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Introduction to CBC and Blood Smear Interpretation
Introduction to CBC and Blood Introduction to CBC and Blood Smear InterpretationSmear Interpretation
Thanyaphong Na Nakorn, MD, PhDDivision of Hematology
Department of MedicineChulalongkorn University
Thanyaphong Na Nakorn, MD, PhDThanyaphong Na Nakorn, MD, PhDDivision of HematologyDivision of Hematology
Department of MedicineDepartment of MedicineChulalongkorn UniversityChulalongkorn University
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Complete blood countComplete blood countComplete blood countThe most common test used in clinical medicineDetermine type and severity of blood cell abnormalitiesNowadays, CBC is fully automated and highly reproducible.Correct interpretation of automated CBC can reduce rate of unnecessary blood smear examinationProvide useful information for provisional diagnosis of RBC and WBC diseases
The most common test used in clinical medicineThe most common test used in clinical medicineDetermine type and severity of blood cell Determine type and severity of blood cell abnormalitiesabnormalitiesNowadays, CBC is fully automated and highly Nowadays, CBC is fully automated and highly reproducible.reproducible.Correct interpretation of automated CBC can Correct interpretation of automated CBC can reduce rate of unnecessary blood smear reduce rate of unnecessary blood smear examinationexaminationProvide useful information for provisional Provide useful information for provisional diagnosis of RBC and WBC diseasesdiagnosis of RBC and WBC diseases
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Component of automated CBCComponent of automated CBCComponent of automated CBC
Blood count basic parameters: Hb, Hct, RBC, WBC, platlet.Red cell indices: MCV, MCH, MCHC, RDWWBC differentialsCytogram or ScattergramReticulocyte count
Blood count basic parameters: Blood count basic parameters: HbHb, , HctHct, , RBC, WBC, RBC, WBC, platletplatlet..Red cell indices: MCV, MCH, MCHC, RDWRed cell indices: MCV, MCH, MCHC, RDWWBC differentialsWBC differentialsCytogramCytogram or or ScattergramScattergramReticulocyteReticulocyte countcount
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Basic principles of automated blood Basic principles of automated blood cell analyzercell analyzer
Electrical impedance:Electrical impedance:Coulter CounterCoulter Counter ,, CellCell--DynDyn®®
Optical impedance and light scatter:Optical impedance and light scatter:
TechniconTechnicon--H seriesH series
VCS (volume, conductivity, light scatter) technology VCS (volume, conductivity, light scatter) technology
BeckmanBeckman--Coulter VCS, MAXM,Coulter VCS, MAXM, STKSSTKS
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• Cell count and size can be measured by electrical impedance
•• Cell count and size can be measured by Cell count and size can be measured by electrical impedanceelectrical impedance
Coulter TechnologyCoulter TechnologyCoulter Technology
©J.Paul Robinson©J.Paul Robinson
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Direction of flowDirection of flow
The Bernoulli EffectThe Bernoulli Effect
Velocity GradientVelocity Gradient
Viscous drag along walls. Viscous drag along walls.
Lower pressureLower pressure
Particles move to low pressure areaParticles move to low pressure area
Laminar Coaxial FlowLaminar Coaxial FlowSheath fluidSheath fluid
Hydrodynamic FocusingHydrodynamic Focusing
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Technicon TechnologyTechniconTechnicon TechnologyTechnology
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VCS TechnologyVCS TechnologyVCS Technology
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Impedance, laser light scatter Impedance, laser light scatter Cell Cell DynDyn 4000 (Abbott)4000 (Abbott)
CytochemistryCytochemistry, flow technology , flow technology ((reticulocytereticulocyte analysis)analysis)
AdviaAdvia 120 (Bayer)120 (Bayer)
CytochemistryCytochemistry, flow technology , flow technology ((reticulocytereticulocyte analysis)analysis)
TechniconTechnicon HH--3 (Bayer)3 (Bayer)
Impedance, light absorption (halogen Impedance, light absorption (halogen light source)light source)
CobasCobas Argos 5Argos 5--diff (Roche)diff (Roche)
