Body Fluid Analysis For Cellular Composition Proposed Guideline

   

H56-P Vol. 25 No. 20

 

Body Fluid Analysis forGuideline Cellular Composition; Proposed PLEASE    

 

   

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 ccomments omments 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@cl [email protected] si.org         

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

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standard or guideline.

 

 

H56-P ISBN 1-56238-575-5

Volume 25 Number 20

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  Body Fluid Analysis for Cellular Composition; Composition; Proposed Clinical and Laboratory Standards Institute document H56-P—  Guideline  provides recommendations for standardization of the collection and transport of body fluids, numeration and identification of cellular components, and guidance for f or qualitative and quantitative assessment of body fluid. Composition; Proposed Guideline. CLSI Clinical and Laboratory Standards Institute (CLSI).  Body Fluid Analysis for Cellular Composition; document H56-P (ISBN 1-56238-575-5). 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@c [email protected]; lsi.org; Website: www.clsi.org

 

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This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system, transmitted, made available in prior any form or by any means (electronic, mechanical, photocopying, recording, oror otherwise) without 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 reproduce or under otherwise use the text this be document an extent the exemptions to granted here or the Copyright Lawofmust obtainedtofrom Clinicalthat andexceeds 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 [ISBN 1-56238-575-5]. Clinical and Laboratory Standards Institute, Guideline.  CLSI document H56-P [ISBN 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

 

Volume 25   Committee Membership

H56-P

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

Samuel J. Machin, MB, ChB, FRCPath Vice-Chairholder The University College London Hospitals London, United Kingdom

Advisors

Dorothy M. Adcock, MD Esoterix Coagulation Aurora, Colorado Frank M. LaDuca, PhD International Technidyne Corporation Edison, New Jersey Ginette Y. Michaud, MDand FDA Center for Devices Radiological Health Rockville, Maryland Albert Rabinovitch, MD, PhD Abbott Laboratories, Hematology Business Unit Santa Clara, California

Francis Lacombe, MD, PhD Laboratoire d’Hematologie Pessac, France Kandice Kottke-Marchant, MD,

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

PhD Cleveland Clinic Foundation The 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

Ian Giles Sysmex America, Inc. Mundelein, Illinois

Diane I. Szamosi, MA, MT(ASCP)SH Greiner Bio-One  North America, Preanalytics

Jan W. Gratama, MD Erasmus University Medical Center-Daniel Den Hoed Rotterdam, Netherlands

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, Zlockie, MBA Vice President, Standards

David E. Sterry, MT(ASCP) Staff Liaison  Donna M. Wilhelm  Editor

Melissa A. Lewis  Assistant Editor  Editor  

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 Number 20   Acknowledgement

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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 Laboratoriumsmedizin..

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Volume 25   Contents

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Abstract....................................................................................................................................................i   Committee Membership...................... Membership.... .................................... .................................... .................................... .................................... .................................... .......................... ........ iii  Foreword.............. Foreword....................... .................. .................. .................. .................. .................. .................. .................. ................... ................... .................. ................. ................. .................. ............. vii  1 

Scope..........................................................................................................................................1  

2 

Standard Precautions..................................................................................................................1 

3 

Definitions ........ ................. .................. .................. ................... ................... .................. .................. .................. .................. .................. .................. .................. .................. ............1 ...1 

4 

Preanalytical Variables ................ .................................. .................................... ................................... ................................... ..................................... .......................3 ....3  

5 

Specimen Collection ................. ................................... ................................... ................................... ................................... ................................... ...........................4 .........4   5.1  5.2  5.3  5.4 

Cerebrospinal Fluid.......................................................................................................4  Serous Fluid .................. ................................... ................................... ................................... ................................... .................................... ..........................5 ........5   Synovial Fluid...............................................................................................................6  Bronchoalveolar Bronchoa lveolar Lavage (BAL) ......... .................. ................. ................. .................. .................. .................. .................. .................. ............7 ...7 

6 

Specimen Handling and Transport.............................................................................................7  6.1  CSF ................. .................................. ................................... ................................... ................................... ................................... ................................... .......................7 .....7   6.2  Serous Fluids.................................................................................................................7  6.3  Synovial Fluids ................. .................................. .................................. .................................. .................................. ................................... ........................8 ......8   6.4  BAL ............... ................................. ................................... ................................... ................................... ................................... ................................... .......................8 ......8  

7 

Quantitative Assessment............................................................................................................8  7.1  7.2 

8 

Morphology Assessment..........................................................................................................14  8.1  8.2  8.3 8.4  8.5 

9 

Cerebrospinal Fluid.....................................................................................................26  Serous (Pleural, Peritoneal, Pericardial) ................. .................................... ..................................... ................................. ...............30 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 .......25 Physician Review........................................................................................................25  Result Reporting ................ .................................. .................................... .................................... .................................... ................................... .................26 26 

