CLSI-H56-Body fluid analysis for cellular composition 2006

Body Fluid Analysis for Cellular
Composition; Proposed Guideline
PLEASE
H56-P
Vol. 25 No. 20
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This proposed document is published for wide and thorough review in the new,
accelerated Clinical and Laboratory Standards Institute (CLSI) consensus-review
process. The document will undergo concurrent consensus review, Board review,
and delegate voting (i.e., candidate for advancement) for 90 days.
Please send your comments on scope, approach, and technical and editorial content
to CLSI.
Comment period ends
17 November 2005
The subcommittee responsible for this document will assess all comments received
by the end of the comment period. Based on this assessment, a new version of the
document will be issued. Readers are encouraged to send their comments to
Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400,
Wayne, PA 19087-1898 USA; Fax: +610.688.0700, or to the following e-mail
address: customerservice@clsi.org
S
SS
SSS
COMMENT
This guideline provides users with recommendations for collection and transport of body
fluids, numeration and identification of cellular components, and guidance for qualitative
and quantitative assessment of body fluid.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
Clinical and Laboratory Standards Institute
Providing NCCLS standards and guidelines, ISO/TC 212 standards, and ISO/TC 76 standards
Clinical and Laboratory Standards Institute (CLSI,
formerly NCCLS) is an international, interdisciplinary,
nonprofit, standards-developing, and educational
organization that promotes the development and use of
voluntary consensus standards and guidelines within the
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development of standards and guidelines for patient
testing and related healthcare issues. Our process is
based on the principle that consensus is an effective and
cost-effective way to improve patient testing and
healthcare services.
In addition to developing and promoting the use of
voluntary consensus standards and guidelines, we
provide an open and unbiased forum to address critical
issues affecting the quality of patient testing and health
care.
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Consequently, conformance to this voluntary consensus
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Address comments to Clinical and Laboratory Standards
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the revision of documents in response to comments
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Volume 25 Number 20
H56-P
ISBN 1-56238-575-5
ISSN 0273-3099
Body Fluid Analysis for Cellular Composition; Proposed Guideline
Diane I. Szamosi, MA, MT(ASCP)SH
Josephine M. Bautista, MS, MT(ASCP)
Joanne Cornbleet, MD, PhD
Lewis Glasser, MD
Gregor Rothe, DrMed
Linda Sandhaus, MD
Marc Key, PhD
Aurelia Meloni-Ehrig, PhD, DSc
Naomi B. Culp, DA, MT(ASCP)SH
William Dougherty
Abstract
Clinical and Laboratory Standards Institute document H56-P—Body Fluid Analysis for Cellular Composition; Proposed
Guideline provides recommendations for standardization of the collection and transport of body fluids, numeration and
identification of cellular components, and guidance for qualitative and quantitative assessment of body fluid.
Clinical and Laboratory Standards Institute (CLSI). Body Fluid Analysis for Cellular Composition; Proposed Guideline. CLSI
document H56-P (ISBN 1-56238-575-5). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2005.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are
listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is
not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax:
610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org
Number 20
H56-P
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 Clinical and Laboratory Standards
Institute, except as stated below.
Clinical and Laboratory Standards Institute 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 document’s text.
Reproduced with permission, from CLSI publication H56-P—Body Fluid Analysis for
Cellular Composition; Proposed Guideline (ISBN 1-56238-575-5). Copies of the current
edition may be obtained from Clinical and Laboratory Standards Institute, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the
exemptions granted here or under the Copyright Law must be obtained from Clinical and Laboratory
Standards Institute by written request. To request such permission, address inquiries to the Executive Vice
President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898, USA.
Copyright ©2005. Clinical and Laboratory Standards Institute.
Suggested Citation
(Clinical and Laboratory Standards Institute. Body Fluid Analysis for Cellular Composition; Proposed
Guideline. CLSI document H56-P [ISBN 1-56238-575-5]. Clinical and Laboratory Standards Institute,
940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.)
Proposed Guideline
August 2005
ISBN 1-56238-575-5
ISSN 0273-3099
ii
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Committee Membership
Area Committee on Hematology
Bruce H. Davis, MD
Chairholder
Maine Medical Center Research
Institute
Scarborough, Maine
Maryalice Stetler-Stevenson, MD,
PhD
National Institutes of Health
Bethesda, Maryland
Advisors
Samuel J. Machin, MB, ChB,
FRCPath
Vice-Chairholder
The University College London
Hospitals
London, United Kingdom
Dorothy M. Adcock, MD
Esoterix Coagulation
Aurora, Colorado
Frank M. LaDuca, PhD
International Technidyne
Corporation
Edison, New Jersey
Ginette Y. Michaud, MD
FDA Center for Devices and
Radiological Health
Rockville, Maryland
Albert Rabinovitch, MD, PhD
Abbott Laboratories, Hematology
Business Unit
Santa Clara, California
Charles F. Arkin, MD
Lahey Clinic
Burlington, Massachusetts
J. David Bessman, MD
University of Texas Medical Branch
Galveston, Texas
Douglas J. Christie, PhD, FAHA
Dade Behring, Inc.
Newark, Delaware
Ian Giles
Sysmex America, Inc.
Mundelein, Illinois
Jan W. Gratama, MD
Erasmus University Medical
Center-Daniel Den Hoed
Rotterdam, Netherlands
Francis Lacombe, MD, PhD
Laboratoire d’Hematologie
Pessac, France
Kandice Kottke-Marchant, MD,
PhD
The Cleveland Clinic Foundation
Cleveland, Ohio
Richard A. Marlar, PhD
Oklahoma City VA Medical Center
Oklahoma City, Oklahoma
Powers Peterson, MD
Weill Cornell Medical College in
Qatar
Doha, Qatar
Diane I. Szamosi, MA,
MT(ASCP)SH
Greiner Bio-One
North America, Preanalytics
Monroe, North Carolina
Luc Van Hove, MD, PhD
Abbott Laboratories
Abbott Park, Illinois
John A. Koepke, MD
Durham, North Carolina
Subcommittee on Body Fluid Analysis for Cellular Composition
Diane I. Szamosi, MA,
MT(ASCP)SH
Chairholder
Greiner Bio-One North America,
Preanalytics
Monroe, North Carolina
Josephine M. Bautista, MS,
MT(ASCP)
FDA Center for Devices and
Radiological Health
Rockville, Maryland
Joanne Cornbleet, MD, PhD
Stanford University Medical Center
Stanford, California
Lewis Glasser, MD
Rhode Island Hospital
Brown Medical School
Providence, Rhode Island
Gregor Rothe, DrMed
Bremer Zentrum für
Laboratoriumsmedizin
Bremen, Germany
Linda Sandhaus, MD
University Hospitals of Cleveland
Cleveland, Ohio
Staff
Clinical and Laboratory Standards
Institute
Wayne, Pennsylvania
John J. Zlockie, MBA
Vice President, Standards
David E. Sterry, MT(ASCP)
Staff Liaison
Donna M. Wilhelm
Editor
Melissa A. Lewis
Assistant Editor
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Acknowledgement
This guideline was prepared by Clinical and Laboratory Standards Institute (CLSI), as part of a
cooperative effort with IFCC to work toward the advancement and dissemination of laboratory standards
on a worldwide basis. CLSI gratefully acknowledges the participation of IFCC in this project. The IFCC
expert for this project is Gregor Rothe, DrMed, Bremer Zentrum für Laboratoriumsmedizin.
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Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1
Scope..........................................................................................................................................1
2
Standard Precautions..................................................................................................................1
3
Definitions .................................................................................................................................1
4
Preanalytical Variables ..............................................................................................................3
5
Specimen Collection ..................................................................................................................4
5.1
5.2
5.3
5.4
6
Specimen Handling and Transport.............................................................................................7
6.1
6.2
6.3
6.4
7
Cerebrospinal Fluid.....................................................................................................26
Serous (Pleural, Peritoneal, Pericardial) .....................................................................30
Synovial Fluid.............................................................................................................36
Bronchoalveolar Lavage Fluid....................................................................................43
Additional Studies....................................................................................................................46
10.1
10.2
10.3
11
Slide Preparation.........................................................................................................14
Identification of Morphologic Constituents................................................................15
Evaluation of Nucleated Cell Subtypes ......................................................................25
Physician Review........................................................................................................25
Result Reporting .........................................................................................................26
Fluid Types ..............................................................................................................................26
9.1
9.2
9.3
9.4
10
Manual Counting ..........................................................................................................8
Automated Methods....................................................................................................10
Morphology Assessment..........................................................................................................14
8.1
8.2
8.3
8.4
8.5
9
CSF ...............................................................................................................................7
Serous Fluids.................................................................................................................7
Synovial Fluids .............................................................................................................8
BAL ..............................................................................................................................8
Quantitative Assessment............................................................................................................8
7.1
7.2
8
Cerebrospinal Fluid.......................................................................................................4
Serous Fluid ..................................................................................................................5
Synovial Fluid...............................................................................................................6
Bronchoalveolar Lavage (BAL) ...................................................................................7
Immunologic Studies ..................................................................................................46
Flow Cytometric Studies ............................................................................................54
Cytogenetic Analysis ..................................................................................................58
Sample Storage After Completion of Testing..........................................................................59
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Contents (Continued)
12
Quality Control and Quality Assurance ...................................................................................59
12.1
12.2
12.3
12.4
Quality Control ...........................................................................................................59
Quality Assurance.......................................................................................................60
Proficiency Testing (External Quality Assessment) ...................................................60
Continuous Education and Training ...........................................................................61
References.............................................................................................................................................62
Appendix A. Reagent Formulations .....................................................................................................67
Appendix B. Interpretation of Cell Types.............................................................................................68
The Quality System Approach..............................................................................................................72
Related CLSI/NCCLS Publications ......................................................................................................73
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Foreword
Clinical data derived from proper body fluid procedures and accurate test results are essential to make the
appropriate diagnosis and administer the proper therapy to patients. Some variables may influence the test
results reported. Because these variables are loosely defined, inconsistency from one institution to another
may exist. This guideline will provide users with recommendations for the collection and transport of
body fluids, procedures for the numeration and identification of cellular components, and guidelines for
the qualitative and quantitative assessment of body fluids.
Invitation for Participation in the Consensus Process
An important aspect of the development of this and all Clinical and Laboratory Standards Institute (CLSI)
documents should be emphasized, and that is the consensus process. Within the context and operation of
CLSI, the term “consensus” means more than agreement. In the context of document development,
“consensus” is a process by which CLSI, its members, and interested parties (1) have the opportunity to
review and to comment on any CLSI publication; and (2) are assured that their comments will be given
serious, competent consideration. Any CLSI document will evolve as will technology affecting laboratory
or healthcare procedures, methods, and protocols; and therefore, is expected to undergo cycles of
evaluation and modification.
The Area Committee on Hematology has attempted to engage the broadest possible worldwide
representation in committee deliberations. Consequently, it is reasonable to expect that issues remain
unresolved at the time of publication at the proposed level. The review and comment process is the
mechanism for resolving such issues.
The CLSI voluntary consensus process is dependent upon the expertise of worldwide reviewers whose
comments add value to the effort. At the end of a 90-day comment period, each subcommittee is obligated
to review all comments and to respond in writing to all which are substantive. Where appropriate,
modifications will be made to the document, and all comments along with the subcommittee’s responses
will be included as an appendix to the document when it is published at the next consensus level.
A Note on Terminology
CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization
wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences
while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the
global metrological community have evolved differently in the United States, Europe, and elsewhere; that
these differences are reflected in CLSI, ISO, and CEN documents; and that legally required use of terms,
regional usage, and different consensus timelines are all obstacles to harmonization. Despite these
obstacles, CLSI recognizes that harmonization of terms facilitates the global application of standards and
is an area that needs immediate attention. Implementation of this policy must be an evolutionary and
educational process that begins with new projects and revisions of existing documents.
Key Words
Body fluids, bronchoalveolar lavage, cerebrospinal fluid, pericardial fluid, peritoneal fluid, pleural fluid,
serous fluid, synovial fluid
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Body Fluid Analysis for Cellular Composition; Proposed Guideline
1
Scope
The intended purpose of this guideline is to explain how to collect, process, examine, store, and report
results for body fluid specimens for the characterization of inflammatory, infectious, neoplastic, and
immune alterations. It will also discuss preanalytical, analytical, and postanalytical variables related to
body fluid cellular analyses. For the purpose of this document, the following body fluids will be
discussed: cerebrospinal, serous (pleural, peritoneal, pericardial), and related fluids (i.e., peritoneal
dialysate, peritoneal lavage, and bronchoalveolar), and synovial fluids.
This guideline describes manual and automated methods to enumerate cellular components and to identify
normal and abnormal elements. It also addresses additional studies that may be used for body fluid testing
in the routine clinical laboratory.
This document is intended for medical technologists, pathologists, microbiologists, cytologists, nurses,
and other healthcare professionals responsible for the collection and transport of body fluid specimens to
the clinical laboratory, as well as the processing, testing, and reporting of results. It is also intended for
manufacturers of products or instruments used for body fluid testing.
2
Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all infectious agents and thus are
more comprehensive than universal precautions which are intended to apply only to transmission of
blood-borne pathogens. Standard and universal precaution guidelines are available from the U.S. Centers
for Disease Control and Prevention (Garner JS, Hospital Infection Control Practices Advisory Committee.
Guideline for isolation precautions in hospitals. Infect Control Hosp Epidemiol. 1996;17(1):53-80). For
specific precautions for preventing the laboratory transmission of all infectious agents from laboratory
instruments and materials and for recommendations for the management of exposure to all infectious
disease, refer to the most current edition of Clinical and Laboratory Standards Institute document M29—
Protection of Laboratory Workers From Occupationally Acquired Infections.
3
Definitions
accuracy (of measurement) – closeness of the agreement between the result of a measurement and a true
value of the measurand (VIM93).1
analytical sensitivity – in quantitative testing, the change in response of a measuring system or
instrument divided by the corresponding change in the stimulus (modified from VIM93)1; NOTE 1: The
sensitivity may depend on the value of the stimulus; NOTE 2: The sensitivity depends on the imprecision
of the measurements of the sample; NOTE 3: In qualitative testing, the test method’s ability to obtain
positive results in concordance with positive results obtained by the reference method; NOTE 4: If the
true sensitivity of a device is better than the reference method, its apparent specificity will be less and the
level of apparent false-positive results will be greater; NOTE 5: For FISH, the percentage of scorable
nuclei or metaphase cells with the expected signal pattern (number of signals, size of signals, and color of
signals).
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analytical specificity – ability of a measurement procedure to measure solely the measurand (ISO
17511).2
antibody – specific immunoglobulin formed by B lymphocytes and plasma cells in response to exposure
to an immunogenic substance and able to bind to the antigen.
anticoagulant (additive) – an agent that prevents coagulation of blood or blood products
arthrocentesis – aspiration of a joint.
arthrocentesis fluid – joint fluid obtained from aspiration of a joint.
carry-over – the discrete amount of analyte carried by the measuring system from one specimen reaction
into subsequent specimen reactions, thereby erroneously affecting the apparent amounts in subsequent
specimens.
cerebrospinal fluid – fluid within the ventricles of the brain and the subarachnoid space.
collection vessel – any tube or container, preferably plastic, which serves to contain the body fluid
specimen.
empyema fluid – the presence of pus in a body cavity; usually refers to pus in the pleural cavity.
epitope – any site on an antigen molecule at which an antibody can bind; the chemical structure of the
site determining the specific combining antibody.
exudate – a fluid with a high concentration of protein or cells that accumulates in a body cavity as a result
of increased capillary permeability.
iatrogenic fluids – fluids introduced into a body cavity by the physician.
immunocytochemical assay//immunohistochemical assay – an immunoassay that detects an antigen
present in a specimen that is contained within intact or histologically sectioned cells or tissues.
immunocytology//immunocytochemistry – localization of immunoreactive substances within cells of a
cytological specimen that have been specifically labeled with an antibody.
immunohistology//immunohistochemistry – localization of immunoreactive substances within cells or
tissues of a histological specimen that have been specifically labeled with an antibody.
measuring range – a set of values of measurands for which the error of a measuring instrument is
intended to lie within specified limits (VIM93).1
peritoneal dialysate fluid – a physiologic synthetic fluid introduced into the peritoneal cavity for the
purpose of normalizing fluid, electrolyte, and solute balance in the body using the principles of
ultrafiltration and diffusion.
peritoneal fluid – a body fluid within the peritoneal cavity.
peritoneal lavage fluid – a physiologic synthetic fluid introduced into the peritoneal cavity for the
purpose of irrigating the cavity and removing the fluid for the purpose of examining its contents.
2
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peroxidase – an enzyme commonly used in immunohistochemistry to label immunoreactive substances
in cells and tissues; NOTE: In immunohistochemistry, the reaction of peroxidase with an appropriate
substrate-chromogen produces a colored reaction product that can be viewed microscopically.
pleural fluid – the serous fluid within the pleural cavity.
precision (of measurement) – closeness of agreement between independent test results obtained under
stipulated conditions (ISO 3534-1).3
Romanowsky type stains – any stain containing methylene blue and/or its products of oxidation (azure
B), and a halogenated fluorescein dye, usually eosin B or Y.
sample – one or more parts taken from a system, and intended to provide information on the system,
often to serve as a basis for decision on the system or its production (ISO 15189)4; NOTE: For example,
a volume of serum taken from a larger volume of serum (ISO 15189).4
specimen – biological material that is obtained in order to detect or to measure one or more quantities,
such as amount or concentration (ISO/CD 18112-1).5
substrate-chromogen – a reagent commonly used in immunohistochemistry that contains both a
substrate and a chromogen; NOTE: When reacted with an appropriate enzyme, the substrate-chromogen
produces a colored reaction product that specifically labels immunoreactive substances in cells and
tissues.
thoracentesis fluid – fluid obtained from removal of pleural fluid from the thoracic cavity.
transudates – fluid with a low concentration of protein that has accumulated in a body cavity.
traumatic tap – contamination of body fluids by extraneous cells or fluid derived from blood or tissue
during the procedure of withdrawal of fluid from a body cavity.
ventricular shunt fluid – ventricular shunts are placed for the treatment of hydrocephalus to remove
fluid from the ventricles and provide drainage to another site (e.g., the peritoneal cavity); NOTE: The
fluid that fills the shunt is designated ventricular shunt fluid.
4
Preanalytical Variables
A path of workflow is the description of the necessary steps needed to deliver a particular product or
service to the organization or entity it defines. This workflow path can be influenced by preanalytical,
analytical, and postanalytical variables. Preanalytical variables can be further exemplified by erroneous
test requests, specimen handling, collection procedures, collection vessels, anticoagulants, specimen
transport, and receipt of specimens.6
The test request procedure is important to address in the quality system model for laboratory testing.1 This
procedure may be affected by preanalytical variables that include an error in the order entry procedure or
in a test request being incorrectly ordered, either as tests added to or deleted from the original test request.
Specimen collection procedures can also be subjected to preanalytical variables. For example, the
techniques used in the collection of body fluids can affect the reportable test results. It is therefore
recommended that procedures be standardized in facilities, so that these collection errors can be
minimized or eliminated. These procedures should also be established as part of an institution’s Standard
Operating Procedures (SOPs).
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The type of collection vessels that are used to collect and transfer body fluids could possibly affect test
results. The material used may possibly absorb or leach constituents, making cellular enumeration and
morphologic identification inaccurate. Cellular adherence, especially in glass tubes, may artificially
change differential cell counts in low protein solutions, such as in BAL. In contrast to glass tubes or
polystyrene tubes, polypropylene tubes are suitable for the collection and mixing of aspirated
broncholavage fluid. It is therefore the responsibility of the facility to select the appropriate collection
vessels by conducting internal studies to evaluate the material selected, and/or obtain such information
from the appropriate manufacturer or published studies.
The type of anticoagulant (additive) used for the collection of specific body fluids may also affect test
results. For example, using an additive when it is not required (cerebrospinal fluid, or CSF) may possibly
affect the enumeration of white and red blood cells. Using the wrong additive (synovial) could possibly
introduce artifacts and therefore interfere with the identification of cellular elements present on a slide.
