Introduction to CBC and Blood Smear Interpretation

Thanyaphong Na Nakorn, MD, PhD Division of Hematology Department of Medicine Chulalongkorn University
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Complete blood count

z z z z

The most common test used in clinical medicine Determine type and severity of blood cell abnormalities Nowadays, CBC is fully automated and highly reproducible. Correct interpretation of automated CBC can reduce rate of unnecessary blood smear examination Provide useful information for provisional diagnosis of RBC and WBC diseases
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Component of automated CBC

Hb, Hct, Hct, z Blood count basic parameters: Hb, platlet. RBC, WBC, platlet. z Red cell indices: MCV, MCH, MCHC, RDW z WBC differentials z Cytogram or Scattergram z Reticulocyte count

Basic principles of automated blood cell analyzer

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Electrical impedance: Coulter Counter , CellCell-Dyn Dyn Optical impedance and light scatter: TechniconTechnicon-H series

VCS (volume, conductivity, light scatter) technology BeckmanBeckman-Coulter VCS, MAXM, STKS

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Coulter Technology
Cell count and size can be measured by

electrical impedance

J.Paul Robinson J.Paul Robinson

Hydrodynamic Focusing
Sheath fluid Laminar Coaxial Flow

The Bernoulli Effect Direction of flow Velocity Gradient

Viscous drag along walls.

Lower pressure

Particles move to low pressure area 6

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Technicon Technology

VCS Technology

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Instrument Coulter STKS Coulter GENGEN-S Sysmex SESE-9000 Cobas Argos 55-diff (Roche) Technicon H-3 (Bayer) Advia 120 (Bayer) Cell Dyn 4000 (Abbott)

Methodology features Impedance, conductivity, laser technology Impedance, flow cytometry, cytometry, conductivity, laser light scatter (reticulocyte analysis) Direct current (resistance to cell volume); radioradio-frequency (cell density, size) Impedance, light absorption (halogen light source) Cytochemistry, Cytochemistry, flow technology (reticulocyte analysis) Cytochemistry, Cytochemistry, flow technology (reticulocyte analysis) Impedance, laser light scatter
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Red cell parameters

z

Direct Measurement

6/ml (RBC ) (RBC) Erythrocyte Concentration (RBC) x 106 Mean Corpuscular Volume (MCV) (MCV ) Femtolitre (fl) (MCV) (Hb ) (Hb) Gram/decilitre (g/dl) Hemoglobin (Hb) Gram/decilitre

Indirect Measurement

(Hct) Hct) = RBC x MCV/10 Hematocrit (Hct) % (MCH ) = HB x 10 / RBC (MCH) RBC (pg) Mean Corpuscular Hemoglobin (MCH) (MCHC ) Hemoglobin Concentration Concentration (MCHC) (MCHC) Mean Corpuscular Hemoglobin = Hb/Hct x 100 (g/dl) (RDW ) Red Cell Distribution Width (RDW) (RDW) % %
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RBC size and MCV

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Red Cell Distribution Width

RBC x 106/ml

20th percentile

80th percentile

RDW

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RDW =

80th percentile - 20th percentile 80th percentile + 20th percentile

X constant

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three-part differentials WBC: three-part

CellCell-Dyn 1700CS
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Five-part differentials WBC: Five-part

Neutrophil Lymphocyte Monocyte Eosinophil Basophil
lymphocyte Baso Monocyte N Eo

Coulter VCS
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Six-part differentials WBC: Six-part

Peroxidase channel
Neutrophil Lymphocyte Monocyte Eosinophil Basophil Large Unstained Cell

(LUC)

Technicon*H2 Technicon*H2
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Six-part differentials WBC: Six-part

Basophil/lobularity channel

Additional parameters in TechniconTechnicon-H analyzer analyzer Technicon-H

1. 1.