Direct current (resistance to cell Direct current (resistance to cell volume); radiovolume); radio--frequency (cell density, frequency (cell density, size)size)
SysmexSysmex SESE--90009000
Impedance, flow Impedance, flow cytometrycytometry, , conductivity, laser light scatter conductivity, laser light scatter ((reticulocytereticulocyte analysis)analysis)
Coulter GENCoulter GEN--SS
Impedance, conductivity, laser Impedance, conductivity, laser technologytechnology
Coulter STKSCoulter STKSMethodology featuresMethodology featuresInstrumentInstrument
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Red cell parametersRed cell parametersRed cell parametersDirect Measurement
Erythrocyte Concentration (RBC) x 106/mlMean Corpuscular Volume (MCV) Femtolitre (fl)Hemoglobin (Hb) Gram/decilitre (g/dl)
Indirect MeasurementHematocrit (Hct) = RBC x MCV/10 %Mean Corpuscular Hemoglobin (MCH) = HB x 10 / RBC (pg)Mean Corpuscular Hemoglobin Concentration (MCHC) = Hb/Hct x 100 (g/dl)
Red Cell Distribution Width (RDW) %
Direct MeasurementDirect MeasurementErythrocyte Concentration (Erythrocyte Concentration (RBCRBC) ) x 10x 1066/ml/mlMean Corpuscular Volume (Mean Corpuscular Volume (MCVMCV) ) FemtolitreFemtolitre (fl)(fl)Hemoglobin (Hemoglobin (HbHb)) Gram/Gram/decilitredecilitre (g/dl)(g/dl)
Indirect MeasurementIndirect MeasurementHematocritHematocrit ((HctHct) = RBC x MCV/10 ) = RBC x MCV/10 %%Mean Corpuscular Hemoglobin (Mean Corpuscular Hemoglobin (MCHMCH) = HB x 10 / RBC (pg)) = HB x 10 / RBC (pg)Mean Corpuscular Hemoglobin Concentration (Mean Corpuscular Hemoglobin Concentration (MCHCMCHC) ) = = Hb/HctHb/Hct x 100 x 100 (g/dl)(g/dl)
Red Cell Distribution Width (Red Cell Distribution Width (RDWRDW) ) %%
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RBC size and MCVRBC size and MCVRBC size and MCV
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RRed Cell ed Cell DDistribution istribution WWidthidthRBC x 106/ml
75 80 85 90
20th percentile80th percentile
RDW
RDW =80th percentile - 20th percentile
80th percentile + 20th percentile X constant
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WBC: three-part differentialsWBC: threeWBC: three--part differentialspart differentials
CellCell--DynDyn 1700CS1700CS
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WBC: Five-part differentialsWBC: FiveWBC: Five--part differentialspart differentials
NeutrophilLymphocyteMonocyteEosinophilBasophil
NeutrophilNeutrophilLymphocyteLymphocyteMonocyteMonocyteEosinophilEosinophilBasophilBasophil
Coulter VCSCoulter VCS
lymphocyte
Monocyte
Baso
NφEo
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WBC: Six-part differentialsWBC: SixWBC: Six--part differentialspart differentials
NeutrophilLymphocyteMonocyteEosinophilBasophilLarge Unstained Cell (LUC)
NeutrophilNeutrophilLymphocyteLymphocyteMonocyteMonocyteEosinophilEosinophilBasophilBasophilLarge Unstained Cell Large Unstained Cell (LUC)(LUC)
TechniconTechnicon*H2*H2
PeroxidasePeroxidase channelchannel
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WBC: Six-part differentialsWBC: SixWBC: Six--part differentialspart differentials
TechniconTechnicon*H2*H2
Basophil/lobularityBasophil/lobularity channelchannel
Additional parameters in Technicon-H analyzer
1. Mean peroxidase activity index (MPXI)
2. Lobularity Index (LI)
Additional parameters in Additional parameters in TechniconTechnicon--H analyzerH analyzer
1.1. Mean Mean peroxidaseperoxidase activity activity index (MPXI)index (MPXI)
2.2. LobularityLobularity Index (LI)Index (LI)
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Platelet parametersPlatelet parametersPlatelet parameters
Platelet count
Mean Platelet Volume (MPV)
Platelet Distribution Width (PDW)
Plateletcrit (Pct)
Platelet countPlatelet count
Mean Platelet Volume (MPV)Mean Platelet Volume (MPV)
Platelet Distribution Width (PDW)Platelet Distribution Width (PDW)
PlateletcritPlateletcrit (Pct)(Pct)
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Reticulocyte countReticulocyteReticulocyte countcount
Reticulocyte = non-nucleated RBC with polyribosomal RNA as stained by supravital stain (new methyleneblue or brilliant cresyl blue)Polychromasia underestimates reticulocytesThree methods of reticulocyteenumeration