Fluid Types ............... ................................. ................................... ................................... .................................... ................................... ................................... .......................26 .....26   9.1  9.2  9.3  9.4 

10 

Manual Counting ......... .................. .................. .................. .................. .................. .................. .................. .................. .................. .................. ................8 .......8  Automated Methods....................................................................................................10 

Immunologic Studies ................. ................................... ................................... ................................... ................................... ............................ ...........46 46  Flow Cytometr Cytometric ic Studies ........ ................. .................. .................. .................. .................. .................. .................. .................. .................. ............54 ...54  Cytogenetic Analysis ................ .................................. .................................... .................................... .................................... ............................ ..........58 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 59  Quality Assurance.......................................................................................................60  Proficiency Testing (External Quality Assessment) .................. .................................... ................................. ...............60 60  Continuous Education and Training ......... ................... ................... .................. .................. .................. .................. .................. ...........61 ..61 

References.............................................................................................................................................62   Appendix A. Reagent Formula Formulations tions ......... .................. .................. ................... ................... .................. .................. .................. .................. .................. .................. ..........67 .67  Appendix B. Interpretation of Cell Types.............................................................................................68  The Quality System Approach..............................................................................................................72  Related CLSI/NCCLS Publications......................................................................................................73 

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Volume 25   Foreword

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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 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 p recautions and body substance su bstance 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 un iversal precaution guidelines are available availab le from the U.S. 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 infectiou in fectiouss disease, refer to the most current edition of Clinical and Laboratory Standards Institute document  document   M29 —  Protection of Laboratory Workers From Occupationally Acquired Infections. 3  Definitions accuracy (of measurement)  accuracy  measurement)  – – closeness of the agreement between b etween the resul resultt of a measurement and a true

value of the measurand (VIM93). (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) 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).

Clinical and Laboratory Standards Institute. All rights reserved. 

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 Number 20  

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analytica analyt icall specificity  – ability of a measurement procedure to measure solely the measurand (ISO

17511).2  17511).

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 o f 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 speci men that is contained cont ained within intact i ntact or histologically histolo gically sectioned cells or tissues. tissue s. 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 measura measurands nds for which the error of a measuring instrument is

intended to lie within specified limits (VIM93). (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 irrig ating the cavity and removing the fluid flu id for the purpose purpos e of examining its contents.

2

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Volume 25  

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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)  – close closeness ness 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  (IS O 15189) 15189)4; NOTE: For example,

a volume of serum taken from a larger volume of serum (ISO 15189). 15189) .4 

specimen  – biological material that is obtained in order order to detect or to measure one or more quantities,

such as amount or concentration (ISO/CD 18112-1). 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 substrate-chromoge n

 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. 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 prean alytical variables that t hat include incl ude an error in the order entry procedure pro cedure 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). Clinical and Laboratory Standards Institute. All rights reserved. 

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 Number 20 H56-P   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 sp ecimens will produce inaccurate test results. However, circumstances may arise ari se 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 specimen transported integrity, to degradation, or deterioration of the constituents of thea fluid. The istransport timecould must affect also betheacceptable 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 sy stem for the transport transpo rt of body fluids. 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 la borat la boratory ory for analysis or 7,8   rejection of the specimen. In addition, a Laboratory Incident Report should be filed. filed.7,8

5  Specimen Collection 5.1  Cerebrospinal Fluid

Cerebrospinal fluid is usu is usually ally collected by lumbar puncture, but may also be obtained by lateral cervical 9 or cisternal puncture. puncture.   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

Clinical and Laboratory Standards Institute. All rights reserved. 

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Volume 25 H56-P   analyzed for chemical and serologic studies. Subsequent tubes should be used for microbial 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  lu mbar   puncture include headache, infection, and brain herniation. Rarer complications may also occur.9  Refer to Section  Section  10.1.1  10.1.1  for collection of samples for cytological examination. Table 1. Specimen Requirements for Cerebrospinal Fluid Test Anticoagulant Volume (mL)

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. 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 hour s. s.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 wide range may be sent the laboratory. As little as 15 to greater thancytology 100 mL specimens, may be senta for analysis. anal ysis.ofA volumes 50-mL specimen is recommended. reto commended. Sterility is not required and 12 11 12   no anticoagulant is necessary. necessary.   However, heparin and EDTA are also used. 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 Tests  Anticoagulant 

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 bacterio static effect

8-10

AFB culture

SPS, none, or anticoagulant without  bactericidal or bacteriostatic bacterio static 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 33..  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 use d as anticoagulants anticoagulants because they 13,14  produce crystalline crystalli ne material that can be confused confuse d with wi th pathologic crystals. crystals.   However, others have used 15 lithium heparin and EDTA without difficulty. difficulty.   Oxalate should not be used because of extensive formation of calcium oxalate crystals. Refer to  to  Section 10.1.1 10.1.1   for collection of samples for cytological examination.