In some body fluids, the proper order of draw is important so that the incidence of cellular contamination
from tube to tube is reduced. It is also necessary so that a microbiology specimen is not contaminated. In
addition, hemolyzed and clotted specimens are not recommended as specimens of choice for analysis
because these types of specimens will produce inaccurate test results. However, circumstances may arise
when it is not possible to acquire another specimen from a patient. These exceptions to standard practice
must be clearly defined in the site’s Standard Operating Procedures.
Specimen transport is also a procedure that may be affected by preanalytical variables. For example, the
temperature at which a specimen is transported could affect the integrity, degradation, or deterioration of
the constituents of the fluid. The transport time must also be acceptable to maintain specimen integrity.
The method of transport may also affect the integrity of the specimen. For example, the use of a
pneumatic tube system must be approached with caution because excessive shaking of body fluids may
result in a breakdown of the cellular constituents. It is therefore recommended that each facility acquire
information from the manufacturer (e.g., sample transportation time, sample transportation method). In
addition, the facility must also establish Standard Operating Procedures regarding the use of the
pneumatic tube system for the transport of body fluids.
The receipt of specimens into a laboratory accessioning department may also be affected by preanalytical
variables. These specimens should be received with proper identification. A bar-code label should include
the name of the patient, the medical record number, the accession number, the location (unit), the date and
time of specimen collection, and the list of the tests ordered. The date and time on the specimens should
be verified against the actual collection time of the specimen. If a significant discrepancy between the
times exists, the unit should be notified to rectify the discrepancy before test analyses. In some situations
(LIS downtime, emergencies), handwritten labels accompanied with requisition slips should be an
acceptable means of specimen identification, providing that the required information is properly
documented. If these specimen identification guidelines are not met either electronically or manually, the
unit will be notified that a new specimen will be required. If specimen identification guidelines are not
met, the laboratory personnel must follow the administrative guidelines of the laboratory for analysis or
rejection of the specimen. In addition, a Laboratory Incident Report should be filed.7,8
5
5.1
Specimen Collection
Cerebrospinal Fluid
Cerebrospinal fluid is usually collected by lumbar puncture, but may also be obtained by lateral cervical
or cisternal puncture.9 Sterile technique is mandatory to avoid introducing bacteria. Manometric
measurements may be done and are the responsibility of the clinical service rather than the laboratory.
Usually, fluid is collected into three or four tubes for chemical, microbiologic, and cellular analysis. The
tubes should be labeled according to the sequence of collection. It is preferable to have the first tube
4
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analyzed for chemical and serologic studies. Subsequent tubes should be used for microbial and cellular
analysis to obtain accurate cell counts and decrease the chance of bacterial contamination. A sterile tube
must be used for microbial studies. No anticoagulant is necessary, since spinal fluid does not clot except
occasionally if the puncture is traumatic. Since the volume of CSF is relatively small, the total amount
collected is limited and usually varies from 10 to 20 mL in adults. Up to 8 mL may be safely removed
from the smallest infant. Complications of lumbar puncture include headache, infection, and brain
herniation. Rarer complications may also occur.9 Refer to Section 10.1.1 for collection of samples for
cytological examination.
Table 1. Specimen Requirements for Cerebrospinal Fluid
Anticoagulant
Volume (mL)
Test
Comments
(e.g., protein, glucose,
other special tests)
None
3-5
Tube #1
If traumatic tap is
suspected, cell count
should also be
performed on Tube 1.
Gram stain and culture
None
3-5
Tube #2
Cell count and
differential
None
3-5
Tube #3 or 4
Other tests as required
(e.g., cytology)
None
3-5
Tube #4
5.2
Serous Fluid
Serous fluids (e.g., pleural, peritoneal) from large volume collections may be aliquoted into smaller
volumes before transport to the laboratory or in the laboratory. Specimens should be gently agitated
during collection, before aliquoting, and before testing for cell counts and differentials.
Ethylenediaminetetraacetic acid (EDTA) is the recommended anticoagulant for cell counts and
differentials. Refrigerated storage is adequate for cell counts and differentials for up to 24 hours.10
Although testing can be done on small volumes of fluid, 5 to 8 mL is recommended in the event followup studies are needed (e.g., flow cytometry). A sterile collection tube must be used for microbial studies.
For cytology specimens, a wide range of volumes may be sent to the laboratory. As little as 15 to greater
than 100 mL may be sent for analysis. A 50-mL specimen is recommended. Sterility is not required and
no anticoagulant is necessary.11 However, heparin and EDTA are also used.12 If clumps of material are
present, they can be processed as a cell block. Refer to Section 10.1.1 for collection of samples for
cytological examination.
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Table 2. Specimen Requirements for Serous Fluids
Anticoagulant
Tests
*
Volume (mL)
RBC, WBC, differential
EDTA
5-8
Total protein, LD, glucose
amylase
Heparin, none
8-10
Gram stain, bacterial culture
SPS*, none, or anticoagulant without
bactericidal or bacteriostatic effect
8-10
AFB culture
SPS, none, or anticoagulant without
bactericidal or bacteriostatic effect
15-50
PAP stain, cell block
None, heparin, EDTA
5-50
SPS = Sodium polyanetholsulfonate
5.3
Synovial Fluid
The amount of fluid removed depends on the size of the joint and effusion. A 3- to 5-mL sample is ideal
for laboratory analysis. However, since this may not be possible in smaller joints, the physician should
prioritize the requested tests and clearly communicate with the laboratory. Specimens should not be
rejected because of small volumes, since even a drop may provide definitive diagnosis in crystalline joint
disease and only small volumes are needed for cell count and differential. Infected fluids may also grow
organisms even if the volume is compromised. Specimen requirements are listed in Table 3. The
following precautions should be noted. The physician must be careful not to express synovial fluid into
tubes using a needle on the collection tray, previously used to remove fluid from a medicinal vial. Fluid
should be thoroughly mixed after collection and before analysis in the laboratory to obtain accurate cell
counts.
Some texts indicate that lithium heparin and EDTA should not be used as anticoagulants because they
produce crystalline material that can be confused with pathologic crystals.13,14 However, others have used
lithium heparin and EDTA without difficulty.15 Oxalate should not be used because of extensive
formation of calcium oxalate crystals. Refer to Section 10.1.1 for collection of samples for cytological
examination.
6
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Table 3. Specimen Requirements for Synovial Fluid*
Test
Anticoagulant
Volume (mL)
Comments
Cell count,
differential, crystals,
inclusions
Heparin, EDTA
3-5
Can be done on a few
drops of fluid. Mix
thoroughly.
Glucose
Protein
CH50
Fluoride or none
None
None
3-5
8-hr. fast preferred
C3, C4
None or EDTA
Culture
SPS, none, or
anticoagulant without
bactericidal or
bacteriostatic effect
*
Freeze if not tested
immediately.
Requires 1 mL
3-5
Sterile tube required
Requirements may change with advances in technology.
5.4
Bronchoalveolar Lavage (BAL)
A fiber-optic bronchoscope is wedged into a midsize segmental bronchus, and aliquots of sterile saline are
instilled and aspirated into the alveolar spaces. In this manner, cells and organisms in the alveoli distant to
the bronchoscope can be sampled. The instillation volume typically is approximately 100- to 300-mL
sterile saline in 20- to 50-mL aliquots. The first aliquot should be discarded. The other aliquots are pooled
for further analysis. In diffuse lung disease, the middle or lingular lobe is used as a standard site for BAL.
If a definite segment has been lavaged, this should be recorded on the request form.
Aspiration of the instilled solution should be carried out with as little trauma as possible. A typical
recovery is in the range of 50 to 70%. A very low recovery of less than 25% of the applied volume may
appear in cases of chronic obstructive lung diseases. Low-volume recovery should be recorded on the
request form. Refer to Section 10.1.1 for collection of samples for cytological examination.
6
Specimen Handling and Transport
Specimens should be transported to the laboratory promptly. Cellular degeneration of CSF can begin
within one hour of collection, so cell counts should be completed as soon as possible.
6.1
CSF
Cerebrospinal fluid (CSF) specimens should be transported at ambient temperature to the testing site as
soon as possible following completion of the collection procedures. CSF for microbiology testing should
never be refrigerated before or after transport; since some organisms are fastidious and temperature
sensitive, they have the capability of becoming nonviable.
6.2
Serous Fluids
It is also recommended that pleural, pericardial, and peritoneal fluids be transported to the testing site at
ambient temperature. To preserve the integrity of these specimens, however, the testing site should be in
receipt of these specimens as soon as possible after the completion of the collection procedures.
Otherwise, cell lysis, cellular degradation, and bacterial growth could occur and possibly affect the test
results.
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Serous fluids for the cytology laboratory should be sent as soon as possible. If storage is necessary, the
specimen should be refrigerated at 4 °C without a fixative. Serous fluids have a high protein content,
cellular detail with Papanicolaou (PAP), H & E, or other stains will be adequately preserved with
refrigeration for several days.11
6.3
Synovial Fluids
Synovial fluid specimens may be transported and analyzed at room temperature.
6.4
BAL
Bronchoalveolar lavage (BAL) samples should be kept at room temperature and transported to the
laboratory immediately after collection. Analysis of cell number, viability, and differential count should
be performed within three hours. Preliminary tests demonstrate a deterioration of cellular characteristics
after approximately six hours. Specimens that cannot be processed within 36 hours should be discarded.
Samples are often filtered using 50- to 70-µ nylon filters before staining to remove phlegm and dust.
7
Quantitative Assessment
7.1
Manual Counting
Manual cell counting is a basic procedure in the evaluation of body fluids. There are variations of the
manual procedure (e.g., cells may be counted by light microscopy using stains to enhance the recognition
of cells or using phase microscopy). Each laboratory should establish its own procedure.
7.1.1
•
•
•
•
•
•
•
•
•
Hemacytometer (e.g., Neubauer or equivalent counting chamber)
Hemacytometer coverslip
3% acetic acid
Acidified crystal violet stain
Saline
Test tubes (for manual dilutions)
White cell and red cell diluting pipettes
Manufactured dilution ampule systems
Calibrated pipettes with tips
7.1.2
•
Reagents and Supplies
Instrumentation
Microscope
7.1.3
Procedure
Mix the specimen well by rotation on an automated mixer for a maximum of two to five minutes
(excessive rocking may damage cells) or hand mix by inverting the tube ten to 15 times. The exception is
synovial fluid, which must be mixed for five to ten minutes due to the viscosity of the fluid. If the fluid is
in a conical tube, flick the bottom of the conical tube several times to dislodge cells before mixing the
specimen. The more turbid the sample, the greater the mixing process impacts cell count accuracy.
8
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7.1.3.1
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Specimen Dilutions
The sample should be well mixed before analysis.16,17 Both erythrocytes and nucleated cells are
enumerated in the same chamber. Specimens are usually counted undiluted, unless they are bloody or
cloudy. Typical dilutions for any fluid can range from 1:10 to 1:200 or higher, depending on the turbidity
of the specimen. Different diluents can be used to dilute the fluids. Isotonic saline can be used for both
white and red cell dilutions while acetic acid or hypotonic saline may be used to lyse red cells for white
cell dilutions. Acetic acid should not be used as a diluent for synovial fluid manual nucleated cell counts,
since mucin will coagulate. If manual nucleated cell counts are performed on synovial fluid samples,
erythrocytes can be lysed, with preservation of nucleated cells, by using a hypotonic saline solution
(0.3%).
Several quality assurance stipulations have been promulgated by regulatory agencies. These include the
use of certified pipettes or commercial dilution systems, periodically checking diluting fluids for
extraneous particles and counting samples in duplicate.18
7.1.3.2
Hemacytometer Preparation and Charging
Before charging the hemacytometer chamber, make sure it is clean and dry. Place a coverslip on the
hemacytometer. Place the hemacytometer in a petri dish lined with moist paper. Elevate the
hemacytometer on two sticks so it does not come in direct contact with the moist paper. Fill both sides of
the hemacytometer, being careful not to overfill. After the hemacytometer is loaded, allow the cells to
settle for five to ten minutes (the amount of time required for the cells to settle depends on the cellularity
of the specimen). Label the petri dish using a crayon or by attaching a computer label. The label must
include the patient’s name or specimen number and the set-up time. Cells must be counted as soon as
possible. If the fluid has drawn back from the sides of the hemacytometer, the sample has begun to dry
out and the counts are invalid. Re-mix the sample and set the hemacytometer counts up again.
The following guidelines are recommended for counting areas16:
a) If less than an estimated 200 cells are present in all nine squares, count all nine squares. This area
counted is 9 mm2.
b) If more than an estimated 200 cells are present in all nine squares, then count the four corner squares.
This area counted is 4 mm2.
c) If more than an estimated 200 cells are present in one square, then count five of the squares within the
center square for an area of 0.2 mm2.
7.1.3.3
Cell Counting Procedures
Place the hemacytometer under the microscope, using low power only (10X), and adjust to see the cells.
Scan the large squares. For accuracy, there should be even distribution of cells (approximately no more
than ten cells variation in the large squares). Cells should not overlap. For diluted samples, a minimum of
200 cells should be counted. Then, switch to high power magnification (40X). The count is performed
under high power. Depending on the number of cells present, an appropriate number of squares should be
counted. The more cells present, the smaller and fewer the numbers of squares that need to be counted.
7.1.3.3.1
Hemacytometer
The hemacytometer is 0.1 mm deep and the etched surface is a total surface area of 9 mm2. The counting
area is divided into nine large squares. The center large square is subdivided into 25 small squares. The
25 small squares are subdivided into 16 smaller squares (see Figure 1 below).
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Figure 1. Hemacytometer Counting Area. Reprinted with permission from Medical Center Laboratory
(www.MedicalCenterLab.org) and Judy Stranak, MAEd, MT(ASCP)SH.
7.1.3.3.2
White and Red Blood Cell Counts
Nucleated cells may be counted in the same chamber as erythrocytes. Count and average the result. Count
the appropriate areas on both sides of the hemacytometer for the dilution and number of cells present as
follows:
•
•
•
•
•
All nine squares if no dilution
All nine squares for 1:10 dilution
Four corner squares for 1:20 dilution
Center square for 1:100 dilution
Red cell area for 1:200 dilution
7.1.4
Calculations
Cells/mm3 = # of cells counted x 104 x dilution factor
# of squares counted
Total cells = cells/mL x volume of original cell suspension
7.2
Automated Methods
Additional information on automated cell counts, for each fluid, can be found under the microscopic
examination headings in Section 9.
Automated methods for body fluid analysis offer the laboratory an alternative to improve the precision of
the results by counting more cells than manual methods. While the coefficient of variation at low cell
counts is high on automated instruments, it is not nearly as high as manual cell counts.19 There are a
number of instruments available to perform body fluid cell counting. Depending on the instrument, the
technologies incorporated can include impedance, digital imaging flow cytometry, flow cytometry, light
scatter, dyes, and fluorescence, or a combination of these technologies. The manufacturer of each
automated device should have a statement of intended use that clearly defines which body fluids have
been approved by a regulatory agency for testing.
10
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7.2.1
H56-P
Processing Body Fluids on Automated Devices
Follow the manufacturer’s guidelines for the proper selection of appropriate body fluids that may be
analyzed on the instrument. Only analyze those body fluids, on any particular instrument, for which
clearance has been obtained and which are identified in the Intended Use statement for the device.
Furthermore, follow the manufacturer’s recommended procedures for any special treatment required for
the specific body fluid specimen to be analyzed.
The key issue in using automated counters is to ensure that the instrument can provide reliable counts at
the low levels of cells encountered in body fluids. Thus, each laboratory must define the lower limits for
counting nucleated cells and erythrocytes, below which the use of automated or semiautomated counters
is not reliable.18 The lower limit for counting should not exceed the limits recommended by
manufacturers.
Once a laboratory establishes specific guidelines for acceptable cell count limits performed by automated
methods, they must identify reflexive methods for specimens with cell count(s) below such limits. When
automated counts are flagged, the laboratory should indicate an alternative method to verify the count(s).
Guidelines should also indicate the need for manual differential cell counting as appropriate for the
automated method.
Care should be taken to identify samples with noncellular particulate material that can falsely elevate
automated counts or clog the orifice of the counter.
7.2.2
Defined Analytical Measurement Range (AMR)
Each manufacturer must state the AMR for which body fluid analysis is acceptable for each particle type.
The laboratory must establish a protocol detailing the steps to be taken when a sample exceeds the AMR
for a given particle type (e.g., dilution for concentrations exceeding the upper limit of the AMR and
alternative analytical methods for particles falling below the lower limit of the AMR).
7.2.3
Defined Sensitivity Limit
In addition to the defined analytical measurement range, the manufacturer must also state the sensitivity
limit—the minimum detectable concentration for each particle type enumerated. The laboratory must
establish a protocol detailing the steps to be taken when a sample is near or below the sensitivity limit for
each particle type (e.g., alternative analytical methods for particles near or below the sensitivity limit).
7.2.4
Performance Testing
Performance testing of automated instruments for body fluid analysis should, at a minimum, assess
imprecision, inaccuracy, correlation to reference methods, and reportable range.20 Local regulatory
requirements may also include carry-over, sensitivity, and specificity as part of the testing for
implementation of new instruments/test methods. Alternatively, consult the manufacturer regarding
recommendations for performance testing. Formulas for performance testing are listed below.
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Imprecision
Short-Term Imprecision:
The formula for short-term standard deviation is:
where: n = number of samples
di = difference between duplicates for sample i
Convert the above standard deviation to coefficient of variation (CV) as follows:
where: Xa is the mean of all values of x.
Long-Term Imprecision:
The formula for long-term standard deviation is:
where: n = number of samples
xi = mean of results for day I
x = mean of days or grand mean of all results
Convert the above standard deviation to coefficient of variation (CV) as follows:
Inaccuracy
The formula for inaccuracy is:
where:
12
T
= estimated variance of the test method
nt
R
= estimated variance of the reference method
nr
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T = p t x qt
pt = mean of yi
100
pr = mean of xi
100
7.2.5
Carry-Over
The effect of one sample on the next sample immediately following it should obviously be minimized.
This is especially true for clear and colorless cerebrospinal fluids (CSF) that may follow a bloody CSF.
Any carry-over that may be present should be of no clinical significance whatsoever. There are two types
of carry-over: 1) positive carry-over; and 2) negative carry-over.
Positive carry-over is the effect of an elevated sample on a subsequent sample of lower
concentration.
Negative carry-over is the effect of a low concentration sample on a subsequent sample of higher
concentration. This condition can be possibly observed in instruments where a dilution effect can
occur by the diluent/rinsing agent during the rinse cycle that occurs between sample analyses.
Several protocols for determining carry-over are available.21,22
7.2.6
Sensitivity and Specificity
Sensitivity – Sensitivity is the ability of the automated method to accurately and reliably detect and
quantify low levels of red blood cells and nucleated cells. Such sensitivity is dependent upon the
instrument’s carry-over, precision, and accuracy (see above). Testing sensitivity must be done at the limit
of clinical performance that is specified by the manufacturer. For detailed information on determination
of limits of detection, refer to the most current edition of CLSI/NCCLS document EP17—Protocols for
Determination of Limits of Detection and Limits of Quantitation.
Specificity – Specificity of the automated method is its ability to accurately identify formed elements in
the body fluid and may be subject to interferences. The instrument manufacturer should clearly identify
any potential interfering substances when performing body fluid analyses. For detailed information on
evaluation of precision performance, refer to the most current edition of CLSI/NCCLS documents EP5—
Evaluation of Precision Performance of Quantitative Measurement Methods and EP15—User
Verification of Performance for Precision and Trueness.
7.2.7
Correlation to Reference Methods
Correlative studies of the automated device to reference methods are best determined through regression
analysis, in which the r2, slope, and y-intercept are established. For detailed information on method
comparison, refer to the most current edition of CLSI/NCCLS document EP9—Method Comparison and
Bias Estimation Using Patient Samples.
7.2.8
Quality Control
Quality control of the automated device provides the operator with reasonable confidence that the
instrument is functioning properly and within the manufacturer’s specifications. It is important that
quality control measurements are performed in the same manner in which the body fluids will be
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processed and analyzed (i.e., quality control materials must be processed through the same fluidic paths as
will the body fluid specimens be processed).
