Body-Fluids-Out-of-the-Dark-Ages-Handouts

Body FluidsOUT OF THE DARK AGESPatti Merritt, MT(ASCP)SHHematology Product SpecialistBeckman Coulter, Inc.OBJECTIVES•Manual vs Automated–Why automate?–How to automate–Suggestions to make it easy•Meeting Automated Body Fluid CAP Requirements –HEM.35414, HEM.35452, HEM.35471, HEM.35490•Instrument Procedures and Validations–What needs to be done?–Additional things to Consider•Quick Overview of BF Cellular MorphologyHemacytometer CountsO•Labor intensive–High complexity, require a skilled laboratorian.•Pre-Analytical Variables–Low counts and high counts have increased tech to tech variation.–Chamber loading technique–Counting area•Susceptible to errors–Dilutional and Calculation errorsWhy Automate??•Easily adapted from routine hematology.•Can be run by less skilled employee and interpreted by aTechnologist.•Fewer Calculation errors.•High linearities on instruments•Only simple dilutional calculations may be needed; ie1:2or 1:5 for high cellular counts.•Standardized•More cells counted•Low and high counts fewer variables•Better reproducibilityInstrument ValidationCLSI GuidelinesJune 2010H26-A2New TermsLoB, LLoD, LLoQ Limit of Blank (LoB) -performed to establish the inherent background of the analyte being reported and representsthe highest result that is likely to be observed for a blank sample i.e. sample that does not have any of the analyte being measured.Lower Limit of Detection LLoD-the lowest amount of analyte that can be detected with stated probability.Lower Limit of Quantitation (LLoQ) -the lowest amountof analyte in a sample that can be quantitatively determined with stated acceptable precision and trueness.InstrumentInstructions For Use (IFU)Check for 510kBody Fluid Clearance Cerebrospinal FluidSerous Fluid•Pleural•Pericardial•Peritoneal•Bronchial LavageSynovial FluidRegulatoryRequirementsCAP RequirementInstrumentBackground Counts HEM.35414Background Checks -Automated Counts Phase IIInstrument background counts are performed each day of testing on the diluent fluid and lysing agent to check for contamination that might affect cell counts.NOTE: This can be done by processing these fluids on the instrument used for cell counting and checking for the presence of significant background in the dilutingfluids and lysing agents.Evidence of Compliance:✓Written procedure defining frequency and method for performing background checks AND✓Records of background checksInstrument BackgroundDecision RulesCAP RequirementBF AMR & Backgrounds HEM.35452Acceptable LimitsPhase IIThe laboratory defines the upper and lower limits for counting body fluid cells (erythrocytes,nucleated cells) outside of which the use of automated or semi-automated cell counters is not reliable.NOTE: The laboratory must have an appropriate protocol that limits the use of automated or semi-automated instruments for cell counting in the very low concentration ranges often seen with body fluids. The lower limit selected must reflect the particular instrument's background count and sensitivity.Evidence of Compliance:✓Written procedure defining the upper and lower reporting limits for automated and/or semi-automated cell counters and actions to be taken if the limits are exceeded AND✓Records of study to validate reportable rangeREFERENCES1) International Committee for Standardization in Haematology(ICSH). Protocol for evaluation of automated blood cell counters.Clin LabHaemat. 1984;6:69-842) Subira D, et al. Flow cytometric analysis of cerebrospinal fluid samples and its usefulness in routine clinical practice.Am J Clin Pathol.2002;117:952-958Linearity TestingSensitivity StudyLower LimitActual ActualExpected Expected ID TNC 1TNC 2TNC Avg TNC RBC 1RBC 2RBC Avg RBC1Undiluted 5.40 5.41 5.41 5.41 1.878 1.885 1.882 1.880 21:2 2.71 2.70 2.71 2.710.9700.9740.9720.940 31:4 1.54 1.46 1.50 1.350.4850.4850.4850.470 41:80.710.720.720.680.2480.2520.2500.235 51:160.340.340.340.340.1240.1250.1250.118 61:320.190.170.180.170.0610.0680.0650.059 71:640.0330.0320.0330.029 81:1280.0160.0170.0170.015 91:2560.0090.0080.0090.007CAP RequirementAutomated LimitationsCounts-AutomatedClumps/DebrisHEM.35471 CellPhase IThe laboratory