Examination of Peripheral Blood Smear

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Examination of Peripheral Blood Smear. Dr S Homathy. Complete blood count The most common test used in clinical medicine Determine type and severity of blood cell abnormalities Nowadays, CBC is fully automated and highly reproducible. - PowerPoint PPT Presentation

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  • Examination of Peripheral Blood SmearDr S Homathy*

  • Complete blood count 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*

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

  • Preparation of blood smearThere are three types of blood smears: The cover glass smear.The wedge smear .The spun smear.The are two additional types of blood smear used for specific purposesBuffy coat smear for WBCs < 1.0109/L Thick blood smears for blood parasites .

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  • Peripheral Blood SmearObjective1. Specimen Collection2. Peripheral Smear Preparation3. Staining of Peripheral Blood Smear4. Peripheral Smear Examination*

  • Specimen CollectionVenipunctureshould be collected on an EDTA (Disodium or Tripotassium ethylene diamine tetra-acetic acid) TubeEDTA liquid form preferred over the powdered formChelates calcium

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  • Specimen 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 temperature*

  • Specimen CollectionPlatelet satellitosis*

  • Peripheral Smear PreparationWedge techniqueCoverslip techniqueAutomated Slide Making and Staining*

  • Peripheral Smear PreparationWedge techniquePush Type wedge preparationPull Type wedge prepartion

    Easiest to masterMost convenient and most commonly used technique

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  • Material neededGlass slide 3 in X 1inBeveled/chamfered edges

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  • 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*

  • Precaution: Too large drop = too thick smear Too small drop = too thin smear*

  • 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

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  • Control thickness of the smear by changing the angle of spreader slideAllow the blood film to air-dry completely before staining.Do not blow to dry. The moisture from your breath will cause RBC artifact*

  • Peripheral Smear Preparation*

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  • 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.

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  • 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 Smear

    Smear is 2/3 or 3/4 the entire slide

    Smear is finger shaped, very slightly rounded at the feathery edge: widest area of examination

    Lateral edges of the smear visible

    Should not touch any edge of the slide.*

  • Should be margin free, except for point of application

    Smear is smooth without irregularities, holes or streaks

    When held up in light: feathery edge should show rainbow appearance

    Entire whole drop of blood is picked up and spread*

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  • **

  • Cover Slip Techniquerarely usedused for Bone marrow aspirate smearsAdvantage: excellent leukocyte distributionDisadvantage: labeling, transport, staining and storage is a problem*

  • 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 4. Mounted on a 3x1 inch glass slide*

  • Precautions:

    Very light pressure should be applied between the index finger and the thumb

    Crush preparation technique

    Too much pressure causes rupture of the cells making morphologic examination impossible

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

  • * tail body head

  • Thin area: Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.

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  • Thick area: Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion.

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  • Common causes of a poor blood smear

    Drop of blood too large or too small.Spreader slide pushed across the slide in a jerky manner.Failure to keep the entire edge of the spreader slide against the slide while making the smear.Failure to keep the spreader slide at a 30 angle with the slide.Failure to push the spreader slide completely across the slide.*

  • 6.Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide

    7.Holes in film: Slide contaminated with fat or grease

    8.Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water

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  • Biologic causes of a poor smear

    Cold agglutinin: RBCs will clump together. Warm the blood at 37 C for 5 minutes, and then remake the smear.Lipemia: holes will appear in the smear. There is nothing you can do to correct this.Rouleaux: RBCs will form into stacks resembling coins. There is nothing you can do to correct this

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  • Automatic Slide Making and StainingSYSMEX 1000i

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

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  • Romanowsky stainingLeishman's stain : a polychromatic stainMethanol : fixes cells to slidemethylene blue stains RNA,DNAblue-grey color Eosin stains hemoglobin, eosin granulesorange-red color pH value of phosphate buffer is very important

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  • Pure Wright stain or Wright Giemsa stainBlood smears and bone marrow aspirate

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  • ProcedureThin smear are air dried.Flood the smear with stain. Stain for 1-5 min. Experience will indicate the optimum time. Add an equal amount of buffer solution and mix the stain by blowing an eddy in the fluid.Leave the mixture on the slide for 10-15 min. Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain.Stand slide on end, and let dry in air.

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  • 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 black

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

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  • Trouble shootingMacroscopicOverall 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 counts

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  • Microscopic: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

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  • *Too acidic Suitable Too basic

  • Too Acid Stain:RBC too pale, WBC barely visibleinsufficient staining timeprolonged buffering or washingold stain

    Correction:lengthen staining timecheck stain and buffer pHshorten buffering or wash time

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  • Too Alkaline Stain:RBC gray, WBC too dark, Eosinophil granules are graythick blood smearprolonged staininginsufficient washingalkaline pH of stain componentsheparinized sample

    Correction :check pHshorten stain timeprolong buffering time

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  • Problem encountered during stainingWater artifact: moth eaten RBC, heavily demarcated central pallor on the RBC surface, crenation, refractory shiny blotches on the RBC

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  • 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

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  • 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 rinses

    Disadvantages:Staining process has begun, no STAT slides can be added in the batchAqueous solutions of stains are stable only after 3-6 hours

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  • QUICK STAINSFast, convenient and takes about 1 minute to be accomplishedModified Wright