Disposable transfer pipettes are recommended to reduce the biohazards associated with resuspending and
transferring the sediment; their reuse is not recommended. Transfer pipettes should be clean and particle-
free. Single-use, glass transfer pipettes may be used.
7.2.2 Equipment
The equipment found in the testing site requires routine quality control checks and preventive
maintenance. Maintenance should be documented on forms kept at the testing site.

Number 5 NCCLS
7.2.2.1 Microscope Slides/Viewing Devices
Commercially available, disposable, standardized microscope slides or viewing devices with calibrated
chambers are preferred. Single-use glass microscope slides and cover slips are acceptable.
NOTE: It is not recommended to reuse slides.
7.2.2.2 Centrifuge
A centrifuge with a self-locking lid (when rotor is spinning) and an ambient temperature (15 to 25 C) is
desirable for urine sedimentation.
Calibrate centrifuges to provide a relative centrifugal force (RCF) of 400g. Consult the manufacturers
instructions for the recommended protocol. Calibration should be performed at periodic intervals
determined by the testing site depending on its use. At a minimum, centrifuge calibration should be
performed annually. Relative centrifugal force (RCF or G) is calculated by using a rotor with a known
radius (R in cm) at a given speed (N is revolutions per minute) where G = 0.00001188RN2.
7.2.2.3 Microscope
A microscope with 10x and 40x bright-field objectives, or a microscope equipped with phase objectives
and condenser, is recommended.
7.2.3 Reagents
All reagents must be properly labeled, dated with expiration date, and stored. When new reagents are
opened or prepared, positive and negative control checks should be performed to ensure proper
performance characteristics of the reagent. Results of the quality control checks with lot numbers and
expiration dates of the reagents should be recorded. Reagents shall not be used after their expiration dates.
7.3 Specimen Collection
7.3.1 Specimen Type
The type and quality of the urine specimen greatly affects the results of the microscopic examination. The
specimen of choice for a microscopic evaluation is the first morning urine. This urine will be the most
concentrated, which maximizes recovery of sediment elements. It is extremely important that information
about the type of specimen, the time of collection, and the physical/chemical results be noted when the
need for a microscopic examination of urine is determined by the provider.
A minimally sufficient quantity of urine to permit both macroscopic and microscopic evaluation is usually
considered to be 12 mL (50 mL is preferred). Urine specimens from infants may necessitate the use of
smaller volumes.
Urine specimens that are held unrefrigerated for more than two hours are not acceptable for microscopic
examination. Casts and red and white blood cells are especially susceptible to lysis in urine specimens
with a low specific gravity (<1.010) and in urine specimens with alkaline pH (>7.0).
The urine specimen should be collected in a clean, leakproof, disposable container. The specimen should
be free of fecal contamination and contain no bathroom tissue or other foreign materials. If these criteria
are not met, the provider performing the test should seek another specimen that meets the criteria.

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7.3.2 Specimen Handling
The accuracy of a urinalysis is dependent on the quality of the specimen submitted; therefore, care should
be taken to submit a properly collected and transported urine specimen. For more detailed information,
see the most current edition of NCCLS document GP16Routine Urinalysis and Collection,
Transportation, and Preservation of Urine Specimens.
To ensure suitability for an analysis, the urine specimen should be inspected as soon as possible (i.e., as
part of the patient examination) following collection. Consider the following points when ensuring the
suitability of the specimen:
the specimen is labeled with the patients name;

acceptability of the elapsed time between collecting the specimen and its receipt at the testing site
(i.e., less than two hours);
suitability of the container and its condition (e.g., closure in place);
adequate volume and absence of contaminating materials; and
presence or absence of chemical preservatives consistent with intended specimen use.
7.3.3 Documentation
Documentation should indicate the type of urine specimen collected, the date and time of collection, and
an indication of specimen refrigeration prior to testing.
The documentation, on a form or in the patients chart, should also include any specific situations that
might influence the results of the analysis (e.g., preservatives for specimens; medications, such as aspirin,
vitamins, or antibiotics; presence of menstrual period; strenuous exercise prior to specimen collection;
additional pertinent clinical information).
7.3.3.1 Label
The container should be designed to accept a label that will adhere during refrigeration. The label should
include sufficient space to include the patients full name, unique identification number, date and time of
specimen collection, and the name of the preservative in the container, if applicable. To ensure proper
specimen identification, place labels on the container, not on the closure.
7.4 Testing Procedure
(1) Pour 12 mL of well-mixed urine specimen into a graduated, disposable centrifuge tube.
(2) Spin tube of urine at 400 g for 5 minutes.
NOTE: Higher RCF and longer centrifugation times, although useful in recovering cells, are apt to
break up cellular casts.1
(3) Carefully decant the supernatant. The final volume used to resuspend the sediment may vary, but
should remain a constant within any given testing site.
(4) Gently resuspend the sediment in the remaining supernatant.
(5) A drop of resuspended sediment is placed on a glass slide and cover-slipped for examination. Allow
urine to settle for 30 to 60 seconds before examining.

Number 5 NCCLS
(6) Examine with low- and high-power objectives. Subdued light (achieved by lowering illumination
intensity) or phase-contrast illumination will be required to detect sediment entities with a low
refractive index. The fine focus should be varied continuously while scanning, systematically
progressing around the entire examination area while being careful to examine along the edges for
casts.1
(7) The urine sediment should be examined microscopically on low power for crystals, casts, and
epithelial cells. Ten fields should be counted and the average number of casts and epithelial cells
reported per low-power field (e.g., two hyaline casts/lpf). The type and number of crystals per low-
power field should also be reported (e.g., few calcium oxalate crystals/lpf, rare uric acid
crystals/lpf).
(8) The urine sediment should be examined microscopically on high power for red cells, white cells,
and renal tubular cells. Ten fields should be counted and the average number of cells per high-
power field (e.g., 20 RBC/hpf) reported.
(9) The presence of bacteria, yeast, trichomonads, and mucus should also be noted during high-power
examination.
(10) Review the entire report, including physical, chemical, and microscopic data and correlate with
available clinical information. Discrepancies should be resolved before releasing the report.
Although values will vary depending on the standardization system used,1 a normal value for the
procedure used should be provided for interpretation of results.
Consistency in all aspects of the microscopic examination is essential to the production of meaningful
results. A properly written and maintained procedure manual helps ensure that all microscopy providers
perform the microscopic examination in the same manner. Each provider should evaluate the sediment
using the same procedure, look for the presence of the same sediment entities, and use the same criteria
for identification.
7.4.1 Methods for Examining Urine Sediment
7.4.1.1 Bright-field Microscopy of Unstained Urine
Subdued light is needed to delineate the more translucent, formed elements of the urine such as hyaline
casts, crystals, and mucous threads. Identification of leukocytes (neutrophils, eosinophils, lymphocytes),
histiocytes, renal epithelial cells, viral inclusion cells, neoplastic cells, and cellular casts may be very
difficult in unstained preparations. Phase-contrast microscopy is strongly recommended for the detection
of casts.1
7.4.1.2 Phase-Contrast Microscopy
Many microscopy providers prefer to use phase-contrast microscopy for the detection of more translucent,
formed elements of the urinary sediment. Such elements, notably casts (but also mucous threads and
bacilli), may escape detection using ordinary bright-field microscopy. Phase-contrast microscopy has the
advantage of enhancing the outlines of formed elements, making detection simple.2 A microscope
equipped with 10x and 40x phase objectives plus a 40x bright-field objective and the appropriate rotation
phase/bright-field condenser is most useful. Scanning time is decreased, and the yield is increased.

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7.4.1.3 Stains
Stains such as Sternheimer stain to facilitate identification of epithelial cells and casts and the Sudan III
stain to assist in identifying oval fat bodies, fat droplets, and fatty casts have gained popularity in many
laboratories. Administer the stain according to manufacturer's instructions.j
7.4.2 Examination of Urine Sediment
Sediment entities that should be identifiable using a urine microscopic examination include those listed in
Table 1.1
NOTE: Advanced microscopy skills can be required for the identification of other elements.
See Appendix A for detailed descriptions of the formed elements to assist in their identification in the
urine sediment.
NOTE: Reference values for the procedure outlined in Section 7.4 are cited in the reference given.
Testing sites using standardized systems (described in Section 7.4.3) should refer to the information
provided by the manufacturer.
Normal or reference values for formed elements will vary from one site to another because of 1) the
variation in concentration of random voided urine specimens; and 2) the different methods used to
concentrate the sediment by centrifugation. Individual testing sites should establish their own reference
values.
7.4.3 Standardized Systems
Standardized urinalysis systems are available commercially. All systems provide a capped centrifuge
tube, transfer pipettes that will retain a specific volume of sediment, supravital stainj, and choice of
standardized slides that will hold a specific volume of concentrated urine sediment. Each manufacturer
also markets a urinalysis control. All systems utilize acrylic plastics that have excellent optical properties.
When choosing a standardized system, each testing site will have to make evaluations about the patient
population and degree of sensitivity desired. Other factors to consider include ease of loading, the number
of focal planes, settling properties, and the optical properties of the slide.1
The importance of standardization in microscopic urinalysis cannot be overemphasized. Standardization

is essential for precision in urinalysis, providing a consistent basis on which the provider can make
interpretive decisions.
7.5 Quality Control
Users must adhere to all applicable regulatory requirements and manufacturers instructions.
Maintenance of the microscope is a quality control activity and should be performed at a frequency
proportional to usage (see Section 5.2.3).
7.5.1 Microscopic Examination Quality Control
Quality control of the microscopic examination has been lacking if not totally overlooked. Controls used
to ensure centrifuge speed and timing are appropriate to obtain an adequate sample for urinalysis.
Commercial solutions are available that provide either stabilized or simulated erythrocytes and
leukocytes. At the time of this printing there are no commercially available products that contain
epithelial cells and casts.
j
In the U.S., stains are not indicated for the microscopic analysis of urine sediment in PPM procedures under CLIA.
Number 5 NCCLS
Duplicate urine testing can be used as a precision check for the identification of casts, renal cells, and
other formed elements.
The use of in-house urine specimens as known controls on a daily basis is strongly suggested.1
If there is a disagreement about the presence or quantity of a microscopic element, the examination
should be repeated. If necessary, referral to a reference laboratory should resolve any discrepancy. Each
testing site should establish criteria for reviewing abnormal sediment results. Each testing site will also
have to establish its own acceptable range of performance. This is difficult when so much of urine
microscopy involves the interpretative description of microscopic elements.
It is also recommended that reference texts, atlases, charts, and posters are available for reference.
7.5.2 Recording Quality Control
All providers should use the same terminology and report results in a standard format. Unexpected control
results should be identified, and appropriate corrective action should be taken and documented.
In establishing a quality assurance program the provider should ensure that the following information is
documented:
specimen collection consistently results in the correct specimen type, adequate specimen volume for
the requested analysis, the use of suitable containers, and proper labeling;
specimen transport to the testing area is timely and in accordance with recommended procedures; and
specimen processing ensures prompt inspection and correct storage procedures (e.g., refrigeration and
protection from heat and light).
7.6 Reporting Results
All providers at a testing site who perform microscopic examinations should use the same terminology,
reporting format, and reference values. The individual provider should make decisions about which
formed elements should be reported and quantitated. Documentation should include name, identification
number, date of testing, and initials of person doing the test.
8 Direct Wet Mount Preparations and Potassium Hydroxide (KOH) Preparations
8.1 Principle
Microscopic observation of unfixed wet mounts of clinical specimens, either stained or unstained, can
be useful for the rapid detection of the presence of bacterial, fungal, and parasitic organisms. Presumptive
identification can be made, based on morphology and motility. The presence or absence of white blood
cells and clue cells may also be demonstrated, and a number of well-recognized pathologic conditions
may be identified.

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Table 1. Interpretation of Microscopic Urinalysis
Finding Abnormal Due To Abnormal Due To Reference Values

Systemic Disease Genitourinary Disease
Crystals Rarely: Rarely None present
Refer to Appendix A Usually not significant
Red Blood Cells Rarely: Glomerulonephritis Less than 5/hpf
(RBCs) Sickle cell trait Tumors More with menstrual
Bleeding disorders Stones period
Anticoagulant use Rarely:
Trauma Infection (with WBCs)
White Blood Cells Rarely: Infection of bladder or Less than 5/hpf
(WBCs) Viral infections urinary tract
Radiation therapy Kidney infection (with
Chronic inflammation WBC casts)
Renal transplant
rejection (especially
large lymphocytes)
Renal Tubular Epithelial Rarely: Acute tubular necrosis Rare
Cells Cytomegalovirus Nephrotic syndrome
disease
Transitional Epithelial Rarely: Infection Small numbers
Cells Radiation therapy Urinary tract lesions
Urethral/Ureteral
catheterization
Squamous Epithelial Vaginal exudate (with No 1-2/lpf
Cells bacteria and/or yeast) More with vaginitis
Hyaline Casts Rarely: Proteinuria Rare
Dehydration Acute tubular necrosis
Heavy exercise
Psychological stress
Granular Casts Rarely: Glomerulonephritis, None
Severe dehydration interstitial
Prerenal azotemia Pyelonephritis
Viral infection
Lead poisoning
Cellular Casts No RBC: nephrotic None
syndrome
WBC: Pyelonephritis
Renal Tubular
Epithelial: Acute tubular
necrosis, transplant
rejection
Fatty: Nephrotic
syndrome
Waxy: Progressive renal
disease
Microorganisms No 100,000/mL: always None
infection*
* Observation of microorganisms, indicating infection, should be confirmed by culture.

Number 5 NCCLS
8.2 Materials
8.2.1 Supplies
Supplies used for the procedure include the following:
frosted-end, glass microscope slides;

cover slips;
cotton-tipped swabs;
capped tubes; and
pH paper.
8.2.2 Equipment
Equipment used for the procedure includes the following:
microscope equipped with 10x and 40x objectives;

ocular micrometer (optional); and
slide warmer.
8.2.3 Reagents
Reagents used for the procedure include the following:
Sterile physiologic saline (i.e., sodium chloride, 9 grams/L [0.9%]); and

potassium hydroxide, 10 grams/dL (10%).
NOTE: Concentrated KOH is a highly corrosive liquid that must be used with appropriate personal
protective equipment including gloves and eye/face protection. It is recommended that for use in the PPM
environment, 10% KOH be purchased commercially or obtained from pharmacy/laboratory sources.
8.3.1 Conditions for Patient Preparation
Specimens for direct wet mount are usually collected during an internal examination of the female genital
tract. Specimens from other sources such as skin scrapings, hair, and nail analyses are collected during an
appropriate exam.
8.3.2 Specimen Type
Specimen types include swabs of the vaginal mucosa and vaginal pool or scraping of suspected skin area.
Swabs used to collect specimens should be placed into tubes containing approximately 0.5 mL sterile
physiologic saline, then resealed. To preserve the motility of Trichomonas, specimens should not be
refrigerated and should be examined as soon as possible following collection. All tubes must be properly
labeled with the patient's name and identification number.

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Specimens from the vaginal tract should be tested with pH paper before being placed in saline. A pH
greater than 4.5 is associated with bacterial vaginosis or trichomoniasis. The pH should be documented
on the report.
(1) Place one drop of saline on one slide, and one drop of 10% KOH on the second slide.
(2) Add specimen to each slide.
(3) Check the KOH slide immediately for a fishy, amine odor. (Note odor on report.)
(4) Cover each specimen with a cover slip to exclude air bubbles.
(5) Examine the saline wet mount with the 10x objective for epithelial cells and any budding yeast or
pseudohyphae.
(6) Examine the saline wet mount with the 40x objective and quantitate organisms and cells per high-
power field (hpf), using the following scheme:
QUANTITATION, DIRECT EXAMS

Rare Less than 10 organisms or cells/slide
Occasional Less than 1 organism or cell/10 hpf
1+ (Very Scanty) Less than 1 organism or cell/hpf
2+ (Scanty) 1 to 5 organisms or cells/hpf
3+ (Moderate) 6 to 30 organisms or cells/hpf
4+ (Profuse) Greater than 30 organisms or cells/hpf
(7) Yeast/fungi can best be demonstrated on a KOH mount. The addition of 10% KOH to the clinical
specimen will dissolve tissue cells and keratinized material to allow better visualization of fungal
elements. Heating is helpful to speed the dissolving process, especially for skin and nail specimens.
Any gentle heat source is usable.
(8) Examine the KOH preparation under low power for yeast pseudohyphae and under high power (40x)
for smaller blastospores. Refer to Appendix B for descriptions to assist in the presumptive
identification of fungi.k
8.4.1 Microscopic Examination of Direct Wet Mount Specimens
Cells and organisms that should be identifiable in a microscopic examination of a direct wet mount
specimen of vaginal fluid and the interpretation of those findings are listed in Table 2. See Appendix B
for a more detailed description of components found in vaginal fluid and KOH preparations.
k
This level of identification is not included in the wet mount exam under the PPM subcategory of moderate complexity in CLIA,
nor does it include enumeration of elements seen microscopically. Under CLIA, the PPM wet mount includes only direct wet
mount preparations for the presence or absence of bacteria, fungi, parasites, and human cellular elements.

