examination of peripheral blood smear - دانشکده پیراپزشکی ... · ppt...
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Examination of Peripheral Blood Smear
A well Made and well Stained Smear can provide:Estimates of cell countProportions of the different types of WBCMorphology
Peripheral Blood SmearObjective
1. Specimen Collection2. Peripheral Smear Preparation3. Staining of Peripheral Blood Smear4. Peripheral Smear Examination
Specimen CollectionVenipuncture
should be collected on an EDTA TubeEDTA liquid form preferred over the powdered formChelates calciumDisodium or Tripotassium ethylenediamine tetra-acetic acid
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
Specimen CollectionDisadvantages:
PLATELET SATELLITOSIScauses pseudothrombocytopenia and
pseudoleukocytosisCause: Platelet specific auto antibodies
that reacts best at room temperature
Specimen CollectionPlatelet satellitosis
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
Peripheral Smear Preparation Wedge technique Coverslip technique Automated Slide Making
and Staining
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
Peripheral Smear Preparation
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
Peripheral Smear PreparationPrecaution: Too large drop = too thick
smear Too small drop = too thin
smear
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
Peripheral Smear Preparation
Peripheral Smear PreparationWedge Technique1. Push Type wedge preparation2. Pull 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 thick
2. Too low Hct: Angle should be raised
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
Peripheral Smear PreparationCover Slip Technique
rarely usedused for Bone marrow aspirate smearsAdvantage: excellent leukocyte distributionDisadvantage: labeling, transport, staining and storage is a problem
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
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
Peripheral Smear PreparationAutomatic Slide Making and StainingSYSMEX 1000i
Peripheral Smear PreparationDrying of Smears
FanHeating 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: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
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
Staining of Peripheral Blood Smear
HEMA-TEK STAINER
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
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
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
Features of a well-stained PBS Troubleshooting:2. RBC too pale, WBC barely visibleCauses: Stain or buffer is too acidic
UnderbufferingOver rinsing
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
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
Peripheral Smear Examination
Microscopic: 40x Objective
1. Correct area where to star counting is determined
2. WBC estimate: internal quality control
Peripheral Smear Examination Microscopic:
100x Objective; OIO1. Highest magnification2. WBC differential counting
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
Peripheral Smear Examination
Too 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 counters
Accuracyof 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 count
200 RBCs/field(200 is the average number of RBC/field)
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
Summarizing WBC parameters
STEP 1WBC increased : leukocytosisWBC decreases: leukopenia
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
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)
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
LEFT SHIFT
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
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
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
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
Summarizing RBC Parameters
Step 5Morphology
1. Size2. Shape3. Inclusions4. Young rbcs5. Color6. Arrangement
Summarizing Platelet ParametersPlatelet count (x 109/L)Mean Platelet Volume MPV, flMorphology