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Examination of Peripheral Blood Smear

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Page 1: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Examination of Peripheral Blood Smear

Page 2: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

A well Made and well Stained Smear can provide:Estimates of cell countProportions of the different types of WBCMorphology

Page 3: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Blood SmearObjective

1. Specimen Collection2. Peripheral Smear Preparation3. Staining of Peripheral Blood Smear4. Peripheral Smear Examination

Page 4: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Specimen CollectionVenipuncture

should be collected on an EDTA TubeEDTA liquid form preferred over the powdered formChelates calciumDisodium or Tripotassium ethylenediamine tetra-acetic acid

Page 5: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Specimen Collection Advantages1. Many smears can be done in just a single

draw2. Immediate preparation of the smear is

not necessary3. Prevents platelet clumping on the glass

slide

Page 6: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Specimen CollectionDisadvantages:

PLATELET SATELLITOSIScauses pseudothrombocytopenia and

pseudoleukocytosisCause: Platelet specific auto antibodies

that reacts best at room temperature

Page 7: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Specimen CollectionPlatelet satellitosis

Page 8: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Specimen CollectionSolution recollect specimen using Sodium Citrate

in a 9:1 dilutionCorrection for dilution

2.7 ml blood0.3 ml anticoagulant9/10 dilution is reciprocal 10/9 = 1.1

all computations for WBC and Platelet should be multiplied to 1.1

Page 9: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation Wedge technique Coverslip technique Automated Slide Making

and Staining

Page 10: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation Wedge technique1. Easiest to master2. Most convenient and most commonly

used technique Material needed1. Glass slide 3 in X 1in2. Beveled/chamfered edges

Page 11: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation

Page 12: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation Procedures:1. Drop 2-3 mm blood at one end of the

slideDiff safe can be used

a. Easy droppingb. Uniform drop

Page 13: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationPrecaution: Too large drop = too thick

smear Too small drop = too thin

smear

Page 14: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation2. The pusher slide be

held securely with the dominant hand in a 30-45 deg angle.- quick, swift and smooth gliding motion to the other side of the slide creating a wedge smear

Page 15: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation

Page 16: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationWedge Technique1. Push Type wedge preparation2. Pull Type wedge prepartion

Page 17: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationPrecautions: Ensure that the whole drop of blood is picked

up and spread Too slow a slide push will accentuate poor

leukocyte distribution, larger cells are pushed at the end of the slide

Maintain an even gentle pressure on the slide Keep the same angle all the way to the end

of the smear.

Page 18: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationPrecautions: Angle correction:

1. In case of Polycythemia: high Hct angle should be lowered- ensure that the smear made is not to thick

2. Too low Hct: Angle should be raised

Page 19: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Feature of a Well Made Wedge Smear

Smear is 2/3 or ¾ the entire slideSmear is finger shaped, very slightly

rounded at the feathery edge: widest area of examination

Lateral edges of the smear visibleSmear is smooth without irregularities,

holes or streaksWhen held up in light: feathery edge

should show rainbow appearanceEntire whole drop of blood is picked up

and spread

Page 20: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral
Page 21: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationCover Slip Technique

rarely usedused for Bone marrow aspirate smearsAdvantage: excellent leukocyte distributionDisadvantage: labeling, transport, staining and storage is a problem

Page 22: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation 22 x 27mm clean coverslipMore routinely used for bone marrow

aspirateTechnique:

1. A drop of marrow aspirate is placed on top of 1 coverslip2. Another coverslip is placed over the other allowing the aspirate to spread.3. One is pulled over the other to create 1 thin smears

Page 23: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Preparation4. Mounted on a 3x1 inch glass slidePrecautions:Very lgiht pressure should be applied

between the index finger and the thumbCrush preparation techniqueToo much pressure causes rupture of the

cells making morphologic examination impossible

Too little pressure prevents the bone spicules from spreading satisfactorily on the slide

Page 24: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationAutomatic Slide Making and StainingSYSMEX 1000i