Number 5 NCCLS
Table 2. Interpretation of Microscopic Observations of Vaginal Fluid3
Finding Abnormal Abnormal Due Abnormal Due Abnormal Due Reference

Due To To Vaginal To To Values
Bacterial Candidiasis Trichomonas Desquamative
Vaginosis Vaginitis Inflammatory
With Vaginitis
Gardnerella
White Blood Rare 3+ to 4+ 2+ to 4+ 3+ to 4+ 2+
Cells
Lactobacilli Rare Present Present/Absent Reduced/ Predominant
Absent
Clue Cells Occasional to Absent Absent/Present Absent
4+
Other Cells Large clumps of Occasional Absent
epithelial cells Parabasal/ (except
Basal cells RBCs during
menses)
Other Predominant Budding Yeast Trichomonas 2+ bacteria Other
Organisms Gardnerella Pseudohyphae Frequently lactobacilli
morphotypes associated with subgroups
other Occasional
organisms yeast
Other pH > 4.5 pH > 4.5 pH > 4.5 pH < 4.5
Findings Amine Any finding Vaginal
Positive associated with erythema
Lava Flow other vaginitis-
on KOH causing
preparation organisms
8.5 Quality Control
Commercial controls are not currently available for wet mount preparations. Multiple examiners of the
same specimen can help determine accuracy of the test results.
Providers should use the same equipment, procedure, reporting format, terminology, and reference values.
The individual provider should make decisions about which elements should be reported and quantitated.
See Appendix B for more detailed descriptions of constituents found in vaginal fluid.
8.7 Limitations of the Procedure
Limitations of the procedure include the following:
Many intravaginal medications will leave oil droplets which can make interpretation of direct wet
mounts difficult. It is often useful in such situations to perform a gram stain.

Volume 23 HS2-A
Many vaginal infections are uncomplicated and can be diagnosed using wet mount/KOH alone. If
there are unusual circumstances, a gram stain can be a very helpful aid.l
9 Pinworm Examinations
9.1 Principle
Enterobiasis or pinworm infestation is the most common intestinal nematode in humans and quite common
among school-aged children. It is very contagious by direct ingestion or inhalation of the ova.
Because only a small percentage of infected persons have demonstrable eggs in their stools, the standard
laboratory examination for ova and parasites is rarely diagnostic. A direct specimen collected from the
perianal region can contain the eggs of Enterobius vermicularis, as it is to this area that the gravid female
migrates and lays large numbers of eggs while the host is resting.
The most common test to detect ova is the cellulose tape test or cellulose acetate cellophane tape test
developed by Graham et al.4
The identification of the adult worms is done by the collection of fecal material by digital rectal
examination.
9.2 Detection of Ova: Cellophane Tape Test
This method consists of applying an adhesive tape onto the perianal surface to capture the eggs. The
adhesive surface is mounted onto a glass slide and then examined under the microscope for identification
of the eggs. The tape captures no adult forms.
The eggs are ovoid, elongated, smooth and transparent, measuring approximately 50 x 30 m. Under the
microscope, eggs appear asymmetric and flattened on one side. (See Figure 2.) Ova are transparent, and
many contain well-developed larvae.
9.2.1 Materials
9.2.1.1 Supplies
adhesive, clear cellophane tape of - or 1-inch width;

wooden applicator/tongue depressor;
clean container/specimen cup; and
microscope slide.
9.2.1.2 Equipment
Equipment used for the procedure includes a microscope with 10x and 40x objectives.
l
In the U.S., under CLIA, gram stains cannot be performed as a PPM procedure.

Number 5 NCCLS
9.2.1.3 Reagents
Reagents are not required for the identification of ova via the cellophane tape test.
9.2.2 Specimen Collection
9.2.2.1 Conditions for Patient Preparation
The specimen should be collected from the anal verge in the morning, right after the person wakes and
before moving around or washing. Written instructions should be provided to the person assisting in
collection, including a precaution to wash hands after collection and handling. Specimens should be
obtained before therapy has begun.
9.2.2.2 Specimen Type
Specimens are collected by adherence of the eggs to the sticky surface of the tape. Commercial paddles
with a sticky surface mounted in a capped tube are also available for specimen collection.
For collecting the eggs with cellophane, one-and-a-half to two inches of transparent, adhesive cellophane
tape are rolled and fixed at the end of a tongue depressor or wooden stick with tape sticky side out.
With gloved hands, spread the anal folds apart and firmly press the adhesive tape or paddle onto the
patients anal skin in several regions for 10 to 15 seconds, and then remove gently.
9.2.2.3 Specimen Handling
The tongue depressor or paddle should be placed in a capped container for transport. The test is to be
performed the same day of collection. Specimens may be kept at room temperature. Pinworm eggs are
very infectious; caution should be used to avoid cross-contaminating individuals involved in handling the
specimen.
9.2.3 Testing Procedure
(1) After collecting, the cellophane tape is mounted onto a glass slide or kept at room temperature rolled
with sticky side out in a covered, clean container or specimen cup. Label slide with the patient's
name and identification number.
(2) Examination of the mounted glass slide is performed in conventional bright-field or phase-contrast
microscopy using a 10x to 40x dry objective.
(3) The eggs are distinguishable by their characteristic shape, size, and transparent appearance. (See
Figure 2.)

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Figure 2. Egg. From the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. Reprinted with
permission.
9.2.4 Quality Control
No direct controls are available except for reference illustrations of E. vermicularis eggs and
differentiating features from other intestinal nematodes.5
9.2.5 Reporting Results
Report positive or negative for pinworm ova. Report adult pinworms if found.
9.2.6 Limitations of the Procedure
Multiple specimens, collected over several days, may be necessary before confirming diagnosis. Only five
to ten percent of infected patients pass demonstrable eggs into their stools. Because the female worms
may not lay eggs every night, three to six consecutive, daily specimens are recommended.
9.3 Detection of Adult Parasites
For this procedure, a wet preparation is made in normal saline by applying the fecal material with a
wooden stick onto a slide.
The adult Enterobius vermicularis is a whitish roundworm, approximately 2 to 5 mm and 8 to 13 mm

long for the male and female, respectively. Under microscopy, it can be recognized by a cuticular, wing-
like alar expansion, caudal alae at the anterior portion, a ventrally curved posterior end for the male,
and a long, pointed tail for the female. The worm has a complete digestive system with a well-developed
esophagus. (See Figure 3.)
9.3.1 Materials
9.3.1.1 Supplies

Number 5 NCCLS
sterile surgical gloves;

wooden applicator;
glass slide and cover slip; and
nail polish or mounting medium (optional).
9.3.1.2 Equipment
Equipment used for the procedure includes a microscope with 10x and 40x objectives.
9.3.1.3 Reagents
The reagent used for the procedure is saline solution.
9.3.2 Specimen Collection
9.3.2.1 Condition for Patient Preparation
Female pinworms can be observed directly on the perianal area at night when they migrate to deposit
eggs.
A parent can be instructed to examine a potentially infected child with a flashlight while the child is
sleeping. Alternately, the provider examines the patient in the practitioner's office. The patient is
positioned lying on his or her side, while gowned, and drawing the knees forward.
9.3.2.2 Specimen Type
The fecal material is collected by inserting a gloved finger into the anus and removing fecal material from
the anus inside rim. Care must be taken to observe the potential for contamination when obtaining a
specimen in this fashion.
9.3.2.3 Specimen Handling
After collecting the fecal material, the provider should put it in a small container and maintain the
specimen at room temperature until examined.
9.3.3 Testing Procedure
(1) Make a wet preparation with a drop of saline on a microscope slide labeled with the patients name,
using a wooden stick. It is recommended that the preparation be covered with a cover slip and sealed
with nail polish.
(2) The specimen should be examined soon after collection before drying.
(3) The examination of the wet preparation is performed under conventional bright-field or phase-
contrast microscopy using 10x and 40x dry objectives.
The adult female E. vermicularis of up to 13 mm is distinguished by its round shape, whitish transparent
appearance, and the marked distention due to egg development. (See Figure 3.) Parasites are easily
disrupted and occasionally surrounded by eggs.

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Female Male
Figure 3. Adult Worms. From the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand.
Reprinted with permission.
9.3.4 Quality Control
No direct controls are available except for reference illustrations of E. vermicularis adult worms and
differentiating features from other intestinal nematodes.5
9.3.5 Reporting Results
Report positive or negative for pinworms.
9.3.6 Limitations of the Procedure
A negative result does not preclude pinworm infestation. The cellophane tape test for detection of the ova
should be pursued if clinically indicated. See also Section 9.2.6.
10 Fern Test
10.1 Principle
The fern test is used to evaluate the pregnant female for the presence of amniotic fluid in vaginal
secretions. The provider can rely on a combination of patient history, vaginal fluid pH of 7.0 or greater,
and a positive fern (crystallization) test in deciding whether the amniotic sac has ruptured. The test is
based on the ability of amniotic fluid to form a fern pattern when air-dried on a glass slide. See Section 11
for postcoital evaluation of cervical mucus.

Number 5 NCCLS
10.2 Materials
10.2.1 Supplies
glass microscope slides; and

transfer pipette.
10.2.2 Equipment
Equipment used for the procedure includes a microscope with a 40x objective.
10.2.3 Reagents
Reagents are not required for the fern test.
The patient is examined in the dorsal lithotomy position.
10.3.2 Specimen Type
A vaginal liquid pool specimen is collected by transfer pipette after insertion of a sterile speculum into the
vaginal vault.
Specimens are tested immediately. Once the specimen on the slide is dry, it does not need to be read
immediately.
(1) Obtain a few drops of vaginal fluid in a clean transfer pipette.
(2) Place a drop of fluid onto a clean glass slide labeled with the patient's name.
(3) Place the slide on a flat surface.
(4) Allow the slide to dry completely in room air.
(5) Examine the slide under the microscope at 40x magnification.
10.5 Quality Control
No direct controls are available for the fern test.

Volume 23 HS2-A
Amniotic fluid will form a tree-like or fern pattern when air-dried. A positive fern test exhibits this
characteristic pattern, indicative of the presence of amniotic fluid and therefore rupture of fetal
membranes. A negative test is reported when no fern pattern is seen on the slide.
A positive fern test should be used in conjunction with the patient's history and vaginal pH exam when
deciding whether or not fetal membrane rupture has occurred.
The greatest accuracy in identifying released amniotic fluid is achieved by Papanicolaou staining and
cytologic evaluation.
Erroneous results may be obtained when the slide is examined before it is completely dried; if the slide is
dried under a circulating air current (near or under a fan); if dirty or detergent-contaminated slides, cover
slips and/or pipettes are used; if the slide is heat fixed; or if cytologic fixatives or preservatives are used
on the slide.
11 Postcoital, Direct, Qualitative Examinations of Cervical Mucus
11.1 Principle
The postcoital test (PCT), also known as the Sims-Huhner Test,6 is part of an infertility evaluation to
determine the receptivity of cervical mucus and the ability of sperm to penetrate the cervical mucus and to
maintain activity.
The infertility assessment may identify contributing factors by the male (identified by a semen analysis)
or by the female (primarily a failure to ovulate or other hormone abnormalities, tubal pathology, or a
cervical factor).
11.2 Materials
11.2.1 Supplies
Recommended supplies used for the procedure include the following:
two glass slides (minimum);

cover slips;
cotton swabs;
transfer pipette;
sterile vaginal speculum;
14-gauge catheter or similar suction device;
12cc syringe;
forceps; and
Petri dish.

Number 5 NCCLS
11.2.2 Equipment
Equipment used for the procedure includes a microscope with 40x objective.
11.2.3 Reagents
Reagents are not required for postcoital testing.
Patients who are to have postcoital testing performed should be using some form of ovulation
determination (BBTs, LH surge testing, etc.).
When the patient determines an ovulation is occurring, she and her partner should have intercourse,
timing the act in such a way that the testing can be performed within 2 to 12 hours following intercourse.
On arrival to the provider, a history should be taken to record the time since intercourse and the length of
time since last previous intercourse.
The patient is examined in the dorsal lithotomy position. A sterile vaginal speculum, lubricated with only
water, is inserted into the vagina and the cervix visualized. Excess vaginal secretions are gently removed
with a cotton swab. A 14-gauge catheter attached to a 12cc syringe, or similar suction device, is then
placed into the cervical os and the cervical mucus is removed by pulling back on the syringe plunger to
create suction in the catheter.
Cervical mucus is the specimen used for this procedure.
The specimen is tested as soon as possible after collection.
(1) The volume of mucus is measured in the syringe.
(2) The mucus is discharged into a Petri dish.
(3) The amount of mucus is then classified as scant, moderate, or profuse.
(4) The color and clarity are noted.
(5) The amount of tenacity (Spinnbarkeit) is then tested by grasping a portion of the mucus with forceps
and noting the distance (cm) which it can be drawn before breaking (6 to 10 cm desirable).
(6) A drop of mucus is placed onto a microscope slide.
(7) A cover slip is placed over a portion of the mucus.

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(8) The mucus is immediately evaluated using high-dry 40x objective. The number and motility of sperm
are then recorded.
No direct controls are available for postcoital testing.
Report volume, color, clarity, Spinnbarkeit, sperm per high-power field, and percentage of mobile sperm.
Any other cellular elements or organisms should be noted.
The amount of mucus is classified as scant, moderate, or profuse, based on the providers estimation.
11.6.1 Interpretation
Midcycle mucus should be clear, watery, and profuse. Good Spinnbarkeit at midcycle is at least 10 cm.
What constitutes a normal number of sperm in a PCT has been a matter of dispute. Within six to eight
hours after coitus, at least five to ten motile sperm should be present per high-power field.1
Greater than 20 sperm/hpf has been positively correlated with pregnancy; no sperm suggests faulty sperm
production in the male. While the former gives a better prognosis for pregnancy, a substantial number of
pregnancies occur even when no sperm are found in the postcoital test.6 A poor result on the postcoital
test can raise a suspicion of an immunologic or other problem and can be of value in determining therapy.
The limitations of the procedure include:
procedure not performed during the midcycle ovulatory stage (the mucus is thick and opaque instead
of thin and clear);
use of dirty or contaminated materials;
patient examined too long after intercourse (greater than 12 hours);
specimen was contaminated by excess vaginal secretions due to failure to wipe the area with a cotton
swab prior to the collection of cervical mucous from the os, causing any sperm present to be
inadequately evaluated due to overlying vaginal debris;
examining the cervical mucous after drying, killing active spermatozoa.
12 Qualitative Semen Analysis
12.1 Principle
Presence and/or motility of sperm are direct microscopic procedures used to evaluate semen samples.
Active motility of spermatozoa is necessary for penetration of the cervical mucous and subsequent
migration up the fallopian tubes to fertilize the ovum. Seminal fluid contains not only spermatozoa, but
also prostatic and other glandular secretions.
Seminal fluid examinations for the presence or absence of spermatozoa are of value in substantiating the
effectiveness of a vasectomy and should be done with great care. To reliably conclude that no sperm are
present, examination should use a concentrated (centrifuged) specimen, and conclusions should not be

Number 5 NCCLS
based on results from a single specimen. These cautions are especially important, considering the
potential for detailed procedural review that may occur in a medico-legal case.
Microscopic examination of seminal fluid for presence and motility of spermatozoa is also utilized in
evaluating couples for infertility. Many cases of male infertility are detectable in seminal fluid samples
using routine methodologies beyond the scope of PPM.
12.2 Materials
12.2.1 Supplies
Supplies used for the procedure include:
glass or plastic container for collection;

glass microscope slides;
cover slips; and
transfer pipette.
12.2.2 Equipment
Equipment used for the procedure includes a microscope with 40x objective.
12.2.3 Reagents
Reagents are not necessary for qualitative semen analysis.
The patient, following a 48- to 72-hour period of abstinence from sexual activity, collects freshly
ejaculated semen. The most satisfactory collection method is obtained by masturbation.
Seminal fluid is the specimen used for this procedure.
12.3.3 Handling Conditions
The specimen should be collected in a clean glass jar or suitable plastic or polyethylene container free of
preservatives or other chemical additives. The specimen container should be warm, at least to room
temperature if not body temperature, prior to collection. The specimen should be labeled with the name of
the patient and the time of collection.
The specimen should be evaluated as soon as possible after ejaculation, within 30 minutes if possible. The
specimen should not be evaluated if it is more than two hours old. If collected away from the testing site,
the specimen should be transported to the testing site immediately after collection, avoiding temperature
changes during transport and keeping the specimen at body temperature until self-liquefaction is complete
(15 to 30 minutes). The sample should then be mixed thoroughly before testing.

