Pactical laboratory Physiology I - 2015

EXPLORATION OF WHITE BLOOD CELLS (WBC)

1. WHITE BOLLD CELLS COUNT

Bone marrow is considered to be one of the biggest „organs” in adult human body, his
dimensions can be compared with the liver.
Bone marrow contains:
 Stem pluripotent cells: are the origin cells for all blood lines; can auto regenerate and
differentiate.
 Hematopoetic progenitor cells, “engaged”, which will transform in different types of blood
cells:
- Myeloid progenitor cells, which transform into erytrocytes, some of leukocytes
(neutrophyls, eozinophyls, basophyls and monocytes) and trombocytes.
- Limfoid progenitor cells, which transform into lymfocytes B and T.
Adult cells from leukocytes series will work together to defense the organism against infection
(viral, bacterial or parasitic), elimination of neoplastic cells and differentiation of self from
nonself.

LEUKOCYTE SERIES has 3 cellular lines:

1. GRANULOCITIC SERIES consists of polinuclear elements with granules:
 Unsegmented granulocyte (young granulocyte) has an horse shoe shaped nucleus;
 segmentated granulocyte (adult granulocyte) has a segmented nucleus. Mature cell has
more segments than young cell.

According to granulation type in cytoplasm there are types of granulocytes:

1.1. Segmented neutrophilic granulocyte – has acidophilic cytoplasm (pink), with specific
neutrophilic granules, small, numerous; nucleus is segmented in 2-5 lobes united by cromatine
bridges. It has the following roles:
 in microphagocytosis – the main function of defense against infections (represents the first
defense line due to inglobation and destruction of extracellular particles).
 Secretory role for: transcobalamine-I (α1-globulin which transports vitamin B12),
interleukins (IL1 induces fever), prostaglandines.

1.2. Segmented eosinophilic granulocyte – has a cytoplasm with large granules, even,
eosinophilic (orange) and the nucleus has 2 segments. It has the following roles:
 in phagocytosis: the factors who determine eosinophilic chemotasis are: imune complexes
antigen-antibody (have the major role in imune reactions), fibrine, proteolitic enzyme,
histamin. Eozinophilic cells can phagocyte bacteria and fungus, but are less atracted by
them in comparison with neutrophiles.
 in antiparasitic defence: even if it cannot phagocyte parasites, the eosinophile attach on
them and eliberates granules with a citotoxic substance (PBM –major basic protein).
 In allergic diseases.

1.3. Segmented basophilic granulocyte – contains characteristic basophilic granules in all the
cytoplasm, sometimes over the nucleus; the nucleus is uncompleted segmented. It has the
following roles:
 in phagocytosis, but in a lower degree.

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 in degranulation, with the release of numerous mediators, phenomenon with importance in
pathogenesis and diagnostic of some allergic diseases, and chemotactic role for neutrophils
and eosinophils.

2. LYMPHOCYTIC SERIES. Acording to size the lymphocytes can be: small, medium and large
acording to maturation and represents the major cellular component from limphatic tissues.
The shape of lymphocytes is round or ovale, with diameter of 6–12 µm. Nucleus is large ocupies
the largest part of the cell and it is intense colored. Cytoplasm is basophilic (light blue). They
havethe following roles:
• in immune response, started by a foreighn cell appeared in the body or a proteic molecule.
This response is characterized by antibody production by lymphocyte B (umoral immunity)
and citotoxic cell molecules produced by lymphocyte T (cellular immunity) .

3. MONOCYTIC SERIES. Monocytes are the largest cells from blood, with diameter between 10 -
25 μm specialised in body defence. The nucleus is unsegmented, renal shape without nucleoles.
The cytoplasm is abundent and basophilic (dark blue). Monocytic series include:
 bone marrow precursors
 monocytes in blood
 macrophages in tissues
It has the following roles:
 in macrophagocitosis coloidic particles, degenerated cells;
 in immune response same as other cells (antigen presenting cells).

Fig.2. Normal peripheral blood. Cloration MGG.

1. segmented neutrophilic granulocyte; 2. eosinophilic granulocyte; 3. basophilic granulocyte;
4. lymphocyte; 5. monocyte

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Methods
 Manual: the WBC are counted directly (in counting chamber); the blood volume is known
and is diluted into a known proportion (dilution is made in order to prevent coagulation of
blood and to enable their counting).
 Automatic: based on the electrical impedance principle – a leukocyte suspension (from a
known volume of reagent) is passing through an opening gated by electrodes to which a
constant current is applied. As each leukocyte is passing through the electrodes, the
electrical power between the two electrodes is dropping (the leukocytes do not conduct
electricity), this being called “pulse”. The device is able to count each pulse, which will be
further expressed as number of leukocytes.