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Volume 25  

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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 chronic obstructive  obstructive  lung diseases. Low-volume recovery should be recorded on the request form. Refer to to Section 10.1.1 10.1.1 f   f or or collection of samples for cytological examination.

6  Specimen Handling and Transport Specimens should be transported to counts the laboratory within one hour of collection, so cell should bepromptly. completedCellular as soon degeneration as possible.  possible.   of CSF can begin 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 soon as possible after growth the completion of and the possibly collectionaffect procedures. Otherwise, cell lysis, cellular as degradation, and bacterial could occur the test results. Clinical and Laboratory Standards Institute. All rights reserved. 

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 Number 20 H56-P   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 Papanicola Papani colaou ou (PAP), H & E, or other stains will be adequately preserved with 11 refrigeration for several days. days. 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 Assessmen Assessmentt 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 

Reagents and Supplies

•  •  •  • 

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 

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.

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Volume 25   7.1.3.1  Specimen Dilutions

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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 can will be coagulate. If manual nucleated cell countscells, are performed synovial saline fluid samples, erythrocytes lysed, with preservation of nucleated by using aonhypotonic solution (0.3%). Several quality assurance stipulations have been promulgated by regulatory agencies. These include the use of certified pipettes or commercial dilution syste s ystems, ms, periodically checking diluting fluids for 18 18   extraneous particles and counting samples in duplicate. duplicate. 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 areas area s16: a) If less than an estimated 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 20 estimated  2000 cells are present in all nine squares, then count the four corner squares. This area counted is 4 mm m m2. c) If more than an estimated 200 cells 2are present in one square, then count five of the squares within the center square for an area of 0.2 mm mm . 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 m m2. 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). Clinical and Laboratory Standards Institute. All rights reserved. 

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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. eryth rocytes. 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 automated cell counts, for each fluid, can be found under the microscopic examination headings in  in Section 9. 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 variatio variationn at low cell 19  There are a counts is high on automated instruments, it is not nearly as high as manual cell counts. counts .19  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.

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Volume 25   7.2.1  Processing Body Fluids on Automated Devices

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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. Thelow keylevels issue in automated counters to ensure instrument candefine provide countsfor at the of using cells encountered in body is fluids. Thus,that eachthelaboratory must thereliable lower limits counting nucleated cells and erythrocytes, below which the use of automated or semiautomated counters is not reliable. 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. 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 d i = 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: 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 n t R  = estimated variance of the reference method n r  Clinical and Laboratory Standards Institute. All rights reserved. 

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Volume 25    

H56-P T = pt 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. 21,22  21,22 

Several protocols for determining carry-over are available. 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 informa tion on  on  determination of limits of detection, refer to the most current edition of CLSI/NCCLS document EP17 document  EP17 — Protocols Protocols for  Determination of Limits Limits of Detection and Li Limits mits of Quantitation. 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 information on on evaluation of precision performance, refer to the most current edition of CLSI/NCCLS documents  documents   EP5 —  and  EP15 — User  Evaluation of Precision Performance of Quantitative Measurement Methods  and  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 r 2, slope, and y-intercept are established. For detail d etailed ed information on method comparison, refer to the most current edition of CLSI/NCCLS document EP9 —  Method Comparison and  Bias Estimation Using Using Patient Sam Samples. ples. 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 Clinical and Laboratory Standards Institute. All rights reserved. 

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 Number 20 H56-P    processed and analyzed analy zed (i.e., quality control materials must be processed through throu gh the same fluidic fluidi c 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 is used for cell  cell  .counting cou 18 nting of blood specimens, there is no need to have separate control runs instrument for body fluid cell counting. counting  

8  Morphology Assessment 8.1  Slide Preparation 8.1.1 

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. cytocentrifugation .23  Even hypocellular samples with a yield, chamber cell count of zero can have h ave a yield of approximately 35 cells per slide slide.23  The quantitative yield, 24 however, varies from 30 to 75%, 75%,   and smaller cells, such as lymphocytes, may be underrepresented. 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: qualit y:23  1.  Fresh, unfixed specimens should be used for slide preparation. Cells may begin deteriorating in a few few 26 hours, particularly in body fluid samples with low protein content, such as cerebrospinal fluid. fluid.   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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Volume 25 H56-P   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 th e sample for washing and cytocentrifugation. cytocentrifug ation. 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 i ncubating at 37 °C ° C for ten minutes. Washing the cells after liquefaction also is helpful. 5.  Cellular samples or bloody samples slides need toare be difficult diluted with saline before to avoid overcrowded slides. Overcrowded to interpret due cytocentrifugation 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 cerebrospi cerebrospinal nal 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 Romanowsky-ty pe 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 inclu i nclude de sedimentation methods, methods,27-31  and centrifugation with smears made from the resuspended sediment. sediment .29  These methods are difficult to standardize and produce smears of variable quality. variable quality. These alternatives are inferior to cytocentrifugation, and are not recommended. Filtration methods 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 description descriptionss apply to properly prepared cytocentrifuge slides optimally stained with Romanowsky stains. 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. Clinical and Laboratory Standards Institute. All rights reserved. 