Volume 23 HS2-A
(1) If the sample has been produced outside of the test site, it should be warmed to 37 C for five to ten
minutes before examination.
(2) Using a transfer pipette, place one drop of well-mixed seminal fluid on a clean glass slide and cover
with a cover slip.
(3) Examine the slide immediately using the high-dry 40x objective. Presence or absence of sperm is
recorded. NOTE: If examining for azoospermia, centrifuge a portion of the specimen following the
directions for urine microscopic testing and reexamine for the presence of sperm.
(4) Examine at least 200 spermatozoa for motility. NOTE: Do not use fields close to cover glass edges.
(5) Estimate the number of motile forms, using the high-dry objective. Only forward motion of the
spermatozoa is considered as true motility. Circular or sluggish motion or just motion of the tail is not
considered true motility. Count all forms of motility in the calculation and comment on type
observed, i.e., sluggish, little or no forward progression.
(6) If RBCs are present, indicate the number per high-power field. Note other cells and miscellaneous or
exogenous components observed.
Commercially available controls have recently been introduced with varying levels of sperm
concentration for quantitative analysis. Currently, there are no quality control materials available for
motility testing.
(1) Record presence or absence of spermatozoa. It is not unusual to find viable sperm in a post-
vasectomy patient. Instruction 3 above is advised to assure a thorough analysis.
(2) Report percent of motile spermatozoa calculated using the following equation:
No. of Motile Sperm

Total No. of Sperm (100 )
Comment on any motility that is other than forward progression.
(3) Report red blood cells per high-power field. Report other cells or debris present.
12.6.1 Interpretation7
Reference Range: >50% motile spermatozoa
Azoospermia: less than one sperm per high-power field
While erythrocytes are not normally found in semen, a few can be present without indicating pathology.
Some debris is typical, as are epithelial cells in low numbers. NOTE: Advanced microscopy skills can be
required for the identification of abnormal morphology.

Number 5 NCCLS
Complete sterilization can be determined as two consecutive monthly centrifuged specimens showing no
spermatozoa.
The limitations of the procedure include:
delayed (greater than two hours) examination of the specimen;
collection in improper container, such as condoms or other receptacles which may be contaminated
with spermicides, lubricants, detergents, or powders;
exposure of the specimen to temperature extremes during transport;
abnormally low sperm count allowing for evaluation of less than 200 spermatozoa; and
use of dirty or contaminated supplies.
Testing should be repeated to confirm azoospermia on a subsequent specimen.
13 Nasal Smears for Inflammatory Cells
13.1 Principle
Testing of a nasal discharge for presence of white blood cells by direct smear. May also be referred to as
nasal smear for eosinophils or nasal WBCs.
Examination of a direct smear is useful in distinguishing the nature of a nasal discharge. In nasal smears,
the identification of eosinophils is correlated with allergic rhinitis. The procedure is useful to provide a
differential diagnosis. When the condition is due to allergy, the predominant cell is the eosinophil. When
the condition is due to nonallergic causes, the discharge will show a predominance of neutrophils or
acellular mucus. Infectious processes show a predominance of neutrophils.
13.2 Materials
13.2.1 Supplies
Supplies used for the procedure include:
nonabsorbent collection material;

microscope slide; and
cotton-tipped swab.
13.2.2 Equipment
Equipment used for the procedure includes a microscope.
13.2.3 Reagents
Commercially prepared Wright-Giemsa or Hansel stain is the reagent used for the procedure.

Volume 23 HS2-A
There is no special preparation for the patient except the ability to produce the symptomatic discharge.
Therefore some withdrawal of antihistamines or decongestants for a brief period may be helpful. The
patient should be present to produce the specimen.
Have the patient discharge their nasal passages into a nonabsorbent material. Paper with a smooth finish,
such as waxed paper, plastic wrap, or an unused laboratory specimen transport bag is acceptable.
The specimen should be maintained at room temperature until examined. To prevent drying, assemble all
necessary equipment to examine the discharge before initiating the specimen collection.
(1) Smears are prepared by transferring a sample of the produced mucus with a cotton swab onto a glass
microscope slide.
(2) A thin smear is essential. A simple test is to check whether standard print can be read through the
smeared material. Identifying elements of cellular detail will be difficult to determine if the smear is
too thick.
(3) The smears should be allowed to air dry.
(4) Stain the smears using either a commercially prepared Wright-Giemsa stain or a Hansel stain. NOTE:
Commercial kits are readily available for the rapid Wright-Giemsa stain technique and are provided
with instructions and references.
(5) Neutrophils are recognized by their segmented or lobulated (two to five lobes) nuclei connected by a
thin filament of chromatin. The abundant cytoplasm is pale pink or colorless and contains many fine,
lilac-colored, neutrophilic granules. Eosinophils are recognized by their bright orange-red, spherical
granules. There is typically a bilobed nucleus separated by a thin filament, but occasionally more than
two lobes may be seen. The granules are larger than neutrophilic granules.
(6) Wright-Giemsa bloodstains may yield bluish granules in eosinophils, while the granules will appear
bright red using a Hansel stain, and the neutrophils and mucus debris will have a blue color.
(7) With low-power scanning, make rough qualitative counts by approximating the average number of
polymorphonuclear cells. Determine whether the leukocytes seen are neutrophils or eosinophils.
No commercially prepared direct controls are currently available; however, positive patient slides may be
used for comparison.
Presence or absence of eosinophils and neutrophils.

Number 5 NCCLS
Poor staining can cause false-negative recognition of eosinophils; therefore, staining of known material
for quality control purposes is essential.
Artifacts, cellular distortion, and cellular degeneration are common in smears, making recognition of the
cell type difficult. Reliance on staining characteristics provides the specificity required.
14 Fecal Leukocyte Examination
14.1 Principle
Testing for fecal leukocytes may also be referred to as stool WBCs, and includes evaluation of fecal
material for presence of white blood cells by direct smear.
A positive stained smear for polymorphonuclear leukocytes has a high sensitivity and specificity for
bacterial diarrhea. Combined with a history of abrupt onset, greater than four stools per day, and no
vomiting before the onset of diarrhea, the identification of leukocytes is an effective presumptive
diagnostic test for bacterial diarrhea. Conditions associated with numerous fecal leukocytes, blood, and
mucus include diffuse antibiotic-associated colitis, ulcerative colitis, shigellosis, salmonellosis,
Campylobacter, and Yersinia infection. Salmonella typhi may evoke a monocyte response. Conditions
associated with modest numbers of fecal leukocytes include early shigellosis involving small bowel,
antibiotic-associated colitis, and amebiasis. Conditions associated with an absence of fecal leukocytes
include toxigenic bacterial infection, giardiasis, and viral infections.
14.2 Materials
14.2.1 Supplies
Supplies used for this procedure include:
plastic specimen container;

microscope slide;
wooden applicator stick; and
cover slips.
14.2.2 Equipment
Equipment used for this procedure includes a microscope.
14.2.3 Reagents
Commercially prepared Loefflers methylene blue or Wright-Giemsa stain is the reagent used for this
procedure.
If the patient is asked to bring a specimen from home, the uninstructed patient can exhibit considerable
ingenuity in collecting a stool specimen. Therefore, a few simple instructions are likely to provide for a

Volume 23 HS2-A
more satisfactory specimen. If the patient does not have a bedpan, a clean glass jar is a satisfactory
alternative. A plastic bag cut halfway down each side seam and taped to each side of the top of the toilet
bowl is a suitable alternative collection device. Patients should be warned against passing urine at the
same time into the container. Tongue depressors or pieces of cardboard are reasonable instruments for
transferring the stool from the bedpan or other collection device to a transport container, e.g., a plastic,
hard paper, or glass container.
At least one gram of a naturally passed fecal specimen should be placed in a plastic specimen container.
Polyvinyl alcohol (PVA) is a recommended preservative. A ratio of one volume of feces to five volumes
PVA is recommended.
The specimen should be maintained at room temperature until examined. Because gas frequently
accumulates in unfixed stool specimens, care should be taken to gradually loosen the cap of a transport
container. Failure to observe this simple precaution can have unpleasant consequences.
(1) Place a small speck of mucus or a drop of liquid stool on a glass microscope slide with a wooden
applicator stick.
(2) Add two drops of commercially prepared Loefflers methylene blue, taking care not to contaminate
the dropper with the specimen. The stain can be placed on the slide first to avoid this possibility.
(3) Mix thoroughly and carefully.
(4) Place a cover slip on the mixture.
(5) Let stand for two or three minutes for good nuclear staining.
(6) With low-power scanning, make a qualitative estimate of the number of leukocytes.
Alternatively, smears are prepared by selecting flecks of mucus from fecal material with a cotton swab
that is then rolled across a glass slide. The smear is allowed to air-dry and is stained with a commercially
prepared Wright-Giemsa stain.
The neutrophil demonstrates a nucleus that is often segmented or lobulated (two to five lobes) and is
connected by a thin filament of chromatin. The abundant pale pink or colorless cytoplasm contains many
fine, lilac-colored, neutrophilic granules with a Wright-Giemsa stain.
Artifacts, cellular distortion, and cellular degeneration are common in smears. The nuclear lobes may
appear eccentric, and the cytoplasm may contain toxic granules or be vacuolated. Neutrophils may show
morphologic changes due to autolysis, including nuclear pyknosis and fragmentation, making recognition
of the cell type difficult.
No direct controls are currently available for fecal leukocyte testing; however, positive patient slides may
be used for comparison. Proper storage and use of the stain is necessary for accurate interpretation.

Number 5 NCCLS
Report results in terms of the presence or absence of WBCs.
Ten to fifteen percent of stools that yield a bacterial pathogen lack fecal leukocytes. The results may not
be sufficiently specific to preclude the use of culture.

Volume 23 HS2-A
References
1
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. 19th ed. Philadelphia: WB Saunders Company;
1996.
2
Brody LH, Webster MC, Kark RM. Identification of elements of urinary sediment with phase-contrast microscopy. JAMA.
1968;206.
3
Metzger GD. Laboratory diagnosis of vaginal infection. Clin Lab Sci. 1998;1(3):47-52.
4
Graham CF. A device for the diagnosis of enterobius infection. Am J Trop Med Hyg. 1941;21:159-161.
5
Koneman EW, Allen SA, Janda WM, Schereckenberger PC, Winn WC. Parasitology. In: Color Atlas and Textbook of
Diagnostic Microbiology. 5th ed. Philadelphia: Lippincott. 1997:1101, plate 20-3.
6
Glass R. Office Gynecology. 4th ed. Baltimore, MD: Williams & Wilkins; 1993.
7
NAFA Andrology Laboratory Quality Group on Semen Analysis. Manual for Basic Semen Analysis. Oslo, Finland: Nordic
Association for Andrology (NAFA); 1998.

Number 5 NCCLS
Additional References
Addison LA, Fisher PM. The Office Laboratory. Norwalk, CT: Appleton and Lange; 1990.
American Society of Internal Medicine. Glossary of Terms For Urine Sediment and Clinical Microscopy. American Society of
Internal Medicine Manual. 1991;Appendix III;48-54.
Baron EJ. Female Genital Tract Microbiology Part 1. Advance for Medical Laboratory Professionals. 1995;7(7).
Baron EJ. Female Genital Tract Microbiology Part 1. Advance for Medical Laboratory Professionals. 1995;7(11).
Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. Philadelphia: WB Saunders Co.; 1994.
Buchanan RE, Gibbons NE, eds. Bergeys Manual of Determinative Bacteriology. 8th ed. Baltimore: Williams & Wilkins; 1974.
Cannon DC. Examination of seminal fluid. In: Henry J, ed. Clinical Diagnosis by Laboratory Methods. 16th ed. 1979:712.
Echeverria P, Sethabuti O, Pitarangsi C. Microbiology and diagnosis of infections with Shigella and enteroinvasive E. coli. Rev
Infect Dis. 1991;13;S220.
Foster B. Optimizing Light Microscopy for Biological and Clinical Laboratories. Dubuque, IA: Kendall Hunt; 1997.
Friedman ML, McElin TW. Diagnosis of ruptured fetal membranes, clinical study and review of the literature. Am J Obstet
Gynecol. 1969;104:544.
Gardner P, Provine HT. Manual of Acute Bacterial Infections. Boston: Little, Brown & Co.; 1979.
Gill GW. Troubleshooting problems in microscopy. Advance for Medical Laboratory Professionals. 1997;24-27.
Hansel FK. Cytologic diagnosis in respiratory allergy and infection. Ann Allergy. 1996;24:564.
Howanitz PJ, et al. Timeliness of urinalysis. Arch Pathol Lab Med. 1997;121:667-672.
Larson E. Clinical Microbiology and Infection Control. Boston: Blackwell; 1984.
Mass D. Lab testing tips: Ensuring correct diagnosis in testing for vaginitis. CAP News. 1998.
Mullarkey MF, Hill JS, Webb DR. Allergic and nonallergic rhinitistheir characterization with attention to the meaning of nasal
eosinophilia. J Allergy Clin Immunol. 1980;65:122.
Ringsrud K, Linne J. Urinalysis and Body Fluids: A Color Text Atlas. New York City: Mosby; 1995.
World Health Organization. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction
4th ed. Cambridge England: Cambridge University Press; 1999.
Website:
http://www.vh.org/Providers/CME/CLIA/UrineAnalysis

Volume 23 HS2-A
Appendix A. Microscopic Components in Urine Sediment

A1 Red Blood Cells (Erythrocytes)
Under high power, unstained red blood cells in wet preparations appear as pale yellow-orange discs. They
vary in size but are usually about 8 in diameter. In old or hypotonic specimens, cells may appear as
faint, colorless circles or ghosts, since the hemoglobin has dissolved out. Dysmorphic RBCs
(acanthocytes, donut shaped, etc.) may be clinically meaningful in supporting the diagnosis of glomerular
bleeding. Red blood cells may become crenated in hypertonic urine and appear as small, rough cells with
irregular edges and surfaces. Smooth, folded, shrunken, and crenated cells may all be seen in the same
urine specimen.
Red blood cells may be confused with oil droplets or yeast cells. Oil droplets (mineral oil or vaginal
creams) show a great variation in size and are usually refractile. Yeast cells are oval to round, generally
smaller than erythrocytes, and often show budding. Surface crenations on red blood cells resemble
granules, and the cells may be confused with small granulocytes.
A2 White Blood Cells (Leukocytes, Neutrophilic)
Under high power, neutrophilic leukocytes in wet preparations appear as colorless, granular cells about
two to three times the size of a red cell. It is not unusual to see dense, granular neutrophils not much
larger than a red cell and swollen, large neutrophils in the same specimen. Occasionally, ingested bacteria
or yeast are seen in the cytoplasm of leukocytes, crowding the nucleus and enlarging the cell two to three
times.
In freshly voided urine, nuclear detail is well defined. With cellular degeneration, nuclear segments fuse
into a single, round nucleus and granules are lost from the cytoplasm, making distinction from renal
tubular cells difficult or impossible. In dilute or hypotonic urine, neutrophils swell. There also may be
small intracytoplasmic vacuoles and loss of nuclear segmentation. Cytoplasmic granules wiggle or
dance due to Brownian movement. Neutrophils containing these refractile dancing granules are
called glitter cells.
A3 Epithelial Cells
A3.1 Renal Tubular Epithelial Cells
Renal tubular epithelial cells are derived from the epithelial lining of all segments of the nephron. They
vary in size from approximately three to five times the size of a red cell, up to twice as large as a
neutrophil. Typically, they are polyhedral in shape and elongated or ovoid with granular cytoplasm. The
single nucleus is round and sometimes eccentric. Renal tubular cells originating from the proximal tubule
may show a microvillus border, which is visible with bright-field microscopy. Disintegrating RTE cells
become swollen and frayed, and the cytoplasm is often indistinct. In wet preparations, RTE cells may be
difficult to distinguish from degenerating neutrophils, mononuclear leukocytes, or transitional epithelial
cells.
A3.1.1 Oval Fat Bodies
Oval fat bodies are renal tubular epithelial cells containing fat globules. Lipid droplets containing
cholesterol are anisotropic in polarized light, but show up brightly against a dark field and appear to be
divided into four quadrants. Their appearance resembles a Maltese cross.
An NCCLS global consensus guideline. NCCLS. All rights reserved 45

