Examination of Peripheral Blood Smear

Examination of Peripheral Blood SmearA well Made and well Stained Smear can provide:Estimates of cell countProportions of the different types of WBCMorphologyPeripheral Blood SmearObjective1. Specimen Collection2. Peripheral Smear Preparation3. Staining of Peripheral Blood Smear4. Peripheral Smear ExaminationSpecimen CollectionVenipunctureshould be collected on an EDTA TubeEDTA liquid form preferred over the powdered formChelates calciumDisodium or Tripotassium ethylenediamine tetra-acetic acidSpecimen CollectionAdvantagesMany smears can be done in just a single drawImmediate preparation of the smear is not necessaryPrevents platelet clumping on the glass slide

Specimen CollectionDisadvantages:PLATELET SATELLITOSIScauses pseudothrombocytopenia and pseudoleukocytosisCause: Platelet specific auto antibodies that reacts best at room temperatureSpecimen CollectionPlatelet satellitosis

Specimen CollectionSolution recollect specimen using Sodium Citrate in a 9:1 dilutionCorrection for dilution2.7 ml blood0.3 ml anticoagulant9/10 dilution is reciprocal 10/9 = 1.1 all computations for WBC and Platelet should be multiplied to 1.1Peripheral Smear PreparationWedge techniqueCoverslip techniqueAutomated Slide Making and StainingPeripheral Smear PreparationWedge techniqueEasiest to masterMost convenient and most commonly used techniqueMaterial neededGlass slide 3 in X 1inBeveled/chamfered edges

Peripheral Smear Preparation

Peripheral Smear PreparationProcedures:Drop 2-3 mm blood at one end of the slideDiff safe can be useda. Easy droppingb. Uniform drop

Peripheral Smear PreparationPrecaution: Too large drop = too thick smear Too small drop = too thin smearPeripheral Smear PreparationThe 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

Peripheral Smear Preparation

Peripheral Smear PreparationWedge TechniquePush Type wedge preparationPull Type wedge prepartion 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. Peripheral Smear PreparationPrecautions: Angle correction:1. In case of Polycythemia: high Hct angle should be lowered- ensure that the smear made is not to thick2. Too low Hct: Angle should be raised Feature of a Well Made Wedge SmearSmear is 2/3 or the entire slideSmear is finger shaped, very slightly rounded at the feathery edge: widest area of examinationLateral 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

Peripheral Smear PreparationCover Slip Techniquerarely usedused for Bone marrow aspirate smearsAdvantage: excellent leukocyte distributionDisadvantage: labeling, transport, staining and storage is a problemPeripheral 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 smearsPeripheral 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 impossibleToo little pressure prevents the bone spicules from spreading satisfactorily on the slidePeripheral Smear PreparationAutomatic Slide Making and StainingSYSMEX 1000i

Peripheral Smear PreparationDrying of SmearsFanHeating pansNo breath blowing of smears may produce crenated RBCs or develop water artifact (drying artifact)

Staining of Peripheral Blood Smear Wright Staing MethodAutomated Slide StainersQuick Stains

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

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

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

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

Staining of Peripheral Blood Smear What contributes to the problem:humidity in the air as you air dry the slides.Water absorbed from the humid air into the alcohol based stainSolution:Drying the slide as quickly as possible.Fix with pure anhydrous methanol before staining.Use of 20% v/v methanol

Staining of Peripheral Blood Smear AUTOMATED SLIDE STAINERSIt takes about 5-10 minutes to stain a batch of smearsSlides are just automatically dipped in the stain in the buffer and a series of rinsesDisadvantages:Staining process has begun, no STAT slides can be added in the batchAqueous solutions of stains are stable only after 3-6 hours

Staining of Peripheral Blood Smear

HEMA-TEK STAINERStaining of Peripheral Blood Smear QUICK STAINSFast, convenient and takes about 1 minute to be accomplishedModified Wrights-Giemsa Stain, buffer is aged distilled waterCost effectiveDisadvantage:Quality of stains especially on color acceptanceFor small laboratories and for physicians clinic only

