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CLS416 - Clinical Hematology CLS416 - Clinical Hematology Day 2Day 2

Blood Smear Examination, Blood Smear Examination, Reticulocyte CountsReticulocyte Counts

Pictures/Diagrams Copyright © University of Nebraska

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Manual Cell CountingManual Cell Counting• Dilution, diluting fluid & counting area Dilution, diluting fluid & counting area variesvaries• Hemocytometer has two counting chambersHemocytometer has two counting chambers• Chamber dimensions: 3mm x 3mm x 0.1mmChamber dimensions: 3mm x 3mm x 0.1mm

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ObjectivesObjectives

• Refer to lecture objectives 1-12 on page 34

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Peripheral Blood Smear ExamPeripheral Blood Smear Exam

• The blood smear is a wonderful tool used to The blood smear is a wonderful tool used to verify instrument ‘numbers’verify instrument ‘numbers’– CBC parameters should agree with findings observed CBC parameters should agree with findings observed

on the blood smear on the blood smear

• The CBC & Differential is the most frequently The CBC & Differential is the most frequently requested analysisrequested analysis– Differentials may be automated or manualDifferentials may be automated or manual

• Microscopic exam of a Wright’s stained smear: Microscopic exam of a Wright’s stained smear: – Manual WBC differential Manual WBC differential – WBC estimatesWBC estimates– Platelet estimatesPlatelet estimates

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CBC & Automated DifferentialCBC & Automated Differential

No band # or cell morphology

‘Normal’ reference range for age and sex

WBC Types

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CBC & Manual DifferentialCBC & Manual Differential

Significant variations from normal are noted on report

Sickle cell Sickle cell anemiaanemia

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Peripheral Blood Smear ExamPeripheral Blood Smear Exam• Macroscopic exam of smearMacroscopic exam of smear

• Microscopic exam of smearMicroscopic exam of smear– Low power (10x)Low power (10x)

• Scan to access overall smear qualityScan to access overall smear quality• Detect big, ‘scary’ cellsDetect big, ‘scary’ cells

– High dry power (40x)High dry power (40x)• WBC estimatesWBC estimates

– Oil immersion (100x)Oil immersion (100x)• WBC differentials, cell morphology, platelet estimatesWBC differentials, cell morphology, platelet estimates

10x10x 100x100x10x10x

Fibrin Fibrin

Patie

nt

Nam

e/#

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Peripheral Blood Smear ExamPeripheral Blood Smear Exam• WBC estimates are done to verify the validity of WBC estimates are done to verify the validity of

automated WBC countsautomated WBC counts– 40x, high dry power = HPF40x, high dry power = HPF– Count WBCs in 10 HPFs where Count WBCs in 10 HPFs where red cellsred cells slightlyslightly overlapoverlap– Include fields with no cells in estimateInclude fields with no cells in estimate– Count broken or smudge cellsCount broken or smudge cells– Take average # per HPF x 2,000Take average # per HPF x 2,000– The WBC estimate is never ‘reported’The WBC estimate is never ‘reported’

40x

WBC estimate = 6,000/cmm

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Peripheral Blood Smear ExamPeripheral Blood Smear Exam

• Manual differentials are done if significant patient Manual differentials are done if significant patient abnormalities exist and/or automated differential results abnormalities exist and/or automated differential results are ‘flagged’are ‘flagged’

• Requires well-made/stained smear Requires well-made/stained smear – Oil immersion (OIFs) and high lightOil immersion (OIFs) and high light– Rainbow area where Rainbow area where red cells red cells just touchjust touch

Rainbow area

Stain precipitateToo thickEdge, too thin Under-stained

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Smear Exam - DifferentialsSmear Exam - Differentials

• Differential includes:Differential includes:– WBC differential that classifies WBC typesWBC differential that classifies WBC types

• Slide is scanned, counting and Slide is scanned, counting and identifying 100 consecutive WBCs identifying 100 consecutive WBCs • Reported in % (and absolute #)Reported in % (and absolute #)