Number 5 NCCLS
A3.1.2 Inclusion Bearing Cells
Inclusions such as those produced by cytomegalovirus, herpes simplex virus, and polyoma virus may
occasionally be detected in the urine; these cells are excreted intermittently, and multiple samples should
be examined. Due to the rapid degeneration of these cells, the urine must be processed as soon as possible
after collection.
A3.1.3 Atypical Urothelial Cells
Urothelial neoplasms shed varying numbers of malignant cells into the urine. The presence of papillary
fragments or urothelial cells with increased nuclear cytoplasmic ratios and cytomegaly should be noted,
and cytologic follow up should be recommended.
A3.2 Transitional Epithelial Cells
Transitional epithelial cells (urothelial cells) vary in size, averaging about four to six times the size of a
red cell. The nucleus is well defined, oval or round, usually central. Binucleate cells may occur.
Transitional epithelial cells can occur singly, in pairs, or in small groups (syncytia). In wet preparations,
they appear smaller and plumper than squamous epithelial cells and have a well-defined cell border.
They may be spherical, ovoid, or polyhedral. The smaller cells resemble renal tubular epithelial cells.
Some, called tadpole cells, have elongated cytoplasmic processes, indicating a direct attachment to the
basement membrane. Small vacuoles and/or cytoplasmic inclusions may be present in degenerating cells.
A3.3 Squamous Epithelial Cells
These large, flat cells are derived from the lining of the female urethra, the distal male urethra, or from
external skin. In wet preparations, squamous cells are about five to seven times as large as a red cell and
larger than most transitional epithelial cells. A single small, condensed, round, polygonal or oval central
nucleus about the size of a small lymphocyte is seen in flat, round, or rectangular cells. Degenerating
squamous cells have granular swollen cytoplasm with a frayed cell border and a pyknotic nucleus.
A4 Spermatozoa
In wet preparations, the sperm head is about 4 to 6 in length, usually tapering anteriorly. It is smaller and
narrower than a red cell. Slender tails are about 40 to 60 in length. The head may be separated from the
tail, making identification more difficult.
A5 Casts
Casts are cylindrical protein bodies that form in the distal tubules and collecting ducts of the kidney. They
are subclassified on the basis of their morphologic appearance and composition. They have delicate
parallel sides, rounded or tapered ends, and may be straight, curved, or convoluted. True casts should not
be confused with long mucus threads, rolled-up squamous epithelial cells, cellulose, or other fibers.
A5.1 Hyaline Casts
Hyaline casts are colorless, homogeneous, translucent, and have a low refractive index. They have a
smooth or finely wrinkled surface and may appear tortuous or coiled. Inclusion granules may
occasionally be seen in the cast matrix.
46 An NCCLS global consensus guideline. NCCLS. All rights reserved

Volume 23 HS2-A
A5.2 Granular Casts
Granular casts may contain many fine or coarse granules that are most often evenly dispersed over the
cast, but may be confined to one area or loosely scattered. They may also include degenerating cell
remnants. Distinction between coarsely and finely granular casts has no clinical relevance.
A5.3 White Blood Cell Casts
The casts may be crowded with cells, or have only a few clearly defined cells present in the matrix, often
at one end. They contain predominantly intact segmented neutrophils, with cell membranes and nuclei
clearly visible in most of the cells. The nucleus of the segmented neutrophil may be degenerated and
rounded, precluding categorization of the cell.
A5.4 Red Blood Cell Casts
The predominant cells are intact erythrocytes, densely or loosely covering the hyaline or granular matrix.
The red cells may be shrunken or crenated when compared with those in the surrounding urine. A yellow
or red-brown color is seen when a large number of red cells fill the cast. Red cells are of uniform size
within the cast, as opposed to fat globules, which vary in size.
A5.5 Cellular Cast, Renal Tubular Epithelial (RTE)
These casts contain RTE cells within their matrix that are usually intact and irregularly dispersed over the
surface. However, in some RTE casts, the cells may be lined up in columns or rows, indicating
sloughing of the epithelium of an entire tubule. RTE cells have a large, single, central nucleus and
relatively sparse agranular cytoplasm. As RTE cells degenerate, their nuclei become pyknotic and dense.
The cast matrix may contain granules thought to arise from degenerating RTE cells. While the cast
matrix may be scant or difficult to visualize due to overlying RTE cells, it must be present in order to
identify a cast.
A5.6 Fatty Casts
Fatty casts contain large numbers of spherical, highly refractile fat droplets of varying size in the cast
matrix or within oval fat bodies in the cast. Casts may appear pale yellow, but fat droplets appear black
with focusing under low power. Fat may be stained with commercially prepared Sudan stain or examined
with polarized light to demonstrate the birefringent Maltese-cross pattern of cholesterol esters.
A5.7 Waxy Casts
Waxy casts are usually broad and stubby with blunt ends that may appear broken off. They have well-
defined parallel margins that may be serrated or notched. The colorless or waxy yellow interior is dense
and homogenous. They are thought to arise from the degeneration of cellular casts.
A6 Crystals
Crystals in the urine are of limited clinical significance. Phosphates, urates, and oxalates are especially
common and occur in normal urine sediment. A prerequisite for the positive identification of crystals is
knowledge of the urinary pH. This helps with the preliminary separation.

Number 5 NCCLS
A6.1 At Acid pH
Cystine - A clear, colorless, hexagonal crystal sometimes pitted and occasionally twinned or
laminated. There may be a wide variation in size in the same specimen from about the size of a red
cell to that of a squamous epithelial cell. Cystine may be classified as a pathogenic crystal.
Uric acid - These crystals appear at low-acid pH. They are usually colored yellow to brown.
Common forms are four-sided, flat, and show a large variation in size and shape. Twinned, thick,
brown rosettes and thick, yellow wedge shapes also occur.
Sulfonamides - These crystals precipitate at low-acid pH. They are colorless to yellow-brown or
green-brown in color. Sulfonamide crystals appear as bundles of long needles with eccentric binding
resembling stacked wheat sheaves. They are also seen as spherical clumps with radiating spikes and
fan shapes. Sulfamethoxazole crystals are dark brown, divided or fractured spheres.
Ampicillin - Following large, intravenous doses it appears in urine as long, slender, colorless crystals
that aggregate into irregular sheaves after refrigeration.
Amorphous urates - These crystals are often referred to as brick dust. These colorless or red-
brown aggregates of granular material must be distinguished from bacteria. Amorphous urates are
generally more pleomorphic than bacteria, and are not associated with increased white blood cells. A
gram stain may be needed for further evaluation.m
A6.2 At Neutral-to-Acid pH
Calcium oxalate - These crystals vary in size and may be much smaller than red blood cells. The
dihydrate form appears as small, colorless octahedrons that resemble stars or envelopes. They are
sometimes described as two pyramids joined at the base. Larger crystals sometimes clump together.
Oval, elliptical, or dumbbell monohydrate forms are less commonly seen. All calcium oxalate crystals
are birefringent.
Cholesterol - These crystals are large, flat, clear, colorless rectangular plates or rhomboids, that often
have one notched corner. They are frequently accompanied by fatty casts and oval fat bodies. They
may be confused with radiographic contrast media, but are not associated with a high urinary specific
gravity.
Tyrosine - This crystal is rarely seen. Silky, fine needles appear colorless to black under low power
depending on focusing. Clumps or sheaves form after refrigeration. Tyrosine may be classified as a
pathogenic crystal.
Leucine - These highly refractive, brown, spherical crystals have a central nidus and spoke-like
striations extending to the periphery. Leucine spherules are birefringent. Leucine may be classified as
a pathogenic crystal.
Bilirubin - Occasionally seen in urine with large amounts of bilirubin, and usually accompanies bile-
stained cells. Small, brown needles cluster in spheres or clumps or on cells or hyaline casts.
m
In the U.S., under CLIA, gram stains cannot be performed as a PPM procedure.

Volume 23 HS2-A
A6.3 At Neutral-to-Alkaline pH
Amorphous phosphates - These crystals form colorless or brown granular aggregates. They are
similar in appearance to amorphous urates, but occur in alkaline urine.
Triple phosphates (ammonium magnesium phosphate) - These are typically colorless, often large
crystals with a coffin-lid appearance. They exhibit birefringence and are often accompanied by
amorphous phosphates and bacteria.
Ammonium biurate - Biurates appear as crystalline, yellow-brown, smooth spheres with radial or
concentric striations. The thorn apple variety has projecting thorns. These should not be confused
with sulfonamide crystals.
A7 Bacteria
Most commonly, gram-negative enteric organisms are identified in wet mounts as rod-shaped organisms
of medium size. Large, longer bacilli seen in urine are likely to be the gram-positive lactobacilli from
vaginal or fecal contamination. Coccoid forms are more difficult to identify in wet urine sediments and
must be distinguished from amorphous phosphates and amorphous urates.
A8 Yeasts
Yeasts are seen as colorless, oval, thick-walled cells, slightly smaller than a red blood cell. A cell with a
single bud is characteristic. The cells stain poorly with aqueous stains in wet preparations but are strongly
gram-positive with gram staining. Candida species form elongated cells up to about 50 long, resembling
mycelia. These are branched and have terminal budding forms.
A9 Parasites
A9.1 Protozoa
Trichomonas vaginalis primarily causes vaginal infection but is also capable of infecting the urethra,
periurethral glands, bladder, and prostate. It ranges up to 30 in length, is oval to pear-shaped with a
single anterior nucleus, four anterior flagellae, and a posterior protruding sharp axostyle. Under high
power, living forms show a short, undulating membrane and jerky movement. Degenerating forms
resemble large, oval cells without visible flagellae and few distinguishing features and may easily be
confused with neutrophils or other leukocytes.
A9.2 Helminths
Enterobius vermicularis is an intestinal nematode or roundworm known as pinworm. Females are white
and threadlike and 5 mm to 13 mm long with a pointed (pin-like) posterior end. Eggs are about 50 to 60
long and about half as wide, forming an elongated, well-defined oval with one flat side. These usually
contain an embryo. Eggs laid in the perianal skin may occasionally be seen in the urine. Adult worms are
rarely seen in urine.
A10 Miscellaneous/Exogenous
A10.1 Fat Droplets
Free, refractile droplets are seen as dark spherules under low power and clear spheres of varying size
under high power. Fat droplets may represent endogenous triglycerides, neutral fats, cholesterol esters, or

Number 5 NCCLS
combinations of all three. Exogenous mineral oil, catheter lubricant, or vaginal cream also appear as fat
globules sometimes assuming large, amorphous, irregular shapes.
A10.2 Fecal Contamination
This may be the result of a fistula between the colon and urinary tract or due to contamination of the urine
specimen with feces at the time of collection. There is an overall yellow-brown color. Plant structures,
muscle fibers, and microorganisms are seen.
A10.3 Fibers
Hair, synthetic and natural fibers from clothing, cotton balls, dressings, and disposable diapers are all
found in urine specimens. Most fibers are long, sometimes twisted and large. Short cellulose fibers from
disposable diapers resemble large, broad, waxy casts. They are well defined, flat, refractile, and colorless
and have fissures, pits, or cross-striations.
A10.4 Mucus
Mucus strands or threads frequently are found in urinary sediments arising from glands in the lower
urinary and vaginal tracts. Translucent, delicate strands are seen in long, wavy, intertwined aggregates.
They are usually in the background of the field and are more obvious with phase microscopy than bright
field.
The material in mucus strands resembles that in hyaline casts. Casts are shorter and cylindrical with
parallel, rounded edges. Casts may appear frayed and fibrillar with degeneration, but these strands are not
as long as the usual mucus strand.
A10.5 Pollen Grains
These contaminate urine and urine containers on a seasonal basis. They are usually large, about 20 or
more in diameter and tend to be rounded or regularly shaped with a well-defined, thick cell wall. Some
resemble worm ova. They may be winged or have short, regular, thorny projections. Some are yellowish-
tan.
A10.6 Starch Granules
They are commonly found in surgical gloves and are a frequent contaminant of body fluids. Size varies
from that of a red cell to four to six times larger. The usual form is colorless and irregularly rounded with
a central slit or indentation.
A10.7 Stainn
Crystal violet-safranin or similar stains used for wet urinary sediments crystallize, especially at alkaline
pH, and form brown-to-purple, needle-shaped crystals. These stains are available from various
commercial suppliers.
n
In the U.S., stains are not indicated for the microscopic analysis of urine sediment in PPM procedures under CLIA.

Volume 23 HS2-A
References for Appendix A
College of American Pathologists. Glossary of terms for urine sediment. 1999 Surveys Hematology
Glossary. 1999;27-36.
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. 19th ed. Philadelphia: WB
Saunders Company; 1996.

Number 5 NCCLS
Appendix B. Microscopic Components in Vaginal Fluid and KOH Preparations

B1 Squamous Cells
Squamous cells measure 25 to 70 m and demonstrate a polygonal flagstone appearance. These cells
are almost always present in vaginal fluid.
B2 White Blood Cells
White blood cells measure 14 to 16 m and exhibit a granular cytoplasm. White blood cells (with
characteristic multilobed nucleus) are usually present in vaginal specimens in rare to scanty numbers. If
they appear to be 3+, abnormal flora may be suspected. Conditions usually associated with 3+ white
blood cells include Trichomonas, Vaginal Candidiasis, N. gonorrhoeae, herpes simplex, and severe
atrophic vaginitis.
B3 Red Blood Cells
Red blood cells appear as biconcave discs measuring 7 to 8 m in diameter. They are normally smooth,
but may be greatly distorted in vaginal and urine specimens. The cytoplasm is clear and does not contain
a nucleus. Red blood cells may be confused with yeast. They will lyse with addition of KOH, which is
useful in distinguishing between RBCs and yeast. RBCs may be present in vaginal fluid as a result of
current or recent menses, or due to desquamative inflammatory process.
B4 Clue Cells
Diagnostic clue cells should have small gram-negative, curved gram-negative, or gram-variable rods,
covering not only on the surface of the cell, but also spread out past the cell boundaries, obscuring the
cytoplasmic margins. The entire cell need not be covered with bacteria, but cells with organisms simply
sticking to the surface without extending past the cytoplasmic margins should not be considered clue
cells. Clue cells may be described as shaggy in appearance. Of note is a phenomenon that occurs
when KOH is added to a slide with moderate to large numbers of clue cells. The lysis of the epithelial
cell membranes causes a massive release of bacteria into the surrounding liquid, creating a lava flow
appearance.
B5 Parabasal Cells
Parabasal cells measure 16 to 40 m in diameter and appear oval to round in shape. They have a nucleus
to cytoplasm ratio of 1:1 to 1:2. Less mature epithelial cells may be found in increased numbers at the
time of menstruation and postmenopause. Parabasal cells, if present with large numbers of WBCs and
altered flora in vaginal fluid, are suggestive of desquamative inflammatory vaginitis.
B6 Basal Cells
Basal cells measure 10 to 16 m and appear round. They have a nucleus to cytoplasm ratio of 1:2. Similar
in size to WBCs, they are distinguished by round rather than lobed nucleus. Basal cells are deep tissue
cells, and their presence with large numbers of WBCs and altered flora in vaginal fluid strongly suggests
desquamative inflammatory vaginitis.