Appropriate quality control measurements will include the performance of a background count of the
fluidic system and any additional fluids required for body fluid analysis, such as any diluents and lysing
reagents that are not routinely used for the primary use of the instrument. Unless analysis of controls is
specified by the manufacturer, verify with your local accrediting agency if any additional control
materials need to be analyzed on a routine basis. The College of American Pathologists (CAP) states that
if the same instrument is used for cell counting of blood specimens, there is no need to have separate
control runs for body fluid cell counting.18
8
Morphology Assessment
8.1
8.1.1
Slide Preparation
Cytocentrifugation
Wedge smears (push smears) should not be used with fluids because of their inferior ability in preserving
intact cells. The cytocentrifuge preparation is recommended for air-dried body fluid slides because this
technique concentrates the cells, minimizes cell distortion, and produces a monolayer of cells.
Romanowsky-type stained slides of cytocentrifuged CSF and other body fluids show excellent
morphologic detail, and cells appear similar to their counterparts in blood or bone marrow. Cells typically
are randomly dispersed in a small circular area, and a microscopic differential can be performed to
subclassify the nucleated cells. When malignancy is suspected, the whole cellular area should be
evaluated microscopically on each prepared slide, since malignant cells may be present in low frequency.
The cytocentrifuge instrument generally contains a centrifugation bowl with multiple slide assembly
units. The assembly consists of a filter card placed upon a slide and a chamber to hold the sample, secured
together by a clip. The outlet arm of the chamber is opposed to a hole in the filter card, exposing a round
area on the glass slide. In the resting position, the fluid specimen in the chamber does not contact the
glass slide. During centrifugation, the fluid and cells are forced out of chamber outlet onto the slide. The
filter absorbs the fluid, while the cells are deposited on the slide.
Cells are concentrated approximately 20-fold by cytocentrifugation.23 Even hypocellular samples with a
chamber cell count of zero can have a yield of approximately 35 cells per slide.23 The quantitative yield,
however, varies from 30 to 75%,24 and smaller cells, such as lymphocytes, may be underrepresented.25
The speed and time of centrifugation, the amount of sample in the chamber, and the filter paper
absorbance are factors that can influence both the cell yield and morphology.
Although the cytocentrifuge is not a complex instrument, some sample processing and instrument
techniques can enhance slide quality:23
1. Fresh, unfixed specimens should be used for slide preparation. Cells may begin deteriorating in a few
hours, particularly in body fluid samples with low protein content, such as cerebrospinal fluid.26 If
there is a prolonged delay in preparing cytocentrifuge slides (i.e., more than eight hours), the report
should include a statement that the differential count may be inaccurate, due to cellular degeneration.
2. Pleural, pericardial, peritoneal, and synovial fluid samples may contain fibrin and other proteins that
can clog the filter card, reducing cell yield and affecting cell distribution on the slide. Washing the
cells before cytocentrifugation, by centrifuging an aliquot of the sample and resuspending in saline,
can improve both the cell yield and morphology.
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3. If clots are present, both the cell count and differential may be inaccurate. However, slides can be
prepared and examined for malignant cells. The clots should be agitated gently to free trapped cells
before aliquoting a portion of the sample for washing and cytocentrifugation.
4. Viscous synovial fluids can be liquefied by adding 400 units of the enzyme hyaluronidase (solution or
powder form) to approximately 1 mL of fluid, and incubating at 37 °C for ten minutes. Washing the
cells after liquefaction also is helpful.
5. Cellular samples or bloody samples need to be diluted with saline before cytocentrifugation to avoid
overcrowded slides. Overcrowded slides are difficult to interpret due to clumping of cells and
distortion of morphology. By using a standardized scheme for sample dilution based on cell counts, a
slide with a uniform monolayer of cells can be obtained on every sample. The appropriate dilution
will depend upon the amount of sample in the chamber and the cytocentrifuge speed and time.
Alternately, for bloody samples, some laboratories prefer to gently lyse the erythrocytes before
cytocentrifugation.
6. Adding a drop of sterile, 22% albumin to the sample chamber before adding the sample enhances
adherence of cells to the glass slide and reduces cell smudging or disintegration, particularly for low
protein specimens, such as cerebrospinal fluid.
7. Proper alignment of the sample chamber outlet port to the hole in the filter card is essential to
optimize cell yield.
8. Residual fluid remaining in the cell chamber after cytocentrifugation must not be allowed to flow
back onto the slide. Air-dried cytocentrifuge slides for Romanowsky-type stain must be kept free of
moisture until fixing and staining. If unfixed slides become wet, artifactual change occurs, resulting in
a “shrunken” or “rounded-up” appearance to the cells.
8.1.2
Other
Alternative methods of cell concentration for morphologic evaluation include sedimentation methods,27-31
and centrifugation with smears made from the resuspended sediment.29 These methods are difficult to
standardize and produce smears of variable quality. These alternatives are inferior to cytocentrifugation,
and are not recommended. Filtration methods27,31-34 that are widely used in cytopathology laboratories are
less practical for the hematology laboratory because they involve prefixation in ethanol, which precludes
Romanowsky-type staining of air-dried smears.
8.2
Identification of Morphologic Constituents
The following descriptions apply to properly prepared cytocentrifuge slides optimally stained with
Romanowsky stains.35-37 Differences from typical blood or bone marrow aspirate morphology are
emphasized. Because cytocentrifugation produces a thin cell monolayer, cells may be slightly larger than
their counterparts in blood or bone marrow aspirate smears. More intense staining of basophilic
cytoplasm and azurophilic cytoplasmic granules also may occur.
8.2.1
Myeloid Series
Neutrophils, Eosinophils, Basophils, Mast Cells: All maturation stages appear similar to those in blood
or bone marrow. The segmented neutrophil and eosinophil show more distinctive lobe separation, and the
nuclear lobes often are peripherally located close to the cell membrane. Toxic granulation and toxic
vacuolization, when present in blood neutrophils, also will be seen in body fluid neutrophils. However,
small vacuoles may occur in many body fluid cells, either due to degenerative change in the fluid or to
cytocentrifugation. Both neutrophils and eosinophils can phagocytose microorganisms in body fluids.
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Degenerated Neutrophils: Neutrophil degeneration frequently is seen in body fluids, particularly
accompanying fluid neutrophilia. The nucleus becomes pyknotic, and appears as a small, dense, round
mass. If toxic granulation is present, the granules can coalesce into azurophilic clusters. These cells may
resemble nucleated red blood cells, but typically have some residual azurophilic granules in the cytoplasm
to identify them as neutrophils.
8.2.2
Erythroid Series
Erythrocytes, Nucleated Red Cells: These cells appear similar to their counterparts in blood and bone
marrow, except that crenation and even lysis of erythrocytes may occur in body fluids.
8.2.3
Lymphoid Series
Lymphocytes: The typical small lymphocyte appears slightly larger than in blood smears, often with
more abundant cytoplasm. A small nucleolus also may be visible. Small numbers of azurophilic granules
sometimes are present in the cytoplasm.
Reactive Lymphocytes: Reactive lymphocytes occur commonly in body fluids and have many different
morphologic variants. They typically have a round to slightly indented (“bean-shaped”) nuclear contour
and abundant cytoplasm, which varies in color from slate to deeply basophilic. Reactive lymphocytes of T
or NK lineage often contain small numbers of azurophilic granules, while B-lymphocytes occasionally
contain multiple small cytoplasmic vacuoles. Immunoblastic forms have less condensed chromatin,
multiple small nucleoli, and a small amount of deeply basophilic cytoplasm, sometimes with scant
azurophilic granules. Plasmacytoid forms show ropey nuclear chromatin, multiple small nucleoli, and
abundant amounts of deeply basophilic cytoplasm; they frequently have a clear Golgi region next to the
nucleus. In contrast to malignant lymphoma cells, reactive lymphocytes have a distinct, smooth nuclear
membrane and regular nuclear contour. Typically, a spectrum of reactive lymphocyte morphology is
present, in contrast to a more homogeneous appearance for lymphomatous infiltrates.
Plasma Cells: These cells appear similar to their counterparts in bone marrow, and frequently occur with
other reactive lymphocyte forms. Plasma cell variants, such as “Mott” cells (plasma cells with abundant
immunoglobulin-laden small vacuoles) also occur in body fluids.
8.2.4
Mononuclear Phagocytic Series
Monocyte: Monocyte morphology varies from the typical appearance in blood to an activated, enlarged
form with copious cytoplasm and a few small vacuoles. At some arbitrary point in this activation process,
the monocyte is called a histiocyte.
Macrophage: When the monocyte/histiocyte shows evidence of phagocytosis (such as ingested material,
remnants of digested products, or large postingestion vacuoles), it is called a macrophage. Macrophages
are large, have dense nuclear chromatin, and can have a round nucleus or a nucleus flattened against one
side of the cell. The cytoplasm is abundant and frequently vacuolated. Occasionally, the vacuoles in the
cytoplasm may coalesce to form a “signet ring” cell. The phagocytic activity of macrophages can be
extraordinary, including ingestion of erythrocytes (erythrophage), neutrophils (neutrophage, “Reiter” cell
in synovial fluid), lipids (lipophage), microorganisms, and crystals. Macrophages also may contain blueblack hemosiderin granules arising from the iron of a digested red cell (siderophage). Hematin crystals
(yellow-brown, rhomboid-shaped) rarely are seen in macrophages, and represent an iron-free pigment of
hemoglobin breakdown from ingested erythrocytes.
16
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8.2.5
H56-P
Lining Cells
Ventricular Lining Cells: Cells lining the ventricles (ependymal cells) or choroid plexus (choroidal
epithelial cells) may be shed into the CSF, particular in neonates, in patients with a ventricular shunt or
Ommaya reservoir, or after brain surgery. These cells may occur singly, or in clusters. Often these cells
have degenerated to such an extent that only naked nuclei remain. The cells have a round nuclear contour,
eccentrically placed nuclei, condensed to finely granular chromatin, and lack nucleoli. The abundant
cytoplasm typically is amphophilic, or blue-pink, and grainy. Choroid cells may have microvilli.
Leptomeningeal Cells: Rarely, cells from the subarachnoid or pia membranes lining the CSF cavity
exfoliate into the CSF. These cells may appear in clusters, with spindle-shaped nuclei and a moderate
amount of gray-blue cytoplasm. Black pigment granules may be present in the cytoplasm.
Mesothelial Lining Cells: Mesothelial cells line the pleural, peritoneal, and pericardial cavities and have
a variety of morphologic appearances. They can proliferate and desquamate into effusions in any disease
process, and may be shed individually or in clusters. However, clusters of mesothelial cells generally have
“windows” between the cells, in contrast to the tight clusters formed by nonhematopoietic malignant
cells. Unstimulated mesothelial cells are smaller than reactive mesothelial cells, with an eccentrically
placed nucleus, round to oval nuclear contour, dense chromatin, no nucleolus, and a moderate amount of
light to moderately basophilic cytoplasm, without cytoplasmic granules. In contrast to the plasma cells, a
Golgi zone is not visible. In chronic effusions, stimulated mesothelial cells proliferate and enlarge,
showing less condensed nuclear chromatin and small nucleoli. Multiple nuclei may occur; in contrast to
malignant cells, these nuclei are approximately equal in size. Degenerative changes in mesothelial cells
include cytoplasmic blebbing and cytoplasmic vacuolization, particularly at the cell periphery.
Mesothelial cells may be phagocytic and transform into macrophages; since intermediate stages occur, it
can be difficult to differentiate mesothelial cells from macrophages.
Synoviocyte (Synovial Lining Cells): Synovial lining cells arise from the synovial membrane lining the
joint capsule and have a similar appearance to mesothelial cells.
8.2.6
Malignant Cells
Blast Cells: Blasts found in body fluids resemble their counterparts in blood and bone marrow. Myeloid
lineage blasts may have more prominently staining cytoplasmic granules. Careful correlation with blood
smear findings is necessary to determine whether the finding of blasts in the body fluid represents true
leukemic involvement, or reflects blood contamination of the body fluid or from inadvertent marrow
puncture.
Lymphoma Cells: Large cell lymphoma resembles immunoblastic reactive lymphocytes, with immature
nuclear chromatin, multiple nucleoli, and moderate amounts of basophilic cytoplasm. However, cytologic
features suggesting lymphoma include: irregular nuclear contour, lack of a prominent nuclear membrane,
large nucleoli, small clear vacuoles covering the nucleus, lack of a clear Golgi region, and a homogeneous
appearance to the infiltrate. Small cell lymphomas are difficult to distinguish from normal lymphocytes
and may require flow cytometry or immunohistochemical studies for definitive identification.
Nonhematopoietic Malignant Cells: A variety of malignant neoplasms can invade the body cavities,
including adenocarcinoma, sarcoma, and primary brain tumors. Cytologic features of nonhematopoetic
malignant cells may include: large size, high nuclear to cytoplasmic ratio, irregular nuclear contour, large
nucleoli, multinuclearity with variable nuclear size and shape, formation of tight clusters with indistinct
cell separation, signet-ring cells in clusters, well-demarcated vacuoles with a clear interior, and nuclear
molding (indentation of the nucleus of one cell by that of an adjacent cell).
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8.2.7
H56-P
Miscellaneous Cells
Squamous Epithelial Cells: Squamous cells from skin may contaminate body fluids. They have a low
nuclear to cytoplasmic ratio, a small round nucleus with dense chromatin, and abundant cytoplasm with
an angulated cell contour.
Endothelial Cells: Endothelial cells that line tissue blood vessels rarely are seen in body fluids, but
occasionally occur in CSF after brain surgery. They have an elongated shape and contain a spindle or
elliptical nucleus with reticular chromatin and with one or more nucleoli. The frayed cytoplasm may
contain a few azurophilic granules.
Chondrocyte (Cartilage Cells): Cartilage cells may inadvertently be obtained during lumbar puncture or
joint aspiration. They have round to oval nuclear contour with condensed chromatin, and a very
distinctive burgundy-colored cytoplasm.
Neural Tissue/Neurons: Neural tissue (fragments of cells, and stroma, sometimes with capillaries), as
well as isolated neurons, sometimes occur in the CSF. The tissue fragments appear as a pink or blue
fibrillar matrix sometimes containing degenerated nuclear material. Intact neurons or ganglion cells have
a pyramidal shape, often with extended processes.
Germinal Matrix Cells: Germinal matrix cells are found beneath the ependymal lining cells in ventricles
of premature neonates; they are primitive pluripotential cells that can give rise to neuronal cells, and are
blast-like in morphology. They frequently form loose clusters within a tissue-like matrix.
8.2.8
Microorganisms
Bacteria: Rod-shaped bacilli, round cocci, branching filamentous bacteria, and acid-fast bacilli all may
be seen in body fluids, occurring both extracellularly and intracellularly. Most bacteria have a basophilic
hue with Romanowsky stain. They can be distinguished from stain precipitate by their relatively uniform
size and shape.
Yeast, Fungi: Most yeast and fungi typically are regular in contour, round to oval shaped with dense
basophilic staining. They also may be seen both extracellularly and intracellularly. In CSF, Cryptococcus
is a large, round to oval yeast-like fungus with a thick capsule. Cytocentrifugation can produce capsule
disintegration, resulting in a “sun-burst” appearance.
Parasites: Toxoplasma, amoebae, and other large parasites rarely are found in body fluids, with typical
characteristic appearances.
NOTE: The following images represent examples of the types of cells that are commonly seen in body
fluids. This document is not intended for use as an atlas of morphology for instructional or diagnostic
purposes.
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Eosinophils
Mast cell (left center)
Autolytic neutrophils
Autolytic neutrophils
Lymphocytes
Reactive lymphocytes
Macrophage
Macrophages
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Plasma cells
Number 20
20
Macrophage with ingested
erythrocyte (erythrophage)
Macrophage with ingested
neutrophil (neutrophage)
Macrophage with ingested
lipid (lipophage)
Macrophage with hemosiderin
(siderophage)
Macrophage with hemosiderin
and hematin crystal
Macrophage with ingested
erythrocytes and hemosiderin
granules
Macrophage with sodium
urate crystals
Ventricular lining cells
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Macrophages (signet ring cells)
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Ventricular lining cells
Mesothelial cells (stimulated)
Mesothelial cells
Mesothelial cells
Mesothelial cells
Mesothelial cells (mitotic)
Mesothelial cells (peripheral
vacuoles)
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Mesothelial cells (binucleate)
Ventricular lining cells
Number 20
22
Blast cells (acute lymphocytic
leukemia)
Lymphoma cells (diffuse
large cell lymphoma)
Nonhematopoietic malignancy
(ovarian adenocarcinoma)
Nonhematopoietic malignancy
(ovarian adenocarcinoma)
Nonhematopoietic malignancy
(breast adenocarcinoma)
Nonhematopoietic malignancy
(oat cell carcinoma)
Nonhematopoietic malignancy
(ovarian adenocarcinoma)
Nonhematopoietic malignancy
(gastric adenocarcinoma)
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Nonhematopoietic malignancy
(breast adenocarcinoma)
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Chondrocyte (cartilage cell)
Germinal matrix cells
Yeast (Candida albicans)
Neural tissue
Yeast, fungi (Cryptococcus
neoformans)
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Parasites (Toxoplasma
trophozoites)
Cartilage
Number 20
24
Cryptococcus in CSF. Note the deeply
staining basophilia of yeasts. A
capsule can be discerned around some
organisms. Wright’s stain.
Monosodium urate crystals in synovial
fluid. Note the elongated needle-like
shape and bright birefringence of the
crystals. The crystals are intracellular.
Polarized light.
Monosodium urate crystals in synovial
fluid. Note the red background, the
blue color of the vertical crystals, and
yellow color of the horizontal crystals.
First order red compensator.
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Calcium pyrophosphate crystals in synovial
fluid. NOTE: The crystals may be
rectangular or rhomboid. Some crystals
may be needle-like and confused with
MSU crystals. Brightfield.
Volume 25
8.3
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Evaluation of Nucleated Cell Subtypes
Morphologic assessment typically includes a quantitative or semiquantitative evaluation of the nucleated
cell composition. A differential count typically includes:
•
hematopoietic cells: segmented/band neutrophils, immature granulocytes (metamyelocytes,
myelocytes, promyelocytes), lymphocytes, reactive lymphocytes, monocytes/macrophages,
eosinophils, basophils, mast cells, plasma cells, nucleated red blood cells;
•
lining cells: ventricular lining cells (CSF), leptomeningeal cells (CSF), germinal matrix cells (CSF),
mesothelial lining cells (pleural, peritoneal, pericardial fluids), synovial lining cells;
•
blasts, lymphoma cells, and nonhematopoietic tumor cells; and
•
atypical cells (with description in a comment).
The report must clearly indicate all cell types included with each numeric percentage. Individual
laboratories may choose to group or separate some of these categories. For example, it is difficult to
distinguish reactive-appearing mesothelial cells from monocytes/macrophages, and these cells may be
combined into one category without compromising clinical interpretation. Nonhematopoietic cells, such
as CSF lining cells and metastatic tumor cells, may be included in a category designated “other cells” and
described in a comments section of the report.
Other significant morphologic findings should also be reported in a comments section, such as the
presence of intracellular or extracellular microorganisms, erythrophages or siderophages (CSF),
lipophages (CSF), and crystals.
Contaminating cells should not be included in the differential, and, if identified on the counting chamber,
also should be excluded from the cell count. These include squamous epithelial cells, endothelial cells,
neuroectodermal cells (CSF), cartilage cells (CSF, synovial fluid), and ciliated epithelial cells
(bronchoalveolar lavage). Degenerating cells also should be excluded from the differential unless their
identity is apparent.
Clumps of lining cells, germinal matrix cells, or tumor cells should be reported in a comments section
rather than as part of the differential. Likewise, cell clumps in the counting chamber should be excluded
from the cell count.
If cells cannot be identified, they may be reported as “atypical” and described in a comment, pending
physician review (see Section 8.4).
Some laboratories may prefer to report a semiquantitative description of cell composition, particularly
when the cell count is low with a scant cell yield on the slides.
Malignant cells may occur in low frequency, even when the cell count is low. Therefore, it is important to
scan all slides carefully in cases of known or suspected malignancy.
