Laboratory Blood smear pr guide

2.  Estimates of cell count
 Proportions of the different types of WBC
 Morphology

3.  Objective
1. Specimen Collection
2. Peripheral Smear Preparation
3. Staining of Peripheral Blood Smear
4. Peripheral Smear Examination

4.  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

5.  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

6.  Disadvantages:
PLATELET SATELLITOSIS
 causes pseudothrombocytopenia and
pseudoleukocytosis
 Cause: Platelet specific auto antibodies that
reacts best at room temperature

7.  Platelet satellitosis

8.  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

9.  Wedge technique
 Coverslip technique
 Automated Slide Making
and Staining

10.  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

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

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

14. 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

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

17. 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.

18. 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

19.  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

21.  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.  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

23. 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

24.  Automatic Slide Making and Staining
 SYSMEX 1000i

25.  Drying of Smears
Fan
Heating pans
No breath blowing of smears – may produce
crenated RBCs or develop water artifact
(drying artifact)

26.  Wright Staing Method
 Automated Slide Stainers
 Quick Stains

27.  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

28.  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

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

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

31.  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

32.  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

33. HEMA-TEK STAINER

34.  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

35.  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

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

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

38.  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

39.  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

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

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

42.  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

43.  Too thin Too thick

44.  Systematic
 Choose the best area for assesment
 Back and forth serpentine or battlement track
patters in preferred

45.  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

46.  Extremely low WBC counts, do the Diff count
under 50X OIO
 Extremely low WBCs: WBC are concentrated,
buffy coat smears are made

47. Anisocytosis
Poikilocytosis
Cellular Inclusions

48.  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)

49. 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

50.  STEP 1
WBC increased : leukocytosis
WBC decreases: leukopenia

51.  STEP 2
Relative
differen
tial
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

52.  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)

53.  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

55.  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

56.  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

57.  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

58.  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

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

60.  Platelet count (x 109/L)
 Mean Platelet Volume MPV, fl
 Morphology