– Cell morphology Cell morphology • WBCs, RBCs and platelets are evaluatedWBCs, RBCs and platelets are evaluated• Significant variations from normal cell appearance are Significant variations from normal cell appearance are

noted or quantitated on report noted or quantitated on report give give cluesclues to cause of to cause of a disordera disorder

– Platelet estimate Platelet estimate • Used to verify the accuracy of automated platelet countsUsed to verify the accuracy of automated platelet counts

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WBC DifferentialsWBC Differentials• Reference percentage ranges vary with ageReference percentage ranges vary with age

• Significance:Significance:– Deviations from normal reference ranges may Deviations from normal reference ranges may

indicate disease indicate disease provide provide cluesclues to diagnosis to diagnosis– No immature cellsNo immature cells should be present should be present– May see smudge, broken or necrotic (dying) cellsMay see smudge, broken or necrotic (dying) cells

• Critical values: Blasts cellsCritical values: Blasts cells

^̂Seg Neutrophils and Lymphs Seg Neutrophils and Lymphs are the most numerous WBCs are the most numerous WBCs in the blood of normal adultsin the blood of normal adults

**Due to low #, count Eos & Due to low #, count Eos & Basos using same counter Basos using same counter key…keep track of # of eachkey…keep track of # of each

^

^

**

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Segmented neutrophil

Eosinophil

Basophil

Identify the cells at 1, 2, and 3 Identify the cells at 1, 2, and 3 Match each cell with the correct WBC listed Match each cell with the correct WBC listed

1

2 3

1

2

3

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Large lymphocyte

Monocyte

Small lymphocyte

Identify the cells at 1, 2, and 3 Identify the cells at 1, 2, and 3 Match each cell with the correct WBC listed Match each cell with the correct WBC listed

1

2

3

1

2

3

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Monocyte

Band neutrophil

Identify the cells at 1 and 2 Identify the cells at 1 and 2 Match each cell with the correct WBC listed Match each cell with the correct WBC listed

1

2

21

15

1

2

4

3

5

6

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Segmented neutrophil

Band neutrophil

Eosinophil

Basophil

Small lymphocyte

Large lymphocyte

Monocyte

Identify the cells at 1 through 7Identify the cells at 1 through 7Match each cell with the correct WBC listed Match each cell with the correct WBC listed

1

4

72

6

5

3

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Other Differential FindingsOther Differential Findings

Necrotic neutrophil w pyknotic nucleus Smudge cell

Broken neutrophil

Eosinophil

Broken eos

Band Neutrophil

Neutrophil

Lymph

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Identify the cells at 1, 2, and 3 Identify the cells at 1, 2, and 3 Match each cell with the correct cell listed Match each cell with the correct cell listed

Which cells are normal blood findings?Which cells are normal blood findings?

Nucleated red cell

Blast cells

Small lymphocyte

1

2

3

2

3

1

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Identify the cell shownIdentify the cell shown

A ‘heart’ cellA ‘heart’ cell

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Platelet Estimates Platelet Estimates

Platelet Platelet estimate = estimate = 120,000 uL 120,000 uL

• PLT estimates are done to verify the validity of PLT estimates are done to verify the validity of automated PLT countsautomated PLT counts– Oil immersion (100x) and high light Oil immersion (100x) and high light – Count platelets in 10 OIFs where Count platelets in 10 OIFs where red cells just touchred cells just touch– Include fields with no cells in estimateInclude fields with no cells in estimate– Find the average # per OIFFind the average # per OIF– The platelet estimate is never ‘reported’ The platelet estimate is never ‘reported’

100x

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Platelet Counting ErrorsPlatelet Counting Errors

SchistocytesPlatelet Clumping

Action?? Action??Have blood

sample redrawnPerform manual

platelet count

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Automated Cell CountingAutomated Cell Counting