8.4
Physician Review
A qualified physician should review all slides with atypical unidentified cells or suspected malignant
cells. Each laboratory should determine additional morphologic findings and/or clinical situations in
which physician slide review is required.
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Separate aliquots of body fluids are frequently analyzed simultaneously in the hematology or core
laboratory and the cytopathology laboratory. Laboratories should have a policy to correlate these results,
particularly when atypical or malignant cells are identified. When nonhematopoietic tumor cells are first
suspected on Romanowsky-stained cytocentrifuge slides, verification by PAP-stained cytopathology may
be appropriate.
8.5
Result Reporting
Nucleated cell differentials are reported in conventional units (%) or SI units (proportion, or %/100). The
number of cells counted should be included in the report only if less than 100 are counted. If absolute
counts are needed, the nucleated cell count is multiplied by the percentage and divided by 100; absolute
counts are reported in conventional units (/µL) or SI units (X106/L).
A comments section may be added to include additional clinically significant morphologic findings. If the
laboratory is aware that malignancy is suspected (e.g., on samples from known oncology patients), it also
is useful to comment specifically on the presence or absence of malignant cells. However, it is important
to understand that body fluid differential counts are not an appropriate screening or diagnostic test for
malignancy in previously undiagnosed patients. If a differential count has not been performed, the
comments section may include a descriptive statement about the cell composition (for example, whether
the fluid is lymphocyte, monocyte/macrophage, or neutrophil predominant, or contains mixed
inflammatory cells).
Physician review of slides should be indicated in the report.
9
Fluid Types
9.1
Cerebrospinal Fluid
9.1.1
Macroscopic Examination
Macroscopic examination of CSF includes observations of clarity, color of the neat specimen, color of the
supernatant, and clot formation. Normal CSF is clear and colorless. Increased cell counts cause turbidity
that is noted when nucleated cell counts approach 200/µL. This number is not sacrosanct and varies with
the cell type.9 Since erythrocytes have less volume than nucleated cells, more cells are required to
produce equivalent turbidity. Grading turbidity seems an unnecessary exercise, since cell counts are
routinely reported. Color should be reported as colorless, yellow, orange, pink, or brown corresponding
to bilirubin, oxyhemoglobin (orange/pink), and methemoglobin respectively. Although the various
pigments can be identified based on their unique spectral absorption “fingerprints” and quantified by
spectrophotometry, this is not necessary in routine clinical laboratory practice. Viscosity is not routinely
reported. CSF does not clot, but clots may be associated with a traumatic tap.
9.1.2
9.1.2.1
Microscopic Examination
Enumeration
The sample should be well mixed before analysis. Both nucleated cell and erythrocyte counts are
manually enumerated using a hemocytometer chamber. Both erythrocytes and nucleated cells are
enumerated in the same chamber. If the specimen is excessively bloody or the nucleated cell count is
markedly increased, the sample should be diluted.
Automated cell counts are limited by their poor sensitivity in pathologic specimens with low cell counts.
In one study using impedance technology, the accuracy of counts below 0.2 x 109/L for nucleated cells
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and 0.01 x 1012/L for erythrocytes was poor.38 A similar limitation was noted using light scatter and
absorption.39
Any flagging of automated counts also requires a manual count. Imaging technology does not appear to
have the same limitations with low counts and enumerates cells with sufficient accuracy to be clinically
acceptable. As expected, the coefficient of variation at low cell counts is high. With automated imaging
technology, the differential cell count and other cytologic analyses must be done using conventional
techniques. A problem with imaging technology is the lack of studies in the scientific literature.
9.1.2.2
Morphology
Cellular constituents of normal CSF include lymphocytes, monocytes, and occasional neuroectodermal
cells. Lymphocytes are primarily T-cells (~97%). In pathologic conditions, the list of cell types becomes
extensive. In normal adults, more than two-thirds of the cells are lymphocytes. Morphologically, they
are similar to cells in blood. When challenged by antigenic stimulation, they transform into activated
lymphocytes, developing more abundant cytoplasm and changes in the nuclear chromatin pattern. Some
cells may transform into immunoblasts. Stimulation of B-cells recapitulates differentiation to plasma
cells.40 Monocytes constitute approximately 14% of nucleated cells in normal adult CSF. The percentage
increases to a mean value of approximately 70% in neonates.41 Monocytes are morphologically similar to
PB-monocytes. They may transform to macrophages. Erythrophages, siderophages, and lipophages are
monocytoid cells or macrophages that have ingested erythrocytes, contain iron, or are filled with lipid.
Neutrophils are not present in normal CSF. The occasional neutrophil in “normal” CSF has been
attributed to the sensitivity of cytocentrifugation capable of detecting the rare cell introduced by
microscopic contamination during lumbar puncture. Thus, when the total nucleated cell count is normal,
the rare neutrophil may not be pathologic.
CSF specimens may contain malignant cells derived from three general sources: primary brain tumors,
metastatic solid tumors, and hematopoietic neoplasms. The morphology is varied depending upon the
cell of origin and stage of differentiation. Examples are illustrated in the accompanying
photomicrographs. Mitoses may be seen, but are also present in nonmalignant and even normal fluids,
since the CSF is a nutrient medium.
Neuroectodermal cells also shed into the CSF of both normal and pathologic fluids. They are derived
from two sources, the meninges and cells lining or in contact with ventricular fluid. Choroidal epithelial
cells are the most frequent neuroectodermal cells observed in CSF. They occur most frequently in
specimens from infants.
Extraneous cell types include hematopoietic cells from bone marrow, chondrocytes from intervertebral
discs, capillaries from the choroid plexus or arachnoid membrane, germinal matrix cells in neonates, and
fragments of neural tissue.
9.1.3
Specimens From Shunts and Reservoirs
Ventricular-systemic shunts are used to reduce intraventricular pressure by facilitating the removal of
cerebrospinal fluid in patients with hydrocephalus. The most frequent drainage site is the peritoneal
cavity. One complication is shunt failure secondary to a foreign body reaction with occlusion of the tube
at either end. Samples of CSF removed from the shunt reflect this reaction and may show increased
mononuclear cells, foreign body multinucleated giant cells, eosinophils, or clusters of ependymal
cells.42,43 Mast cells may be seen rarely, probably from the peritoneal end of the shunt.
The Ommaya reservoir is a subcutaneous reservoir attached to a catheter that empties into the lateral
ventricle. It is used for the delivery of drugs to the CNS.
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Specimens from ventricular-systemic shunts and Ommaya reservoirs are sent for evaluation of infection.
Thus, cell counts, differentials, and microbiologic cultures are the tests of interest. Approximately 90%
of the infections are secondary to coagulase-negative staphylococci.
9.1.4
9.1.4.1
Result Reporting
Reporting Terminology
Laboratory reports should note the specimen type, the sequence of the tube in the collection process,
color, clarity, red cell count, nucleated cell count, differential cell count, and unusual cells, including cells
from primary and secondary malignancies. The latter are best mentioned in a comments section. A
description of the supernatant should be included in all colored and cloudy fluids.
Colors should be reported as colorless, yellow, orange, pink, or brown. Rarely will other colors be seen.
When other colors are seen, they should be reported (e.g., black). It is best to limit the reporting of colors
to the previous categories to provide some intralaboratory consistency. Xanthochromia literally means
yellow fluid, but has been expanded to include pink, orange, or yellow. Indicating the actual color has
more interpretive value. Regrettably, xanthochromia is interpreted as synonymous with a pathologic
bleed; however, it may also occur in other conditions. Thus, avoiding the term and reporting the actual
color is recommended.
Turbidity should be routinely reported. It does not need to be graded, since cell counts are routinely done.
Nucleated and red cell counts are reported in conventional units (µL) or SI units (106/L). Differentials are
reported as a percentage in conventional units or the percentage multiplied by 0.01 to express the number
as a fraction in SI units.
In the nucleated differential, all cells derived from the hematopoietic system should be included. The
term mononuclear cell should be avoided, since the term does not adequately distinguish monocytes from
lymphocytes, a distinction that has diagnostic significance. Siderophages, erythrophages, histiocytes from
lysosomal storage diseases, lipophages, neuroectodermal cells, microorganisms, neoplastic cells,
chondrocytes, LE-cells, as well as others should be reported in a comments section.
All cerebrospinal fluids should be treated as stats, not only because of medical necessity but also because
of the instability of cellular constituents. Immediate reporting to the clinician is mandatory.
9.1.4.2
Normal Values
Normal values are age-dependent. Accurate values are problematic in neonates because of difficulty
obtaining normal specimens. Differential counts from the literature obtained from chamber counts or push
smears of centrifuged pellets lack the cytologic detail and preservation of cells for accurate identification
and differential counts. Cytologically, one age-related difference is a predominance of monocytes in the
neonate that is gradually replaced by lymphocytes. Values in Table B1 represent combined data collated
from various reports.9,41,44-47
9.1.5
Analytic Significance
Color. The usual alteration of color in the cerebrospinal fluid is secondary to a pathologic bleed in the
central nervous system or a traumatic lumbar puncture. Initially, pathologic bleeds result in extravasation
of erythrocytes that are lysed, with eventual catabolism of hemoglobin to bilirubin. The latter occurs
approximately 12 hours after a bleed and persists for two weeks. The various hues depend on the
admixture of hemoglobin and bilirubin pigments. Examination of the supernatant is helpful in
differentiating between a traumatic tap and pathologic bleed. A colorless supernatant is associated with a
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traumatic tap and a pink or yellow color with a pathologic bleed. Although the term xanthochromia
literally means yellow color, it has become synonymous with a pathologic bleed in common medical
usage. However, a pink supernatant occurs initially in pathologic bleeds and a yellow color may be seen
in the absence of a pathologic bleed. Other causes of “xanthochromia” include an elevated total protein
above 150 mg/dL and elevated serum bilirubin usually above 7 mg/dL. Rarer causes include
hypercarotenemia and drugs.
Turbidity. Normal CSF is clear. Various degrees of turbidity occur as either the red cell count or white
cell count increases. In a traumatic tap, the supernatant fluid will be clear.
Erythrocyte Counts. Red cells reflect either central nervous system bleeding or a traumatic puncture.
Comparison of cell counts between the first tube collected and the third or fourth tube is an excellent way
to distinguish the two, the last tube showing a marked decrease in the count if the puncture is traumatic.
In addition, if the tap is traumatic, the number of blood leukocytes added to the sample can be calculated
from the CSF red cell count and the relative numbers of leukocytes and erythrocytes in the blood:
WBC added = WBCB
RBCCSF
RBCB
WBC added = WBCB x RBCCSF
RBCB
The number of WBC added is subtracted from the leukocyte count of the CSF sample, to determine what
the true WBC count should have been if there was no contamination of the CSF from the traumatic
bleeding:
true CSFWBC = CSFWBC hemocytometer count – WBC added.
Nucleated Cell Counts. Elevated nucleated cell counts are present in numerous conditions and their
value assumes special significance in the diagnosis of meningitis. Nucleated cell counts are elevated in
both viral and bacterial meningitis. Lymphocytes predominate in the former and neutrophils in the latter.
In meningitis, there is a correlation between increasing cell counts and positive bacterial cultures.48 Other
infectious causes of increased CSF nucleated cells include fungi, mycobacteria, and parasites.
Cytology. Pathologic fluids have a variety of cell types. Their interpretation is summarized in Table B1.
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Table 4. Cerebrospinal Fluid Reference Intervals*
Volume
10-60 mL
Babies
60-100 mL
Children
57-286 mL
Adults16
Color
Colorless
Cells
Erythrocytes46,47
Newborn preterm
Newborn term
Neonate
> 3 months
Adults
Leukocytes47,49,50
0-1 month
2 months to 16 years
Adults
0-27/µL (0-27 x 106/L)
0-7/µL (0-7 x 106/L)
0-5/µL (0-5 x 106/L)
Leukocyte Differential44,50,51
Neonates
Lymphocytes
Monocytes
Histiocytes
Neutrophils
Neuroectodermal
2-38% (.02-.38)
50-94% (.50-.94)
1-9% (.01-.09)
0-8% (0.00-.08)
rare
0-1000/µL (0-1000 x 106/L)
0-800/µL (0-800 x 106/L)
0-50/µL (0-50 x 106/L)
0-5/µL (0-5 x 106/L)
0-5/µL (0-5 x 106/L)
Adults
Lymphocytes
Monocytes
Histiocytes
Neutrophils
Neuroectodermal
*
SI units are in parentheses.
9.2
63-99% (.63-.99)
3-37% (.03-.37)
rare
0-2% (0.00-0.02)
rare
Serous (Pleural, Peritoneal, Pericardial)
This section will cover fluids of the pleural, pericardial, and peritoneal cavities (see Figure 2). They
include serous, chylous, hemorrhagic, and iatrogenic fluids. The word serous is derived from serum,
emphasizing that serous fluids are normally formed by the simple mechanism of plasma ultrafiltration.
9.2.1
Macroscopic Examination
Color and clarity of the fluid should be routinely reported by the laboratory. Pathologic fluids may have a
variety of colors depending on the etiology of the effusion. Transudates are straw colored and clear.
Other colors in pathologic fluids include red, brown, green, white, and black. Clarity may be described as
clear, cloudy, or opalescent. If the fluid is viscous, it should be noted on the report.
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9.2.2
9.2.2.1
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Microscopic Examination
Enumeration
Cell counts may be done manually or on automated counters. Few instruments have been cleared or
approved for counting cells in serous fluids by regulatory agencies. Claims of linearity below 200
nucleated cells/µL should be verified by the laboratory, since results in this range may compromise
clinical decisions with peritoneal dialysate fluids that require accuracy between 1 and 100/µL.
Manual counts are done using a hemocytometer chamber. If fluid is clear, it can be counted undiluted.
Cloudy or bloody fluids can be diluted using isotonic saline or other appropriate fluids. All nucleated
cells should be counted, since it is difficult to accurately distinguish cell types in the chamber (e.g., a
mesothelial cell from a histiocyte). Commercial controls are available.
9.2.2.2
Morphology
Differential nucleated cell counts are usually done on stained preparations, primarily using Romanowsky
stains. One study indicates that pleural fluid differentials obtained by an automated cell counter was not
sufficiently accurate for clinical use.10
Cell types include leukocytes, macrophages, mesothelial cells, and metastatic cells from solid tumors.
Leukocytes include neutrophils, eosinophils, basophils, monocytes, lymphocytes, plasma cells, immature
granulocytes, and blasts. Their morphology is described in a previous section. Although morphology in
serous fluids is similar to blood or bone marrow, degenerative changes are more frequent.
Microorganisms may be visualized. They may be bacteria or fungi. Identification relies on the use of
Gram’s, methenamine silver, periodic acid Schiff, acid-fast stains, and culture.
9.2.3
Other Fluids From Serous Cavities
These fluids include peritoneal lavage and peritoneal dialysate fluids. Neither is a true body fluid; they
are extraneous fluids introduced into the peritoneal cavity for diagnosis or treatment.
9.2.3.1
Peritoneal Lavage Fluids
Peritoneal lavage fluids are sterile physiologic fluids introduced into the peritoneal cavity, originally used
in emergency medicine to diagnose intra-abdominal bleeding from a ruptured organ following blunt
trauma to the abdomen. Evaluation of the erythrocyte count determines the need for exploratory
laparotomy. The technique has largely been supplanted by radiologic techniques (e.g., ultrasound of the
abdomen). However, the technique and the nucleated cell count may also be used to diagnose intestinal
perforation.
9.2.3.2
Peritoneal Dialysate Fluids
Patients with chronic renal failure may be treated with chronic ambulatory peritoneal dialysis (CAPD)
rather than hemodialysis to control the adverse effects of renal failure.
Fluids are sent to the laboratory to evaluate the nucleated cell count, differential, and microbiologic
culture to determine infection and the offending organism.
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9.2.4
9.2.4.1
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Result Reporting
Reporting Terminology
Specimens received by the laboratory are designated by a variety of names. Pleural fluids are also labeled
as chest, thoracentesis, or empyema fluid. A strict definition of the latter means pus in any sac, but in
practice is usually used to denote pus in the pleural cavity. Peritoneal fluids may be labeled as abdominal,
ascitic, or paracentesis fluid. The latter term actually means withdrawal of fluid from a cavity, but in
common parlance has become shorthand notation for abdominal paracentesis. It is preferable that the
laboratory report designate the fluid by its proper anatomic term (i.e., pleural, peritoneal, or pericardial
fluid). The right or left side should be designated for pleural fluids.
Reports should include both color and clarity of the fluid. A notation should be made if the specimen has
clots.
Quantitative counts include nucleated and red blood cell counts. The term nucleated cell count is
preferred to leukocyte or white blood cell count, since the former is more inclusive. On manual counts, it
may not be possible to differentiate macrophages from mesothelial cells. Not all laboratories will agree
on whether to designate macrophages as leukocytes. To avoid these ambiguities and for uniform
interlaboratory reporting, the term “nucleated cell count” is preferable. Units of measurement are
uniformly metric, but vary widely between laboratories from SI units to conventional units (cells/µL). SI
units are expressed as 109/L for nucleated cell counts and 1012/L for red blood cell counts. The former
should be expressed to two and the latter to three decimal places. It is not unusual for laboratories to use
different units of measurement for cell counts in serous fluids compared with cell counts in blood. No
recommendation is made in this regard, although uniformity would be preferable.
Differential counts should include all cell types and be reported as percentages. Absolute cell counts have
limited value. It is not necessary to distinguish between band neutrophils and segmented neutrophils.
Monocytes and macrophages may be counted in a single category, since transitional forms cause
difficulty in exact classification and categorizing the cells separately serves no medical purpose.
Neoplastic cells from solid tumors should be recognized and be reported after confirmation by a
pathologist, cytologist, or other certified personnel deemed qualified to diagnose malignant cells. Fluids
suspected of malignancy should be correlated with specimens sent to the cytology laboratory, and
evaluated using Papanicolaou or histologic stains. Cooperation between hematology and cytology
laboratories is necessary, and slides should be correlated to provide optimal diagnosis. The techniques
and expertise in the hematology laboratory maximize the diagnosis of hematologic malignancies, whereas
the techniques and expertise of the cytology laboratory maximize the diagnosis of nonhematologic
malignancies.
9.2.4.2
Reference Intervals
In pleural fluid, reference intervals for normal fluids in humans have been inferred from studies on other
animals. Only two studies have been done on normal humans. One normal value study was done on
Japanese soldiers by puncturing the intercostal space and attempting to recover fluid.52 These results had
total cell counts ranging from 1700 to 6200/µL with mean differential counts of 53.7% monocytoid cells,
10.2% lymphocytes, 3.0% mesothelial cells, 3.6% granulocytes, and 29.5% unidentified cells. More
recently, a more sophisticated study was done using a minimally invasive pleural lavage technique on 34
adults.53 Volume, cell counts, and differential counts were analyzed. Since it is not possible for
laboratories to determine their own reference range, this study of pleural fluid provides a convenient
normal reference range derived from the literature as summarized in Table 5.
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Normal values for peritoneal fluid have not been determined. Although not completely satisfactory,
“sterile” ascitic fluid has been used to distinguish “normal” ascites from peritonitis and other serious
intra-abdominal disorders, as discussed in Section 9.2.5.
Reference intervals for peritoneal dialysate fluid are listed in Table 6.
9.2.5
Analytic Significance
9.2.5.1
Macroscopic Examination
Transudates are typically straw colored and clear, as are some exudates.54 A red color indicates blood. If
erythrocytes lyse, hemoglobin may be oxidized to methemoglobin, imparting a brown color. If fluid is
grossly bloody, a hematocrit will distinguish between gross bleeding and a serosanguineous effusion.
Green indicates bile. Fluids that are white indicate pus or chyle, and may be distinguishable
macroscopically by the quality of the color and centrifugation. The supernatant of the former will be
clear after centrifugation. Ascitic fluids may have a pronounced yellow color in jaundiced patients. A
black color has been reported in melanoma.
Cloudy fluids are due to increased numbers of cells or increased triglycerides, and chylous effusions may
have an opalescent appearance.