Mean peroxidase activity index (MPXI)

2. Lobularity Index (LI)

Technicon*H2 Technicon*H2

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Platelet parameters
z z z z z z z z

Platelet count Mean Platelet Volume (MPV) Platelet Distribution Width (PDW) Plateletcrit (Pct)

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Reticulocyte count
z z

z z z z

nonnon-nucleated RBC Reticulocyte = non-nucleated with polyribosomal RNA as stained by supravital stain (new methylene blue or brilliant cresyl blue) Polychromasia underestimates reticulocytes Three methods of reticulocyte enumeration Manual count on slide per 1,000 RBC Automated CBC with counter (Coulter reticulocyte counter VCS, Cell-Dyne CellCell-Dyne 4000, TechniconTechniconH3) Flow cytometry with fluorescent dyes

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Coulter Report from Coulter

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Technicon-H Report from Technicon-H

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Clinical Utility of automated CBC

z Differential diagnosis of RBC disorders
Microcytic RBC

z Use WBC differential cytogram to guide

WBC morphology and phenotypes

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What is your diagnosis?

WBC RBC Hb HCT MCV MCH MCHC RDW HDW PLT 5.00 3.56 6.0 21.0 59.0 17.0 28.8 19.4 4.02 427 x103/L x106/L g/dL % fL pg g/dL % g/dL x103/L

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Differential diagnosis of anemia using MCV and RDW

Low MCV Thalassemia trait Heterozygous HbE, HbE, HbC, HbC, etc. RDW <15 Anemia of chronic disease (ACD) Iron deficiency anemia Thalassemia intermedia Sideroblastic anemia RDW >15 Severe ACD RBC fragmentation Normal MCV ACD Heterozygous HbS, HbS, HbCS, HbCS, HbE, HbE, etc. Hereditary spherocytosis Acute hemorrhage Early or combined nutritional deficiency Myelodysplasia Myelophthisis Sickle cell anemia or Homozygous HbCS High MCV Aplastic anemia MDS Myeloma Liver disease Hyperthyroidism B12 deficiency Folate deficiency AIHA Drugs: HU, ARV, AZA, etc.

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Factors known to cause spurious laboratory results in hematology analyzers.

Parameter RBCs Hb MCV MCHC RDW WBCs Platelets Spuriously increased WBC >50 000/mm3 hyperlipidemia, hyperlipidemia, hyperbilirubinemia Cold agglutinins, hyperglycemia, WBC >50 000/mm3 Hyperlipidemia, Hyperlipidemia, cold agglutinins Post transfusion Nucleated red cells, platelet clumps, unlysed red cells, cryoglobulins WBC fragmentation, severe microcytosis, microcytosis, cryoglobulins Spuriously decreased Clotting Clotting Cryoglobulins WBC >50 000/mm3 Clotting Satellitism, Satellitism, clumping
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Basic Principles for Blood Smear Interpretation

z z

z z

Assess quality of smears Specimen preparation & staining Estimate cell numbers RBC: evenly dispersed with minimal intercellular space 10-20/LPF WBC: 10-20/LPF 7-20/OF Platelets: 7-20/OF Determine predominant cell populations Carefully examine cellular morphology
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RBC disorders
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Hypochromic microcytic anemia Iron deficiency anemia Thalassemia and hemoglobinopathy Macrocytic anemia Megaloblastic anemia Non Non-megaloblastic Non-megaloblastic macrocytic anemia Hemolytic anemia Immune hemolytic anemia: AIHA, DHTR Microangiopathic hemolytic anemia (MAHA) G Red cell enzymopathies: enzymopathies: G-6-PD G-6-PD deficiency RBC membrane defects: spherocytosis, spherocytosis, ovalocytosis, elliptocytosis, elliptocytosis, stomatocytosis ovalocytosis, RBC inclusion bodies and parasites

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Area of blood film examination

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RBC Size and MCV

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WBC disorders
Leukopenia with absolute neutropenia: neutropenia: bone marrow failure, agranulocytosis with atypical lymphocytes: viral infection, chronic lymphoproliferative disorders with immature myeloid cells: acute leukemia, MDS or myelopthisis z Leukocytosis z Reactive leukocytosis: leukocytosis: leukemoid reaction Acute leukemia: AML vs. ALL Chronic myeloproliferative disorders disorders Chronic lymphoproliferative lymphoproliferative disorders disorders z Leukoerythroblastosis z
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Platelet disorders
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Quantitative disorders non Isolated thrombocytopenia: Immune vs. non-immune non-immune Thrombocytopenia Thrombocytopenia associated associated with with other other hematologic abnormalities Thrombocytosis Qualitative disorders (megathrombocytes ) Giant platelets (megathrombocytes) (megathrombocytes) Platelet inclusion or granule abnormality Bizarre in shape and size Megakaryocytes or megakaryoblasts megakaryoblasts

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