Volume 23 HS2-A
B7 Bacteria and Parasiteso
B7.1 Gardnerella vaginalis
Gardnerella vaginalis appears as small, nonmotile, coccobacillus that attaches to the epithelial cell (gram-
negative/gram-variable, facultative anaerobe). It is one of the major bacterial species associated with
bacterial vaginosis (see Section B4, Clue Cells). It produces a characteristic amine odor when KOH
is added to the specimen.
B7.2 Mobiluncus spp.
Mobiluncus spp. appears as thin, curved, gram-negative anaerobic bacilli, demonstrating corkscrew
motility in wet preparations. An amine odor is produced with addition of KOH. This bacterium is best
demonstrated on gram stain. If the specimen is amine-positive and clue cell-negative, a gram stain may
identify Mobiluncus.
B7.3 Trichomonas vaginalis
The typical Trichomonas vaginalis trophozoite measures approximately 7 x 15 m and is oval in shape.
Organisms can be twice this size, and the shape may vary greatly. Other features include a supporting
axostyle that bisects the trophozoite longitudinally and protrudes from the posterior end; and five anterior
flagella, one of which bends back along the outer edge of the undulating membrane creating jerky
motility. The pointed tip may be instrumental in attachment and may also be responsible for tissue
damage. It should not be confused with sperm that have a single tail, much smaller head, and no axostyle.
T. vaginalis may be found in vaginal specimens and urine specimens (especially those contaminated with
vaginal secretions), as well as secretions from the urethra, prostate, and epididymis.
B7.4 Lactobacilli
Lactobacilli are present as normal flora in the vaginal fluid of postpubescent females and produce lactic
acid. It is this metabolic waste which helps maintain the pH in the acidic range of 3.8 to 4.2 in the normal
vaginal environment. They appear as relatively large, nonmotile rods (gram-positive on gram stain).
Hydrogen peroxide-producing strains are thought to be responsible for protection against pathogenic
organisms. If lactobacilli are absent or rare relative to squamous cells, abnormal flora may be suspected.
B8 Yeast and Fungij
The two basic structures used to confirm the presence of yeast and fungi are:
1) Hyphae long filaments which grow and form a mat (mycelium); and
2) Blastospores buds formed during the reproductive process of yeasts.
Multiple buds that do not detach can form chains known as pseudohyphae.
Some fungi will produce both forms (although usually not both at the same time) in tissue depending
upon environmental conditions these are the dimorphic fungi; others will produce only one form or the
other. Most pathogenic fungi are dimorphic.
o
In the U.S., identification of bacteria or parasites is considered high-complexity testing, and is not included in PPM under
CLIA regulations. The general groups of bacteria, fungi, or parasites can be detected as part of the moderate-complexity
procedures in this subcategory of tests.

Number 5 NCCLS
Fungal hyphae, spores, and other fungal forms can be distorted in clinical specimens by the hosts
inflammatory response.
Reference to Appendix B
Koneman EW, Allen SA, Janda WM, Schereckenberger PC, Winn WC. Parasitology. In: Color Atlas and
Textbook of Diagnostic Microbiology. 5th ed. Philadelphia: Lippincott. 1997.

Volume 23 HS2-A
NCCLS consensus procedures include an appeals process that is described in detail in Section 9 of
the Administrative Procedures. For further information, contact the Executive Offices or visit our
website at www.nccls.org.
Summary of Comments and Subcommittee Responses

HS2-P: Provider-Performed Microscopy Testing; Proposed Guideline
General
1. The tone of this document is somewhat colloquial. I have indicated examples of text I suggest be removed or
rephrased. I emphasize this, because I anticipate that this guideline, when finalized, will be widely cited. The
revision, to a more stringent editorial style, will establish the tone that quality assurance in PPM testing is not
optional, but essential. Also the words should and must, must be carefully chosen. If the authors determine
that an activity is essential, the word must is to be used. If the authors are making a recommendation that may or
may not be adapted, then should is to be used.
Full consideration was given to the suggestion that the document reflect a firmer tone with regard to
essential quality criteria. The text has been modified, where appropriate, to reflect the suggested change.
2. The document does not cite the need for quality assurance plan under U.S. federal regulation CLIA '88.
Although the document is intended for use outside the U.S. also, certainly the need to conform to regulatory
agencies should be addressed. In fact, the document does use OSHA regulations as an example of workplace
safety regulations.
Where appropriate, the document has been modified to cite regulations such as CLIA and credentialing
and accreditation requirements as examples for conformance to quality standards.
3. The model procedures in this guideline do not confirm totally to NCCLS document GP2-A3. The outline of
each PPM should be the same as for the test procedure. Have you considered each procedure in NCCLS format
as an appendix, which could be more easily updated than the final guideline? I suggest using the body of the
guideline to identify essential quality assurance practices for each test. For example, quality control for urine
sediments should include not only the description of sources of QC materials but also the frequency of
performing the QC testing, documentation (log sheets), how and who must do a QC material review.
NCCLS document GP2-A3Clinical Laboratory Technical Procedure ManualsThird Edition; Approved

Guideline is currently being revised. By their nature, PPM procedures are not as analytical as the next
complexity level of laboratory procedures, and fewer portions of GP2 are applicable. Future reviews of
the PPM guideline can consider recommended formats for the procedures when those formats have been
revised.
4. Identification of adult Enterobius vermicularis worms requires a level of technical expertise, which may not be
available in the typical pediatrician's office or local health department setting. I suggest that CLIA program be
consulted to determine if this examination falls under the subspecialty of parasitology. I would consider
removing this section for issues related to technical expertise and regulatory compliance.
Pinworm examinations by light microscopy have been determined by the CLIA advisory committee to be
within the scope of PPM procedures and, therefore, are the basis for the procedures included in this
guideline.
5. Throughout the document there are several references made to the economics of testing (choice of equipment)
and the use of an adequate QA program to decrease the burden of liability. Are these issues consistent with the
NCCLS mission? If the goal of this guideline is to promote quality testing, this side, albeit valid, interest should
be omitted.

Number 5 NCCLS
Value is an equation of quality given cost. Attention was paid to the practicality of the recommendations
made for the expected setting. These procedures were given separate regulatory status, because they have
value in patient care. The purpose of the document is to set forth uniform practices that will ensure
accurate results. Medical decisions made with inaccurate results do carry the risk of increased liability
for the practitioner.
6. I suggest that this document also be reviewed by accrediting agencies like COLA, regulatory programs like
CLIA, and subject matter experts.
Accrediting and regulatory agencies are members of NCCLS and have the opportunity to participate in
all NCCLS document reviews.
Foreword
7. I disagree with the statement that market demands are leading to more office-based testing. The number of POL
certifications has decreased since the implementation of CLIA '88, in part due to what providers consider
adequate reimbursement for the additional activities required under a comprehensive QA program.
The text has been modified as suggested. However, a recent national survey by the U.S. Centers for
Medicare and Medicaid Services (CMS) notes that 74% of the CLIA-certified laboratories perform only
waived or waived and microscopy testing, primarily in physicians offices. Therefore, office-based testing
represents a significant portion of the test market. Perhaps they are more focused on the lesser complex
tests, but nevertheless the numbers are substantial enough to warrant attention.
8. My first comment is on what is not in the guideline. As I went through the index, I noted there was no mention
of gram stain. This was not clarified until the footnote on page 49. For international users of the document, it
may be of value to comment in the Foreword, Introduction, or Scope on why gram stains are excluded from this
guideline.
An additional line of clarification has been added to the Scope to reflect the types of specimens and
procedures that are within the scope of the guideline. The following text and footnote have been added
after the first sentence: "It does not include procedures which require the use of stains to determine the
results of the testing. In the U.S., the CLIA certification for provider-performed microscopy (PPM)
described a set of procedures that may be performed under that designation.
Introduction
9. The second line of the first sentence should be rewritten to read, reliable results intended to be used by the
provider to immediately impact patient care decisions.
The text has been modified as suggested.
10. It is not recommended that nurse practitioners, physicians' assistants, and nurse midwives read KOH
preparations for fungal elements or methylene blue preparations for fecal leukocytes. It is recommended that
only physicians that are properly trained read these preparations.
The proposal for the document identified a lack of proper training in physician's office laboratory
procedures in medical residency programs. It was also noted that changes in scope of practice expanded
the need to other levels of providers. Clinical practitioners need resources such as this guideline to make
accurate and appropriate use of these procedures. Recognizing changes in scope of practice, the PPM
CLIA category was changed from "physician-performed microscopy" to "provider-performed
microscopy." In making this change, DHHS specifically noted that in rural areas, among low-income
populations, and in other areas where there is a shortage of physicians, the midlevel practitioners are the
only available healthcare providers.

Volume 23 HS2-A
Section 3.1
11. In the first sentence, reference to all human body fluid specimens should have the term fluid removed.
Universal safety applies to fluid and nonfluid specimens, such as fecal samples.
Section 3.2
12. Personal Protective Equipment: Under the bullet point "recommendations" include gloves. I encourage the
authors to indicate that certain practices are required if the testing site is to operate under the laboratory industry
standards of practice. The heading indicates that the following bullet points are situations when PPE should be
used, but the last point recommends availability, not actually the use, of face masks. Recommending availability
is weak. These high-risk aerosol procedures usually do not apply to PPM procedures and could be omitted.
13. One of the infection control challenges of people wearing gloves is that they often touch charts, light switches,
and microscope controls before they take their gloves off, thereby contaminating everything. We prefer that
glove use is "task-specific," that they are taken off as soon as they are not required. With that in mind, while I
support the need for gloves in sample preparation, I challenge their need while examining the sample under the
microscope. While this section doesn't specifically say to wear gloves during microscopy, the second bullet can
be interpreted in that manner. Would you consider adding a bullet or phrase such as "Glove use should be task
specific, and be removed to avoid unnecessarily contaminating the microscope?
The subcommittee agrees with the comment. The recommended text has been added to the second bullet
point to address the commenters concerns.
14. The second bullet should read: "Gloves must be worn whenever collecting, handling, and testing specimens, to
provide barrier protection for cuts, scratches, or breaks in the skin against potential pathogens, and to provide
barrier protection for the samples under investigation (i.e., protection of the integrity of the sample, as well as
the individual performing the test)."
See response to Comment 13.
15. Rewrite third bullet to read, gown, apron, or laboratory coat which is resistive or impervious to liquids
Section 3.3
16. The use of should in the hand washing and basic laboratory safety considerations is too weak.
The following statement has been added as a footnote to Section 3.3: "In the U.S., employees must wash
their hands with soap and running water as soon as feasible, as recommended above." Also, a reference
to NCCLS document M29Protection of Laboratory Workers from Occupationally Acquired Infections,
has been included for completeness.
17. From a laboratory biosafety perspective the single most important safety procedure is hand washing. It is very
helpful that you have included this section. Would you consider expanding it slightly by including a brief
description on hand washing? "Hands should preferably be thoroughly washed for 20 to 30 seconds under
running water either with soap or antiseptic soap. Alternatively, an alcohol-based, waterless, rapid-kill gel can
be rubbed thoroughly over the surface of the hands."

Number 5 NCCLS
18. Combine second and fourth bullet, i.e., after completing work and/or before leaving the work area to eat, drink,
smoke, apply cosmetics, handling contact lenses, and using restroom facilities.
Section 3.5
19. While bleach is generally accepted as the agent of choice for cleaning up blood spills, it is not necessarily the
only effective agent. Most of the samples being tested in this guideline are not bloody. Would you consider
recommending use of common household, phenol-based disinfectants as an alternative to bleach? Generally
they are easy to purchase, easy to use, and for many, they have a less irritating odor.
The subcommittee has considered other disinfectants for this section and believes the reference to
NCCLS document M29Protection of Laboratory Workers from Occupationally Acquired Infections
addresses the issue of disinfectant options.
20. Is bleach solution, which can be corrosive, the best disinfectant for microscopes? Other disinfectants such as
pure alcohol could be better for microscopes with stainless steel components.
The text in Section 3.5 contains a note suggesting the use of other disinfectants that are less corrosive
than sodium hypochlorite. Users are referred to NCCLS document M29Protection of Laboratory
Workers from Occupationally Acquired Infections for additional information.
21. The microscope stage is the only part of the microscope we routinely disinfect with an alcohol pad (70%); all
other parts are cleaned with a microscope cleaner. I havent heard of using bleach on a microscope; perhaps it is
recommended by vendors.
Section 3.6
22. It is recommended that only designated provider-personnel handle medical waste and should never be handled
by general housekeeping personnel.
The following text has been added to address the commenters concern: Therefore, handling and
disposal of medical waste should be by trained personnel, utilizing materials and procedures which
protect the employees, patients, and the environment.
23. It is important to specify that infectious waste containers should be changed regularly, even if they are not full,
to prevent possible contamination.
Section 4
24. Replace intensify with optimize or maximize in the definition for immersion oil, i.e., used to optimize
the resolution of the image being magnified.
25. The definition of "precision" is not listed. Understanding of this term is essential in discussing good laboratory
practice. The term is used in the document.

Volume 23 HS2-A
26. The definition for qualitative should use the phrase "...that detect the presence or absence of a particular
analyte, constituent, or condition. Specific identification may be performed."
27. I understand within ISO that the phrases "preanalytic" and "postanalytic" in other countries are referred to as
"preexamination" and "postexamination." Since this document is intended for an international audience,
consider including these synonyms within the definitions.
28. Spinnbarkeit should be included in the Definitions section.
A definition for Spinnbarkeit has been added to Section 4.
Section 5.1
29. The fourth bullet should read, cover slip(s).
Section 5.2
30. Once a person has been trained properly to use the microscope, that knowledge and skill is very unlikely to
degenerate. However, if a new technique were to be introduced to the field, then it would be beneficial to place
within the provider-performed microscopy manual a mechanism to make an addendum.
The subcommittee agrees with the comment. The document production process has a mechanism in place
to make changes or additions to the content. This is achieved through the consensus process, as stated on
the inside of the cover, and during the review of the documents by the area committee, which occurs
every three years or earlier if necessary.
31. I do agree that a binocular-head microscope with a built-in light source and mechanical stage are very nice to
have, but a monocular-headed microscope with a reflective light source would work just as well. Within an
office setting with practitioners literally doing a test just a few times per day, a monocular scope would be
adequate and would not create any environmental or ergonomic types of problems. Lastly, nothing is stated
about the quality of the optics, the resolution of the optics. This characteristic is arguably the most important for
a microscope.
The subcommittee agrees with the comment; however, the text states a binocular-head microscope is
desirable and does not exclude the use of a monocular microscope.
With regard to the quality of the optics, the recommended equipment is a modern, high-quality
microscope with the basic set of objectives and ocular lenses to assure the quality of the optics.
32. There is an extensive presentation of how microscopes work and operation of microscopes. This information is
adequately cited. Is it really necessary to paraphrase references in this document? A more definite
recommendation of preventative maintenance activities and intervals, documentation, and training includes
information that could be presented in this document that is not stated in other references.
The subcommittee appreciates the comment; we believe, however, that the recommendations are useful
and appropriate.

Number 5 NCCLS
Section 5.2.1.1
33. Please note that in Section 5.2.3 you use the phrase "back lens," but you have not included this in either the
figure or this section.
The text has been modified to read underside lenses.
Section 5.2.1.4
34. Does the condenser "control the angle of light striking the specimen" or the amount of light?
The text has been modified to read amount of light.
Section 5.2.2
35. On the use of oil immersion objective, the last line of number (4) should read, cover slip.
36. The method of setting the condenser described here is not consistent with Koehler illumination.
The text has been modified to better reflect the use of Koehler illumination during operation of the
microscope.
37. Oil immersion examination is not indicated in PPM testing. This testing should be performed at 40x. This
section could be omitted.
The subcommittee believes that the oil immersion lens is used in microscopic examination procedures and
should be included for the benefit of those who would have an individual procedure that used the oil lens.
38. The proposed guidelines have neglected to mention that the basic requirement for optical performance is the
Koehler illumination. It should precede the explanations of how to focus a specimen. When we adjust the
microscope, the condenser is fixed in an upright position and must not be used to control the contrast. The
diaphragm controlling the condenser's aperture plays this role during examination. The diaphragm of the field
aperture must be open just enough to illuminate the microscopic field. It is fixed during the adjustment of the
microscope (Koehler illumination). Therefore, the first step of "how to focus a specimen" should be the actual
step (3). The actual steps (1) and (2) should be deleted. (See Clancy, M. N., M. Cohen and L. S. Garcia. 1992.
Bright-Field Microscope. In: H. Isenberg (ed.). Clinical Microb. Proc. Handbook, ASM, Washington, D.C. p.
12.14.1; 12.14.812.14.9.)
See the response to Comment 36.
Section 5.2.3
39. Include a specific recommendation for maintenance interval for professional microscope service to be rendered.
The text has been modified to include the COLA requirement, at a minimum, of annual maintenance.
This timeframe is dependent on the number of providers and the number of procedures performed on
the microscope.
Section 6
40. The quality assurance plan is the backbone of a testing program. This topic should be covered directly after
safety issues due to its importance. This section should also clearly state in bullet points the essential

Volume 23 HS2-A
components of an adequate quality assurance plan. To the text presented in Section 6, an additional factor for
error detection, investigation, correction, and documentation should be included.
The subcommittee appreciates the comment; however, the format of HS2 is consistent with other NCCLS
documents and has been maintained for consistency.
The following components of a quality assurance plan have been added, as bullet points, following the
first sentence: identifying problems and errors, assessment of the causes, designing corrective actions,
and monitoring to assure correction.
The fourth sentence has been modified to include, error detection, investigation and implementation
of corrective actions.
Section 6.1.1
41. I suggest rephrasing this section so that, according to accreditation or regulatory requirements, the person
responsible for testing must determine first who can perform testing, and then identify individuals in the
organization who meet these requirements.
To address the commenters concerns this section has been modified to read, The person responsible for
oversight of the testing should first establish the necessary qualifications (education and experience)
required for someone performing microscopic examinations, and then train qualified individuals.
Training should be developed and provided to all providers who conduct the PPM testing.
Section 6.1.2
42. The actual training of the providers occurs mostly while they are seeing patients. There are very few, if any,
formalized courses with regard to training and the various testing. With regard to competency testing, once it
has been ascertained that the person does know how to do the test properly, the likelihood of losing that
competency is very small, if not nonexistent. Ongoing competency testing would yield very few inadequacies at
a great cost in time and effort. The process would be very inefficient. On the other hand, recorded results should
be monitored. Tests need to be charted properly. This would document whether the entire test is being done.
For a variety of reasons, individuals can lose competency and must be refreshed from time to time.
Depending on frequency of test performance, adequacy of the initial training, ongoing monitoring of
performance, etc., competency can be lost. The issue may be the term testing in the Competency
Testing section. Testing sounds formal, rigid, and demanding. The intent of Section 6.1.2 is to describe
possibilities for assessing competency on a periodic basis. Therefore, the subcommittee has changed the
heading of Section 6.1.2 to Competency Assessment.
43. The bulleted remarks regarding method of competency testing are reasonable. However, the list is not complete.
There are many instances where collections of photos, textbooks, or educational software are used to help with
competency testing. Competency assessment exams based on software, books, or photos are complementary to
(not necessarily replacements for) the bulleted list of competency assessment methods listed in Section 6.1.2. It
is important to include these types of competency assessment exams in the list of bulleted items, since these
methods have distinct advantages over the other methods listed.
The subcommittee agrees with this suggestion. The following text has been added: assessment
examination using microscopy images in atlases or textbooks, photographs, or computer software.
44. During the first year of testing, newly trained personnel must receive competency evaluation twice and annually
thereafter.
The first sentence has been modified to include a parenthetical statement, i.e., "(such as twice during the
first year of testing and annually thereafter)," to address competency evaluation intervals.