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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 cont rast to a more homogeneous homogen eous appearance for lymphomatous ly mphomatous infiltrates. infil trates. 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 blue black 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.

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Volume 25   8.2.5  Lining Cells

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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 b e shed individu individually ally or in clusters. clu sters. However, clusters clust ers of mesothelial cells cel ls generally have h ave “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 ov al nuclear nucl ear contour, cont our, dense den se chromatin, chromati n, 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 an d have a similar appearance appear ance 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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 Number 20   8.2.7  Miscellaneous Cells

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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 with reticular chromatin and with one or more nucleoli. The frayed cytoplasm may contain a nucleus 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. morp hology. They frequently form loose loos e clusters within a tissue-like matrix. matri x. 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. i ntracellularly. Most bacteria b acteria have h ave 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 bot h extracellularly extracellu larly 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.

18

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 C  l    i    n  i    c  a  l    a  n  d  L   a  b   o r   a  t    o r   y  S   t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s  r   e  s   e r   v  e  d   .

 

Eosinophils

Mast cell left center 

Autolytic neutrophils

Autolytic neutrophil neutrophilss

Lymphocytes

Reactive lymphocytes

 

Plasma cells

Macrophage

Macrophages

H  6   5  P 

1   9 

 

  2   0 

 N  u m  b   e  r  2   0 

 

 

Macrophages (signet ring cells)

Macrophage with ingested erythrocyte (erythrophage)

Macrophage with ingested neutrophil (neutrophage)

Macrophage with ingested lipid (lipophage)

Macrophage with ingested erythrocytes and hemosiderin granules

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 C  l    i    n  i    c  a  l    a  n  d  L   a  b   o r   a  t    o r   y  S 

Macrophage with sodium urate crystals

 t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s  r   e  s   e r   v  e  d   .

Macrophage with hemosiderin (siderophage)

Macrophage with hemosiderin and hematin crystal

Ventricular lining cells

H  5   6  P 

 

   V  o l    u m  e  2   5 

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 C  l    i    n  i    c  a  l    a  n  d  L   a  b   o r   a  t    o r   y  S   t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s  r   e  s   e r   v  e  d   .

 

Ventricular lining cells

Mesothelial cells (stimulated)

Ventricular lining cells

Mesothelial cells

Mesothelial cells

Mesothelial cells

Mesothelial cells (mitotic)

Mesothelial cells (peripheral vacuoles)

 

Mesothelial cells (binucleate) 2  1 

H  5   6  P 

 

  2  2 

 

 N  u m  b   e  r  2   0   

Blast cells (acute lymphocytic leukemia)

Lymphoma cells (diffuse large cell lymphoma)

 Nonhematopoiet ic malignancy  Nonhematopoietic (ovarian adenocarcinoma)

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 C  l    i    n  i    c  a  l    a  n  d  L   a  b   o r   a  t    o

 y r   S   t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s  r   e  s   e r   v  e  d   .

 Nonhematopoieti c malignancy  Nonhematopoietic (breast adenocarcinoma)

 Nonhematopoie tic malignancy  Nonhematopoietic (ovarian adenocarcinoma)

 Nonhematopoiet ic malignancy  Nonhematopoietic (breast adenocarcinoma)

 Nonhematopoieti c malignancy  Nonhematopoietic (oat cell carcinoma)

 Nonhematopoieti c malignancy  Nonhematopoietic (ovarian adenocarcinoma)

H  5   6  P 

 Nonhematopoieti c malignancy  Nonhematopoietic (gastric adenocarcinoma)

 

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 a  l    a  n  d  L   a  b   o r   a  t    o r   y  S   t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s 

 e  2   5 

 C  l    i    n  i    c

 

Chondrocyte (cartilage cell)

Cartilage

Neural tissue

 e r   s   e r   v  e  d   .

Germinal matrix cells

Yeast (Candida albicans)

Yeast, fungi (Cryptococcus neoformans)

 

2   3 

Parasites (Toxoplasma trophozoites)

H  5   6  P 

 

  2  4 

 N  u m  b   e  r  2   0 

 

 

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 cr crystals ystals are intracellular. Polarized light.

Monosodium urate crystals in synovial fluid. Note the red background, the  blue color of the vertical crystals crystals,, and yellow color of the horizontal crystals. First order red compensator.

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 b   a  o r   a  t    o r   y  S   t    a  n  d   a r   d   s  I    n  s   t    i    t    u  t    e  . A   l    l   r   i    g  h   t    s  r   e  s   e r   v  e  d   .