In the rare circumstance of a mesothelioma, the fluid may be viscous because of a high concentration of
hyaluronic acid.54 A foul odor suggests infection and if the odor of urine is detected, it indicates a
ruptured urinary bladder or urine if the sample is inadvertently collected from the bladder during
abdominal paracentesis.
9.2.5.2
9.2.5.2.1
Cell Counts and Differentials
Pleural Fluid
Red blood cell counts are of little significance. As indicated previously, a hematocrit can distinguish
between a serosanguineous effusion and hemothorax. The latter may be secondary to trauma, pulmonary
emboli, or malignancy.54 The nucleated cell count is of some significance, but chemical tests are the
primary studies used to distinguish transudates from exudates. Approximately 80% of transudates will
have cell counts less than 1000/µL and most of the remainder are less than 2000/µL. Cell counts above 10
000/µL are usually associated with parapneumonic effusions. Differential counts are useful and in
exudative lymphocytic effusions, immunophenotyping can distinguish between benign and malignant
lymphoproliferative disorders.
Differential cell counts are important in determining the etiology of an effusion. The interpretation of
differential cell counts in pleural fluid is summarized in Table B2. Neutrophilia (>50%) indicates an acute
inflammatory process (e.g., parapneumonic effusions). Eosinophilia (>10%) is seen in many conditions
including pneumothorax, pulmonary emboli, traumatic hemothorax, possible immunoallergic reaction to
chest tubes, parasitic diseases, and Churg-Strauss syndrome.54
9.2.5.2.2
Peritoneal Fluid
Red blood cell counts have limited diagnostic value. Pink ascitic fluid has red blood cell counts of at least
10 000/µL. With counts >20 000/µL, the fluid appears red. A traumatic tap must be distinguished from
hemoperitoneum. Malignancy may be associated with bloody fluids.
Normal values for nucleated cell counts, differentials, and biochemical tests have not been established for
peritoneal fluid. Thus, medical decision levels are based on comparison with sterile ascitic fluid in
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cirrhotic patients without other intra-abdominal disease. In one reported study of sterile uncomplicated
ascitic fluids, cell counts ranged from 0 to 2610/µL. When the skewed distribution was corrected, 0 to
562/µL was the reference range.55
Total cell counts may increase markedly following diuretic therapy. In sterile uncomplicated ascitic fluid,
neutrophils ranged from 0 to 100%, with a mean value of 27%.55 Absolute neutrophil counts ranged from
0 to 2532/µL with a mean of 82/µL. The total nucleated cell count and absolute neutrophil count are the
“standards for diagnosing spontaneous bacterial peritonitis,” with an absolute neutrophil value of
>250/µL suggestive of peritonitis.56 In tuberculous peritonitis, cell counts are typically greater than
1000/µL and lymphocytes predominate.
As indicated previously, 10% of cases of ascites are secondary to malignancy. The morphologic criteria
for identifying malignant cells from solid tumors has been mentioned previously. Mesothelial cells must
be distinguished from malignant cells.
Some miscellaneous observations include LE cells, Reed-Sternberg cells, mast cells, and megakaryocytes.
Mast cells are shed from the omentum and have no pathologic significance. Megakaryocytes have been
reported in myeloproliferative disorders.
9.2.5.2.3
Peritoneal Lavage Fluid
As indicated previously, this technique was initially used for the diagnosis of bleeding following intraabdominal trauma and has been supplanted by radiologic techniques when available. Red blood cell
counts are used to determine the need for exploratory laparotomy. The procedure introduces some red
blood cells and values up to 10 000/µL are considered consistent with the technique. Each hospital will
determine appropriate values for exploratory surgery. Originally, 100 000/µL was considered an
appropriate level. This was then decreased to 50 000/µL. The lower the selected cutoff value, the higher
the percentage of negative laparotomies.
9.2.5.2.4
Peritoneal Dialysate Fluid
Normally, dialysate fluid is clear and colorless. If peritonitis develops, the fluid becomes cloudy and the
diagnosis is obvious. Representative cell counts and differentials of noninfected fluids in patients on
continuous ambulatory peritoneal dialysis are shown, in Table 6. Noninfected fluids usually have
nucleated cell counts of 50/µL or less.57,58 Cell counts are routinely done on infected fluids to monitor the
effectiveness of antimicrobial therapy. Following infection, neutrophils significantly increase from mean
values of 18% in noninfected fluids to mean values greater than 70%.57 In addition to the neutrophilia
seen in acute inflammation, eosinophilia (≥10%) may be present in some fluids. The pathogenesis is
speculative. Possible etiologies include immunoallergic reactions to the plastic catheter, additives to the
fluid (e.g., antibiotics), or the introduction of air into the peritoneal cavity.
9.2.5.2.5
Pericardial Fluid
Most pericardial effusions are serosanguineous or hemorrhagic. Red blood cell counts have little clinical
significance. In contrast, nucleated cell counts differ significantly in transudates compared with exudates.
In one study, mean values for transudates were 2210/µL, compared with values of 14 116/µL for
exudates.59 The standard deviations are large and there is considerable overlap. Bacterial and rheumatoid
effusions had the highest percentage of neutrophils with mean values approximately 70% or greater.59
Monocytosis was secondary to hypothyroid or malignant effusions, with mean values of 75% or greater.59
The incidence of malignant effusions varies in reported series from 10 to 25%, depending on the
geographic location.60,61 Identification of malignant cells is similar to that described in other fluids, and
mesothelial cells must be distinguished from neoplastic cells. LE cells have also been reported in
pericardial effusions.59
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Table 5. Reference Intervals for Pleural Fluid. (From Noppen M, De Waele M, Li R, et al. Volume and cellular
content of normal pleural fluid in humans examined by the pleural lavage. Am J Respir Crit Care Med. 2000;162:1023-1026.
Official Journal of the American Thoracic Society. ©American Thoracic Society. Reprinted with permission.)
*
Volume (mL/cavity)
4.1-12.7 mL
Nucleated cell count
1395-3734/µL
Macrophages
64-80%*
Lymphocytes
18-36%*
Neutrophils
0-1%*
Mesothelial cells
0-2%*
Results expressed as interquartile range.53
Table 6. Peritoneal Dialysate Cell Count and Differential in Noninfected Drainage Fluids (n=29).
(Modified from Rubin J, et al. Peritonitis during continuous ambulatory peritoneal dialysis. Ann Intern Med. 1980;92:7-13.
Reprinted with permission from the American College of Physicians.)
*
©
Red blood cells/µL
24 ± 48*
Total nucleated cells/µL
36 ± 48
Leukocytes/µL
21 ± 27
Neutrophils (%)
18 ± 15.8
Lymphocytes (%)
24 ± 26
Monocytes (%)
35 ± 26
Eosinophils (%)
7±7
Basophils (%)
3±2
Results expressed as mean ± SD.
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Figure 2. Relationship of Serous Membranes, Body Cavities, and Viscera. The heart is enclosed in
the pericardial sac. The inner surface of the pericardial sac is the parietal pericardium, and the firmly
attached membrane lining the exterior surface of the heart is the visceral pericardium. Parietal pleura
lines the inner surface of the wall of the thoracic cavity and the visceral pleura covers the lung. Parietal
peritoneum covers the walls of the abdominal and pelvic cavities. Visceral peritoneum lines the surfaces
of the abdominal organs. (Reprinted from Glasser L. Extravascular biological fluids. In: Kaplan LA, Pesce AJ. Clinical
Chemistry: Theory, Analysis, and Cellular Composition. St. Louis: CV Mosby Co; 1996, with permission from Elsevier.)
9.3
9.3.1
Synovial Fluid
Macroscopic Examination
Macroscopic analysis includes color, clarity, and viscosity. Normal synovial fluid is colorless or pale
yellow and clear. Print can be clearly read through a tube containing synovial fluid. Pathologic specimens
may be colored yellow, white, or red, and the clarity may be translucent, cloudy, or opaque. As with
other fluids, breakdown products of heme cause a yellow color, leukocytes make the fluid white,
erythrocytes impart a red color, and cells (nucleated cells or erythrocytes) cause a cloudy appearance. If
particles are present, they should be noted. These may be fragments of cartilage (wear particles) or
particles containing collagen or fibrin (rice bodies). Particles may also be seen in metallosynovitis from a
prosthetic implant.
Viscosity can be measured at the bedside by the physician placing a finger at the tip of the syringe and
stringing out the fluid or determining the length of the string after expressing it from the syringe. Normal
fluids will form a string greater than four centimeters. Clinically, there is no need for a sophisticated
measurement of viscosity.
Like the gross observation of viscosity, the mucin clot test may reflect the degree of hyaluronate
polymerization. Hyaluronidase derived from neutrophils most likely has a pathogenetic role in decreasing
viscosity. The test is qualitative and involves the addition of 2% acetic acid to synovial fluid. Mucin
clots are graded as good, fair, or poor. Since other tests provide similar or more definitive information, a
critical re-evaluation of the test would be of value to determine if it is obsolete.
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9.3.2
9.3.2.1
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Microscopic
Enumeration
Cell counts may be done manually or by automated methods. Manual methods require a hemacytometer.
Clear fluids usually require no dilution. Isotonic saline is an adequate diluent. With most fluids, the
nucleated cell and erythrocyte counts can be done in the same chamber. If desired, erythrocytes can be
lysed using 0.3% saline as a diluent. Solutions containing acetic acid should not be used, since they
coagulate hyaluronate. Noninflammatory fluids are viscous and create problems in loading the chamber.
This can be resolved using hyaluronidase, if desired. Approximately 400 units of hyaluronidase are added
to 1 mL of synovial fluid and incubated for ten minutes at 37 °C.13 However, since viscous fluids are
either normal or noninflammatory, approximate cell counts are clinically acceptable, and excessive
personnel time is not justified.
Automated cell counts have been validated for total nucleated cells and erythrocytes on impedance-based
and laser-based optical systems.62,63 Lower limits of detection should both follow analytically acceptable
standards and provide clinically relevant information.62-64 Acceptable lower limits of detection were set
as >0.150 or 0.200 x 109/L for nucleated cells and 0.01 or 0.03 x 1012/L for erythrocytes.62,63 Samples
flagged for cellular interference should be enumerated manually.62 If automated instruments are used,
pretreatment of samples with hyaluronidase was considered necessary adding an additional 20 minutes of
processing time.62,63 NOTE: Flow imaging technology has also been validated for automated cell counts
for nucleated cells and erythrocytes.65-69
9.3.2.2
Morphology
Differential cell counts are done using manual or automated methods. The former has many advantages
that include identification of unusual cell types, crystals, or microorganisms.
Differential counts using automated instruments are problematic. One study indicates that the percentages
of neutrophils and mononuclear cells can be reliably measured. However, the latter category includes
lymphocytes, monocytes, immature granulocytes, and blasts.63 In addition, automated methods discourage
cytological observations that may have clinical relevance or important clinical consequences (e.g.,
bacteria).
Normal cellular constituents of synovial fluid include neutrophils, lymphocytes, monocytes, histiocytes,
and synovial lining cells.
Neutrophils normally constitute less than 25% of all nucleated cells. In addition to intact neutrophils, it is
not unusual to see necrobiotic changes, many characteristic of apoptotic cells with single or multiple
dense, hyperchromatic, homogenous nuclear masses. In pathologic specimens, neutrophils may have dark
cytoplasmic inclusions of immune complexes in wet preparations with light microscopy. Such cells are
called ragocytes or R.A. cells, the latter name because of the association with rheumatoid arthritis. In
collagen vascular diseases, typical L.E. cells may be seen on Romanowsky-stained smears. L.E. cells are
neutrophils that have engulfed large, round, purple hyaline homogeneous nuclear masses.
In normal fluids, cells in the monocyte/macrophage category normally constitute the majority of cells
with a mean value of 48%.13 On stained smears, monocyte/macrophages with basophilic cytoplasmic
inclusions have been designated Reiter cells.
Lymphocytes range from few to many in normal fluids (see Table 7), with a mean value of approximately
25%.13 They have similar morphologic features to blood lymphocytes or may show reactive changes in
pathologic fluids.
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Normally, synovial lining cells on average constitute only 4% of nucleated cells. Many other cell types
have been described in pathologic fluids. These include eosinophils, basophils, mast cells, plasma cells,
bone marrow cells, chondrocytes, Gaucher cells, platelets, and sickle cells. Unlike the other body fluids
discussed, malignant cells are so rarely seen that it does not impact the routine clinical laboratory.
The morphologic appearance of cells is illustrated in the accompanying photomicrographs.
9.3.2.3
9.3.2.3.1
Crystals
Polarization Microscopy
Polarization microscopy is one of the cornerstones in the laboratory analysis of synovial fluids, and is
essential for the diagnosis of crystalline joint disease. Because the crystal has two different indices of
refraction, the material is said to be birefringent, a property detected by polarization microscopy.
A polarizing microscope is a light microscope that has two additional filters, designated a polarizer and
analyzer. The substage light source emits light vibrating in all planes. The light is then screened by the
polarizer, a grid that filters out all rays of light except the ray vibrating parallel to the direction of the lines
of the grid. The polarized light then passes through the condenser and the specimen slide to the analyzer,
similar to the grid of the polarizer, and then through the eyepiece lens to the eye. If the analyzer grid lines
are at right angles to the lines of the grid of the polarizer, all the rays of light are screened out and the
field is black. If a birefringent crystal is present, it rotates the polarized light so that the light can pass
through the grid of the analyzer and appear white against a black background (see Figure 3). The physical
characteristics of a crystal can be exploited further for exact identification using a first order red
compensator. The background appears red and the crystals yellow or blue, depending upon the orientation
of the axes of the crystal.
Some suggestions regarding technique for crystal identification include the following: 13,70,71
1. Both wet and stained cytocentrifuge preparations should be examined.
2. Crystals may be missed with light microscopy by bright light. Lowering the condenser improves
contrast.
3. No examination for crystals is complete without polarization microscopy.
4. Dust, scratches, and debris must be distinguished from pathologic crystals.
5. With polarization microscopy, it may be difficult to keep the plane of focus because of the black
background. Introducing more light and using the first order red compensator will help.
6. Scan the slide using a 10X objective and use higher power objectives (40X, 100X) for definitive
identification.
9.3.2.4
Crystal Identification
Although several types of crystals have been noted in synovial fluid, monosodium urate and calcium
pyrophosphate dehydrate are the most frequent. Other crystals of pathologic significance include basic
calcium phosphate, steroid crystals, and cholesterol.
Monosodium urate (MSU) crystals are associated with gout. They may be difficult to visualize with
bright light. With polarization microscopy, they are 2 to 10 µ thin, needle-shaped, bright crystals with
negative birefringence. Identification is enhanced using the first order red compensator.70 When its long
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axis is perpendicular to the direction of slow vibration of the light in the compensator, it appears blue, and
when its long axis is parallel to this direction, it appears yellow.13 A fixed preparation from a known case
of gout can be used as a control to determine the orientation of the crystal using the first order red
compensator. Numerous monosodium urate intraleukocyte crystals are seen in acute gout. If gout is
suspected clinically but crystals are not detected, some studies suggest that repeat examination after 24
hours of storage at 4 °C improves the diagnostic yield.72 However, others question the diagnostic validity
of this finding.13
Urate crystals may on rare occasions form a spherulite with a beach ball appearance. They are
extracellular and in some cases, the only form of the crystal.73 They must be distinguished from globules
of fat.
Calcium pyrophosphate dehydrate (CPPD) crystals are associated with pseudogout but may also be
present in lesser amounts in noninflammatory fluids. 15 Their shape, birefringence, and dichroism contrast
with MSU. Thus, they are usually rhomboid; rarely, needle shaped; easily detected on stained preparation
with light microscopy; weakly birefringent; positively birefringent; and with the first order red
compensator, demonstrate yellow and blue dichroism opposite that of MSU. The crystals are
phagocytized by both neutrophils and monocytes. CPPD can also be detected by staining air-dried
cytocentrifuge preparations with alizarin red S.74
Basic calcium phosphates (BCP) include several chemical forms of calcium, including hydroxyapatite.
The crystals are not usually detected in the routine clinical laboratory because they are at the limit of
resolution of the light microscope. By electron microscopy, they are rhomboid or needle like and form
aggregates that can be suspected by the expert light microscopist. Their association with arthritis is well
documented.75,76,77 In one study, BCP crystals were only associated with osteoarthritis or rheumatoid
arthritis.15
Steroid crystals have protean morphology.78,79 Intra-articular injection of corticosteroids is a wellestablished clinical practice. Steroids may crystalize and synovial fluids may contain crystals from a
previous injection or inadvertently, if joint fluid was aspirated through a needle used to withdraw fluid
from a medicinal vial. Like other crystals, steroids may cause an acute inflammatory synovitis in 0.6 to
2% of patients, beginning several hours after injection and lasting up to 72 hours.80,81 The crystals may
mimic CPPD or MSU. They have been described as needles, rods, amorphous, branched, and
agglutinated. Birefringence is bright and the sign (+ or -) depends on the steroid. Interpretation of
crystals should be guarded following intra-articular therapy.
Cholesterol crystals are seen in chronic effusions of joints or bursae. They are seen in rheumatoid arthritis
and suggest a chronic severe persistent synovitis. Cholesterol crystals are extracellular rectangles with
notched corners and bright birefringence.
Other crystals or particles include hematoidin, Charcot-Leyden crystals, metal, and artifacts. Hematoidin
is a breakdown product of hemoglobin indicative of extravasation of erythrocytes but with no additional
pathologic significance. Charcot-Leyden crystals have been reported in eosinophilic synovitis associated
with urticaria.13 Fragments of metal from a prosthesis may cause a metallosynovitis. They may be extraor intracellular. Artifacts include crystals of calcium oxalate, dry K2EDTA, lithium heparin, starch
granules, and dust. A useful chart has been previously published (see Table 8).71
9.3.3
9.3.3.1
Result Reporting
Reporting Terminology
Laboratory reports should include the type of fluid, the joint or bursa, side of the body, color, clarity,
presence of particulate material, presence or absence of crystals, type of crystals, erythrocyte count,
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nucleated cell count, differential count, and special morphologic findings. Viscosity is best evaluated at
the time of aspiration and should be recorded in the physician’s notes. The mucin clot test is also a
measure of viscosity. Results should be recorded as good, fair, or poor. Cell counts are reported in
standard units (µL) or SI units, 109/L for nucleated cells and 1012/L for erythrocytes. If SI units are used,
the former should be reported to the second decimal point and the latter to the third. Laboratories make
several observations on crystals that include morphology, birefringence, strength of birefringence, sign of
birefringence, and extinction angle.13 However, these parameters do not need to be in the laboratory
report; only the type of crystal needs to be reported. The frequency (rare, numerous) has diagnostic
significance and should be noted. For example, calcium pyrophosphate deposition disease has been
defined as containing an average of more than one CPPD crystal per 50X oil immersion field in an
unstained preparation.15
The differential count is reported in percentage. Segmented and band neutrophils should be reported
together. Monocytes and macrophages should also be reported as a single category. The term
mononuclear cells should not be used, since lymphocytes should be reported as a separate category. One
question that needs to be addressed is the reporting of R.A. cells, Reiter cells, tart cells, Döhle bodies, and
toxic granules. In the author’s opinion, this places an unnecessary burden on the clinical laboratory for
information that is either nonspecific or has no clinical relevance. Others may disagree.15 Miscellaneous
observations should be noted in a comments section. These include particulate matter, LE cells,
siderophages, fat, bone marrow, and tumor cells.
Bacteria are a critical observation and should be reported immediately to the physician with the names of
the persons reporting and receiving the report, and the time should be documented on the report. If
bacteria are present, a Gram stain should be done and results reported.
9.3.3.2
Reference Intervals
Reference intervals for synovial fluid are shown in Table 7.
9.3.4
Analytic Significance
The macroscopic, microscopic, and bacteriologic examination of synovial fluid are the keystones to
diagnosis. Unlike other fluids of the parental body cavities, chemical determinations play a secondary
role. Synovial fluids can be divided into five groups by their gross appearance at the time of aspiration:
normal, noninflammatory, inflammatory, purulent (septic), and hemorrhagic. Inflammatory fluids can be
further subdivided into a broad category of inflammatory diseases of diverse etiology and crystalline joint
disease. Characteristic findings of each group are listed in Table B3.82
The diagnostic significance of crystals, tissue fragments, microorganisms, and cell types are discussed
below.