has a procedure to detect clumps of cells or debris that may give spurious cell counts.NOTE: The procedure should include performing macroscopic assessment of body fluid samples processed on cell counting instruments. Instrument generated flags and findings on microscopic examination that suggest the presence of debris are important observations and may require the performance of a wet mount. Marked clumping or clots precludes reporting an automated count. The laboratory report should note the limited accuracy of cell counts in these situations, and include a description of the specimen problem.InstrumentLimitationsFlags / CodesCAP RequirementControls HEM.35490Stabilized ControlsPhase IITwo different stabilized control specimens are analyzed and results recorded at least daily.NOTE: Manufacturers recommendations for control material selection should be followed, and the selected control should be compatible with the methodology used by the instrument.ManufacturerRecommendationsBody Fluid Controls COULTER DxH800 Body Fluid Control is recommended for monitoring system performance of the body fluid cycle’s RBC and TNC count parameters. Additionally, COULTER Body Fluid Control can be used for verification of the reportable range and linearity of the TNCand RBC in the body fluid panel.AUTOMATED ANALYSISWHAT DO I HAVE TO DO??Instrument Manuals Know Your Analyzer Limits•Hyaluronidase is recommended to pretreat synovial fluidsThese enzymes randomly cleave β-N-acetylhexosamine-[1→4] glycosidic bonds in hyaluronic acid, chondroitin, and chondroitin sulfates .•This is a powder that does not require calculations to manipulate data for dilutional effectsSynovial Fluids….LiquifySynovial FluidBCI Protocoltreat synovial fluids prior to analysis according to your laboratory standards. Add in the ratio of 1 mL of synovial fluid to 5 mg of hyaluronidase. Mix for 5 minutes.Body FluidsTo reduce body fluid sample viscosity, use hyaluronidase to treat synovial fluids prior to analysis according to your laboratory standards . Add in the ratio of 1 mL of synovial fluid to 5 mg of hyaluronidase. Mix for 5 minutes.Sample AnalysisEasy to FollowInstructionsMethod ComparisonData Gathering •Work the data gathering into routinetesting.•Whenever a Body Fluid is received, counts are done both ways, and copies of allresults are saved for review and statistical comparison.Method ComparisonData Gathering •Set up a File Folder so that the Tech caninsert hard-copies of the manual results with the instrument print-outs for the Automated Counts.•If you will be using more than one instrument, make sure to analyze all specimens on each analyzer you intend to evaluate.–Correlate Hemacytometer to Primary Unit–Correlate Primary Unit to Secondary UnitMethod ComparisonData Gathering •Periodically, monitor data collection to ensure all Fluid Types you intend to validate are included.•Continue the study with additional specimensto ensure distribution between body fluidtypes and instruments, if necessary.•Identify “problem” fluids and look for common causes.Body FluidData WorksheetMethod ComparisonProcedure Manuals •Evaluation data and documentation must be available that the method was found to be acceptable by a Medical Director / Pathologist.•Append test data summary to the Body Fluid procedure; be sure to hold onto raw data!•ALL other Regulatory (CAP) guidelines forBody Fluids must be addressed within theBody Fluid procedures.What else do you need to consider?AdditionalMaintenance? Coulter: No additional maintenance must be performed when running fluids.RESULT CONVERSIONHemacytometer vs Instrument Units•Many labs result Hemacytometer counts in mm3•Many Hematology Analyzers report:–WBC (TNC) x 103–RBC x106•If a lab wishes to continue resulting out body fluids in mm3, they will have to convert the Body Fluid counts•Some Computer systems may have the capability to do this conversion for you or you may wish to create a worksheet to document. Check with your IT person.RESULT CONVERSIONHemacytometer vs Instrument Units•TNC counts must be multiplied by 1,000or the decimal moved to the right 3spaces to convert from103to mm3.