Peripheral blood
Practical lesson WS 10 – group 1051
Teacher: Tomáš Kučera
Preparation, staining and evaluation of the blood
smear
Blood cells are studied in smears prepared by spreading of a drop of peripheral
blood in a thin layer on the microscopic slide.
Procedure:
Disinfection of the skin of the fingertip of the left fourth or third finger
(at right-handers)
Puncture into the ball of fingertip with sterile lancet or single-use needle
The first drop of blood is wiped off because blood is diluted by tissue fluid
The second drop of blood is placed near an end of a glass (microscopic) slide and
spread using another slide (called “spreader” slide)
Spreading: spreader slide is moved over the glass slide at an
angle of 45º, when slide edge touches the blood drop, the
blood spreads by capillary forces along its edge. A thin film of
blood is obtained by a smooth quick motion of the spreader
slide across a glass slide. The air dried blood smear is fixed
and stained.
Blood smears are stained with mixtures of acidic (eosin) and
basic dyes (methylene blue and its oxidation products –
methylene violet and azure)
PAPPENHEIM´S METHOD
Method is used for staining of peripheral blood and bone marrow smears
Fixation of blood smear with May-Grünwald solution
(eosinate of methylene blue in methanol) .......... 3 min.
Staining with diluted May-Grünwald solution
(the same amount of distilled water was added) ......... 1 – 2 min.
Pour off mixture
Staining with Giemsa-Romanowsky
(eosinate of the methylene azure, blue and violet) …………….. 15 min.
Washing (distilled water), air-drying
Results of staining:
red blood cells – pink/red, nuclei – purple/blue, neutrophilic granules –
salmon pink, eosinophilic granules – brick-red, basophilic granules –
blue/violet, cytoplasm of lymphocyte – sky blue, cytoplasm of monocyte
– pale blue (grayish or greenish), azurophilic granules - purple red
Evaluation of the blood smear
Blood smear is observed in light microscope using HI objective
(oil.im.,100x) and immersion oil
RBC evaluation: size, shape, structure
anisocytosis (microcytosis, macrocytosis),
poikilocytosis - variation in RBC shape:
spherocytosis, ovalocytosis, sickle cells
WBC evaluation: size and morphology
Leukogram (differential leukocyte count) - proportional incidence (%)
Arneth formula - reflects ratio of the neutrophilic granulocytes based on the
number of nuclear lobes. Shift to left - predominance of the young forms (band
and two lobes). Shift to right - predominance of the old forms (four and five lobes).
FORMED ELEMENTS - blood cell count (number per μL or L)
ERYTHROCYTES - Male: 4.5-6.0 x 1012 / L
Female: 4.0-5.2 x 1012 / L
LEUKOCYTES – 4.000 - 10.000 per μL (4 – 10 x 109 / L)
LEUKOGRAM (differential leukocyte count)
Neutrophils: 60-70 % (band form: 2-5 %)
Eosinophils: 2-5 %
Basophils: 0-1 %
Lymphocytes: 20-40 %
Monocytes: 2-10 %
THROMBOCYTES - 150.000 - 400.000 per μL
NG
Ly
band
form
NG
Eos
Ly
Mo
NG
BG
Mo