Manual count on slide per 1,000 RBC Automated CBC with reticulocyte counter (Coulter VCS, Cell-Dyne 4000, Technicon-H3)Flow cytometry with fluorescent dyes
ReticulocyteReticulocyte = non= non--nucleated RBC nucleated RBC with with polyribosomalpolyribosomal RNA as stained RNA as stained by by supravitalsupravital stain (new stain (new methylenemethyleneblue or brilliant blue or brilliant cresylcresyl blue)blue)PolychromasiaPolychromasia underestimates underestimates reticulocytesreticulocytesThree methods of Three methods of reticulocytereticulocyteenumerationenumeration
Manual count on slide per 1,000 Manual count on slide per 1,000 RBC RBC Automated CBC with Automated CBC with reticulocytereticulocyte counter (Coulter counter (Coulter VCS, CellVCS, Cell--Dyne 4000, TechniconDyne 4000, Technicon--H3)H3)Flow Flow cytometrycytometry with fluorescent with fluorescent dyesdyes
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Report from Coulter™Report from CoulterReport from Coulter™™
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Report from Technicon-H™Report from Report from TechniconTechnicon--HH™™
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Clinical Utility of automated CBCClinical Utility of automated CBCClinical Utility of automated CBC
Differential diagnosis of RBC disordersMicrocytic RBC
Use WBC differential cytogram to guide WBC morphology and phenotypes
Differential diagnosis of RBC disordersDifferential diagnosis of RBC disordersMicrocyticMicrocytic RBCRBC
Use WBC differential Use WBC differential cytogramcytogram to guide to guide WBC morphology and phenotypesWBC morphology and phenotypes
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WBCWBC 5.005.00 x10x1033//µµLLRBCRBC 3.563.56 x10x1066//µµLLHbHb 6.06.0 g/dLg/dLHCTHCT 21.021.0 %%MCVMCV 59.059.0 fLfLMCHMCH 17.017.0 pgpgMCHCMCHC 28.828.8 g/dLg/dLRDWRDW 19.419.4 %%HDWHDW 4.024.02 g/dLg/dLPLTPLT 427427 x10x1033//µµLL
What is your diagnosis?What is your diagnosis?What is your diagnosis?
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Differential diagnosis of anemia using MCV and RDW
Differential diagnosis of anemia Differential diagnosis of anemia using MCV and RDWusing MCV and RDW
B12 deficiencyB12 deficiencyFolateFolate deficiencydeficiencyAIHAAIHADrugs: HU, ARV, Drugs: HU, ARV,
AZA, etc.AZA, etc.
Early or combined Early or combined nutritional nutritional deficiencydeficiency
MyelodysplasiaMyelodysplasiaMyelophthisisMyelophthisisSickle cell anemia or Sickle cell anemia or
Homozygous Homozygous HbCSHbCS
Iron deficiency anemiaIron deficiency anemiaThalassemiaThalassemia intermediaintermediaSideroblasticSideroblastic anemiaanemiaSevere ACDSevere ACDRBC fragmentationRBC fragmentation
RDW >15RDW >15
AplasticAplastic anemiaanemiaMDSMDSMyelomaMyelomaLiver diseaseLiver diseaseHyperthyroidismHyperthyroidism
ACDACDHeterozygous Heterozygous HbSHbS, ,
HbCSHbCS, , HbEHbE, etc., etc.Hereditary Hereditary
spherocytosisspherocytosisAcute hemorrhageAcute hemorrhage
ThalassemiaThalassemia traittraitHeterozygous Heterozygous HbEHbE, , HbCHbC, ,
etc.etc.Anemia of chronic Anemia of chronic
disease (ACD)disease (ACD)RDW <15RDW <15
High MCVHigh MCVNormal MCVNormal MCVLow MCVLow MCV
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SatellitismSatellitism, clumping, clumpingWBC fragmentation, severe WBC fragmentation, severe microcytosismicrocytosis, , cryoglobulinscryoglobulins
PlateletsPlatelets
ClottingClottingNucleated red cells, platelet clumps, Nucleated red cells, platelet clumps, unlysedunlysed red cells, red cells, cryoglobulinscryoglobulins
WBCsWBCsPost transfusionPost transfusionRDWRDW
WBC >50 000/mmWBC >50 000/mm33HyperlipidemiaHyperlipidemia, cold agglutinins, cold agglutininsMCHCMCHC
CryoglobulinsCryoglobulinsCold agglutinins, hyperglycemia, Cold agglutinins, hyperglycemia, WBC >50 000/mmWBC >50 000/mm33
MCVMCV
ClottingClottinghyperlipidemiahyperlipidemia, , hyperbilirubinemiahyperbilirubinemiaHbHb
ClottingClottingWBC >50 000/mmWBC >50 000/mm33RBCsRBCs
Spuriously increasedSpuriously increased Spuriously decreasedSpuriously decreasedParameterParameter
Factors known to cause spurious laboratory Factors known to cause spurious laboratory results in hematology analyzers. results in hematology analyzers.