–0.2 x 103= 200 mm3•RBC counts must be multiplied by 1,000,000 or the decimal moved to the right 6spaces to convert from 106to mm3.–0.010 x 106= 10,000 mm3LabWorksheetDxH800 Analytical UnitsAbnormal Body Fluid Cell MorphologyA QUICK ReviewCAP RequirementObservation Assessment –Body FluidHEM.35566 MorphologicPhase IIThe laboratory at least annually assesses morphologic observations among personnel performing body fluid cell differentials, to ensure consistency.NOTE: Suggested methods to accomplish this include:1.Circulation of body fluid smears with defined nucleated cell differential distributions, and/or2.Multi-headed microscopy, and/ore of body fluid photomicrographs with referee and consensus identifications (e.g.former CAP Surveys photomicrographs)e of digital imagesThe procedure manual should include definitions of semi-quantitative measurements such as 1+,2+, 3+, etc.Evidence of Compliance:✓Written procedure defining the method and criteria used for evaluation of consistency AND ✓Employee records documenting morphology assessmentCELL TYPES•Myeloid–Mature –Seg, Eo, Baso–Immature –Meta, Myelo, Blast–Degenerating–Phagocytic•Lymphoid–Lymphocyte–Lymphoma Cell–Plasma Cell•Lining Cells–Mesothelial–Synovial–Choroid •Mononuclear / Phagocytic–Monocyte–Macrophage–Neutrophage–Erythrophage–Siderophages–Lipophages•Malignant Cells–Tumors–Mesothelioma–Meduloblastoma–Neuroblastoma–Metastatic Carcinoma–Metastatic Melanoma •Crystals, Microorganisms and MiscellaneousCELL TYPES Myeloid Series•Degenerating Cells–Pyknotic, dense nuclei–Pale cytoplasms–May be confused with NRBCsCELL TYPESMyeloid Series•Phagocytic Neutrophils–Bacteria•Cocci –Strep & Staph•Rods –Gram negative bacilli–Fungi•Candida•HistoplasmaCELL TYPESLymphoid Series•Plasma Cells / Plasmacytoid Lymphs–Forms of B-reactive Lymphs–Round to bean-shaped, eccentric nucleus–Deeply basophilic cytoplasm–Often Golgi region apparent–Aggressive myeloma, morphology malignant•Cells 2 –4 times bigger•Bi-or multi-nucleated Plasma Cells / Plasmacytoid Lymphs•Mitotic figuresCELL TYPESLymphoid Series •Lymphoma Cell -Dependent upon the nature of the process•Typically appear singly but cytocentrifugation may cause smallaggregates–Large Cell Lymphoma•Immature nuclear chromatin; high N/C ratio•Small, well-defined, punched out vacoules–Follicular Lymphoma (aka small cleaved lymphoma)•Slightly larger than mature, normal lymphs; high N/C ratio•Nucleus may show large clefts / irregularities–Lymphoblastic Lymphoma•May have more convoluted or folded nucleusCELL TYPESLining Cells •Mesothelial–Normally lines pleural, pericardial and peritoneal–12 to 20u–Can be found individually or clustered–Low N/C ratio–ROUND to OVAL ECCENTRIC nucleus–May have multiple nuclei which overlap–May have irregularly shaped vacuolesLINING CELLSMesothelialNormal vs Abnormal Malignant MesotheliomaCELL TYPESMononuclear / Phagocytic•Macrophage–Large cells 15 to 30u–Abundant cytoplasm–One or more round / oval nuceli–Evidence of phagocytosis•Signet Ring form•Blood cells•Bacteria•Remnants of digested material•Cytoplasmic vacuolesCELL TYPES Mononuclear / Phagocytic•Neutrophage–Macrophage with one or more phagocytosedneutrophils–Initially entire neutrophil is evident–Digestion leaves apyknotic nucleus •Erythrophage–Macrophage withingested RBCs–Does not always imply invivo erythrophagocytosisCELL TYPESMononuclear / Phagocytic•Siderophages–Macrophage arising from iron (hemosiderin) in thedigested RBCs–See approximately 48hrs after CSF hemorrhage –Diagnostic aid todifferentiate truehemorrhage fromtraumatic tap •Lipophages–Macrophage containingfat–May be from extracellular fatty material or a resultof ingested cellsCELL TYPESMalignant Cells Dependent on the underlying malignancyHigh N/C ratioIrregularly shaped nucleiTendency to form LARGE clustersNUCLEAR MOLDING•Tumors•Mesothelioma•Meduloblastoma•Neuroblastoma•Metastatic Carcinoma•Metastatic MelanomaSUMMARY •Automation of cellular Body Fluids is easier and more accurate than Hemacytometer counts•Getting started, gathering data and satisfying regulatory requirements can be accomplished with planning and preparation•Body Fluid Cell Identification takes practice (and a good reference book!)。