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. H  5   6  P 

 

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8.3  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 lympho ma cells, and nonhematopoietic non hematopoietic 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 should not the be included in the differential, and, if identified on cells, the counting chamber, also should be cells excluded from cell count. These include squamous epithelial 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, identified, they  they  may be reported as “atypical” and described in a comment, pending  physician review revi ew (see Section 8.4) 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. id entified. When nonhematopoietic tumor cells are first fi rst suspected on Romanowsky-stained cytocentrifuge slides, verification by PAP-stained cytopathology may  be appropriate. 8.5  Result Reporting

 Nucleated cell differentials different ials are reported in conventional units (%) or SI units unit s (proportion, (proporti on, 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 wh noted when en nucleated cell counts approach 200/µL. This number is not sacrosanct and varies with 9 the cell type.   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 spectrophotometry reported. CSF does not clot, but clots may be associated with a traumatic tap. 9.1.2 

Microscopic Examination

9.1.2.1  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 10 9/L for nucleated cells 26

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12/L for erythrocytes was poor .38  A similar limitation was noted using light scatter and and 0.01 x39 10   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, 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,  photomicrographs 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 mononucle mon onuclear ar cells, foreign body multinucleated giant cells, eosinophils, or clusters of ependymal 42,43 cells. cell s.   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 

Result Reporting

9.1.4.1  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 count s are reported in conventional c onventional units (µL) or SI units (10 6/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 differ ence ence is a pr edominance edominance of monocytes in the neonate that is gradua gradually lly replaced replaced by lymphocytes. Values in in Table B1  B1  represent combined data collated 9,41,44-47 from various repor tts. s.   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 Clinical and Laboratory Standards Institute. All rights reserved. 

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Volume 25 H56-P   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 vi ral and bacterial meningitis. meni ngitis. Lymphocytes predominate in the former and neutrophils neut rophils in i n the th e llatter. atter. 48 In meningitis, there is a correlation between increasing cell counts and positive bacterial cultures .  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 Fluid Reference Intervals* 

Volume Babies Children Adults16

10-60 mL 60-100 mL 57-286 mL

Color

Colorless

Cells Erythrocytes 46,47  Newborn preterm  Newborn term  Neonate > 3 months Adults Leukocytes 47,49,50  0-1 month 2 months to 16 years Adults

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) 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

Adults Lymphocytes Monocytes Histiocytes  Neutrophils  Neuroectodermal * SI units are in parentheses.

63-99% (.63-.99) 3-37% (.03-.37) rare 0-2% (0.00-0.02) rare

9.2  Serous (Pleural, Peritoneal, Pericardial)

This section will cover fluids of the pleural, pericardial, and peritoneal cavities (see Figure 2). 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 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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Microscopic Examination

9.2.2.1  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 ple ural fluid  fluid differentials obtained by an   automated cell counter was not 10 sufficiently accurate for clinical use. use .  

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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Result Reporting

9.2.4.1  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 us ed to denote deno te pus in the pleural cavity. Peritoneal Periton eal fluids may be labeled lab eled as abdominal, abdo minal, 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 blo od cell count, coun t, since the former is more inclusive. inclusi ve. On manual counts, it 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 o ther certified personnel deemed qualified q ualified 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 value study was done on Japanese soldiers by puncturing the intercostal space and attempting to recover fluid. 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, rece ntly, 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 in  Table 5. 5.

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Volume 25 H56-P    Normal values for peritoneal fluid have not been determined. Although not completely satisfactory, “sterile” ascitic fluid has been used to distinguish distinguish “n “normal” ormal” ascites from peritonitis and other serious intra-abdominal disorders, as discussed in  in Section 9.2.5. 9.2.5. Reference intervals for peritoneal dialysate fluid are listed in  in Table 6. 6. 9.2.5 

Analytic Significance

9.2.5.1  Macroscopic Examination  A   red color indicates blood. If Transudates are typically straw colored and clear, as are some exudates. exudates .54 A 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 b een reported in melanoma. melano ma.

Cloudy fluids are due to increased numbers of cells or increased triglycerides, and chylous effusions may have an opalescent appearance. In the rare circumst circumstance ance of a mesothelioma, the fluid may be viscous because of a high concentration of 54 hyaluronic acid. acid.   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  Cell Counts and Differentials 9.2.5.2.1  Pleural Fluid Red blood cell counts are of little significance. As indicated previously, a hematocrit can distinguish  between a serosanguineous serosanguin eous effusion  effusion and hemothorax. hemothorax. The latter may be secondary to trauma, pulmonary 54 emboli, or malignancy. malignancy.   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 Eosinophilia Eosinophil (>10%) immunoallergic is seen in many reaction conditions including pneumothorax, pulmonary emboli,effusions). traumatic hemoth he mothorax, orax,ia possible to 54 chest tubes, parasitic diseases, and Churg-Strauss syndrome. syndrome.   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 distinguish ed from hemoperitoneum. Malignancy may be associated with bloody fluids.  Normal values for nucleated cell counts, counts , differentials, and biochemical biochemi cal tests have not been established establi shed for  peritoneal fluid. Thus, medical decision levels are based on comparison with sterile ascitic fluid in ©