Page 25: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear PreparationDrying of Smears

FanHeating pansNo breath blowing of smears – may produce crenated RBCs or develop water artifact (drying artifact)

Page 26: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear Wright Staing MethodAutomated Slide StainersQuick Stains

Page 27: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear Pure Wright stain or Wright Giemsa stainBlood smears and bone marrow aspiratePolychrome stains: Eosin and Methylene

blue stainsPurpose: see and evaluate cell morphology

Page 28: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear Eosin + Methylene Blue = thiazine

eosinate complexThe complex will not stain any color unless

a buffer is added: 0.05M sodium phosphate (pH 6.4) and aged distilled water (pH 6.4-6.8)

Methanol is added to fix the cells on the slide

Page 29: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

Free Methylene Blue: - basic- stains acidic cellular

components such as RNAFree Eosin

- acidic- stains basic cellular components

such as Hgb and eosinophilic granules

Page 30: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

Problem encountered during stainingWater artifact: moth eaten RBC, heavily

demarcated central pallor on the RBC surface, crenation, refractory shiny blotches on the RBC

Page 31: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

What contributes to the problem:1. humidity in the air as you air dry the slides.2. Water absorbed from the humid air into the

alcohol based stain Solution:1. Drying the slide as quickly as possible.2. Fix with pure anhydrous methanol before

staining.3. Use of 20% v/v methanol

Page 32: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

AUTOMATED SLIDE STAINERS1. It takes about 5-10 minutes to stain a batch of

smears2. Slides are just automatically dipped in the stain in

the buffer and a series of rinses Disadvantages:1. Staining process has begun, no STAT slides can

be added in the batch2. Aqueous solutions of stains are stable only after

3-6 hours

Page 33: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

HEMA-TEK STAINER

Page 34: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Staining of Peripheral Blood Smear

QUICK STAINS Fast, convenient and takes about 1 minute to be

accomplished Modified Wrights-Giemsa Stain, buffer is aged

distilled water Cost effective Disadvantage:Quality of stains especially on color acceptanceFor small laboratories and for physician’s clinic only

Page 35: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Features of a well-stained PBS

Macroscopically: color should be pink to purple

Microscopically:RCS: orange to salmon pinkWBC: nuclei is purple to blue

cytoplasm is pink to tan granules is lilac to violet

Eosinophil: granules orange Basophil: granules dark blue to black

Page 36: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Features of a well-stained PBS Troubleshooting:1. RBC gray, WBC too dark Eosinophil

granules are grayCause: stain or buffer is to alkaline

inadequate rinsing Prolonged staining heparinized sample

Page 37: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Features of a well-stained PBS Troubleshooting:2. RBC too pale, WBC barely visibleCauses: Stain or buffer is too acidic

UnderbufferingOver rinsing

Page 38: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination

Macroscopic1. Overall bluer color: increased blood

proteins (multiple myeloma, rouleaux formation)

2. Grainy appearance: RBC agglutination (cold hemagglutinin diseases)

3. Holes: increased lipid4. Blue specks at the feathery edge:

Increased WBC and Platelet counts

Page 39: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination

Microscopic: 10x Objective

1. Assess overall quality of the smear i.e feathery edge, quality of the color, distributin of the cells and the lateral edges can be checked for WBC distribution

2. Snow-plow effect: more than 4x/cells per field on the feathery edge: Reject

3. Fibrin strands: Reject4. Rouleaux formation, large blast cell

assessment

Page 40: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination

Microscopic: 40x Objective

1. Correct area where to star counting is determined

2. WBC estimate: internal quality control

Page 41: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination Microscopic:

100x Objective; OIO1. Highest magnification2. WBC differential counting

Page 42: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination

Optimal Assessment Area:1. RBCs are uniformly and singly distributed2. Few RBC are touching or overlapping3. Normal biconcave appearance4. 200 to 250 RBC per 100x OIO

