Examination of Peripheral Blood Smear

Report
Examination of Peripheral Blood
Smear
A well Made and well Stained Smear can
provide:
 Estimates of cell count
 Proportions of the different types of WBC
 Morphology
Peripheral Blood Smear
 Objective
1.
2.
3.
4.
Specimen Collection
Peripheral Smear Preparation
Staining of Peripheral Blood
Smear
Peripheral Smear Examination
Specimen Collection
 Venipuncture
should be collected on an EDTA Tube
EDTA liquid form preferred over the
powdered form
Chelates calcium
Disodium or Tripotassium ethylenediamine tetra-acetic acid
Specimen Collection

1.
2.
3.
Advantages
Many smears can be done in just a single draw
Immediate preparation of the smear is not necessary
Prevents platelet clumping on the glass slide
Specimen Collection
 Disadvantages:
PLATELET SATELLITOSIS
 causes pseudothrombocytopenia and pseudoleukocytosis
 Cause: Platelet specific auto antibodies that reacts best at
room temperature
Specimen Collection
 Platelet satellitosis
Specimen Collection
 Solution
recollect specimen using Sodium Citrate in a 9:1 dilution
Correction for dilution
2.7 ml blood
0.3 ml anticoagulant
9/10 dilution is reciprocal 10/9 = 1.1
all computations for WBC and Platelet should be multiplied
to 1.1
Peripheral Smear Preparation


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Wedge technique
Coverslip technique
Automated Slide Making
and Staining
Peripheral Smear Preparation

1.
2.
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1.
2.
Wedge technique
Easiest to master
Most convenient and most commonly used technique
Material needed
Glass slide 3 in X 1in
Beveled/chamfered edges
Peripheral Smear Preparation
Peripheral Smear Preparation

1.
Procedures:
Drop 2-3 mm blood at one end of the slide
Diff safe can be used
a. Easy dropping
b. Uniform drop
Peripheral Smear Preparation
Precaution: Too large drop = too thick
smear
Too small drop = too thin
smear
Peripheral Smear Preparation
2.
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 Preparation
Wedge Technique
1. Push Type wedge preparation
2. Pull Type wedge prepartion
Peripheral Smear Preparation
Precautions:
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 Preparation
Precautions:
Angle correction:
1. In case of Polycythemia: high Hct
should be lowered
- ensure that the smear made is not
2. Too low Hct: Angle should be raised
angle
to thick
Feature of a Well Made Wedge Smear
 Smear is 2/3 or ¾ the entire slide
 Smear is finger shaped, very slightly rounded at the




feathery edge: widest area of examination
Lateral edges of the smear visible
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
Peripheral Smear Preparation
 Cover Slip Technique
rarely used
used for Bone marrow aspirate smears
Advantage: excellent leukocyte distribution
Disadvantage: labeling, transport, staining and storage is a
problem
Peripheral Smear Preparation
 22 x 27mm clean coverslip
 More routinely used for bone marrow aspirate
 Technique:
1. A drop of marrow aspirate is
placed on top of 1
coverslip
2. 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
Preparation
4. Mounted on a 3x1 inch glass slide
Precautions:
 Very lgiht 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
Peripheral Smear Preparation
 Automatic Slide Making and Staining
 SYSMEX 1000i
Peripheral Smear Preparation
 Drying of Smears
Fan
Heating pans
No breath blowing of smears – may produce crenated RBCs
or develop water artifact (drying artifact)
Staining of Peripheral Blood Smear
 Wright Staing Method
 Automated Slide Stainers
 Quick Stains
Staining of Peripheral Blood Smear
 Pure Wright stain or Wright Giemsa stain
 Blood smears and bone marrow aspirate
 Polychrome stains: Eosin and Methylene blue stains
 Purpose: see and evaluate cell morphology
Staining of Peripheral Blood Smear
 Eosin + Methylene Blue = thiazine eosinate complex
 The 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 RNA
 Free Eosin
- acidic
- stains basic cellular components such as Hgb and
eosinophilic granules
Staining of Peripheral Blood Smear
Problem encountered during staining
Water artifact: moth eaten RBC, heavily demarcated central pallor
on the RBC surface, crenation, refractory shiny blotches on the
RBC

Staining of Peripheral Blood Smear

1.
2.
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1.
2.
3.
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 stain
Solution:
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

1.
2.