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 Number 20 H56-P   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. range.55 Total cell counts may increase markedly following diuretic therapy th erapy.. In sterile uncomplicated ascitic fluid, neutrophils ranged from 0 to 100%, with a mean value of 27%. 27%.55 A bsolute 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 spontaneo spontaneous us bacterial peritonitis,” with an absolute neutrophil value of 56 >250/µL suggestive of peritonitis.   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 distingui shed 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 co nsidered 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 i s clear and colorless. colorl ess. If peritonitis periton itis develops, d evelops, the fluid flu id becomes cloudy and the th e diagnosis is obvious. Representative cell counts and differentials of noninfected fluids in patients on continuous ambulatory peritoneal dialysi dialysiss are shown, in Table 6. Noninfected fluids usually have   nucleated cell counts of 50/µL or less. less.57,58  Cell counts are routinely done on infected fluids to monitor the effectiveness of antimicrobial therapy. Following infection, neutrophils neutrophil s significantly si gnificantly increase from mean 57  57  values of 18% in noninfected fluids to mean values greater than 70%. 70%. 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, study, mean values for transudates were 2210/µL, compared with values of 14 116/µL for exudates. exudate s.59 The  T he standard deviations are large and there is considerable overlap. Bacterial and rheumat oid effusions had the highest percentage of neutrophils with mean values approximately 70% or greater . greater .59  Monocytosis was secondary to hypothyroid or malignant effusions, with mean values of 75% or greater .59  The incidence of malignant malignant effusions varies in reported series from 10 to 25%, depending on the geographic location. location.60,61  Identification of malignant cells is similar to that described in other fluids, and mesothelial cells must must   be distinguished from neoplastic cells. LE cells have also been reported in  pericardial effusions effusion s.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 co unt

1395-3734/µL

Macrophages

64-80%* 

Lymphocytes

18-36%* 

 Neutrophils

0-1%*  *

Mesothelial cells Results expressed as interquartile range. range.53

*

0-2%  

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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 Number 20  

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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 t he walls of the abdominal and pelvic pelv ic cavities. cavities . Visceral peritoneum lines l ines the th e 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 m may ay 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 cont aining collagen collag en or fibrin (rice bodies). Particles may also be seen in metallosynovitis metallosy novitis from fro m a  prosthetic implant. impl ant. Viscosity can be measured at the bedside by the physician placing a finger at the tip of the syringe and stringing the fluid or determining length of the string after expressing it from Normal fluids willoutform a string greater thanthe four centimeters. Clinically, there is no needthe forsyringe. a sophisticated measurement of viscosity. Like the gross observation of viscosity, the mucin clot test may reflect the degree of hyaluronate  polymerization. Hyaluronidase derived deri ved from neutrophils neutrophi ls most likely has h as 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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Volume 25   9.3.2 

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Microscopic

9.3.2.1  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 Approximately 400 400 units of hyaluronidase are added 13 to 1 mL of synovial fluid and incubated for ten minutes at 37 °C.   However, since viscous fluids are either normal or noninflammatory, approximate cell counts are clinically acceptable, and excessive  personnel time is i s not justified. justified . Automated cell counts have been been validated validated for total nucleated cells and erythrocytes on impedance-based detection should both follow analytically acceptable and laser-based optical systems. systems.62,63  Lower limits of detection standards and provide clinically relevant information.62-64  Acceptable lower limits of detection detec tion were set 9 12 as >0.150 or 0.200 x 10 /L for nucleated cells and 0.01 or 0.03 x 10 /L for erythrocytes.62,63  Samples 62 62  

flagged for cellular interference should be enumerated manually. manually. If   automated instruments are used,  pretreatment of  o f  samples  samples with hyaluronidase was considered necessary adding an additional 20 minutes of  processing time.62,63  NOTE: Flow  Flow imagi  imaging ng technology has also been validated for automated cell counts 65-69  65-69  for nucleated cells and erythrocytes. erythrocytes. 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 measure measured. d. However, the latter category includes 63 lymphocytes, monocytes, immature granulocytes, and blasts. blasts .  I  Inn 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, neutrophils , lymphocytes, monocytes, histiocytes, and synovial lining cells.  Neutrophils normally constitute cons titute 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 R.A. cells, theL.E. lattercells name association with rheumatoid arthritis. In collagen vascularor diseases, typical maybecause be seenofonthe Romanowsky-stained smears. L.E. cells are neutrophils that have engulfed large, round, purple hyaline homogeneous nuclear masses. In normal fluids, cells in the the monocyte/macrophage category normally constitute the majority of cells 13   On stained smears, monocyte/macrophages with basophilic cytoplasmic with a mean value of 48%. 48%.13 inclusions have been designated Reiter cells. Ly Lympho mphocytes cytes range from few to many in normal fluids (see (see  Table 7) 7), with a mean value of approximately 13 25%..   They have similar morphologic features to blood lymphocytes or may show reactive changes in 25%  pathologic fluids. flu ids. ©