Number 5 NCCLS
Section 6.1.3
45. Published books and software should be mentioned as sources of continuing education. Well-tested, well-
designed, accredited software is at least as good as the homemade PT slide/picture albums mentioned, and
certainly much more organized.
The subcommittee agrees with this comment and has modified the text to include textbooks and journals,
as well as commercially available educational packages.
Section 6.2.1
46. Having the appropriate initial training with a new piece of equipment is prudent. If equipment requires
calibration checks, those should be performed as part of the operational manuals of the equipment.
The text has been modified to address the commenters concern.
47. There are several statements that, although good, practical advice, do not address the issue of quality testing. I
suggest deleting these, as indicated. (delete - last paragraph of Section 6.1.3; last part of first sentence of first
paragraph in 6.2.1 beginning with "because it is expensive"; from the end of first sentence in second
paragraph in 6.2.1 ("both practical and wise") and the entire second sentence of the second paragraph; the last
sentence of the second paragraph of 6.2.1; the fourth sentence of the first paragraph of 6.4; and the second
sentence of the first paragraph in Section 6.5.)
Section 6.2.2
48. I am having difficulty envisioning a situation where glassware used in PPM testing could be reused with a
blank. Microscope slides and tubes should simply be single use.
In an international setting, there may be instances in the world where glass slides and other equipment
are at a premium.
49. Regarding the sentence: "If glassware is reused, a blank must be run to detect carryover." With perhaps the
single exception of using a counting chamber, the reuse of glassware in microscopy is totally unacceptable. By
including this alternative, you detract from the clear, unambiguous statement in the first sentence, and imply
that reuse is OK if you run a blank. Since you have no reason to believe that one reused slide provides any
information on another, this alternative gives no assurance. I strongly urge you to delete this sentence.
The text has been modified as suggested. The sentence has been deleted.
Section 6.3
50. The difference between the test result (which is part of the patient's permanent medical record) and the quality
assurance documentation used to derive that result is not clear in this section. The need to establish a document
retention schedule is part of the QA plan. Under CLIA, documents must be available for two years.
The term data has been replaced with test results.
With regard to document retention, the first sentence of the third paragraph has been modified to read,
The results should be retained for a minimum of two years; however, retention times may vary
depending on local and regional regulatory requirements and standards of practice for the medical
community.
51. First paragraph, last sentence. Reword sentence to read, criteria and recording of unacceptable specimens.

Volume 23 HS2-A
The sentence has been modified to read, recording of unacceptable specimens, recording of test
results, and reporting
Section 6.4
52. The bulleted statements beginning Initial Approval Process are very important. This paragraph should be
emphasized with regard to competency.
The subcommittee agrees with the comment. The text has been modified to read, A procedure manual is
also a valuable resource for training new providers, verifying provider competency (see Section 6.1.2), or
troubleshooting testing problems when they arise.
Section 6.5
53. The purpose of QC testing is to determine that the testing system is operating within pre-established limits.
The subcommittee agrees with the comment. The text has been modified to read, The purpose of quality
control (QC) is to monitor the analytical process to determine that the test system is operating within pre-
established limits, thus ensuring the accuracy of each examination or measurement performed on a
patient specimen.
54. This section addresses one of the challenges in quality control extremely well, i.e., the availability of control
materials for microscopy. However, one aspect needs more emphasis. Collection of multiple samples from
positive and negative patients is helpful, but if they remain unfixed, or if they are heat-fixed or alcohol-fixed,
the survival of morphologic structures beyond a few weeks is very limited. Once the typical positive
morphology has decayed, the value of the sample as a positive control is lost. Laboratories depending on fresh
materials for positive controls need to implement a program that is constantly on the look-out for positive
materials. Alternatively, commercial sources can be sought.
The following sentence has been added to address the commenters concern: Because the survival of
positive morphological structures is limited, testing sites that choose to use fresh patient specimens for
control material need to implement a program that is constantly 'on the look-out' for positive materials.
55. The paragraph beginning "The most common causes of unexpected control results are:" is excellent. This
provider-performed microscopy manual is an excellent training and teaching guide, and I would encourage it to
be included as one of the manuals that all people should read as part of their quality control testing.
The subcommittee appreciates the comment.
Section 6.6
56. It is a requirement of CLIA, ISO, and ILAC that proficiency-testing materials should duplicate clinical samples.
It is fair to say that looking at pictures is nothing like testing clinical samples. While it may be beyond the scope
of NCCLS, and while I would not incorporate this into this document, I strongly believe that standard format
pictures are an unacceptable substitute, and that accrediting bodies do a disservice by accepting the results as a
valid reflection of laboratory performance. In this situation "something" is not better than "nothing."
The subcommittee agrees with the comment. The text has been modified.
57. All testing personnel should have PT testing responsibilities. There should be a schedule to assure that all
testing personnel have the opportunity and responsibility to participate in PT.
The seventh sentence of the first paragraph has been modified to read, PT samples should be tested in
the same manner as patient specimens; therefore, the handling, preparation-processing, examination,

Number 5 NCCLS
and documentation of results should be performed by those providers who routinely perform the patient
testing.
58. All PT results should be reviewed with testing personnel.
The following sentence has been added to the end of the first paragraph: "The grades should be reviewed
and shared with all providers as part of the testing sites QA, continuing education, and competency
evaluation systems.
59. This section is most important with regard to initial training and evaluation. Further, the suggestions regarding
documentation would be prudent to do initially. Once all of this is done, very few people would then lose
competency.
60. When PT programs are unavailable, providers should be required, just as clinical laboratories, to demonstrate
proficiency by exchanging blind samples with a neighboring laboratory every six months.
The following text has been added in response to the commenters concern: If no PT program is
available, the testing site may choose to monitor providers proficiency by exchanging blind samples with
other test sites twice each year.
Section 6.7
61. This is a particularly difficult section, especially when the clinician, specimen processor, and microscopist are
the same person. To be critical and objective about the diagnostic process in this setting may be impossible. As
an alternative to trying to foster self-discipline, did the committee consider introducing the concept of voluntary
internal audit as an approach to nonconformity detection?
The text has been revised to better clarify this step in the diagnostic process as it relates to QA.
Section 7
62. The sentence, "Interpretation of urine sediment requires time, skill, training, and experience acquired through
constant use of various microscopic methods and continuous pathophysiologic correlation of the sediment
findings with the macroscopic results and clinical status of the patient" may be overstated. In this field one
develops an expertise within a short time. The "learning curve" is not long, and once someone has gotten to the
top or close to the top of the curve, the likelihood of losing that skill becomes small. Continuing medical
education can easily fine-tune the knowledge, or tweak skills.
Section 7.1.1
63. Edit the third line to read as a proactive statement. How many normal urine specimens have abnormal
microscopic exams? Under what clinical circumstances would a clinician encounter this situation? Include
literature citation that has documented those clinical situations having bonafide, clinically relevant abnormal
microscopic exam results from properly collected, transported, and processed urine specimens having normal,
physicochemical results?
The subcommittee is unaware of recent publications dealing with normal urine showing abnormal
microscopic findings. The text has been edited to remove the second paragraph of this section, which
relates to microscopic examination of urines with normal physicochemical results.
64. The automated urine sediment analyzer should be noted, as the use of these instruments is now prevalent
worldwide. Their usefulness in screening tests (as well as their limitations of performance) should be added.

Volume 23 HS2-A
While the subcommittee agrees that automated urine sediment analyzer use is now prevalent, discussion
of automated technology is beyond the scope of this document.
Section 7.2.1.2
65. I don't think "is not recommended" is strong enough.
The note has been modified to read, Do not reuse centrifugation tubes.
Section 7.2.2.2
66. Note that in this section the term 400g, which I think is correct, is used. In Section 7.4 (2) the term is replaced
by 400-x g. Would the committee consider putting in a table that gives rotor arm length, rotor speed, and RCF?
While this is available in other places, it may be convenient for the reader to have it in this guide.
A description of relative centrifugal force (RCF) and a formula for its calculation have been added to this
section. Section 7.4 (2) has been revised for consistency.
67. Consider recommending a set calibration interval, e.g., performed every four months, every six months.
The following recommendation has been incorporated in Section 7.2.2.2 to address the commenters
concern: At a minimum, centrifuge calibration should be performed annually.
Section 7.2.3
68. Last sentence: replace should with shall, i.e., reagents shall not be used after their expirations.
Section 7.4
69. In Section 7.4 (6), the condenser should not be lowered to control the contrast.
The parenthetical statement has been corrected to read, (achieved by lowering illumination intensity).
Section 7.4.1.2
70. Third line: replace hardening with augmenting or enhancing.
The term hardening has been replaced with the term enhancing.
71. Sternheimer stain and Sudan III stain should be inserted in the document. In Japan, Sternheimer stain is popular
for routine urine tests for the purpose of the identification of epithelial cells and casts. Sudan III is also very
useful for the identification of oval fat bodies, fat droplets, and fatty casts.
Section 7.4.1.3 has been added to include a description of Sternheimer stain and Sudan III stain.
Section 7.4.2
72. In Table 1, the only diagnosis associated with finding of crystals is cancer. This is inaccurate and misleading.
More importantly, it is inconsistent with the comments in Appendix A, Section A6. I suggest removing the term
"cancer" in the table and replacing it with "refer to Appendix A."

Number 5 NCCLS
73. I note that you include antibody-coated bacteria in Table 1. Is this a procedure that is included under
provider-performed microscopy? If so, labeling as being synonymous with pyelonephritis is not correct (also
prostatitis, chronic infection, catheterization).
The text "antibody-coated: pyelonephritis" has been removed from Table 1 for clarity.
74. In Table 1, under Finding: Crystals, cystine and bilirubin crystals should be added. Also, the meaning of
"cancer (uric acid)" is not clear.
75. In Table 1, under Finding: Renal Tubular Epithelial Cells, nephrotic syndrome should be included.
The committee agrees with the comment. Nephrotic syndrome has been added to the table as
recommended.
76. In Table 1, Finding: Cellular Casts, nephrotic syndrome should be included.
77. In Table 1 under "Finding: microorganisms: 100,000/mL: always infection"A culture is usually necessary to
quantitate bacteria present in that way. By direct microscopy, the number of cells seen by high-power field or
oil-immersion field is usually reported. Is it the same for bacteria?
The text has been modified for clarity as follows: Observation of microorganisms, indicating infection,
should be confirmed by culture.
Section 7.4.3
78. Reword second paragraph, first sentence as follows: The importance of standardization in microscopic
urinalysis cannot be overemphasized.
Section 7.5.1
79. I do not understand the sentence, "The unavailability of commercially prepared quality control solutions does
not justify the neglect of microscopic quality control." I believe that once someone has learned how to identify
the various types of cells or findings for a specific test, that there is actually very little further subtlety. Clearly,
if someone does not have competency initially to identify certain items, then their initial competency testing
should not be rated as "satisfactory." That person would need to be continually trained until he/she has
demonstrated competency.
The second paragraph of this section has been deleted to clarify the purpose of this section.
Section 8.2.3
80. "Sterile physiologic saline" is not "9 grams/dL" but 9 grams/L or 0.9 gm/dL.
The text has been corrected to reflect the proper preparation of physiological saline.
81. I do agree that concentrated potassium hydroxide is a highly corrosive liquid, but to state specifically that one
should wear gloves or eye/face protection might overstate the problem when one is using small amounts of it
from a small container. We must encourage safety, but we must also encourage people to use common sense
and to be responsible for their actions. Taking away a persons responsibility totally might make sense from a

Volume 23 HS2-A
medical/legal point of view or from a work/legal point of view, but to encourage workers to be sloppy or not to
take responsibility for their actions would be much more dangerous.
The following text has been added to the Note in this section: "It is recommended that for use in the PPM
environment, 10% KOH be purchased commercially or obtained from pharmacy/laboratory sources."
Section 8.4
82. In Section 8.4 (8), the last part of the first sentence should read, "and under high power (40x) for smaller
blastospores." Furthermore, the last sentence is an incomplete repetition of the first one.
Section 8.4.1
83. A typographical error appears in the second line of text. It should read, "See Appendix B for a more detailed..."
This typographical error has been corrected.
84. Table 2, in row other organisms under column abnormal due to desquamative inflammatory vaginitis, there
appears to be an over-call for gram-positive cocci when a gram stain was not performed.
Table 2 was included as authored; it has been modified, to remove gram-positive cocci, in response to the
comment.
85. Table 2, last item should read, "vaginal erythema."
Section 8.7
86. A typographical error appears in the first bullet. It should read, "... which can make interpretation of direct wet
mounts difficult.
Section 9
87. Parasite testing: Why is only the pinworm test described in this document? Moreover, the anal cellophane tape
method is more efficient for pinworm detection than fecal analysis.
The scope of the prescribed provider-performed microscopy procedures was limited to those in the CLIA
PPM category of testing. That category specified the performance of pinworm examination, not parasite
testing. While the cellophane tape method is more common, the wet preparation was not excluded.
88. Figures 2 and 3 are hand-drawn renderings. With the availability of electronic imaging and almost universal
access to authoritative texts, the use of these quality illustrations is unnecessary.
Better quality illustrations have been provided for Figures 2 and 3.
Section 9.2
89. In the cellophane tape test results for medical checks of school children in Japan, the adult pinworm is
sometimes found. Therefore, it is recommended that suggested reporting not be limited to none, but should
include rare as a reporting option.

Number 5 NCCLS
Section 9.2.2.2
90. Third paragraph, edit first sentence to read, With gloved hands, spread the anal folds apart
Section 9.2.3
91. Item (2) should read, "... using a 10x to 40x dry objective."
92. Figure 2. This drawing is not absolutely representative. Another drawing may be more accurate. See Murray,
P.R., E. J. Baron, M.A. Pfaller, F.C. Tenover and R.H. Yolken. 1999. Manual of Clinical Microbiology, 7th ed.
ASM Press, Washington, D.C.
93. It is not possible to avoid contamination by such procedure; moreover, it is more infectious. We recommend
using the cellophane tape method.
It is not clear to which procedure the commenter refers. Section 9.2 is the cellophane tape method.
Section 9.3 covers Detection of Adult Parasitesincluding the wet preparation of fecal materialand is
within the scope of the document. Additional mention is included regarding precautions to avoid
contamination.
Section 9.3
94. The description would be more precise if it were presented in the following manner: "... a ventrally curved
posterior end for the male, and a long, pointed tail for the female.
Section 9.3.1.2
95. For the identification of worms 2 to 13 mm in length, stereoscopic microscopy examination is more appropriate
than that of ordinary bright-field microscopy.
Stereoscopic microscopic equipment is not generally available in the settings described; therefore, the
original recommendation for (worm) identification has been maintained.
Section 9.3.2.1
96. The cellophane tape method is sufficient and convenient. The labor-consuming procedure, described in the
document, is not needed.
The procedure may be undertaken to confirm parental observation under some circumstances.
Section 9.3.2.3
97. Where do we put the fecal material after collection and before examination? (Suggestion: The fecal material is
then put in a small container and maintained at room temperature until examined.)