Rice bodies are polished white fragments of tissue containing collagen and fibrin. They are seen in joint
fluid of patients with many arthritides (e.g., rheumatoid arthritis). Fragments of fibrocartilage are
described in meniscal or cruciate ligament tears and cartilage in osteoarthritis.15
Crystal analysis leads to a specific diagnosis in gout. CPPD crystals are associated with inflammatory
fluids in pseudogout, but may also be seen in noninflammatory fluids with a coincidental
chrondocalcinosis.15 In the same study, BCP crystals were found only in osteoarthritis and rheumatoid
arthritis. Others consider their presence a crystalline deposition disease.83 Metallic debris has been noted
from titanium implants. The metallic fragments are both extra- and intracellular.
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Table 7. Normal Synovial Fluid Values. (Modified from McCarty DJ. Synovial fluid. In: Koopman WJ, ed. Arthritis
and Allied Conditions. 14th ed. Philadelphia: Lippincott Williams and Wilkins. 2001:83-104. Reprinted with permission from
Lippincott Williams and Wilkins (http://lww.com)).
Color
Clarity
Viscosity
Mucin clot
Nucleated cells
Differential (%)
neutrophils
lymphocytes
monocytes
histiocytes
synoviocytes
Erythrocytes
Crystals
©
Colorless or pale yellow
Transparent
Very high
Good
13-180/µL
0-25
0-78
0-71
0-26
0-12
0-2000/µL
None
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Table 8. Birefringent Material That May Be Found in Synovial Fluid. (From Judkins JW, Cornbleet PJ.
Synovial fluid crystal analysis. Lab Med. 1997;28:774-779. ©1997 American Society for Clinical Pathology. Reprinted with
permission.)
Material
Crystals
Calcium oxalate
Calcium pyrophosphate
dihydrate (CPPD)
Cartilage, collagen
Cholesterol
Hydroxyapatite
Monosodium urate (MSU)
Steroids
Betamethasone acetate
Cortisone acetate
Methyl prednisone acetate
Prednisone tebulate
Triamcinolone acetonide
Triamcinolone hexacetonide
Other Materials
Debris
EDTA (dry, dipotassium)
Fat (cholesterol esters)
Lithium heparin (not
sodium)
Starch granules
Shape
Birefringence
Bipyramidal
Often rhomboid, may be
rodlike, diamond, or square,
usually <10 µm long
Irregular shaped, rodlike
Flat, platelike, with notch in
corner, occasionally
needlelike, often >100 µm
Small (<1 µm), only
aggregates seen
Needle, rodlike, with parallel
straight edges, usually 8-10
µm long
Strong (no axis)
Weak (+)
Rods, 10-20 µm, blunt ends
Large rods
Pleomorphic, small fragments,
tending to clump
Small, pleomorphic with
branched and irregular
configuration
Pleomorphic, small fragments,
often clumped
Large (15-60 µm) rods with
blunt, squared, or tapered end
Strong (-)
Strong (+)
Strong (no axis)
Small, irregular with jagged,
rounded nonparallel edges
Small, amorphous
Globules
May resemble CPPD
Variable
Weak
Strong (Maltese cross)
Weak (+)
Varying size, round
Strong (Maltese cross)
Strong (+)
Strong
Plates (no axis)
Needles (-)
Weak (no axis)
Strong (-)
Strong (+)
Strong (no axis)
Strong (-)
+ indicates positive, - negative
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Figure 3. A. Incident light vibrates in all planes. B. The vibration of light can be resolved into a
vertical and horizontal vector. C. Polarizer is oriented to allow passage of the horizontal vector. D.
Polarized light vibrating in the horizontal plane. E. Analyzer oriented to allow passage of vertical
vector only. If no light passes, the microscopic field is black. A crystal is placed between the polarizer
and analyzer. In this example, it is anisotropic and rotates the horizontal vector 90°, so that light passes,
through the analyzer and the image of the crystal appears light against a black background. (Figure
contributed by Benjamin Glasser.)
9.4
Bronchoalveolar Lavage Fluid
Bronchoalveolar lavage (BAL) is defined as the bronchoscopic procedure to retrieve cells and soluble
substances from the lining fluid of the distal airways and alveolar units, containing immunologic
components of the lung’s epithelial surface.84 The procedure thus provides a sample that represents a
correlate to an endobronchial or transbronchial biopsy tissue specimen and cellular and immunologic
components in the vascular circulation. It provides a specimen that is involved with a disease process or
in close approximation with it.
BAL is a safe and minimally invasive diagnostic procedure for patients with interstitial lung disease,
whether an infectious, noninfectious immunologic, or malignant etiology is suspected.85-87 In addition, the
bronchoscopic technique reveals specific information in disorders, such as pulmonary alveolar
proteinosis, Langerhans cell histiocytosis, alveolar hemorrhage, or dust exposure.85 It may also be
complementary to high-resolution computerized tomography (CT) or at least useful for diagnosis by
exclusion, and may help to decide whether or not to do surgical biopsy.86
Determination of rather simple laboratory parameters from BAL fluid allows conclusions about cellular
and morphological changes in lung parenchyma, which can hardly be made with other methods.
Differentiation of cells contained in BAL, as well as subtyping of lymphocytes, has gained special
importance for differential diagnosis and analysis of activity-status of interstitial lung diseases.
9.4.1
Macroscopic Examination
The macroscopic examination of BAL fluid will identify the following characteristic states:
•
•
©
gray-brown BAL fluid typical for smokers;
blood (hemorrhagic BAL);
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lipids and lipoproteins (milky BAL); and
mucous flakes.
9.4.1.1
Hemorrhagic BAL
A fresh alveolar hemorrhage results in a BAL fluid looking like a diluted suspension of erythrocytes. A
hemorrhage secondary to bronchoscopy typically results in increasing contaminations during the
fractionated sampling, and this should be reported on the request form. In an older hemorrhagic syndrome
(i.e., when a hemorrhage has taken place earlier and is partially resorbed), BAL-fluid looks orange red to
russet. This macroscopic finding is an indication to look at intracellular iron content by cytochemistry.
9.4.1.2
Lipids and Lipoproteins
A milky aspect of recovered BAL fluid is an indication of alveolar proteinosis. Accumulation of
phospholipid-protein complexes, which are derived from pulmonary surfactant in lung alveoli, is
characteristic for an alveolar proteinosis. An aliquot should be centrifuged if the BAL looks milky. If
typical lipid-protein complexes exist, a creamy layer forms on top of the rest of the BAL after
centrifugation.
9.4.2
Microscopic Examination
Due to its nature as an artificial lavage fluid, the interpretation of results is highly dependent on the
procedure for obtaining the fluid and the laboratory aims to determine the representation through a
number of quality parameters. Thus, improper lavage technique may result in bronchial, rather than
alveolar, sampling, indicated by observing many ciliate bronchial cells. If ciliated bronchial cells are
present, no further tests (e.g., immunophenotyping) should be performed. Contamination by blood
obscures results in case of a hemorrhagic BAL.
9.4.2.1
Enumeration
Determination of cell number in BAL can be performed both with a hemacytometer and with an
automated cell counter. The advantage of automated cell counting is the fast quantification of cell number
and the simultaneous discrimination between erythrocytes and nucleated cells. By using a hemacytometer,
a simultaneous testing of cell viability by adding trypan blue is possible. When cell number is < 50/µl, the
method of hemacytometer cell count should be preferred.
9.4.2.2
Morphology
Evaluation of the cell pattern with cell phenotype is used in clinical practice to distinguish the various
forms in interstitial lung diseases. In addition to examining cell differentials, observing the morphologic
appearances of cells and particles is diagnostic. Examples are the different morphology of macrophages in
extrinsic allergic alveolitis and sarcoidosis, or the detection of dust particles in occupational exposure
conditions.85
The following parameters are typically determined in BAL:
•
•
•
•
•
44
neutrophils;
lymphocytes;
ratio of CD4+ and CD8+ lymphocytes (CD4/CD8 ratio);
eosinophils;
macrophages (including the determination of hemosiderin-loading, golden, brown, or black
pigment inclusions resembling smoker’s pigment, or foamy cells); and
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other cells (red cells, atypical reactive type II pneumocytes, and fragments of hyaline
membranes).
Slides for the differential count may be prepared using a cytocentrifuge. If BAL contains a large number
of contaminating erythrocytes, a “wedge” smear may be preferred. In addition to smears or
cytocentrifuge, filter preparations can be prepared from BAL specimens.88
As a standard staining method, Romanowsky-type stain is recommended. For additional staining,
unstained samples should be prepared and stored. The following staining methods are typically applied
for special purposes: iron stain, PAS, silver, Nile red, or Ziehl-Neelsen.
9.4.2.3
Further Studies
The quantitative determination of the ratio CD4 and CD8 positive T-lymphocytes is the most frequently
requested immunological study in BAL, specifically with relevance to the detection of sarcoidosis. This is
typically performed by flow cytometry, which allows the rapid screening of many cells. Dual staining
with the T-cell-specific antigen CD3 is typically performed to discriminate CD4 and CD8 expression by
monocytes or macrophages and NK cells, respectively.
The heterogeneity in the size and granularity of cells in BAL may lead to difficulties in analysis. Specific
gating techniques employing CD45 and side scatter, however, result in reliable identification of cells in
good correlation to immunocytochemical techniques.89 Specific gating strategies may also allow the use
of less antibody combinations.90 Immunocytochemical techniques allow the use of a conventional light
microscope; however, double labeling is time consuming.88
The analysis of intracellular cytokine profiles by flow cytometry allows the detection of hallmarks of
lymphocyte activation (e.g., increased interferon-gamma production in allergic asthma), which occurs in
the absence of numeric changes in CD4 or CD8 T-cells.91
The determination of CD1a-positive cells is used for diagnosis in the case of Langerhans cell
histiocytosis, although antigen expression also occurs in alveolar macrophages under pathological
conditions.92
9.4.3
9.4.3.1
Result Reporting
Reporting Terminology
Laboratories should report total cell counts per mL, as well as the macroscopic appearance of the BAL
sample. A differential cell count is given together with a description of cytological abnormalities and
particular contents of the sample. Cells that should be routinely reported in the differential count as a
percentage include: macrophages/monocytes, neutrophils, eosinophils, and lymphocytes. Unusual
findings, such as neoplastic cells, dust particles, asbestos particles, or microorganisms, should be noted.
The presence of any ciliated epithelial cells should always be noted on the report. In addition, lymphocyte
subpopulations are quantified and reported as a ratio (helper/cytotoxic cells). Laboratories should report
the site of the BAL fluid (e.g., right or left upper or middle lobe).
9.4.3.2
Reference Intervals
Reference intervals highly depend on age,93,94 sex,95 and smoking,96 but also the standardization of
sampling, and should be determined for each methodology.
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9.4.4.1
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Analytic Significance
Clinical Information Required for Interpretation of Results
Information on prior tests facilitates the interpretation of findings. A previous bronchoalveolar lavage
(within the past two weeks) may lead to local inflammation of the lower airways, resulting in the presence
of neutrophils.
Changes in morphology of macrophages (e.g., basophilic or soot inclusions) have been reported in
smoking. Also, details on a possible occupational exposure to minerals (e.g., stone dust, coal dust,
asbestos, mineral fibers) should be recorded. Treatment with steroids or other immunosuppressive drugs
will affect the composition of BAL fluid and should be indicated.
For interpretation of findings, the macroscopic aspect during the bronchoscopic investigation should be
known. Information on suspicion of neoplastic infiltration, purulent secretion, or hemorrhage is
important. Moreover, the anatomic site of lavage is important and should be noted on all requisitions.
10 Additional Studies
10.1 Immunologic Studies
Cytologic examination of single cells and small groups of cells provides a wealth of diagnostic and
prognostic information to laboratory professionals with specialized training in deciphering complex
morphological information. These interpretations are usually based on the characteristics of cells stained
with a variety of organic and inorganic dyes that can differentially highlight various cellular and
subcellular components. Immunocytology adds an additional dimension to cytology by further providing
the means for molecular analysis. By employing specific antibodies that target well-characterized
molecular targets, it is possible to combine molecular analysis with cellular and subcellular analysis.
The principles of immunocytology borrow many of their methods from those of general cytology, as well
as from general immunohistology. The true strength of immunocytology lies in its unique ability to
integrate these two methodologies.
10.1.1 Sample Collection
In sample collection, there are three primary methods: 1) collecting the sample into a transport or
collection medium containing a fixative; 2) collecting the sample into a container without fixative; or 3)
collecting the sample directly onto the microscope slide in the unfixed state. The latter two methods are
similar in that the cells are not initially exposed to a fixative before being placed on the microscope slide.
10.1.1.1 Sample Collection With Fixative
Cytology samples may be collected directly into a medium containing fixative. This is particularly true
when using an automated monolayer preparation instrument where this method of collection is required
by the manufacturer. Although these methods have been optimized for ease of collection, transport, and
morphological analysis, they have not been extensively tested for compatibility with immunocytology.
Because the manufacturer’s transport medium is proprietary, little information is available on its effects in
preserving epitopes for subsequent antibody staining. Many of these transport media contain mixtures of
ethanol and polyethylene glycol. Such fixatives are generally compatible with immunocytology
procedures, whereas fixatives containing high amounts of methanol, isopropanol, or formalin may cause
denaturation of certain antigens, thus producing weak immunostaining.
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In order to ensure optimal immunostaining, samples collected into fixatives should be processed and
stained as soon as possible. Samples held up to 48 hours are suitable for immunocytochemical analysis.
10.1.1.2 Sample Collection Without Fixative
Samples collected without fixative should be processed and stained as soon as possible. Table 9 provides
a preferred schedule for sample collection, processing, and staining.
Table 9. Schedule for Processing Cytology Specimens
Step
Cell smears
Cell imprints
Red blood cell removal
Cell wash and resuspension
Cell enrichment methods
Preparation of monolayer
Slide storage
Stain slides
Time from initial sampling
Prepare immediately after sample collection
Prepare immediately after sample collection
Perform within 24 hours
Perform within 24 hours
Perform within 24 hours
Perform within 24 hours
Up to 48 hours (room temperature)
Perform within 48 hours
The optimal schedule requires that all steps up through preparation of the microscope slide should be
completed within the first 24 hours, and all subsequent staining steps should be completed within 48
hours.
10.1.1.3 Comparison of Prefixed to Unfixed Specimens
The process of fixation renders the cell membranes rigid. If cells are fixed in suspension, as is the case
when cells are collected into transport medium, the cells retain their three-dimensional shapes as freefloating cells. For squamous epithelial cells, this shape is generally elongated and flattened, whereas most
other cell types, particularly white blood cells and many types of tumor cells, retain a spherical
conformation. When these fixed preparations are deposited onto the slide, they tend to retain a rounded
appearance with densely staining nuclei and scant cytoplasm. In contrast, when cells are applied to a
microscope slide in the unfixed state, they tend to flatten and spread, providing more nuclear and
cytoplasmic detail. Thus, the morphology of the same cell type can be vastly different depending on how
the sample was processed.
The choice of whether to fix before or after application of the cells to the slide depends on the sample
type and also on the manufacturer’s requirements when using an automated monolayer device. Both
methods are compatible with antibody staining. However, fixation after application of cells to the slide
frequently provides better morphological detail.
10.1.2 Sample Preparation
10.1.2.1 Microscope Slides
In order to ensure adequate cellular adhesion, the slides must be chemically treated to promote cell
adhesion. Positively charged slides or silanized slides are available from several commercial sources and
are preferred for immunocytology applications.
10.1.2.2 Application of Specimens to Microscope Slides
Cells may be applied to slides manually, using cell smear methods, or with the aid of an automated
monolayer device or cytocentrifuge. For automated methods, follow the manufacturer’s instructions.
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After applying samples to the slides, the slides should be either dry or nearly dry without excess liquid.
Rapidly air dry any slides containing residual liquid. If storage of slides is required before staining, store
slides in the unfixed state.
10.1.2.3 Slide Storage
Stain slides as soon as possible after preparation. If it is necessary to delay staining:
-
Store unfixed slides at room temperature in a sealed container for no more than 24 hours.
-
If longer storage is necessary, slides may be stored for up to seven days at -20 °C or up to 30 days at 70 °C.
o
Individually wrap slides with two layers of aluminum foil, securely sealing all seams.
Special care is required to avoid scratching or otherwise damaging the area of cellular
deposition.
o
Place wrapped slides in a plastic bag, expel excess air, and seal the bag.
o
Store at -20 °C to -70 °C.
o
When slides are removed for staining, first equilibrate slides to room temperature for 30
minutes before removal from the plastic bag. In order to prevent condensation on the
unfixed cells, it is important that the slides reach room temperature before unwrapping
aluminum foil.
o
Unwrap slides and proceed immediately to fixation and staining.
10.1.2.4 Fixation
The method of fixation is perhaps the most critical step in achieving optimal results. For optimal
morphology, a strong fixation is preferred in order to preserve cellular detail. In contrast, for antibody
staining, weak fixation is preferred in order to retain protein molecules in their native conformation. The
precise balance between these two opposing requirements is critical for optimal staining. Fixatives
containing ethanol and propylene glycol are commonly used for cytology and are generally compatible
with antibody staining. A further consideration is that immunocytology procedures are generally harsher
than standard cytology methods, making the balance between over- and under-fixation particularly
challenging. While the goal for immunocytology is to achieve both acceptable morphology and highsensitivity immunostaining, in general practice morphology is frequently compromised in order to
achieve the high sensitivity of the latter.
Fixatives are generally divided into two categories depending on their mode of action. Agents which
combine with proteins are called additive fixatives, and agents that precipitate proteins are called
coagulating fixatives. Because of the harsh nature of immunocytology, strong fixation is required in order
to achieve optimal morphology. A fixative combining both the additive properties of formalin and the
coagulating properties of ethanol provides an ideal solution. A general fixative for immunocytology is
located in Appendix A.
10.1.2.5 Fixation Procedure
The following procedure is applicable for all samples, whether or not they have been prefixed:
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Place slides in fixative for ten minutes at room temperature.
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-
Rinse briefly in buffered saline (phosphate-buffered saline, PBS, or Tris-buffered saline, TBS; see
Appendix A).
-
Without allowing slides to dry out, proceed to staining.
10.1.3 Immunostaining Methods
10.1.3.1 Permeabilization and Antigen Retrieval
Cells must be permeabilized to allow antibodies and visualization reagents to penetrate the cell
membranes. A suitable permeabilization reagent can be prepared from a buffer containing detergent (see
Appendix A).
Formalin is a cross-linking fixative that can denature epitopes by forming methylene bridges. These cross
links may be broken, restoring the epitopes to their native configuration, by application of heat. A
suitable antigen retrieval reagent can be prepared from a buffer containing detergent. A combined
method for simultaneously performing permeabilization and antigen retrieval is outlined below.
Permeabilization/Retrieval Procedure
-
Place permeabilization/retrieval reagent into a Coplin jar and heat to 95 °C.
Add slides to Coplin jar and incubate for five minutes at 95 °C.
Rinse slides with buffered saline.
10.1.4 Blocking Endogenous Enzymes
Peroxidase and alkaline phosphatase are the two enzymes most frequently employed in immunocytology.
However, both of these enzymes occur naturally in a variety of cells and tissues. In order to avoid falsepositive staining, these endogenous enzymes must be blocked before immunocytochemical analysis.
Blocking methods for peroxidase generally employ solutions of hydrogen peroxide up to 3%. However,
for cytology specimens, 3% hydrogen peroxide can severely damage cellular morphology. Therefore,
weaker concentrations of hydrogen peroxide containing sodium azide are recommended. Commercial
blocking reagents are available. However, the blocking reagent should specify that it is intended for use
with cytology samples.
Endogenous peroxidase blocking reagent
-
0.03% hydrogen peroxide in deionized water
0.2% (w/v) sodium azide
Endogenous alkaline phosphatase blocking reagent
-
0.1N HCl in deionized water
Procedure for blocking endogenous enzymes
-
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Incubate slides with endogenous enzyme blocking reagent for five minutes at room temperature.