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 Number 20 H56-P    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, cel ls, 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  Crystals 9.3.2.3.1  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 lig ht 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 the light can pass through the grid of the analyzer and appear white against a black background (see  Figure 3) 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. 13,70,71 Some suggestions regarding technique for crystal identification include the following:  13,  

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 distinguish ed 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 l ight and usi using ng 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 bright crystals with negative birefringence. Identification is enhanced using the first order red compensator. 70 W  When hen its long 38

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Volume 25 H56-P   axis is perpendicular to the direction of slow vibration of the li ght in the in the compensator, it appears blue, and  A fixed  fixed preparation from a known case when its long axis is parallel to this direction, it appears yellow. yellow .13 A 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 so me studies suggest that repeat examination after 24 hours of storage storage at a t 4 °C improves the diagnostic yield.72 However, others question the diagnostic validity 13 of this finding. finding.   Urate crystals may on rare occasions form a spherulite  spherulite   with a beach ball appearance. They are extracellular and in some cases, the only form of the crystal. crystal .73  They must be distinguished from globules of fat. Calcium pyrophosphate dehydrate (CPPD) crystals crystals are associated with pseudogout but may also be  present in lesser amounts amount s 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 monocyt monocytes. es. CPPD can also be detected by staining air-dried 74

cytocentrifuge preparations with alizarin red S. 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 can  be suspected su spected by the t he expert light microscopist. Their association with arthritis art hritis is well wel l 75,76,77  75,76,77  documente docume ntedd. In one study, BCP crystals were only associated with osteoarthritis or rheumatoid 15 arthritis.   Steroid crystals have protean morphology. 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 inflammatory synovitis synovitis in 0.6 to 80,81 2% of patients, beginning several hours after injection and lasting up to 72 hours. hours.   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 hemoglobin indicative extravasation of erythrocytes butsynovitis with no associated additional  pathologic si signif  gnif product icance. icance. ofCharcot-Leyden crystals of have been reported in eosinophilic 13 with urticaria. urticaria. Fragments of metal from a prosthesis may cause a metallosynovitis. They may be extraor intracellular. Artifacts include crystals of calcium oxalate, dry   K 2EDT EDTA, A, lithium heparin, starch granules, and dust. A useful chart has been previously published (see  (see Table 8) 8).71  9.3.3 

Result Reporting

9.3.3.1  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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 Number 20 H56-P   nucleated cell count, differential count, and special morphologic findings. Viscosity is best evaluated at the time of aspiration and should be recorded 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 inc that include lude morphology, birefringence, strength of birefringence, sign of 13   13  birefringence, and extinction angle. angle. 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 containing an average of more than one CPPD crystal per 50X oil immersion field in an unstained preparation. 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 clinical   laboratory for information that is either nonspecific or has no clinical relevance. Others may disagree. 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 result s reported. 9.3.3.2  Reference Intervals Reference intervals for synovial fluid are shown in Table 77.. 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  diverse etiology and crystalline joint 82 disease. Characteristic findings of each group are listed in Table B3.   The diagnostic significance of crystals, tissue fragments, microorganisms, and cell types are discussed  below. Rice bodies are polished white arthritides fragments of tissue containingarthritis). collagen and are seen in joint fluid of patients with many (e.g., rheumatoid Fragfibrin. Fragmen ments ts They of fibrocartilage are 15 described in meniscal or cruciate ligament tears and cartilage in osteoarthritis. osteoarthritis.   Crystal analysis leads to a specific diagnosis in gout. CPPD crystals are associated with inflammatory fluids in pseudogou pseudogout,t, but may also be seen in noninflammatory fluids with a coincidental only in  in  osteoarthritis and rheumatoid chrondocalcinosis..15  In the same study, BCP crystals were found only chrondocalcinosis 83 arthritis. Others consider their presence a crystalline deposition disease. disease.   Metallic debris has been noted from titanium implants. The metallic fragments are both extra- and intracellular.