Features of a well-stained PBSMacroscopically: color should be pink to purpleMicroscopically:RCS: orange to salmon pinkWBC: nuclei is purple to bluecytoplasm is pink to tan granules is lilac to violet Eosinophil: granules orange Basophil: granules dark blue to blackFeatures of a well-stained PBSTroubleshooting:RBC gray, WBC too dark Eosinophil granules are grayCause: stain or buffer is to alkaline inadequate rinsing Prolonged staining heparinized sampleFeatures of a well-stained PBSTroubleshooting:RBC too pale, WBC barely visibleCauses:Stain or buffer is too acidicUnderbufferingOver rinsingPeripheral Smear ExaminationMacroscopicOverall bluer color: increased blood proteins (multiple myeloma, rouleaux formation)Grainy appearance: RBC agglutination (cold hemagglutinin diseases)Holes: increased lipidBlue specks at the feathery edge: Increased WBC and Platelet countsPeripheral Smear ExaminationMicroscopic:10x ObjectiveAssess 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 distributionSnow-plow effect: more than 4x/cells per field on the feathery edge: RejectFibrin strands: RejectRouleaux formation, large blast cell assessment Peripheral Smear ExaminationMicroscopic:40x ObjectiveCorrect area where to star counting is determinedWBC estimate: internal quality control

Peripheral Smear ExaminationMicroscopic:100x Objective; OIOHighest magnificationWBC differential counting

Peripheral Smear ExaminationOptimal Assessment Area:RBCs are uniformly and singly distributedFew RBC are touching or overlappingNormal biconcave appearance200 to 250 RBC per 100x OIO

Peripheral Smear ExaminationToo thin

Too thick

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

Performance of a White Blood Cell Differential Count100 WBCs are counted using a push down counters (Clay Admas Laboratory counters,Biovation diff countersAccuracyof Diff Count:Count 200 WBC if WBC>40 x 109/LExtremely low WBC counts, do the Diff count under 50X OIO

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

RBC MorphologyAnisocytosisPoikilocytosisCellular Inclusions

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 count200 RBCs/field(200 is the average number of RBC/field) Summarizing WBC parametersTotal WBC counts per (WBC x 109/L)WBC differential counts are percentagesWBC differential count values expressed as actual number of each type of cellWBC morphologySummarizing WBC parametersSTEP 1WBC increased : leukocytosisWBC decreases: leukopeniaSummarizing WBC parametersSTEP 2Relative differential count

Cell TypeIncreasesDecreasesNeutrophilNeutrophiliaNeutropeniaEosinophilEosinophilia

N/A

BasophilBasophilia

N/ALymphocyteLymphocytosisLymphopeniaMonocyteMonocytosisMonocytopeniaSummarizing WBC parametersSTEP 3Absolute Cell CountsEx. WBC 13.6PMNs 67Lym 26Eos 3Baso 3Mono 1Absolute 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)

Summarizing WBC parametersSTEP 4Examination for immature cellsYoung cells should not be seen in the peripheral blood smearImmature cells: possess a nucleusdo not lyse during testingcan be counted as WBC and falsely elevate WBC results

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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 distributionMorphologySummarizing 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 cellsSummarizing RBC ParametersStep 3Examine MCHCDescribes how well the cells are filled with HbHypochromic, normochromic2 conditions when MCHC should be evaluated:1. spherocytosis: slight elevation2. lipemia/icterus: markedly increaseSummarizing RBC ParametersStep 4Examine MCHCDescribes how well the cells are filled with HbHypochromic, normochromic2 conditions when MCHC should be evaluated:1. spherocytosis: slight elevation2. lipemia/icterus: markedly increaseSummarizing RBC ParametersStep 5MorphologySizeShapeInclusionsYoung rbcsColorArrangement

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