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10.1.5 Antibody Preparation
Several manufacturers sell antibodies that have been titered and optimized for tissue
immunohistochemistry. These antibodies are frequently referred to as prediluted antibodies or ready-touse antibodies. Generally, these prediluted antibodies can be used, without modification, for
immunocytology as well.
When preparing antibodies from concentrated stock, it is important to dilute the antibody to an
appropriate titer in an acceptable antibody diluent. An acceptable antibody diluent usually contains a
buffer, a carrier protein, and optionally an antimicrobial agent. A formula for an acceptable antibody
diluent can be found in Appendix A.
10.1.6 Visualization Chemistries
The methods described in this section are optimized for brightfield microscopy utilizing enzymatic
detection methods.
The preferred methods for immunocytology employ an enzymatic reaction that catalyzes the conversion
of a soluble substrate-chromogen into an insoluble colored precipitate that can be viewed microscopically.
The two enzyme systems most commonly employed are peroxidase and alkaline phosphatase. In the
peroxidase method, the peroxidase enzyme, in the presence of hydrogen peroxide (substrate), catalyzes
the oxidative polymerization of a chromogen such as diaminobenzidine (DAB), forming a colored
precipitate at the site of the enzyme. In the case of the alkaline phosphatase method, the alkaline
phosphatase enzyme causes the hydrolytic cleavage of a phosphate group from a substrate such as a
naphthol phosphate. The free naphthol reacts with an appropriate chromogen, such as Fast Red, to
produce a colored precipitate at the enzyme site.
The choice of peroxidase or alkaline phosphatase methodology is primarily a matter of personal
preference. However, historically, alkaline phosphatase methods have been preferred for
immunocytology. Regardless of the enzyme system employed, the principle is the same. The primary
antibody, bound to its cellular antigen, must next be linked to the enzyme. A variety of linking systems
are available from several commercial sources.
A commonly used method for immunocytology involves the application of a secondary antibody
conjugated to an enzyme. If the primary antibody is of murine origin (most monoclonal antibodies), the
secondary antibody must be an antimouse immunoglobulin conjugated to either peroxidase or alkaline
phosphatase. If the primary antibody is of rabbit origin (most polyclonal antibodies), the secondary
antibody must be an antirabbit immunoglobulin conjugated to the appropriate enzyme.
Several other detection methodologies are available, and most are compatible with immunocytology.
However, detection systems based on (strept)avidin-biotin methods are not recommended, due to
potential interference from endogenous biotin, unless an appropriate biotin-blocking method is used.
Other detection methods may employ a polymer-based system that includes a polymer backbone to which
multiple secondary antibodies and enzyme molecules are conjugated. Although in theory, these polymers
should provide enhanced sensitivity by virtue of their large number of enzymes, in practice these
polymers have difficulty penetrating fixed cellular membranes. With vigorous cell permeabilization
methods such as those previously listed, polymer-based systems can perform satisfactorily in
immunocytology applications. Polymer-based immunostaining systems are available from various
manufacturers.
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10.1.6.1 Chromogens
A variety of chromogens are available for immunocytochemistry. However, for optimal performance, a
chromogen compatible with permanent mounting should be employed. Chromogens that are dissolved in
organic solvents will not withstand permanent mounting and should be avoided. For peroxidase enzymes,
diaminobenzidine is the most commonly used chromogen. Diaminobenzidine produces a brown stain at
the site of the peroxidase enzyme.
For alkaline phosphatase, a permanent Fast Red chromogen provides the best sensitivity and best
resistance to organic solvents. Permanent Fast Red chromogens are not as widely used as soluble Fast
Red chromogens, so care should be exercised in selecting the appropriate Fast Red. It should not be
assumed that all Fast Red chromogens are equal.
10.1.6.2 Immunostaining Procedure
After slides have been fixed, permeabilized, and blocked, the next step is the immunostaining procedure.
Several manufacturers provide automated slide staining equipment. When staining is performed using
one of these instruments, the manufacturers’ recommended protocols should be followed. The method
listed below can be used for manual staining or can be programmed into an automated stainer.
-
Apply appropriately diluted primary antibody to the slide and incubate for ten minutes at room
temperature.
Rinse slides in phosphate buffered saline.
Apply secondary antibody/enzyme conjugate and incubate for ten minutes at room temperature.
Rinse slides in phosphate buffered saline.
Apply substrate/chromogen solution and incubate for ten minutes at room temperature.
Rinse slides in deionized water.
10.1.7 Counterstaining
A nuclear counterstain, such as hematoxylin, is applied to the immunostained specimen to provide nuclear
detail. The nuclear counterstain should be light enough so as not to obscure any specific immunostaining,
particularly nuclear immunostaining, but strong enough to provide unambiguous nuclear morphology.
Mayer’s hematoxylin (Lillie’s modification containing 5g/L of hematoxylin) provides a suitable
counterstain. With this, hematoxylin slides should be stained for about 30 seconds.
10.1.8 Controls
10.1.8.1 Positive Control
In every staining procedure, a positive control must be run in order to establish the proper performance of
the staining reagents and methods. The most appropriate positive controls are cytology samples
containing known positive cells of interest. Once a positive sample is identified, it is possible to make a
repository of positive slides that can serve as future positive controls for up to two months. Positive
control cell slides can be stored frozen at -20 °C, as previously described, and used for up to two months.
After prolonged storage, a decrease in staining intensity is frequently observed. If staining becomes
noticeably weaker, the slides should be discarded even if they are less than two months old.
In the absence of appropriate cytology material, a tissue section containing known positive elements may
be used to verify the performance of the reagents. However, the procedural elements of the protocol
cannot be verified with tissue sections.
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10.1.8.2 Negative Control
An appropriate negative control is performed on a second identical cytology sample collected and
prepared at the same time as the patient test sample. An isotype-matched negative control reagent, diluted
to the same concentration as the primary antibody, is used in place of the primary antibody.
10.1.8.3 Interpretation of Controls
Nonspecific staining, if present, will be visible in the negative control. If the negative control reagent
demonstrates positive staining, results with test specimens should be considered invalid.
The positive control should only be utilized to determine correct performance of the reagents and
procedures and should not be used to aid in formulating a specific diagnosis. If the positive controls fail
to demonstrate positive staining, results with test specimens should be considered invalid.
10.1.9
Staining Interpretation
Stained slides are optimized for viewing and analysis via light microscopy. Positive reactivity will be
indicated by the presence of a brown (peroxidase) or red (alkaline phosphatase) reaction product within
the stained cells along with a blue-stained nucleus. In contrast, negative stained cells will exhibit only a
blue nuclear stain.
The quality of the nuclear stain should also be examined to ensure cells are morphologically intact and
have not been damaged by the cell preparation or immunostaining procedure.
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10.1.10 Troubleshooting
Table 10. Troubleshooting
Problem
Probable Cause
Suggested Action
No staining
Reagents not used in proper
order
Repeat experiment and refer to procedure.
Ensure that appropriate species-specific
linking antibody is used.
Weak staining
Slides retained too much
solution after wash bath
Gently tap off excess solution after wash bath.
Slides under-incubated
Review staining procedures.
Inappropriate chromogen
preparation
Review chromogen preparation procedure.
Chromogen expired or used outside its
recommended working stability.
Excessive
background
Slides not thoroughly rinsed
Use fresh solutions in buffer baths
and wash bottles.
Specimen dried during
staining procedure
Use humid chamber. Verify that the
appropriate volume is applied to each
slide completely covering the entire cell area.
Nonspecific binding of
reagents to slides
Check for proper fixation.
Cells detach from
slides
Use of incorrect slides
Use positively charged or silanized slides.
Slides display
precipitation
Inappropriate substratechromogen preparation
or used beyond the working
shelf life
Prepare fresh substrate-chromogen solution
and use within specified working shelf life.
Excessively
strong specific
staining
Reagent incubation times
too long
Review staining procedures.
Primary antibody too
concentrated
Make appropriate dilutions
of primary antibody.
10.1.11 Limitations
1. Immunocytochemistry is a multistep process that requires specialized training in the selection of
appropriate reagents, slide preparation, fixation, staining, and interpretation.
2. The cytologic staining is dependent on the proper handling and processing of the slides before
staining. Improper fixation, washing, drying, heating, or contamination may produce artifacts,
antibody trapping, or false-negative results. Inconsistent results may be due to variations in sample
processing methods, or to inherent irregularities within the cell preparation.
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3. Excessive or incomplete counterstaining may compromise proper interpretation of results.
4. False-positive results may be seen due to nonimmunologic binding of proteins or substrate reaction
products. They may also be caused by endogenous enzyme activity. Sometimes nonspecific staining
may be associated with bacteria in the sample.
5. The presence of mucous on the slides may inhibit the interpretation of the staining results. Mucous
may also produce nonspecific staining.
10.2 Flow Cytometric Studies
The multiparametric characterization of single cells in heterogeneous cell suspensions by flow cytometry
represents a reliable and routinely applicable technique for the quantitative characterization of cells of the
immune and hematopoietic system within different body fluids (see Table 11). According to the
principles of flow cytometry, cells are immunophenotypically identified based on the analysis of the
co-expression of antigens using fluorochrome-conjugated monoclonal antibodies.97,98 This allows the
identification of specific lymphocyte subsets, clonal expansions in lymphoma or leukemia, as well as
differential white blood cell analysis. Alternatively, a white blood cell differential also can be achieved
simultaneously with absolute counts using hematology analyzers based on the analysis of physical
characteristics or cytochemical staining.99-101
Cell surface and intracellular antigens can be analyzed at the same time after permeabilization of cells.
Such intracellular antigens facilitate the detection of malignancies, including those of epithelial or
mesothelial origin. Cytokine-directed antibodies and biochemical probes also allow the characterization
of functional responses of the immune system.102-104 Ploidy analysis following DNA staining can be
analyzed in order to identify malignant cells.105
Flow cytometric testing can be performed as a highly standardized, rapid, and quantitative procedure
using small sample volumes (e.g., in pediatric samples).106 Cells can be stabilized before analysis99,107 and
the immunological phenotype of cells often is more stable than morphologic characteristics. Finally,
electronic storage of list-mode data, which are acquired during flow cytometric measurement, allows the
documentation and expert review of analysis.
The following applications have been examined for flow cytometric analysis and are listed in Table 11.
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Table 11. Applications of Flow Cytometry in the Analysis of Body Fluids
Body Fluid
Application
Methodology
Absolute counting of
Cerebrospinal fluid
• Automated analysis of light
erythrocytes and nucleated cells
scatter or
cytochemistry99,100,101
Quantitative determination of
• Light scatter analysis
lymphocytes, monocytes, and
combined with
neutrophils
immunophenotyping106
• Automated analysis of light
scatter or cytochemistry 99,101
Determination of CD4 and CD8 • Immunophenotyping of
T cells, B cells, and NK cells
constitutive lymphocyte
antigens106,108
Detection of lymphoid or
• Immunophenotypic analysis
myeloid neoplasm infiltration
of clonality (κ/λ light chains,
T cell receptor α/βrepertoire) and aberrant
antigen expression109,110
Functional characterization of
• Immunophenotypic analysis
cells of the immune system
of cytokines, activation
antigens, and functionally
regulated receptors111
Detection of lymphoma
Serous (pleural, peritoneal,
• Immunophenotypic analysis
infiltration
pericardial)
of clonality (κ/λ light chains,
T cell receptor α/βrepertoire), and aberrant
antigen expression112
Detection of malignant
• Immunophenotypic analysis
epithelial cells
of epithelial and tumor
antigens107
• Analysis of aneuploidy105,113
Absolute counting of nucleated • Automated analysis of light
Synovial fluid
cells
scatter114
Characterization of the specific • Immunophenotypic analysis
immune response
of regulatory T-cells115
• Characterization of antigenspecific cells103
Determination of CD4 and CD8 • Immunophenotyping of
Bronchoalveolar lavage fluid
T cells, B cells, and NK cells
constitutive lymphocyte
antigens89,90
Functional characterization of
• Immunophenotypic analysis
cells of the immune system
of cytokines, activation
antigens, and functionally
regulated receptors91,92
Flow cytometry often is used as an adjunct to microscopy. Both methods are competitive tools for the
counting and differentiation of nucleated cells with a higher precision of flow cytometry. Microscopy in
comparison has advantages in acute infectious diseases where microbial agents can be assessed during
nucleated cell analysis. In addition, contamination by nonhematopoietic cells, such as malignant epithelial
cells or reactive mesothelial cells, is more easily observed by microscopy. Flow cytometry, in contrast, is
a sensitive tool for the characterization of chronic inflammatory processes, due to the specific
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characterization of proliferative responses in lymphocytes as well as monocytes and macrophages.
Furthermore, the characterization of infiltration by hematopoietic malignancies is a specific indication for
flow cytometric analysis.
10.2.1 Sample Preparation
Flow cytometry in general only requires that cells are available in suspension. Preparation of samples,
therefore, is similar to that for microscopy. Filtering, washing, or treatment with hyaluronidase is only
applied in the case of contamination by large debris particles, fibrin, or a high viscosity. As a general rule,
any manipulation including washing steps should be reduced to a minimum.
The stability of the material depends on the cell types and the parameters that are analyzed. Thus,
constitutive antigens on lymphocytes (e.g., CD3 or CD4) are typically stable in expression while antigen
expression densities on phagocytic cells may rapidly change (e.g., following cooling and rewarming).
Storage on ice, which is performed in some procedures, therefore, cannot be generally recommended.
Stabilizing solutions or fixatives can be alternatively applied for extended sample storage, but this is not
possible for all procedures due to interferences with the detection of certain antigens and functional
assays.
10.2.1.1 Preparation of Aliquots for Staining
For labeling, typically samples with 20 000 to 100 000 cells in 100 µL are prepared. In case of samples
with a lower cell concentration, this is performed by concentration of cells though centrifugation and
resuspending in autologous medium or staining buffer, which contains protein. Even significantly lower
numbers of cells can be processed successfully in the case of cell-poor cerebrospinal fluid samples.116
Antibodies are usually titrated for staining of up to 1 000 000 cells in 100 µL. Samples with such a high
concentration of cells, therefore, can be processed without dilution.
Filtration through a 50- to 70-µm nylon filter can be applied if needed (e.g., in the case of BAL fluid with
a high amount of mucus).
Analysis can also be performed on frozen or stabilized material.107
10.2.1.2 Selection of Antibodies
In comparison to the analysis in blood, the identification of cells in body fluids is complicated by often
lower cell counts, cells with a high autofluorescence (e.g., macrophages), cells of nonhematopoietic origin
(e.g., epithelial or mesothelial cells), and high amounts of cellular debris or microorganisms. The
identification of cellular subsets based on scatter, therefore, often is less reliable than in blood and
immunological gating procedures (e.g., based on CD45 expression), or multidimensional assays are
performed in order to improve the precision of the identification of cells.89,90,116 The redundant analysis of
the same antigen in different tubes allows the correlation of marker expression across tubes through
immunological gating.
Counterstaining of cells, which are outside of the interest of analysis and which obscure the analysis due
to high autofluorescence or nonspecific binding of antibodies, is another technique to improve the
identification of cells. Nonspecific binding is a special problem when secondary antibodies are used.
Thus, macrophages can be counterstained (e.g., using CD14), and dead cells can be counterstained using
fluorescent DNA dyes, which are excluded by vital cells.
Direct fluorochrome-conjugated antibodies and single-step staining is preferred as cell losses through
washing are reduced and nonspecific staining is lower. The choice of fluorochromes affects the sensitivity
for the detection of antigens with a low expression of antigens. A higher sensitivity is reached using
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fluorochromes, such as R-phycoerythrin, allophycocyanin, or tandem conjugates thereof in comparison to
fluorescein isothiocyanate or peridinin chlorophyll, which lead to a lower signal-to-noise ratio for
antibody-labeled cells.
Cellular autofluorescence can be a major problem in the characterization of the antigen profile of
macrophages or epithelial cells. The use of fluorochromes that do not overlap with autofluorescence in
emission spectrum and quenching of autofluorescence (e.g., with trypan blue) are techniques that improve
the fluorescent detection of antigens on these cells.
10.2.1.3 Staining
In media with a high immunoglobulin content similar to human serum, antibodies can be directly added
without blocking of Fc receptors. Preincubation of samples with rabbit serum in the case of indirect
staining or murine immunoglobulin preparations in the case of direct staining may be applied to reduce
background staining in other media.
Incubation with antibodies can be either performed in native body fluids or staining buffers. These buffers
should be of neutral pH (7.4) and contain proteins, which reduce cell losses during sample preparation.
In case of the staining of intracellular antigens, fixation and permeabilization of cells typically in a twostep procedure is needed. Methods differ in the preservation of antigen structure and expression, as well
as light scatter characteristics of cells.117
Isotype controls cannot be generally recommended for the control of nonspecific staining, as different
preparations of directly conjugated antibodies differ in their nonspecific binding.
10.2.1.4 Lysis, Washing, and Fixation
Lysis of red blood cells only needs to be performed in the case of significant contamination by these cells.
Washing is needed after lysis and, therefore, cell losses may occur.
If lysis is performed, methods which will fix and lyse cells in one step may lead to higher cell losses
during subsequent washing steps, while lysis without fixation (e.g., using ammonium chloride solutions)
often leads to a limited stability of scatter characteristics during prolonged storage. This can be addressed
by fixing cells after washing.
Washing or dilution of samples are alternative methods to reduce the nonspecific fluorescence of
antibodies, which are not tightly bound by cells during analysis. Washing is more effective in reducing
background fluorescence and concentrates cells in the sample, which facilitates analysis in case of low
cell counts. Cell losses during washing depend on the protein content of the buffer, stringency of
centrifugation, as well as the material and the shape of sample preparation tubes.
Paraformaldehyde (0.5 to 1.0%) allows the prolonged storage of stained cell samples for up to seven days
until later flow cytometric analysis.
10.2.2 Measurement and Analysis
10.2.2.1 Instrument Settings
The same flow cytometric instrument settings as those used for the immunological analysis of cells in
blood or bone marrow can be used for the analysis of cells in body fluids (see the most current edition of
CLSI/NCCLS documents H42—Clinical Applications of Flow Cytometry: Quality Assurance and
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Clinical and Laboratory Standards Institute. All rights reserved.
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Immunophenotyping of Lymphocytes,
Immunophenotyping of Leukemic Cells).
H56-P
and
H43—Clinical
Applications
of
Flow
Cytometry:
Logarithmic amplification of scatter signals may facilitate the analysis of light scatter characteristics of
macrophages or epithelial cells at the same time with lymphocytes. Analysis of macrophages may also
require a lower amplification of fluorescence signals, due to their high autofluorescence.
The analysis speed needs to be increased in case of a low cell concentration in the sample.
Acquisition of analysis data in list-mode storage is required to allow offline analysis of flow cytometric
measurements.
10.2.2.2 Controls
Currently, no stable control materials are available for control of the whole process of sample preparation,
staining, and analysis of extracellular body fluid samples. Blood controls or fresh, normal blood samples
that are processed in parallel will allow control of parts of the analysis process.
10.2.2.3 Gating and Analysis
The identification of cells in body fluids is complicated by often lower cell counts, cells with a high
autofluorescence, cells of nonhematopoietic origin, and high amounts of cellular debris or
microorganisms. Immunological gating is, therefore, preferred as described in Section 10.2.1.2. 89,90,116
10.2.3 Reporting and Interpretation
10.2.3.1 Technical Information
Information on the method of cell preparation, cell counts and viability, all antibody combinations and
fluorochromes including lot numbers, testing of controls, instrument quality control, number of cells
analyzed, date of the analysis, and names and storage of list-mode files should be kept available in the
laboratory. A listing of the CD designation of the antibodies used should be provided in the final report.
10.2.3.2 Interpretation
The interpretation should comment on the representativity of the sample for the expected body fluid.
Suspected contamination through hemorrhage should be indicated. Depending on the request, percentages
or absolute counts should be given for the normal cellular elements addressed with the staining procedure.
Reference ranges should be given for these normal cellular elements. Abnormal cellular elements should
be precisely described in their immunophenotype. The description of abnormal staining intensities for
certain antigens is desirable, but cannot be standardized, currently.