Volume 23 HS2-A
Section 9.3.3
98. Figure 3. A drawing of the female worm should be added to that of the male worm illustrated as it is referred to
in Section 9.3 and in Section 9.3.3. It is the most frequently seen.
An illustration of the female worm has been included.
99. For the purpose of detecting worms measuring 2 to 13 mm in length, magnification 40x seems to be too high.
The text has been modified to clarify use of both 10x and 40x magnifications.
Section 9.3.6
100. The rationale for the limitations of the procedure should be described (as is done in Section 9.2.6).
Limitations of the procedure have been incorporated. The cellophane tape test has been recommended
if clinically indicated.
Section 10.1
101. The fern test certainly is one of the tests used to determine whether there is amniotic fluid within the vagina.
However, the fern test is also used to determine the level of estrogen affect when cervical mucus is being
evaluated at the time that a woman is ovulating. Both of these indications should be included.
The procedure for postcoital testing includes evaluation of cervical mucus for estrogen effect at
ovulation. A cross-reference to Section 11 has been included for clarity.
Section 10.3.2
102. Certainly, vaginal fluid can be picked up with a transfer pipette or a cotton-tipped applicator and spread out
onto a slide. Why state so specifically how the specimen is collected?
No prior level of technique was assumed. There is a caution that no interfering materials be
introduced. Certain types of swabs may differently absorb constituents of the specimen; therefore, a
simpler direction can be established with the liquid transfer pipette than specifying the type of swab.
Section 10.3.3
103. The specimen must be dry. Drying can take some time depending on the amount of fluid. Once the specimen
is dry, it does not need to be read immediately. Crystals tend to keep.
Section 10.4
104. Why include the statement "confirmation of test results using the 100x oil immersion objective is
acceptable"? This does not need to be done unless one enjoys looking at the crystal formation. It is not helpful
to determine ferning.
The text has been modified. The reference to confirmation using 100x objective has been deleted.

Number 5 NCCLS
Section 10.6
105. Besides the presence of a fern pattern, there is also the quality or characteristics of the crystal pattern. The
ferning pattern is also a function of the various cellular and chemical elements co-mingled within the vagina.
The quality and characteristics of the crystal pattern are not part of a PPM work-up for the presence
of amniotic fluid.
Section 11
106. More sophisticated laboratories are not using this test. We abandoned the assay over ten years ago, along with
many other laboratories. A retrospective analysis of our own patient population showed that its value in
patient care was next to worthless. I really dont know if the practitioner in the field is using this test.
Assuming that this section is of use to the practitioner, I think that the section adequately covers the topic and
points out some of the pitfalls of this test.
The HS2 guideline is intended for the practitioner in the field, not the sophisticated laboratory;
therefore, the recommended procedure for postcoital testing has been included.
Section 11.2.1
107. The listing of supplies might be too specific. Different people will do things differently. One might state
merely that the supplies should be single-use and sterile.
Section 11.2.1 lists recommended supplies for use in postcoital testing procedures. If an item is essential
for testing, mention of its use is included in the procedure.
108. I prefer a transfer pipette. Why even use any of these terms? Why not state things very simply?
Use of a syringe has been recommended, as it doubles as a volume measurement device. However, if
sufficient suction can be generated to remove mucus, a transfer pipette could serve as an alternative.
The phrase or similar suction device has been added to address the commenters concern.
Section 11.4
109. I would add to the testing procedure the fern test that determines the quality of the estrogen effect in the
cervical mucus.
Interpretation of the fern test as a measure of estrogen effect was not available in the references
reviewed.
Section 11.6
110. This section has similar descriptors to the ones in Section 11.4. Maybe one of these two sections could be
eliminated/combined?
The separation of testing procedures and reporting results has been maintained for consistency.
Section 11.6.1
111. The postcoital test is a test which is interpreted differently by different people. There is not a lot of agreement
on particular numbers or what a particular number means. Some are happier with less, others are happier with
more. This manual should be far more concerned about the techniques and less concerned about the
interpretations. I would suggest leaving the interpretations out. Certainly if one wants to include what should
be part of the interpretation, one might again incorporate this section with Section 11.6.

Volume 23 HS2-A
The intent of Section 11.6.1 is to provide general guidance on result interpretation. Figure 4 has been
deleted, as detailed explanation and interpretation of fertility testing is beyond the scope of this
document.
112. I am not sure that I agree that this guide is the best place to be providing information about diagnostic and
therapeutic approaches to infertility. While interesting, I recommend the committee re-evaluate the need for
the decision tree in this section.
Section 12
113. With regard to semen analysis, I would advise against collection of the semen sample into anything other than
clean, sterile, plastic specimen cups provided by the office or clinic. Avoid the mention of glassware. There
are all sorts of hazards when glassware is used (e.g., recycling, reuse).
There are reports of decreased motility in specimens remaining in plastic containers for more than 60
minutes. The choice of container is less of a concern with qualitative analysis for presence or absence of
sperm.
114. The recommended time for liquefaction of an ejaculate is 30 minutes (room temperature to 37 C). I would
advise the practitioner to mix the sample thoroughly after liquefaction to obtain a good sample aliquot.
The phrase well-mixed has been added to Step 2 for clarity.
Section 12.3.1
115. Instead of three-day period of abstinence, the World Health Organization Guidelines (1999) suggests 48 to
72 hours of sexual abstinence prior to collection.
Section 12.4
116. According to WHO guidelines, all forms of motility should be included. A comment can be made concerning
the type of motility, for example: "sluggish," "little to no forward progression," etc.
The text has been modified in accord with WHO guidelines.
Section 12.6.1
117. The reference range for sperm motility is >50% according to WHO guidelines.
The text has been modified in accord with WHO guidelines.
Section 14.1
118. Is this test still useful and significant? It seems that there is something contradictory between the principle
and the limitations of the procedure. In the principle, the test has "a high sensitivity and specificity for
bacterial diarrhea"; in the limitations of the procedure, however, "the method is not sufficiently sensitive or
specific to preclude the use of culture."
The test is in frequent use in the primary care office as a rapid diagnostic tool. The limitations should
lead the practitioner to use the test as an adjunct to culture, and in association with clinical symptoms
to determine treatment. The text has been modified to address the commenters concern.

Number 5 NCCLS
Section 14.3.2
119. In addition to PVA, is 10% buffered formalin acceptable? We use two gram stain smears made from 10%
formalin for a "quick" read for preserved specimens, i.e., no fresh stool submitted. It also avoids spreading
mercury chloride throughout the laboratory. The PVA, however, appeared much more sensitive when stained
with our standard O&P stain. For fresh, unpreserved stools, we gram-stain them, i.e., never stained with
methylene blue.
If the opportunity is available to provide the patient with a preservative, leukocytes will be maintained
for same-day testing in any preservative compatible with cytology, tissue, or parasitology examinations
(e.g., formalin).
120. Could formalin 10% also be used as a good preservative as used for intestinal parasites (in combination with
PVA)? Its use could be easier for direct examination of fecal leucocytes (a ratio of one volume of feces to
three volumes of formalin 10% is usually recommended).
See response to Comment 119. Many kits for collection of O&P (ova and parasites) specimens
include polyvinyl alcohol (PVA) and formalin. PVA is usually used for preserving trophozoites and
10% formalin for ova.
Section 14.3.3
121. Gas is usually produced in unfixed specimens. The precision should be made as follows: "because gas
frequently accumulates in unfixed stool specimens
Section 14.4
122. Methylene blue should be placed first onto the slide to prevent contamination of the dropper, i.e., step (2)
should come before step (1).
The addition of stain should be via a dropper without coming in contact with the specimen. Text has
been added to reflect that caution as well as the alternative suggested in the comment.
123. It could be useful to briefly describe the protocol for staining with Wright-Giemsa stain in this section.
The protocol for Wright-Giemsa staining is described immediately following the numbered steps of the
methylene blue protocol.
References
124. References on pinworms should be updated.
The references have been updated as suggested.
Appendix A
125. Section A1, Red Blood Cells (Erythrocytes), dysmorphic RBC (acanthocyte, donut shape, etc.), which are
clinically meaningful for the diagnosis of glomerular bleeding, should be inserted in the document.
Text has been added to indicate the significance of dysmorphic red blood cells.
126. In Section A3, subsections regarding oval fatty body, inclusion-bearing-cells, and atypical cells should be
added respectively.

Volume 23 HS2-A
Three subsections have been added to address the recommended topics.
127. Section A5.3 Cellular Cast, Neutrophil: The description sounds like "cellular casts are equal to epithelial
casts." We recommend describing WBC casts individually.
The title has been changed as recommended.
128. Bilirubin crystals should be mentioned in Section A6.2, Crystals At Neutral to Acid pH.
The section has been modified as recommended.
129. Although it may be a rare case, we recommended mentioning 2, 8,-Dihydroxyadenine crystals in Section
A6.3.
The subcommittee appreciates the input; however, due to the rarity of these crystals, they have not
been included in this section.
Appendix B
130. Table B1 may dangerously oversimplify or understate the difficulty of identifying fungal species by direct
microscopy, even presumptively. Pictures may help, as well as a warning that identification of the organisms,
several of which may cause life-threatening infections, based on a direct smear examination, can be extremely
difficult and should be referred, whenever possible, to personnel with more extensive experience.
Picture references are a requirement of both CLIA and COLA and should supplement any written
description. Table B1 has been deleted in response to this comment.
131. Section B6 contains a typographical error. The statement should read: "They have a nucleus to cytoplasm
ratio of 1:2."
132. Table B1. There is a typographical error that should be corrected accordingly: Blastomyces dermatitidis and
Coccidioides immitis.
Table B1 has been deleted in response to Comment 130.
133. Appendix B, Table B1 is unrelated to the appendix and should not be included. Microscopic examination of
Aspergillus species, Cryptococcus species, and especially Histoplasma, Blastomyces, and Coccidiomyces do
not belong here.
Table B1 has been deleted in response to Comment 130.

Number 5 NCCLS
Summary of Delegate Comments and Subcommittee Responses

HS2-A: Provider-Performed Microscopy Testing; Approved Guideline
Appendix A
1. Regarding the statement that amorphous urates must be distinguished from bacteria: Is it within the scope
of this document to clarify that distinction? I suggest adding the following or something similarly appropriate:
Amorphous urates are generally more pleomorphic than bacteria, and are not associated with increased white
blood cells. A gram stain may be needed for further evaluation.
The text has been modified as suggested with the addition of an applicable footnote.
2. Radiographic crystals in urine are described in this document. The CAP Hematology & Clinical Microscopy
Resource Committee, at its last meeting, deleted this definition from the Hematology Glossary, because these
crystals no longer appear with new contrast media in use.
The text has been edited to remove the section describing radiographic media.

Volume 23 HS2-A
Related NCCLS Publications*

GP2-A4 Clinical Laboratory Technical Procedure Manuals; Approved GuidelineFourth Edition (2002). This
document provides guidelines that address the design, preparation, maintenance, and use of technical
procedure manuals (whether they are in paper or electronic formats) for use by the patient-testing community.
GP5-A2 Clinical Laboratory Waste Management; Approved GuidelineSecond Edition (2002). Based on U.S.
regulations, this document provides guidance on safe handling and disposal of chemical, infectious,
radioactive, and multihazardous wastes generated in the clinical laboratory.
GP16-A2 Routine Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved
GuidelineSecond Edition (2001). This guideline offers descriptions of routine urinalysis test procedures
that address materials and equipment, macroscopic examinations, clinical analyses, and microscopic
evaluations.
GP21-A Training Verification for Laboratory Personnel; Approved Guideline (1995). This document provides
background and recommends an infrastructure for developing a training verification program that meets
quality/regulatory objectives.
M29-A2 Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline
Second Edition (2002). This document provides guidance on the risk of transmission of hepatitis viruses and
human immunodeficiency viruses in any laboratory setting; specific precautions for preventing the laboratory
transmission of blood-borne infection for laboratory instruments and materials; and recommendations for the
management of blood-borne exposure.
*
Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should
refer to the most recent editions.

