Documente Academic
Documente Profesional
Documente Cultură
Method
Coversli
p
Advantage
Bone Marrow preparation
Disadvantage
Difficult to master and to label
To thin which can break easily
Slip has to mount to the side
Easy to label
Immature WBC (abnormal cells) can be
easily observed at the edges of the
smear
Beacons Method
Lymphocytosis
- increased lymphocytes viral
- normal: 10-47%
a. Absolute/True Lymphocytosis
b. Relative/False Lymphocytosis
- false increase
- lymphocytes is about 50%
- large cells are pushed at the edge of
the smear
Different Poikilocytes:
Smudge cells
-
Nuclear remnants/fragments of
lymphocyte
looks like a finger
Basket cells
-
Cytoplasmic remnants/fragments of
granulocyte
looks like a net
Echinocyte
- no pathologic relevance
- diagnosis: equidistant, regular-sized
pointed spicules
Acanthocyte
has pathologic relevance
diagnosis: irregularly spaced,
irregular-sized pointed spicules
JOsephine Aluyen
Stains:
o
o
o
o
Leishman
May Grunwald
Jenner-Giemsa
Wright-Giemsa
- crispier granules
- most used
Romanowsky
- modified Wright-Giemsa
- polychrome stain
Methylene Blue
- stains acidic (nucleic acid)
property of the cell
Eosin Y
- acidic
- stains basic (cytoplasm)
property of the cell
stains the protein part,
hemoglobin, eosinophilic
granules
- pink stain
Staining Process
1. Fixation
10% volumes/volume Methanol
- prevent water contamination
- 40% can attract humidity in the air
2. Addition of buffer solution
pH 6.4-6.8 aged distilled water
pH 6.4 0.05 M NaPO4
pH 7.2 observe Malarian
pigments/stiplings
3. Staining
Methylene Blue
Eosin Y
4. Washing remove excess stain
5. Drying
- airdry
- tissue paper - can contaminate
Microscopic:
Neutrophil
granules
Eosinophil
granules
Basophil
granules
blue-black
graying-blue = improper
stain
Monocyte
Lymphocyte
Platelets
RBC
Too Blue
Thick smear
Alkaline stain
Inadequate washing
Too Red
Thin smear
Acid stain
Excessive washing
Heparinized blood:
heparin + Wright stain
Hyperproteinemic
patients
-
Ideal Smear
Macroscopic:
ideal: purple
blue: slides stained after 1 week or
longer
Macroscopic
- bluer overall than normal
due to hyperproteinemia
Roleux Formation
(Pseudoagglutination)
- holes all over the film (lipids)
check hemoglobin
- grainy appearance
agglutinated/clump RBC
(destroyed)
caused by antibodies against
RBC antigen
accumulation of nucleated cells
false elevated: leukocytes
- blue specks out at the feathered edge
increase WBC & platelet
100x OIO
Morphology is evaluated
WBC is generally performed
100 WBC is counted & classified to
obtain percentages of types
Segmenters can be distinguished
from Bands
Inclusions are seen
RBC: Howell-Jolly
WBC: Dohle bodies
Reactive or Abnormal cells are seen
RBC Morphology
o
Microscopic
-
10x objectives
overall film quality, color, distribution
feather edge should be checked quickly
for WBC distribution
presence of Rouleaux formation
scanned quickly for any large abnormal
cells
presence of fibrin strands
specimen must be rejected
snowplow effect
presence of more than four times
the number of cells per field at the
edges / feather compared with the
monolayer area of the film
Size
discoid with central pallor
Anicocytosis
- reference: Lymphocytes
Shape
Poikilocytosis
- depends on severity of abnormal
shape
- can be reported using grading chart
- Skistocyte
- Target cell/Codocyte
- Sickle Cell
Grading Chart
Norma
l
Slight
+1
+2
+3
+4
WBC estimate
select an area in which the RBCs are
separated from one another with
minimal overlapping
average WBC per field x2000
(x40 magnification)
average WBC per field x3000
(x50 magnification)
5%
5-10%
1025%
2550%
5075%
>75%
Hemoglobin concentration
JOsephine Aluyen
2/3
Thin
ring
Distribution
not overlapping because of the zeta
potential that repels the RBC from
each other
Grading Chart
+1
+2
+3
o
-
Polychromasia
representing Reticulocyte
blue-red color
stains to see inclusions
New Methylene Blue
Brilliant Cresil Blue (BSB)
Grading chart
Sligh
t
+1
+2
+3
+4
1%
3%
5%
10%
>11
%
Inclusion
no inclusions
not graded, reported as negative
and positive
Sickle Cell
Popenheimer bodies Sideroblastic
Howell-jolly
Basophilic stiplings
Burr cells
Ovalocyte
Target cell
Stomatocyte
Bizzare RBC
Poikilocytes
Dacrocytes
(tear drop
cell)
Acanthocytes
Schistocytes
Spherocytes
Polychromato
philia
3-4/hpf
510/hpf
>10/hp
f
+1
1-5/hpf
+2
610/hpf
+3
>10/hpf
310/hpf
1120/hpf
>20/hpf
Platelet
o
o
WBC
Distribution
o Roleaux formation
- high protein (except Albumin)
- high ESR reading
+1
+2
+3
o
-
34/hp
5-10
>10
Agglutinated RBC
discoid shape is disrupted
grainy appearance of the smear
Diseases:
Poikilocytosis
Myelogenous Leukmia
- increased Basket cells
- especially in patients undergoing
chemotherapy
all cells (especially leukemic
cells) are fragile
Pyruvate-Kinase Deficiency
- presence of Burr Cells
Acanthocytosis
Abetalipoproteinemia
- presence of Acanthocytes
- absence of B-Lipoprotein
important for lipid
transportation
Normocytic
Leukemia
Myelofibrosis
Diamond Blackfan
Fanconi Anemia
other bone marrow problem
Macrocytic, Hyperchromic
Megaloblastic Anemia
- hypersegmented neutrophil
H. Spherocytosis
Microcytic, Hypochromic
Anemia of chronic disease
Thalassemia
- Thalassemia Minor male with
Fe deficiency
Iron deficiency
Sideroblastic (dimorphism)
Reticulocytosis
Hemolytic Anemia
McLeod Phenotype
- low Kell Antigen
affects RBC shape
causes Neuroacanthocytosis
Hyperproteinemia
Multiple Myeloma
- plasma defect
Macroglobulinemia (Waldens
Strongs)
with Agglutination (Antibody against
RBC Antigen)
Auto-immune Hemolytic Anemia
Hemolytic Transfusion Reaction
Hemolytic Disease of the Fetus &
the Newborn
Cold Heme Agglutinin Disease
JOsephine Aluyen
Problems/Troubleshooting
Bullet-shaped smear
- not ideal
- has a narrow area for observation
- caused when blood was spread before
the drop was fully spread along the
spreader slide
High accumulation of leukocytes
along the side & tail edges of the
film
Thick smear
overlapping of RBC causing falsely
elevated count
remedy:
o smooth spreading
o frosted slide may be used
caused by:
o not smooth/dirty spreading
o chipped off/rough edge
must be avoided
Lymphocyte is >40%
-
Artifacts
Thick areas
EDTA-Anticoagulant samples
Thin areas
-
o
o
o
o
Water artifacts
-
JOsephine Aluyen