1.
2.
AUTOMATED SLIDE STAINERS
It takes about 5-10 minutes to stain a batch of smears
Slides 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 batch
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 acceptance
For 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 pink
WBC:
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

1.
Troubleshooting:
RBC gray, WBC too dark Eosinophil granules are gray
Cause: 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 visible
Causes:Stain or buffer is too acidic
Underbuffering
Over rinsing

Peripheral Smear Examination

1.
2.
3.
4.
Macroscopic
Overall bluer color: increased blood proteins (multiple
myeloma, rouleaux formation)
Grainy appearance: RBC agglutination (cold hemagglutinin
diseases)
Holes: increased lipid
Blue specks at the feathery edge: Increased WBC and
Platelet counts
Peripheral Smear Examination

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Microscopic:
10x Objective
1.
2.
3.
4.
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
Snow-plow effect: more than 4x/cells per field on the
feathery edge: Reject
Fibrin strands: Reject
Rouleaux formation, large blast cell assessment
Peripheral Smear Examination

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Microscopic:
40x Objective
1.
2.
Correct area where to star counting is determined
WBC estimate: internal quality control


Peripheral
Smear
Examination
Microscopic:
100x Objective; OIO
1.
2.
Highest magnification
WBC differential counting

1.
2.
3.
4.
Peripheral Smear Examination
Optimal Assessment Area:
RBCs are uniformly and singly distributed
Few RBC are touching or overlapping
Normal biconcave appearance
200 to 250 RBC per 100x OIO
Peripheral Smear Examination
 Too thin
Too thick
Performance of a White Blood Cell
Differential Count
 Systematic
 Choose the best area for assesment
 Back and forth serpentine or battlement track patters in
preferred
Performance of a White Blood Cell
Differential Count
 100 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/L
Extremely low WBC counts, do the Diff count under 50X
OIO
Performance of a White Blood Cell
Differential Count
 Extremely low WBC counts, do the Diff count under 50X
OIO
 Extremely low WBCs: WBC are concentrated, buffy coat
smears are made
RBC Morphology
Anisocytosis
Poikilocytosis
Cellular Inclusions
Platelet Estimate
 Choose an area where RBC barely touch
 No. of platelet in 10 OIO fields is counted multiplied by 20,000
 Anemia or Erythrocytosis
 Average No. of Plts/field x total RBC count
200 RBCs/field
(200 is the average number of RBC/field)
Summarizing WBC parameters
1.
2.
3.
4.
Total WBC counts per (WBC x 109/L)
WBC differential counts are percentages
WBC differential count values expressed as actual number
of each type of cell
WBC morphology
Summarizing WBC parameters

STEP 1
WBC increased : leukocytosis
WBC decreases: leukopenia
Summarizing WBC parameters
 STEP 2
Relative
differential
count
Cell Type
Increases
Decreases
Neutrophil
Neutrophilia Neutropenia
Eosinophil
Eosinophilia N/A
Basophil
Basophilia
N/A
Lymphocyte Lymphocyto Lymphopeni
sis
a
Monocyte
Monocytosis Monocytope
nia
Summarizing
WBC parameters
STEP
3
Absolute Cell Counts
Ex.
WBC 13.6
PMNs 67
Lym 26
Eos
3
Baso 3
Mono 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)
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Summarizing
WBC parameters
STEP
4
Examination for immature cells
Young cells should not be seen in the peripheral blood smear
Immature cells: possess a nucleus
do not lyse during
testing
can be counted as WBC
and falsely elevate
WBC results
LEFT SHIFT
Summarizing RBC Parameters
 RBC 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 distribution
 Morphology
Summarizing RBC Parameters
 Step1
Examne Hb an Hct for anemia or polycythemia
If the RBC morphology is normal: Use rule of three to
estimate the Hct
 Step 2
MCV: to check and correlate to the morpholic
apperance of the cells
Summarizing RBC Parameters
 Step 3
Examine MCHC
Describes how well the cells are filled with Hb
Hypochromic, normochromic
2 conditions when MCHC should be evaluated:
1. spherocytosis: slight elevation
2. lipemia/icterus: markedly increase
Summarizing RBC Parameters
 Step 4
Examine MCHC
Describes how well the cells are filled with Hb
Hypochromic, normochromic
2 conditions when MCHC should be evaluated:
1. spherocytosis: slight elevation
2. lipemia/icterus: markedly increase
Summarizing RBC Parameters

1.
2.
3.
4.
5.
6.
Step 5
Morphology
Size
Shape
Inclusions
Young rbcs
Color
Arrangement
Summarizing Platelet Parameters
 Platelet count (x 109/L)
 Mean Platelet Volume MPV, fl
 Morphology

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