Normal value
 mean value : 4,000-10,600/ mm3
 adults: 5,000 – 9,000/mm3 (5 – 9 x 103/μl )
 children – different values according to age (until age 18):
 newborn: 8,000-30,000/mm3
 1-2 years : 6,000-17,500/mm3
 3-7 years: 5,500-15,500/mm3
 8-16 years: 4,500-13,500/mm3

Physiological variations
 the number of leukocytes increases (leukocytosis) in children, pregnancy, after physical
effort, after meal, in altitude, emotional stress;
 the number of leukocytes decreases (leukopenia) in elderly, during sleep, prolonged
physical effort;

Pathological variations
 the number of leukocytes increases (leukocytosis) in infectious diseases, leukemia, allergies.
 the number of leukocytes decreases (leukopenia) in bone marrow disorders after ionic
radiation treatment, cytostatic medication.

2. CONCENTRATION OF DIFFERENT TYPES OF LEUKOCYTES

Definition. The leukocytic formula represents the percentage distribution of different types of
leucocytes in a blood sample. Can be determined classical or automated method.

Methods
 Manual: 200 leukocytes from peripheral blood are counted in light microscopy on a sample
slide stained May-Grunwald-Giemsa and a drop of cedar oil, and the ratio between different
types of leukocytes is established based on morphological characteristics (nucleus,
cytoplasma granules) and expressed by percentage.
 Automatic: leukocytes from peripheral blood are automatically separated by a device based
on their size, granularity and nucleus segmentation (flow-cytometric analysis) and obtain
histograms.

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Normal values
Leukocytes Adult Children
Neutrophyls 56-68% 25-40%
Eosinophils 1-3% 1-4%
Basophils 0-1% 0-1%
Lymphocytes 20-40% 50-70%
Monocytes 4-10% 4-8%

Observations
 Normal values can present variations between laboratories due to method or reactives
used;
 In small childrern, in formula we found more lymphocytes than segmentated neutrophyls.
The percent equals around 5-6 years old, then invert until reaching normal values for adult.

Variations
 Neutrophilia represent the increase of neutrophyls over normal value. The presence of
young neutrophyls in peripheal blood represents an intense medullar activity or intense
cytolysis. Neutrophilia could be:
- physiological: stress, exercise, pregnancy;
- pathological: in acute infections, endogenus intoxications (uremia) and exogenous,
chronic myelocytic leukemia, physical agents (trauma, burns).
 Neutropenia is defined as decrease neutrophylsunder 1000/mm3 in the first yer of life and
under 1500/mm3 after one year.
Can be:  congenital
 aquired
 intra- and post-infection,
 after some medication (cytostatics, anticonvulsivants, tranchilisers,
antiinflamatory, antibyotics)
 trough imune mechanisms (anti-neutrophyls antibody)
 Eosinophilia is increased number of eosinophils; found in parasitic infections, allergic
diseases, and dermatological diseases.
 Eosinopenia is decreased number of eosinophils; after corticosteroid therapy.
 Basophilia increased number of basophils. Is found in contagious diseases, chronic
myelocytic leukemia.
 Lymphocytosis is the increase of lymphocytes. Lymphocytosis can be:
- Phisiological: in children;
- pathological: in acute viral infections, tuberculosis, infectious mononucleosis, chronic
lymphocytic leukemia.
 Lymphocytopenia is the decreased lymphocytes under 3000/mm3 in the first 3 years of life
and under 1500/mm3 after 3 years; appears in viral infections, congenital or aquired
imunodeficiency (SIDA), etc.
 Monocytosis is increased number of monocytes. Is found in tuberculosis, infectious
mononucleosis, malaria, subacute bacterial endocarditis, leukemia, neoplasms of breast or
kidney, lymphomas.

In the following table there are some types of leucograms:

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1. PHYSIOLOGICAL Nr.L NE EO BA LY MO
Children   
2. PATHOLOGICAL
Acute bacterial infections   
Parasitosis
Alergy N, 
Dermatological diseases
Mononucleosis
TBC    

Acute viral infections    

Contagious diseases N 

3. FUNCTIONAL INVESTIGATION OF GRANULOCYTES

The main function of granulocytes is phagocytosis, preceded by diapedesis (the passage of

granulocytes from blood through vascular wall), which is started by numerous chemotactic
factors (attract the leucocytes to inflammation, or tissue lesion). In medical praxis we can
test, in vitro, phagocytic function of granulocytes with: latex phagocytic index, NBT test.