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Basic Principles for Blood Smear Interpretation
Basic Principles for Blood Smear Basic Principles for Blood Smear InterpretationInterpretation
Assess quality of smears Specimen preparation & staining
Estimate cell numbers RBC: evenly dispersed with minimal intercellular spaceWBC: 10-20/LPFPlatelets: 7-20/OF
Determine predominant cell populations Carefully examine cellular morphology
Assess quality of smears Assess quality of smears Specimen preparation & stainingSpecimen preparation & staining
Estimate cell numbers Estimate cell numbers RBC: evenly dispersed with minimal RBC: evenly dispersed with minimal intercellular spaceintercellular spaceWBC:WBC: 1010--20/LPF20/LPFPlatelets: 7Platelets: 7--20/OF20/OF
Determine predominant cell populations Determine predominant cell populations Carefully examine cellular morphologyCarefully examine cellular morphology
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RBC disordersRBC disordersRBC disorders
Hypochromic microcytic anemiaIron deficiency anemiaThalassemia and hemoglobinopathy
Macrocytic anemiaMegaloblastic anemiaNon-megaloblastic macrocytic anemia
Hemolytic anemiaImmune hemolytic anemia: AIHA, DHTRMicroangiopathic hemolytic anemia (MAHA)Red cell enzymopathies: G-6-PD deficiencyRBC membrane defects: spherocytosis, ovalocytosis, elliptocytosis, stomatocytosis
RBC inclusion bodies and parasites
HypochromicHypochromic microcyticmicrocytic anemiaanemiaIron deficiency anemiaIron deficiency anemiaThalassemiaThalassemia and and hemoglobinopathyhemoglobinopathy
MacrocyticMacrocytic anemiaanemiaMegaloblasticMegaloblastic anemiaanemiaNonNon--megaloblasticmegaloblastic macrocyticmacrocytic anemiaanemia
Hemolytic anemiaHemolytic anemiaImmune hemolytic anemia: AIHA, DHTRImmune hemolytic anemia: AIHA, DHTRMicroangiopathicMicroangiopathic hemolytic anemia (MAHA)hemolytic anemia (MAHA)Red cell Red cell enzymopathiesenzymopathies: G: G--66--PD deficiencyPD deficiencyRBC membrane defects: RBC membrane defects: spherocytosisspherocytosis, , ovalocytosisovalocytosis, , elliptocytosiselliptocytosis, , stomatocytosisstomatocytosis
RBC inclusion bodies and parasitesRBC inclusion bodies and parasites
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Area of blood film examinationArea of blood film examinationArea of blood film examination
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RBC Size and MCVRBC Size and MCVRBC Size and MCV
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WBC disordersWBC disordersWBC disordersLeukopenia
with absolute neutropenia: bone marrow failure, agranulocytosiswith atypical lymphocytes: viral infection, chronic lymphoproliferative disorderswith immature myeloid cells: acute leukemia, MDS or myelopthisis
LeukocytosisReactive leukocytosis: leukemoid reactionAcute leukemia: AML vs. ALLChronic myeloproliferative disordersChronic lymphoproliferative disorders
Leukoerythroblastosis
LeukopeniaLeukopeniawith absolute with absolute neutropenianeutropenia: bone marrow failure, : bone marrow failure, agranulocytosisagranulocytosiswith atypical lymphocytes: viral infection, chronic with atypical lymphocytes: viral infection, chronic lymphoproliferativelymphoproliferative disordersdisorderswith immature myeloid cells: acute leukemia, MDS with immature myeloid cells: acute leukemia, MDS or or myelopthisismyelopthisis
LeukocytosisLeukocytosisReactive Reactive leukocytosisleukocytosis: : leukemoidleukemoid reactionreactionAcute leukemia: AML vs. ALLAcute leukemia: AML vs. ALLChronic Chronic myeloproliferativemyeloproliferative disordersdisordersChronic Chronic lymphoproliferativelymphoproliferative disordersdisorders
LeukoerythroblastosisLeukoerythroblastosis
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Platelet disordersPlatelet disordersPlatelet disorders
Quantitative disordersIsolated thrombocytopenia: Immune vs. non-immuneThrombocytopenia associated with other hematologicabnormalitiesThrombocytosis
Qualitative disordersGiant platelets (megathrombocytes)Platelet inclusion or granule abnormalityBizarre in shape and sizeMegakaryocytes or megakaryoblasts
Quantitative disordersQuantitative disordersIsolated thrombocytopenia: Immune vs. nonIsolated thrombocytopenia: Immune vs. non--immuneimmuneThrombocytopenia associated with other Thrombocytopenia associated with other hematologichematologicabnormalitiesabnormalitiesThrombocytosisThrombocytosis
Qualitative disordersQualitative disordersGiant platelets (Giant platelets (megathrombocytesmegathrombocytes))Platelet inclusion or granule abnormalityPlatelet inclusion or granule abnormalityBizarre in shape and sizeBizarre in shape and sizeMegakaryocytesMegakaryocytes or or megakaryoblastsmegakaryoblasts
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