Blood_Smear_Prep__eaf7622f.pptx

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. Specimen Collection
2. Peripheral Smear Preparation
3. Staining of Peripheral Blood Smear
4. 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
 Advantages
1. Many smears can be done in just a single draw
2. Immediate preparation of the smear is not
necessary
3. 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
 Wedge technique
 Coverslip technique
 Automated Slide Making
and Staining

 Wedge technique
1. Easiest to master
2. Most convenient and most commonly used
technique
 Material needed
1. Glass slide 3 in X 1in
2. Beveled/chamfered edges

 Procedures:
1. Drop 2-3 mm blood at one end of the slide
Diff safe can be used
a. Easy dropping
b. Uniform drop

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

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

Wedge Technique
1. Push Type wedge preparation
2. Pull Type wedge prepartion

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.

Precautions:
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 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

 Cover Slip Technique
rarely used
used for Bone marrow aspirate smears
Advantage: excellent leukocyte distribution
Disadvantage: labeling, transport, staining and
storage is a problem

 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

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

 Automatic Slide Making and Staining
 SYSMEX 1000i

 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

 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

 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

 Free Methylene Blue:
- basic
- stains acidic cellular components such
as RNA
 Free Eosin
- acidic
- stains basic cellular components such as
Hgb and eosinophilic granules

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

 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

 AUTOMATED SLIDE STAINERS
1. It takes about 5-10 minutes to stain a batch of smears
2. 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 batch
2. Aqueous solutions of stains are stable only after 3-6
hours

HEMA-TEK STAINER

 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

 Troubleshooting:
1. RBC gray, WBC too dark Eosinophil granules
are gray
Cause: stain or buffer is to alkaline
inadequate rinsing
Prolonged staining
heparinized sample

 Troubleshooting:
2. RBC too pale, WBC barely visible
Causes: Stain or buffer is too acidic
Underbuffering
Over rinsing

Peripheral Smear Examination
 Macroscopic
1. Overall bluer color: increased blood proteins
(multiple myeloma, rouleaux formation)
2. Grainy appearance: RBC agglutination (cold
hemagglutinin diseases)
3. Holes: increased lipid
4. Blue specks at the feathery edge: Increased
WBC and Platelet counts

 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: Reject
4. Rouleaux formation, large blast cell assessment

 Microscopic:
 40x Objective
1. Correct area where to star counting is determined
2. WBC estimate: internal quality control

 Microscopic:
 100x Objective; OIO
1. Highest magnification
2. WBC differential counting

 Optimal Assessment Area:
1. RBCs are uniformly and singly distributed
2. Few RBC are touching or overlapping
3. Normal biconcave appearance
4. 200 to 250 RBC per 100x OIO

 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

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

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. Total WBC counts per (WBC x 109/L)
2. WBC differential counts are percentages
3. WBC differential count values expressed as
actual number of each type of cell
4. WBC morphology

 STEP 1
WBC increased : leukocytosis
WBC decreases: leukopenia

 STEP 2
Relative
differentia
l count
Cell Type Increases Decreases
Neutrophil Neutrophilia Neutropenia
Eosinophil Eosinophilia N/A
Basophil Basophilia N/A
Lymphocyte Lymphocyto
sis
Lymphopeni
a
Monocyte Monocytosis Monocytope
nia

 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)

 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

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

 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

 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

 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

 Step 5
Morphology
1. Size
2. Shape
3. Inclusions
4. Young rbcs
5. Color
6. Arrangement

Summarizing Platelet Parameters
 Platelet count (x 109/L)
 Mean Platelet Volume MPV, fl
 Morphology

Blood_Smear_Prep__eaf7622f.pptx

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