• Two main principlesTwo main principles– Coulter electronic impedance principleCoulter electronic impedance principle– Optical light scatter principleOptical light scatter principle

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Auto Instrument ExamplesAuto Instrument Examples

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Checks done to prevent errorChecks done to prevent error

• The most fundamental responsibility of a The most fundamental responsibility of a laboratory is to laboratory is to ensure quality test resultsensure quality test results– Assure specimen integrity to prevent pre-analytic errorsAssure specimen integrity to prevent pre-analytic errors– Monitor reagents/instruments with maintenance & QC Monitor reagents/instruments with maintenance & QC

samples to prevent analytic errorssamples to prevent analytic errors

• Checks done to prevent erroneous results:Checks done to prevent erroneous results:– Critical values are confirmed, called and/or redrawnCritical values are confirmed, called and/or redrawn

– ‘‘Flagged’ parameters are verified by smear review or Flagged’ parameters are verified by smear review or manual method…e.g., estimatesmanual method…e.g., estimates

– Patient results are compared to previous resultsPatient results are compared to previous results

– Inconsistent data is verified…instrument values should Inconsistent data is verified…instrument values should correlate with findings observed on the blood smearcorrelate with findings observed on the blood smear

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Example of Hemodilution:Example of Hemodilution:

Line draw @ 04:45 from ICU patient…critically low Hgb & Hct results are obtained

Redraw to confirm was requested

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Correlation of CBC & Blood SmearCorrelation of CBC & Blood Smear

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Reticulocyte CountsReticulocyte Counts• Measures the rate of RBC production by the Measures the rate of RBC production by the

bone marrowbone marrow– Retics appear as polychromasia on a Wright’s stained Retics appear as polychromasia on a Wright’s stained

blood smear…a retic count is done for numberblood smear…a retic count is done for number

• Automated or manual to confirm auto resultsAutomated or manual to confirm auto results– Supravital ‘living cell’ stain Supravital ‘living cell’ stain New methylene blue New methylene blue– Precipitates RNA into filaments or granules Precipitates RNA into filaments or granules

Wright’sWright’s stained smearstained smear

NRBC

Polychromasia

Supravital stained smearSupravital stained smear

Reticulocytes

Supravital Stain

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Reticulocyte CountsReticulocyte Counts• Two “dots” or more is a reticTwo “dots” or more is a retic

– Count 500 red cells, separating mature RBCs vs retics Count 500 red cells, separating mature RBCs vs retics • 1000 total red cells are counted, # of retics/500 RBCs 1000 total red cells are counted, # of retics/500 RBCs

must agree must agree ++ 2 retics on two different slides 2 retics on two different slides• Another slide must be counted if results do not agreeAnother slide must be counted if results do not agree

• Reported in relative number (%), to nearest tenth Reported in relative number (%), to nearest tenth and absolute #, to nearest thousandand absolute #, to nearest thousand– Absolute # is more reliable than %Absolute # is more reliable than %

Reticulocytes

Supravital stainSupravital stain

Supravital stainSupravital stain

Artifact

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Reticulocyte CountsReticulocyte Counts

• Sources of error for manual retic countsSources of error for manual retic counts– Improper blood to stain ratio or inadequate mixingImproper blood to stain ratio or inadequate mixing– Improper countingImproper counting

• Reference rangeReference range– Adult 0.5-2.0%Adult 0.5-2.0% 25-100,000/cmm Absolute#25-100,000/cmm Absolute#– Newborn 2.0-6.0%Newborn 2.0-6.0%

• SignificanceSignificance– ↑ ↑ absolute Retic count….absolute Retic count….ReticulocytosisReticulocytosis

• Increased RBC production Increased RBC production • Will observe increased amount of polychromatophilic red Will observe increased amount of polychromatophilic red

cells on Wright’s stained blood smearcells on Wright’s stained blood smear

– ↓↓ absolute Retic count….Reticulocytopeniaabsolute Retic count….Reticulocytopenia• Decreased RBC productionDecreased RBC production

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