Number 5 NCCLS
NOTES

Volume 23 HS2-A
NOTES

Active Membership
(as of 1 January 2003)
Sustaining Members Deutsches Institut fr Normung F. Hoffman-La Roche AG Albemarle Hospital (NC)
(DIN) Fort Dodge Animal Health Allegheny General Hospital (PA)
Abbott Laboratories FDA Center for Devices and General Hospital Vienna (Austria) Allina Health System (MN)
American Association for Clinical Radiological Health Gen-Probe Alton Ochsner Medical Foundation
Chemistry FDA Center for Veterinary Medicine GlaxoSmithKline (LA)
Beckman Coulter, Inc. FDA Division of Anti-Infective Greiner Bio-One Inc. Anne Arundel Medical Center (MD)
BD and Company Drug Products IGEN Inc. Antwerp University Hospital
bioMrieux, Inc. Iowa State Hygienic Laboratory Immunicon Corporation (Belgium)
CLMA Massachusetts Department of Public Instrumentation Laboratory Arkansas Department of Health
College of American Pathologists Health Laboratories International Technidyne ARUP at University Hospital (UT)
GlaxoSmithKline National Center of Infectious and Corporation Armed Forces Research Institute of
Ortho-Clinical Diagnostics, Inc. Parasitic Diseases (Bulgaria) IntraBiotics Pharmaceuticals, Inc. Medical Science (APO, AP)
Pfizer Inc National Health Laboratory Service I-STAT Corporation Associated Regional & University
Roche Diagnostics, Inc. (South Africa) Johnson and Johnson Pharmaceutical Pathologists (UT)
National Institute of Standards and Research and Development, L.L.C. Atlantic Health System (NJ)
Professional Members Technology LAB-Interlink, Inc. Aurora Consolidated Laboratories
New York State Department of Laboratory Specialists, Inc. (WI)
American Academy of Family Health Labtest Diagnostica S.A. AZ Sint-Jan (Belgium)
Physicians Ohio Department of Health LifeScan, Inc. (a Johnson & Johnson Azienda Ospedale Di Lecco (Italy)
American Association for Clinical Ontario Ministry of Health Company) Baxter Regional Medical Center
Chemistry Pennsylvania Dept. of Health Lilly Research Laboratories (AR)
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Laboratory Science Environment Nichols Institute Diagnostics Bo Ali Hospital (Iran)
American Society of Hematology Swedish Institute for Infectious (Div. of Quest Diagnostics, Inc.) Brooks Air Force Base (TX)
American Society for Microbiology Disease Control NimbleGen Systems, Inc. Broward General Medical Center
American Type Culture Collection, Nissui Pharmaceutical Co., Ltd. (FL)
Inc. Industry Members Nippon Becton Dickinson Co., Ltd. Cadham Provincial Laboratory
Asociacion Mexicana de Bioquimica Norfolk Associates, Inc. Calgary Laboratory Services
Clinica A.C. AB Biodisk Novartis Pharmaceuticals Cape Breton Healthcare Complex
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Assoc. Micro. Clinici Italiani- Abbott Laboratories, MediSense Ortho-Clinical Diagnostics, Inc. Carilion Consolidated Laboratory
A.M.C.L.I. Products (Rochester, NY) (VA)
British Society for Antimicrobial Acrometrix Corporation Ortho-McNeil Pharmaceutical Cathay General Hospital (Taiwan)
Chemotherapy Alifax S.P.A. Oxoid Inc. Central Peninsula General Hospital
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Laboratory ScienceSocit Anaerobe Systems Pfizer Inc Central Texas Veterans Health Care
Canadienne de Science de A/S ROSCO Pharmacia Corporation System
Laboratoire Mdical Assssor Philips Medical Systems Centro Diagnostico Italiano (Milano,
Clinical Laboratory Management AstraZeneca Powers Consulting Services Italy)
Association AstraZeneca R & D Boston Premier Inc. Champlain Valley Physicians
COLA Avant Immunotherapeutics, Inc. Procter & Gamble Pharmaceuticals, Hospital (NY)
College of American Pathologists Aventis Inc. Chang Gung Memorial Hospital
College of Medical Laboratory Axis-Shield POC AS QSE Consulting (Taiwan)
Technologists of Ontario Bayer Corporation Elkhart, IN Quintiles, Inc. Changi General Hospital
College of Physicians and Surgeons Bayer Corporation Tarrytown, NY Radiometer America, Inc. (Singapore)
of Saskatchewan Bayer Corporation West Haven, Radiometer Medical A/S The Charlotte Hungerford Hospital
ESCMID CT Replidyne (CT)
International Council for Bayer Medical Ltd. Roche Diagnostics GmbH Childrens Hospital (LA)
Standardization in Haematology BD Roche Diagnostics, Inc. Childrens Hospital (NE)
International Federation of BD Biosciences San Jose, CA Roche Laboratories (Div. Hoffmann- Childrens Hospital & Clinics (MN)
Biomedical Laboratory Science BD Consumer Products La Roche Inc.) Childrens Hospital Medical Center
International Federation of Clinical BD Diagnostic Systems Sarstedt, Inc. (Akron, OH)
Chemistry BD Italia S.P.A. SARL Laboratoire Carron (France) Childrens Hospital of Philadelphia
Italian Society of Clinical BD VACUTAINER Systems Schering Corporation (PA)
Biochemistry and Clinical Beckman Coulter, Inc. Schleicher & Schuell, Inc. Childrens Medical Center of Dallas
Molecular Biology Beckman Coulter, Inc. Primary Care Second Opinion (TX)
Japan Society of Clinical Chemistry Diagnostics Streck Laboratories, Inc. Clarian HealthMethodist Hospital
Japanese Committee for Clinical Beckman Coulter K.K. (Japan) Synermed Diagnostic Corp. (IN)
Laboratory Standards Bio-Development SRL Sysmex Corporation (Japan) Clinical Laboratory Partners, LLC
Joint Commission on Accreditation Bio-Inova Life Sciences Sysmex Corporation (Long Grove, (CT)
of Healthcare Organizations International IL) CLSI Laboratories (PA)
National Academy of Bio-Inova Life Sciences North The Clinical Microbiology Institute Columbia Regional Hospital (MO)
Clinical Biochemistry America The Toledo Hospital (OH) Commonwealth of Kentucky
National Association of Testing BioMrieux (NC) Theravance Inc. Community Hospital of Lancaster
Authorities Australia bioMrieux, Inc. (MO) Transasia Engineers (PA)
National Society for Biometrology Consultants Trek Diagnostic Systems, Inc. CompuNet Clinical Laboratories
Histotechnology, Inc. Bio-Rad Laboratories, Inc. Tyco Kendall Healthcare (OH)
Ontario Medical Association Quality Bio-Rad Laboratories, Inc. - France Versicor, Inc. Cook County Hospital (IL)
Management Program-Laboratory Blaine Healthcare Associates, Inc. Vetoquinol S.A. Cook Childrens Medical Center
Service Bristol-Myers Squibb Company Vysis, Inc. (TX)
RCPA Quality Assurance Programs Canadian External Quality Wyeth-Ayerst Covance Central Laboratory
PTY Limited Assessment Laboratory Xyletech Systems, Inc. Services (IN)
Sociedade Brasileira de Analises Chiron Corporation YD Consultant Danish Veterinary Laboratory
Clinicas ChromaVision Medical Systems, YD Diagnostics (Seoul, Korea) (Denmark)
Sociedad Espanola de Bioquimica Inc. Danville Regional Medical Center
Clinica y Patologia Molecular Chronolab Ag Trade Associations (VA)
Taiwanese Committee for Clinical Clinical Design Group Inc. Dean Medical Center (WI)
Laboratory Standards (TCCLS) Cognigen AdvaMed Department of Health & Community
Turkish Society of Microbiology Community Medical Center (NJ) Japan Association Clinical Services (New Brunswick,
Copan Diagnostics Inc. Reagents Ind. (Tokyo, Japan) Canada)
Government Members Cosmetic Ingredient Review Medical Industry Association DesPeres Hospital (MO)
Cubist Pharmaceuticals of Australia Detroit Health Department (MI)
Association of Public Health Dade Behring Inc. - Deerfield, IL Diagnosticos da Amrica S/A
Laboratories Dade Behring Inc. - Glasgow, DE Associate Active Members (Brazil)
Armed Forces Institute of Pathology Dade Behring Inc. - Marburg, Dr. Everett Chalmers Hospital (New
BC Centre for Disease Control Germany 31st Medical Group/SGSL (APO, Brunswick, Canada)
Centers for Disease Control and Dade Behring Inc. - Sacramento, CA AE) Doctors Hospital (Bahamas)
Prevention Dade Behring Inc. - San Jose, CA 67th CSH Wuerzburg, GE (NY) Duke University Medical Center
Centers for Medicare & Medicaid David G. Rhoads Associates, Inc. 121st General Hospital (CA) (NC)
Services/CLIA Program Diagnostics Consultancy Academisch Ziekenhuis-VUB Dwight David Eisenhower Army
Centers for Medicare & Medicaid Diagnostic Products Corporation (Belgium) Med. Ctr. (GA)
Services Eiken Chemical Company, Ltd. Acadiana Medical Laboratories, E.A. Conway Medical Center (LA)
Chinese Committee for Clinical Elan Pharmaceuticals LTD (LA) East Side Clinical Laboratory (RI)
Laboratory Standards Electa Lab s.r.l. Advocate Healthcare Lutheran Eastern Health (Vic., Australia)
Commonwealth of Pennsylvania Enterprise Analysis Corporation General (IL) Elyria Memorial Hospital (OH)
Bureau of Laboratories Essential Therapeutics, Inc. Akershus Central Hospital and AFA Emory University Hospital (GA)
Department of Veterans Affairs EXPERTech Associates, Inc. (Norway)
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(MN) (FL) Pikeville Methodist Hospital (KY) Health
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Florida Hospital East Orlando Langley Air Force Base (VA) Presbyterian Hospital of Dallas (NY)
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Foothills Hospital (Calgary, AB, Center (TN) Providence Health Care Center (KS)
Canada) L'Hotel-Dieu de Quebec (Canada) Queen Elizabeth Hospital (Prince Sun Health-Boswell Hospital (AZ)
Fresenius Medical Care/Spectra Libero Instituto Univ. Campus Edward Island, Canada) Swedish Medical Center
East (NJ) BioMedico (Italy) Quest Diagnostics Incorporated Providence Campus (WA)
Fresno Community Hospital and Louisiana State University (CA) Temple University Hospital (PA)
Medical Center Medical Center Quintiles Laboratories, Ltd. (GA) Tenet Odessa Regional Hospital
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Geisinger Medical Center (PA) Malcolm Grow USAF Medical Services (IN) Touro Infirmary (LA)
Grady Memorial Hospital (GA) Center (MD) Research Medical Center (MO) Trident Regional Medical Center
Guthrie Clinic Laboratories (PA) Martin Luther King/Drew Medical Rex Healthcare (NC) (SC)
Hahnemann University Hospital Center (CA) Rhode Island Department of Health Tripler Army Medical Center (HI)
(PA) Massachusetts General Hospital Laboratories Truman Medical Center (MO)
Harris Methodist Fort Worth (TX) (Microbiology Laboratory) Riyadh Armed Forces Hospital UCLA Medical Center (CA)
Hartford Hospital (CT) MDS Metro Laboratory Services (Saudi Arabia) UCSF Medical Center (CA)
Health Network Lab (PA) (Burnaby, BC, Canada) Robert Wood Johnson University UNC Hospitals (NC)
Health Partners Laboratories (VA) Medical College of Virginia Hospital (NJ) University College Hospital
Highlands Regional Medical Center Hospital Royal Columbian Hospital (New (Galway, Ireland)
(FL) Medicare/Medicaid Certification, Westminster, BC, Canada) University Hospital (Gent)
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Presbyterian (CA) Memorial Hospital at Gulfport (MS) Arabia) University Hospitals of Cleveland
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Holmes Regional Medical Center Memorial Medical Center (LA) Saint Marys Regional Medical University of Alabama-Birmingham
(FL) Jefferson Davis Hwy Center (NV) Hospital
Holzer Medical Center (OH) Hopital Memorial Medical Center (LA) St. Alexius Medical Center (ND) University of Alberta Hospitals
du Sacre-Coeur de Montreal Napoleon Avenue St. Anthony Hospital (CO) (Canada)
(Montreal, Quebec, Canada) Mercy Medical Center (IA) St. Anthonys Hospital (FL) University of Colorado Health
Hpital Maisonneuve Rosemont Methodist Hospital (TX) St. Barnabas Medical Center (NJ) Science Center
(Montreal, Canada) MetroHealth Medical Center (OH) St-Eustache Hospital (Quebec, University of Chicago Hospitals
Hpital Saint-LUC (Montreal, Michigan Department of Canada) (IL)
Quebec, Canada) Community Health St. Francis Medical Ctr. (CA) University of Illinois Medical Center
Hospital for Sick Children (Toronto, Mississippi Baptist Medical Center St. John Hospital and Medical University of the Ryukyus (Japan)
ON, Canada) Monte Tabor Centro Italo - Center (MI) University of Virginia Medical
Hospital Sousa Martins (Portugal) Brazileiro de Promocao (Brazil) St. John Regional Hospital (St. Center
Hotel Dieu Hospital (Windsor, ON, Montreal Childrens Hospital John, NB, Canada) UZ-KUL Medical Center (Belgium)
Canada) (Canada) St. Johns Hospital & Health Center VA (Denver) Medical Center (CO)
Huddinge University Hospital Montreal General Hospital (Canada) (CA) Virginia Department of Health
(Sweden) MRL Pharmaceutical Services, Inc. St. Joseph Hospital (NE) VA (Hines) Medical Center
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Indiana University Nassau County Medical Center (NY) Clinic (WI) (MO)
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Institute of Medical and Veterinary Naval Hospital Corpus Christi TX) St. Joseph Mercy Hospital (MI) System
Science (Australia) Nebraska Health System St. Jude Children's Research VA (San Diego) Medical Center
International Health Management New Britain General Hospital (CT) Hospital (TN) (CA)
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(CA) North Shore Long Island Jewish Canada) West Jefferson Medical Center (LA)
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Kantonsspital (Switzerland) North Shore University Hospital San Francisco General Hospital Wilford Hall Medical Center (TX)
Kenora-Rainy River Regional (NY) (CA) William Beaumont Army Medical
Laboratory Program (Ontario, Northwestern Memorial Hospital Santa Clara Valley Medical Center Center (TX)
Canada) (IL) (CA) William Beaumont Hospital (MI)
Kimball Medical Center (NJ) O.L. Vrouwziekenhuis (Belgium) Seoul Natl University Hospital William Osler Health Centre
Kings Daughter Medical Center Ordre professionnel des (Korea) (Brampton, ON, Canada)
(KY) technologists mdicaux du South Bend Medical Foundation Williamsburg Community Hospital
Klinini Center (Slovenia) Qubec (IN) (VA)
Laboratoire de Sant Publique du Ospedali Riuniti (Italy) Southwest Texas Methodist Hospital Winn Army Community Hospital
Quebec (Canada) The Ottawa Hospital (TX) (GA)
Laboratrio Fleury S/C Ltda. (Ottawa, ON, Canada) South Western Area Pathology Winnipeg Regional Health
(Brazil) Our Lady of Lourdes Hospital (NJ) Service (Australia) Authority (Winnipeg, Canada)
Laboratory Corporation of America Pathology and Cytology Southern Maine Medical Center Wishard Memorial Hospital (IN)
(MO) Laboratories, Inc. (KY) Spartanburg Regional Medical Yonsei University College of
Laboratory Corporation of America Pathology Associates Medical Center (SC) Medicine (Korea)
(NJ) Laboratories (WA) Specialty Laboratories, Inc. (CA) York Hospital (PA)
LAC and USC Healthcare The Permanente Medical Group Stanford Hospital and Clinics (CA)
Network (CA) (CA)
OFFICERS BOARD OF DIRECTORS
Donna M. Meyer, Ph.D., Susan Blonshine, RRT, RPFT, FAARC Tadashi Kawai, M.D., Ph.D.
President TechEd International Clinical Pathology Center
CHRISTUS Health
Wayne Brinster J. Stephen Kroger, M.D., MACP
Thomas L. Hearn, Ph.D., BD COLA
President Elect
Centers for Disease Control and Prevention Kurt H. Davis, FCSMLS, CAE Willie E. May, Ph.D.
Canadian Society for Medical Laboratory Science National Institute of Standards and Technology
Emil Voelkert, Ph.D.,
Secretary Lillian J. Gill, M.S. Gary L. Myers, Ph.D.
Roche Diagnostics GmbH FDA Center for Devices and Radiological Health Centers for Disease Control and Prevention
Gerald A. Hoeltge, M.D., Robert L. Habig, Ph.D. Barbara G. Painter, Ph.D.

Treasurer Habig Consulting Group Pharma Micro Consultancy, LLC
The Cleveland Clinic Foundation
Carolyn D. Jones, J.D., M.P.H. Judith A. Yost, M.A., M.T.(ASCP)
F. Alan Andersen, Ph.D., AdvaMed Centers for Medicare & Medicaid Services
Immediate Past President
Cosmetic Ingredient Review
John V. Bergen, Ph.D.,

Executive Director
NCCLS T 940 West Valley Road T Suite 1400 T Wayne, PA 19087 T USA T PHONE 610.688.0100
FAX 610.688.0700 T E-MAIL: exoffice@nccls.org T WEBSITE: www.nccls.org T ISBN 1-56238-487-2

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What is the purpose of NCCLS?

What is the purpose of NCCLS?

What are the different levels of consensus for NCCLS documents?

What are the different levels of consensus for NCCLS documents?

HS2-A

Reproduced with permission, from NCCLS publication HS2-AProvider-Performed

Copyright 2003. The National Committee for Clinical Laboratory Standards.

(NCCLS. Provider-Performed Microscopy Testing; Approved Guideline. NCCLS document HS2-A

Mina L. Harkins, MBA, Michael J. Wajda, M.S., J.D. Staff

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

The Quality System Approach............................................................................................................ viii

8.7 Limitations of the Procedure .......................................................................................26

Appendix A. Microscopic Components in Urine Sediment.................................................................45

Appendix B. Microscopic Components in Vaginal Fluid and KOH Preparations...............................52

Summary of Comments and Subcommittee Responses........................................................................55

Summary of Delegate Comments and Subcommittee Responses.........................................................74

Related NCCLS Publications................................................................................................................75

The Quality System Approach

Documents & Records Equipment Information Management Process Improvement

Preanalytic Analytic Postanalytic

Also, it is the subcommittees intention to include, as appropriate, photomicrographs of various

Analytic, microscopy, postanalytic, preanalytic, provider

Provider-Performed Microscopy Testing; Approved Guideline

potassium hydroxide (KOH) preparations to detect yeast and fungal elements;

pinworm examinations to detect infestation by the Enterobius vermicularis parasite;

postcoital, direct, qualitative examinations of cervical mucus to investigate infertility;

qualitative semen analysis to test for effective vasectomy or to investigate infertility;

fecal leukocyte examinations to suggest or exclude the diagnosis of a number of pathologic

An NCCLS global consensus guideline. NCCLS. All rights reserved. 1

3.1 Standard Precautions

3.2 Personal Protective Equipment

Personal protective equipment should be utilized in the following situations:

3.3 Hand Washing

after removing gloves;

3.4 Food, Drink, Cigarettes, and Cosmetics

3.5 Routine Cleaning

3.6 Infectious Waste Disposal

Exogenous - Developed or originating outside an organism; caused by external factors.

Measurand A particular quantity subject to measurement.

Postanalytical//post-examination process - All processes following laboratory examination including

Preanalytical//pre-examination process - Steps starting in chronological order from the clinicians

Provider - In the medical context, a clinical practitioner.

An NCCLS global consensus guideline. NCCLS. All rights reserved. 5

proper illumination and use of the objectives;

5.2.1 Parts of the Microscope (See Figure 1.)

Characteristics of the stand include the following:

rests on the base of the microscope; and

Characteristics of the stage include the following:

horizontal platform upon which the specimen slide is placed;

Characteristics of the condenser include the following:

An NCCLS global consensus guideline. NCCLS. All rights reserved. 7

Figure 1. Standard Laboratory Microscope

8 An NCCLS global consensus guideline. NCCLS. All rights reserved.

To use the high (40x) objective:

To use the 100x oil immersion objective:

5.2.3 Care of the Microscope

do not expose the microscope to extreme heat, cold, or temperature changes;

identifying problems and errors;

QA is an important part of a comprehensive quality system consisting of policies, procedures, and