Page 43: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Peripheral Smear Examination

Too thin Too thick

Page 44: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Performance of a White Blood Cell Differential CountSystematicChoose the best area for assesmentBack and forth serpentine or battlement

track patters in preferred

Page 45: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Performance of a White Blood Cell Differential Count100 WBCs are counted using a push down

counters (Clay Admas Laboratory counters,Biovation diff counters

Accuracyof Diff Count:Count 200 WBC if WBC>40 x 109/LExtremely low WBC counts, do the Diff count under 50X OIO

Page 46: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Performance of a White Blood Cell Differential CountExtremely low WBC counts, do the Diff

count under 50X OIOExtremely low WBCs: WBC are

concentrated, buffy coat smears are made

Page 47: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

RBC MorphologyAnisocytosisPoikilocytosisCellular Inclusions

Page 48: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Platelet EstimateChoose an area where RBC barely touchNo. of platelet in 10 OIO fields is counted

multiplied by 20,000Anemia or ErythrocytosisAverage No. of Plts/field x total RBC count

200 RBCs/field(200 is the average number of RBC/field)

Page 49: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing WBC parameters

1. Total WBC counts per (WBC x 109/L)2. WBC differential counts are percentages3. WBC differential count values expressed

as actual number of each type of cell4. WBC morphology

Page 50: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing WBC parameters

STEP 1WBC increased : leukocytosisWBC decreases: leukopenia

Page 51: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing WBC parametersSTEP 2

Relative differential count

Cell Type Increases Decreases

Neutrophil Neutrophilia Neutropenia

Eosinophil Eosinophilia N/A

Basophil Basophilia N/A

Lymphocyte Lymphocytosis

Lymphopenia

Monocyte Monocytosis Monocytopenia

Page 52: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing WBC parameters STEP 3Absolute Cell CountsEx. WBC 13.6

PMNs 67Lym 26Eos 3Baso 3Mono 1

Absolute Neutrophil Count: 9.1 (NV: 2.4-8.2)Absolute Lymphocyte Cout: 3.5 ((NV: 1.4-

4.0)Absolute Monocyte Count: 0.4 (NV: 0.1-1.2)

Page 53: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing WBC parameters STEP 4Examination for immature cellsYoung cells should not be seen in the peripheral blood smearImmature cells: possess a nucleus

do not lyse during testing

can be counted as WBC and falsely elevate WBC results

Page 54: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

LEFT SHIFT

Page 55: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing RBC ParametersRBC Count )RBC x 1012/L)Hb (g/dl)Hct (5 or L/L)Mean Cell Volume (MCV. Fl)Mean Cell Hb (MCH, pg)Mean Cell Hb Concentration (MCHC. %,

g/dl)RBC distributionMorphology

Page 56: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing RBC ParametersStep1

Examne Hb an Hct for anemia or polycythemiaIf the RBC morphology is normal: Use rule of three to estimate the Hct

Step 2MCV: to check and correlate to the morpholic apperance of the cells

Page 57: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing RBC Parameters

Step 3Examine MCHCDescribes how well the cells are filled with HbHypochromic, normochromic2 conditions when MCHC should be evaluated:1. spherocytosis: slight elevation2. lipemia/icterus: markedly increase

Page 58: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing RBC Parameters

Step 4Examine MCHCDescribes how well the cells are filled with HbHypochromic, normochromic2 conditions when MCHC should be evaluated:1. spherocytosis: slight elevation2. lipemia/icterus: markedly increase

Page 59: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing RBC Parameters

Step 5Morphology

1. Size2. Shape3. Inclusions4. Young rbcs5. Color6. Arrangement

Page 60: Examination of Peripheral Blood Smear - دانشکده پیراپزشکی ... · PPT file · Web view · 2012-12-22Peripheral Blood Smear. Objective. 1. Specimen Collection. 2. Peripheral

Summarizing Platelet ParametersPlatelet count (x 109/L)Mean Platelet Volume MPV, flMorphology