40

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Volume 25  

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Table 7. Normal Synovial Fluid Values. (Modified from McCarty DJ. Synovial fluid. In: Koopman WJ, ed.  Arthritis th and Allied Conditions.  14   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 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

Shape

Birefringence

Bipyramidal Often rhomboid, may be rodlike, diamond, or square, usually 100 µm Small (10%)

Basophils

H56-P

Condition

Infectious: bacterial, bacterial , tuberculous, fungal, fu ngal, early viral  Noninfectious (modest (mod est increases): hemorrhage, he morrhage, intrathecal injections, infarction Infectious: parasites, C. immitis   Noninfectious: lymphoma, leukemia, ventricular peritoneal shunts, blood contamination Rare, nonspecific, occasional cases of lymphoma

Lymphocytes

Monocytes

Infectious: viral, tuberculous, fungal; partially treated bacterial meningitis  Noninfectious: multiple sclerosis, sclerosi s, neurosyphilis, cerebral neoplasmas, lymphoproliferative disorders (e.g., lymphoma) Infectious: tuberculous, fungal  Noninfectious: nonspecific n onspecific response respons e 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 positiv e cytology) Metastatic tumors (20% positive if cerebral  parenchyma involved, involv ed, 50% if meninges involved)

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Volume 25   Appendix B. (Continued) 

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Table B2. Interpretation of Pleural Fluid Cell Types  Cell Type

 Neutrophilia (>50% PMN) Eosinophilia Eosinophil ia (>10%) Lymphocytosis (>50%)

Monocytes/Macrophages Monocytes/Macroph ages

Condition

Acute inflammation inflammatio n (e.g., parapneumonic parapneumoni c effusion) Pneumothorax, pulmonary emboli, traumatic hemothorax, reaction to chest tubes,  parasitic diseases, Churg-Strauss Ch urg-Strauss syndrome synd rome Transudates, tuberculosis, carcinoma, coronary artery bypass surgery, lymphoproliferative disorders, chylous effusions Limited diagnostic significance, erythrophages and siderophages are useful in distinguishing pathologic fluids from traumatic

Blasts Plasma cells Mesothelial cells

 Neoplastic cells from solid tumors Miscellaneous LE-cells

taps. Hematopoietic malignancies Reactive conditions, plasma cell myeloma (rare)  Normal constituent constitu ent (≥5%), markedly decreased in tuberculous effusions (5000

%  Neutrophils

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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Volume 25  

H56-P NOTES

Clinical and Laboratory Standards Institute. All rights reserved. 

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71

 

 Number 20

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  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

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.

  s   s    t   n   d   e   r   m  o   c   e   u   c   R   o    D   &

  n   o    i    t   a   z    i   n   a   g   r    O

   t   n   e   m   p    i   u   q    E

   l   e   n   n   o   s   r   e    P

GP2

   &   g   n   y    i   s   r   o   a   t    h   n   c   r   e   u   v   n    P   I

   t   n   n   e   o    i    t   m   a   e   g   m   r   a   o   n   a    f   n I    M

   l   s   s   o   r   e   t   c   n   o   r   o    P   C  

GP21

   t   n   e   c   e   n   m   e   e   r   r   g   u   a   n   c   c   a    O   M

X EP5 EP6 EP9

   t   n   e   m   s   s   e   s   s    A

GP29

   t   n   e   m   e   s   v   s   e   o   c   r   p   o   r   m    P   I

  n   o    i    &   t   e   c   a   c   f    i   s   v    i    t   r   e   a    S   S

   &   s   e    i    t   y    i    l    i    t   e   c    f   a   a    F   S

GP27

Adapted from CLSI/NCCLS document HS1—  A Quality Management System Model for Health 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 Model for Laboratory Services 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 deliver the laboratory’s laboratory’s services, namely namely quality laboratory laboratory information. 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. Preexamination    t   s   e   u   q   e    R    t   s   e

   t   n   e    t   m   s   n   s   e   e    i    t   s   a   s    P   A

 

 

T

Examination

  n   n   o   e   i   m   t   c    i   e   c    l   e   l   p   o  

S   C

X

  n   t   e   r   o   m  p    i   s   c   e   n   r   p   a  

S   T

X

  n   e   t   p   m    i    i   e   c   e   c   p   e  

S   R

X GP16

  g   w   n   i   e    i    t   s   v   e   e  

T   R

X

  n   o    i   y    t   r   a   o   t    t   r   a   e   r   r   o   p   e    b   t   a   n   L   I

Postexamination

 

  s    t    t    l   r   o   u   s   p   e   e

   t   n   e   n   m    t   e   s   e   e   i   m  g    t   -   c   a    t   s   e   n   o   p   a

R   R

   P   S   M

X

Adapted from CLSI/NCCLS document HS1—  A Quality Management System Model for Health Health Care.

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Volume 25

H56-P

 Related CLSI/NCCLS Publications*  EP5-A2

Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline— Second Edition (2004).  This document provides guidance for designing an experiment to evaluate the

 precision performance of quantitative measurement methods; recommendations on comparing the resulting  precision estimates with manufacturers’ precision performance claims and determining when such comparisons are valid; as well as manufacturers’ guidelines for establishing claims.   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.