3.1. NBT TEST (NITROBLUETETRAZOLIUM).

Principle: Nitrobluetetrazolium (NBT) is a yellow dye, which is inglobated and reduced by
normal neutrophils at nitroformazan (dark blue). Are determined the percentage of NBT
positive cells, the granulocytes which inglobated and reduced NBT to nitroformazan, dark
blue precipitate, visible on Giemsa dyed cells.

Clinical utility:
1. To evaluate oxidative metabolism of granulocytes, oxygen free radicals production,
which assures bactericide function of phagocytes.
2. The diagnostic of congenital disfunctions of neutrophil

3.2. Flowcitometry
A new and important method of investigation of oxidative methabolism of neutrophils, but also
their adhesion and phagocytic capacity is flux citometry (flowcitometry).

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BULLETINS

1. M.G. ♀, 30 years, 4 months pregnant 2. P.B. ♂, 3 years

Leucocytes = 11.000/mm3 Leucocytes = 8.000/mm3
Neutrophils = 77% Neutrophils = 38%
Eosinophils = 1% Eosinophils = 4%
Bazophils = 0% Bazophils = 1%
Lymphocytes = 17% Lymphocytes = 52%
Monocytes = 5% Monocytes = 5%

3. S.C. ♂, 22 years 4. A.M. ♂, 27 years

Leucocytes = 10.000/mm3 Leucocytes = 20.000/mm3
Neutrophils = 58% Neutrophils = 74%
Eosinophils = 12% Eosinophils = 2%
Bazophils = 0% Bazophils = 0%
Lymphocytes = 25% Lymphocytes = 20%
Monocytes = 5% Monocytes = 4%

5. R.P. ♀, 22 years 6. B.C. ♀, 2 years

Leucocytes = 4.000/mm3 Leucocytes = 14.000/mm3
Neutrophils = 68% Neutrophils = 19%
Eosinophils = 4% Eosinophils = 1%
Bazophils = 6% Bazophils = 0%
Lymphocytes = 15% Lymphocytes = 71%
Monocytes = 7% Monocytes = 9%

MULTIPLE CHOICE QUESTIONS (ONE CORECT ANSWER)

1. The normal values for leukocytes in adults are:

A. <10,000/mm3
B. 150,000 – 300,000/mm3
C. 5000 – 9000/mm3
D. 25%

2. Which of the following are true:

A. Increased of leukocyte number is called se numeşte leukocytosis
B. Leukopenia can be phisiologic in sleep and phisical effort
C. Leukocytosis means the decrease of leukocytes in blood
D. Leukocytosis occurs phisiological in elderly

3. Leukocytosis is phisiological in:

A. Babies
B. Sleep
C. In elderly
D. After phisical effort

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4. Leukocytic formula in children (5 – 6 years) presents:

A. Neutrophilia
B. Limphocytosis
C. Monocytosis
D. Limphopenya

5. Segmented neutrophilic granulocyte:

A. Has a segmented nucleus due to nuclear cromatine condensation
B. Represent adult granulocyte
C. Belongs to limphocytic series
D. Belongs to granulocytic series

6. Monocyte characteristics are:

A. Renal shape nucleus
B. Segmented nucleus in 2-5 lobes with large basophile cytoplasm
C. His role is in microphagocytosis of colloidal particles
D. His role is in immune response as a accessory cell (antibody presenter)

7. Which of the following statements are true:

A. Eosinophils intervene in phagocytosis of fungus, bacteria, antigen-antibody
complexes
B. Monocytes intervene in inflamatory proceses (defence capacity isreduced)
C. Lymphocytes secret IL-1 (pyrogenic substance) and enzyme
D. Segmentated neutrophils intervene in antimicrobial defence by
microphagocytosis

8. Choose the correct answer:

A. Eritrocytes come from myeloid progenitor cells
B. Eritrocytels come from limphoid progenitor cells
C. Some leukocytes come from myeloid progenitor cells, so as trombocytes
D. Eosinophils, basophils and monocytes come from myeloid progenitor cells

9. About leukocytic formula the following statement is true:

A. Can be read on leukocyte distribution curve
B. Can be interpreted with histograms
C. Normal value for adults is 25-40 %
D. Normal value of eosinophils in adult is higher than in children

10. Choose the correct answer:

A. Lekopenia indicate an adaptation stage of the body to altitudes
B. Monocytosis tipicaly occurs in intestinal parasitosis at children
C. Leukocytosis is found in bone marrow destructions after ionic treatment,
citostatics.
D. Basophilia is found in contagious diseases, chronic myeloid leukemia

Answers: 1-C, 2-B, 3-A, 4-B, 5-D, 6-D, 7-D, 8-C, 9-B, 10-D