Finally, a written interpretation of the results obtained and an explanation of their significance should be
provided. This includes the differential diagnosis consistent with the flow cytometric results.
10.3 Cytogenetic Analysis
Cytogenetic analysis of body fluids, specifically cerebrospinal, serous (pleural, peritoneal, pericardial),
and synovial specimens, is performed to determine their chromosomal complement and, therefore, aid in
the clinical diagnosis of a suspected malignant acquired disorder.118
The sample should be collected aseptically and immediately placed in a sterile plastic screw-capped
container or a sterile plastic test tube. The optimum sample should contain 30 x 106 malignant cells;
58
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however, cultures will be attempted on most samples submitted. The sample should be kept at ambient
temperature (preferable) or refrigerated, but not frozen.
The ability to obtain cells for cytogenetic analysis is dependent on the presence of viable cells in the
specimen submitted for culture and the ability to derive mitotically active cells from the cultures
established from the specimen.
The culturing procedures, harvesting, slide preparation, and metaphase analysis for body fluids are the
same as the ones established for blood and bone marrow specimens.119 Briefly, the medium of choice for
culturing is RPMI 1640 enriched with fetal bovine serum, L-glutamine, and antibiotics. Depending on the
amount of specimen submitted and on the cellularity, one or two short-term culture(s), are established by
inoculating approximately 10 000 000 cells into each 10-mL culture.
Depending on the suspected clinical diagnosis, mitogen-stimulated cultures for T- and B-cell lymphoid
disorders (72 and 96 hours, respectively) may be also set up at a final concentration of 1 x 106/mL per
culture.120 Harvest of the cultures is performed following Colcemid treatment, by hypotonic shock, and
subsequent fixations using Carnoy’s fixative. Banding of the slides is performed by the use of trypsin and
Wright’s or Giemsa stain.121
Metaphases from more than one culture, if possible, are used. If available, at least 20 metaphases are
completely analyzed. Very complex chromosome analyses or hyperdiploid analyses may be performed best
from photographs.
Karyotypes are constructed and interpreted according to the International System for Human Cytogenetic
Nomenclature (ISCN, 1995).122
If indicated, fluorescence in situ hybridization (FISH) can also be performed on these samples to further
characterize abnormalities not detectable during conventional chromosome analysis. FISH techniques are
well established for various hematologic and solid tumor malignancies, and are performed using a large
variety of commercially available probes.123
11 Sample Storage After Completion of Testing
All fluids should be refrigerated upon completion of testing. Refer to the manufacturer’s product insert for
specimen stability if additional tests are ordered.
12 Quality Control and Quality Assurance
12.1 Quality Control
Each facility is responsible for analyzing the appropriate quality control material and being in compliance
with their regulatory, state, and inspection agencies.
12.1.1 Quality Control Material
Quality control practices must be standardized and instituted through a documented procedure. These
practices must also meet the site’s regulatory and accreditation requirements. Control material containing
white and red cells are commercially available. Material should be purchased, evaluated, and integrated
into either the site’s manual test or instrument procedure. Duplicate testing can also be used as a check on
precision for the manual or automated procedures.
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59
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12.1.2 Reporting Quality Control Results
All personnel should use the standardized reporting format instituted in their facility. Unacceptable results
must be identified and documented. An examination of the user, test, and instrument system must be
investigated and corrective action instituted and documented.
12.2 Quality Assurance
12.2.1 Introduction and Purpose
A quality assurance program continuously assesses the components of its system to ensure that it
optimally delivers the best standard of care. Quality assurance includes not only the testing of quality
control material, but also the development, implementation, and review of policies and procedures for
specimen collection, transport, and processing; record keeping; technical competence; standardization;
continuing education; and a scheduled, documented review process.
12.2.2 Recordkeeping
Recordkeeping is an important aspect of quality assurance in the laboratory. Employees should have
access to all current quality control and documentation records. Corrective action policies should be
clearly explained in written, departmental procedures.
Detection and correction of errors, out-of-control results, and review of test results should be components
included in these procedures. In addition, patient results should be reported with accompanying reference
intervals. Records of patient results, reagent and quality control material lot and catalog numbers, as well
as evaluation data, should be kept in accordance with regulatory and accrediting agencies. These results
should also be periodically reviewed by the section supervisor or designee.
12.2.3 Procedures
Procedures should be organized in a way that can be easily followed by laboratory personnel, and should
contain the following elements:
•
•
•
•
•
•
title;
purpose or principle;
procedure instructions;
references;
author; and
approval signatures.
Refer to the most current edition of CLSI/NCCLS document GP2—Clinical Laboratory Technical
Procedure Manuals for additional information.
12.3 Proficiency Testing (External Quality Assessment)
External proficiency testing programs that are sponsored by manufacturers, professional and medical
organizations, and some state public health laboratories are available as a check on accuracy. Unknown
specimens are distributed several times a year for evaluation by the individual laboratory. Results are recorded
and a summary is sent to the participating laboratories to compare performance. Transparencies included in
some proficiency surveys assess the ability of technologists to correctly identify body fluid constituents, but
they do not assess the reproducibility of slide preparation nor cell-finding ability. For more details, refer to the
most current edition of CLSI/NCCLS documents GP27—Using Proficiency Testing (PT) to Improve the
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Clinical Laboratory; and GP29—Assessment of Laboratory Tests When Proficiency Testing is Not Available.
12.4 Continuous Education and Training
The foundation of all good quality systems is effective documentation, coupled with appropriate training
for all persons who work within the system. Consistent, predictable, and high-quality outcomes in
delivery of services, and accurate laboratory testing can be provided only if laboratory personnel have
been appropriately trained. In the present regulatory and quality environment, all training must be
documented. Additionally, scheduled, periodic assessments should be done to verify that performance of
procedures remains consistent.
To ensure the quality of body fluid testing, independent of the site of performance, the qualifications of
testing personnel should match the complexity of the testing performed. Test reliability can be
demonstrated, regardless of employee qualifications and complexity of testing, through proficiency
testing and blind sample studies. Only properly trained personnel should perform a body fluid
examination.
Training and training verification are key factors in a successful laboratory operation. The process of
training and periodic performance verification complements the laboratory’s quality assessment program.
Training is used to train new employees, to introduce new methods, to retrain employees, when
assessments have shown less than satisfactory performance, and for periodic reverification, which is
required to document that an employee remains at an acceptable level of performance.
To verify an employee’s performance, he/she must be measured against an established performance
standard as defined by laboratory management/supervision. A variety of measuring “tools” may be used
to verify performance. For detailed information on training and competence assessment, refer to the most
current edition of CLSI/NCCLS document GP21—Training and Competence Assessment.
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Czader M, Ali SZ. Flow cytometry as an adjunct to cytomorphologic analysis of serous effusions. Diagn Cytopathol. 2003;29:74-78.
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Saha I, Dey P, Vhora H, Nijhawan R. Role of DNA flow cytometry and image cytometry on effusion fluid. Diagn Cytopathol. 2000;22:8185.
114
de Jonge R, Brouwer R, Smit M, et al. Automated counting of white blood cells in synovial fluid. Rheumatology. 2004;43:170-173.
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Cao D, van Vollenhoven R, Klareskog L, Trollmo C, Malmstrom V. CD25brightCD4+ regulatory T cells are enriched in inflamed joints of
patients with chronic rheumatic disease. Arthritis Res Ther. 2004;6:R335-46.
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Subira D, Castanon S, Aceituno E, et al. Flow cytometric analysis of cerebrospinal fluid samples and its usefulness in routine clinical
practice. Am J Clin Pathol. 2002;117:952-958.
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Kappelmayer J, Gratama JW, Karaszi E, et al. Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction
between monoclonal antibody clones, fluorochromes and sample preparation protocols. J Immunol Methods. 2000;242:53-65.
118
Mitelman F, Johansson B, Mertens F, eds. Mitelman database of chromosome aberrations in cancer. Available at:
http://cgap.nci.nih.gov/Chromosomes/Mitelman. Accessed on April 4, 2005.
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Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Vol. 1, 16th ed. Philadelphia: W.B. Saunders Co; 1979:25.
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Prosad RK, et al. Cytogenetic and histologic correlations in malignant lymphoma. Blood. 1987;69:97-102.
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Seabright M. A rapid banding technique for human chromosomes. Lancet. 1971;2:971.
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Mitelman F, ed. ISCN: an international system for human cytogenetic nomenclature. Basel, Switzerland: S. Karger Publishers; 1995.
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Weier HU, Greulich-Bode KM, Ito Y, Lersch RA, Fung J. FISH in cancer diagnosis and prognostication: From cause to course of disease.
Expert Rev Mol Diagn. 2002;2:109-119.
66
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H56-P
Appendix A. Reagent Formulations
A1.
Buffered Saline
Phosphate Buffered Saline, pH 7.2
- Na2HPO4 dibasic sodium phosphate, anhydrous
- NaH2PO4 monobasic sodium phosphate, monohydrate
- NaCl
sodium chloride
- Add deionized water up to 1 L.
Tris Buffered Saline, pH 7.6
- TRIS base trishydroxymethyl aminomethane
Dissolve in deionized water.
- 1 N HCl 1 N hydrochloric acid
- Dilute to a total volume of 1 L in deionized water.
- Adjust pH as needed to pH 7.6 ± 0.2 at 25 °C.
6.8 g
2.6 g
8.0 g
6.10 g
500 mL
37 mL
A2.
General Fixative for Immunocytology
- 50 mL of absolute (100%) ethanol
- 5 mL of 40% (w/v) solution of polyethylene glycol in deionized water
- 5 mL of formalin from 37% formaldehyde stock
- 40 mL of deionized water
A3.
Permeabilization/Retrieval Reagent
- 1.92 g citric acid, anhydrous
- Dissolve in 900 mL deionized H2O.
- 0.1% nonionic detergent (e.g., octylphenoxy polyethoxyethanol)
- pH to 6.0 with concentrated NaOH
- Bring up to 1000 mL with deionized H2O.
A4.
Antibody Diluent
- buffered saline
- 1% bovine serum albumen
- 0.1% sodium azide
- pH to 7.2
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67
Number 20
H56-P
Appendix B. Interpretation of Cell Types
Table B1. Interpretation of Cerebrospinal Fluid Cell Types
Cell Type
Condition
Neutrophils
Infectious: bacterial, tuberculous, fungal, early
viral
Eosinophils (>10%)
Basophils
Lymphocytes
Monocytes
Noninfectious (modest increases): hemorrhage,
intrathecal injections, infarction
Infectious: parasites, C. immitis
Noninfectious: lymphoma, leukemia,
ventricular peritoneal shunts, blood
contamination
Rare, nonspecific, occasional cases of
lymphoma
Infectious: viral, tuberculous, fungal; partially
treated bacterial meningitis
Noninfectious: multiple sclerosis,
neurosyphilis, cerebral neoplasmas,
lymphoproliferative disorders (e.g., lymphoma)
Infectious: tuberculous, fungal
Noninfectious: nonspecific response to mass
lesions (e.g., tumor)
Macrophages
erythrophage, siderophage
lipophage
Plasma cells
Blasts
Immature granulocytes and/or erythroblasts
Neoplastic cells
Indicators of pathologic bleed if no previous
tap
Parenchymatous destructive process
Indicator of antigenic stimulation of Blymphocytes, seen in a variety of conditions
(e.g., multiple sclerosis, viral
meningoencephalitis, cysticercosis, syphilis)
Rarely seen in plasma cell myeloma
Hematopoietic malignancies (e.g., precursor Bor T-cell lymphoblastic leukemia/lymphoma,
Burkitt’s (B-ALL) lymphoma/leukemia, acute
myeloid leukemias)
Bone marrow contamination
Primary CNS tumors (15% have positive
cytology)
Metastatic tumors (20% positive if cerebral
parenchyma involved, 50% if meninges
involved)
68
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H56-P
Appendix B. (Continued)
Table B2. Interpretation of Pleural Fluid Cell Types
Cell Type
Condition
Neutrophilia (>50% PMN)
Acute inflammation (e.g., parapneumonic
effusion)
Eosinophilia (>10%)
Pneumothorax, pulmonary emboli,
traumatic hemothorax, reaction to chest tubes,
parasitic diseases, Churg-Strauss syndrome
Lymphocytosis (>50%)
Transudates, tuberculosis,
carcinoma, coronary artery bypass surgery,
lymphoproliferative disorders, chylous
effusions
Monocytes/Macrophages
Limited diagnostic significance,
erythrophages and siderophages are useful in
distinguishing pathologic fluids from traumatic
taps.
Blasts
Hematopoietic malignancies
Plasma cells
Reactive conditions, plasma cell myeloma
(rare)
Mesothelial cells
Normal constituent (≥5%), markedly decreased
in tuberculous effusions (<0.1%)
NOTE: They must be distinguished from
tumor cells.
Neoplastic cells from solid tumors
Metastatic carcinoma, etc.
Miscellaneous
Systemic lupus erythematosis
LE-cells
Reed-Sternberg cells
Hodgkin’s disease
Megakaryocytes
Myeloproliferative disorders
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69
Number 20
H56-P
Appendix B. (Continued)
Table B3. Pathologic Classification of Synovial Fluids
Test
Normal
Group I
Group II
Noninflammatory
Inflammatory
Group III
Septic
Group IV
Hemorrhagic
Color
Pale
yellow
Yellow
Yellow-white
Yellow-green
Red-brown
Viscosity
High
High
Low
Low
Decreased
Mucin Clot
Firm
Firm
Friable
Friable
Friable
Leukocyte
count (cells
µL)
<200
200-2000
2000-100 000
10 000->100 000
>5000
%
Neutrophils
<25
<25
>50
>75
>25
Glucose
(mg/dL)
~Blood
~Blood
>25 mg/dL
lower than blood
>25 mg/dL lower
than blood
~Blood
Culture
Negative
Negative
Negative
Often positive
Negative
70
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H56-P
NOTES
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H56-P
The Quality System Approach
Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The approach is based on the model presented in the
most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The
quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any
organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how
a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or
service, serving as a manager’s guide. The quality system essentials (QSEs) are:
Documents & Records
Organization
Personnel
Equipment
Purchasing & Inventory
Process Control
Information Management
Occurrence Management
Assessment
Process Improvement
Service & Satisfaction
Facilities & Safety
GP2
GP21
X
EP5
EP6
EP9
GP29
Facilities &
Safety
Service &
Satisfaction
Process
Improvement
Assessment
Occurrence
Management
Information
Management
Process
Control
Purchasing &
Inventory
Equipment
Personnel
Organization
Documents
& Records
H56-P addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other documents
listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
GP27
Adapted from CLSI/NCCLS document HS1—A Quality Management 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, CLSI/NCCLS document GP26⎯Application of a Quality
Management System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of
three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow these
processes to deliver the laboratory’s services, namely quality laboratory information.
H56-P addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
X
Post-test
Specimen
Management
X
GP16
Postexamination
Results
Report
X
Laboratory
Interpretation
Specimen
Receipt
X
Testing
Review
Specimen
Transport
Examination
Specimen
Collection
Test Request
Patient
Assessment
Preexamination
X
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
72
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Related CLSI/NCCLS Publications*
EP5-A2
Evaluation of Precision Performance of Quantitative Measurement Methods; Approved
Second Edition (2004). This document provides guidance for designing an experiment to
precision performance of quantitative measurement methods; recommendations on comparing
precision estimates with manufacturers’ precision performance claims and determining
comparisons are valid; as well as manufacturers’ guidelines for establishing claims.
Guideline—
evaluate the
the resulting
when such
EP6-A
Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach;
Approved Guideline (2003). This document provides guidance for characterizing the linearity of a method
during a method evaluation; for checking linearity as part of routine quality assurance; and for determining
and stating a manufacturer’s claim for linear range.
EP9-A2
Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Second Edition
(2002). This document addresses procedures for determining the bias between two clinical methods, and the
design of a method comparison experiment using split patient samples and data analysis.
GP2-A4
Clinical Laboratory Technical Procedure Manuals; Approved Guideline—Fourth Edition (2002). This
document provides guidance on development, review, approval, management, and use of policy, process, and
procedure documents in the laboratory testing community.
GP16-A2
Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved
Guideline—Second Edition (2001). This guideline describes routine urinalysis test procedures that address
materials and equipment, macroscopic examinations, clinical analyses, and microscopic evaluations.
GP21-A
Training and Competence Assessment; Approved Guideline—Second Edition (2004). This document
provides background information and recommended processes for the development of training and
competence assessment programs that meet quality/regulatory objectives.
GP27-A
Using Proficiency Testing (PT) to Improve the Clinical Laboratory; Approved Guideline (1999). This
guideline provides assistance to laboratories in using proficiency testing as a quality improvement tool.
GP29-A
Assessment of Laboratory Tests When Proficiency Testing is Not Available; Approved Guideline
(2002). This document offers methods to assess test performance when proficiency testing (PT) is not
available; these methods include examples with statistical analyses. This document is intended for use by
laboratory managers and testing personnel in traditional clinical laboratories as well as in point-of-care and
bedside testing environments.
*
Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process;
therefore, readers should refer to the most recent editions.
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OFFICERS
Thomas L. Hearn, PhD,
President
Centers for Disease Control and Prevention
Robert L. Habig, PhD,
President Elect
Abbott Laboratories
Wayne Brinster,
Secretary
BD
Gerald A. Hoeltge, MD,
Treasurer
The Cleveland Clinic Foundation
Donna M. Meyer, PhD,
Immediate Past President
CHRISTUS Health
Glen Fine, MS, MBA,
Executive Vice President
Tenet Odessa Regional Hospital
(TX)
The Children’s University Hospital
(Ireland)
The Permanente Medical Group
(CA)
Touro Infirmary (LA)
Tri-Cities Laboratory (WA)
Tripler Army Medical Center (HI)
Truman Medical Center (MO)
Tuen Mun Hospital (Hong Kong)
UCLA Medical Center (CA)
UCSF Medical Center (CA)
UNC Hospitals (NC)
Unidad de Patologia Clinica
(Mexico)
Union Clinical Laboratory (Taiwan)
United Laboratories Company
(Kuwait)
Universita Campus Bio-Medico
(Italy)
University of Chicago Hospitals
(IL)
University of Colorado Hospital
University of Debrecen Medical
Health and Science Center
(Hungary)
University of Maryland Medical
System
University of Medicine & Dentistry,
NJ University Hospital
University of MN Medical Center Fairview
University of the Ryukyus (Japan)
The University of the West Indies
University of Virginia Medical
Center
University of Washington
US LABS, Inc. (CA)
USA MEDDAC-AK
UZ-KUL Medical Center (Belgium)
VA (Tuskegee) Medical Center
(AL)
Virginia Beach General Hospital
(VA)
Virginia Department of Health
Washington Adventist Hospital
(MD)
Washington State Public Health
Laboratory
Washoe Medical Center
Laboratory (NV)
Wellstar Health Systems (GA)
West China Second University
Hospital, Sichuan University (P.R.
China)
West Jefferson Medical Center (LA)
Wilford Hall Medical Center (TX)
William Beaumont Army Medical
Center (TX)
William Beaumont Hospital (MI)
Winn Army Community Hospital
(GA)
Winnipeg Regional Health
Authority (Winnipeg, Canada)
York Hospital (PA)
BOARD OF DIRECTORS
Susan Blonshine, RRT, RPFT, FAARC
TechEd
J. Stephen Kroger, MD, MACP
COLA
Maria Carballo
Health Canada
Jeannie Miller, RN, MPH
Centers for Medicare & Medicaid Services
Kurt H. Davis, FCSMLS, CAE
Canadian Society for Medical Laboratory Science
Gary L. Myers, PhD
Centers for Disease Control and Prevention
Russel K. Enns, PhD
Cepheid
Klaus E. Stinshoff, Dr.rer.nat.
Digene (Switzerland) Sàrl
Mary Lou Gantzer, PhD
Dade Behring Inc.
James A. Thomas
ASTM International
Lillian J. Gill, DPA
FDA Center for Devices and Radiological Health
Kiyoaki Watanabe, MD
Keio University School of Medicine
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: customerservice@clsi.org T WEBSITE: www.clsi.org T ISBN 1-56238-575-5