Normal Physiology

LABORATORY WORK 1
BLOOD PHYSIOLOGY

UNIT 2

Determination of the main indicators of red blood:

1 Determination of hemoglobin concentration
2 Determination of the number of red blood cells
3 Calculation of the color index
4 Definition of ESR and its interpretation
5 Determination of hematocrit

Name_________________________________________________________________________

Group________________________________________________________________________

Teacher_________________________Signature_________________Date_________________

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Plan of class
1. Lab. w: Sahli method (measuring of Hb)
2. Lab. w: Calculation of RBCs number concentration
3. Lab. w: Calculation of color index
4. Lab. w: Estimation of ESR
5. Lab. w: Determination of Hematocrit

LAB. W: SAHLI METHOD (MEASURING OF Hemoglobin)

Background
Hemoglobin the main component of erythrocytes, serves as the vehicle for the transportation of
oxygen and carbon dioxide. It is composed of amino acids that form a single protein called globin,
and a compound called heme, which contains iron atoms and the porphyrin. The iron pigment is
that portion of the hemoglobin that combines readily with oxygen and gives blood its characteristic
red color. Each gram of hemoglobin can carry 1.34 ml of O2. The oxygen-combined capacity of
the blood is directly proportional to the hemoglobin concentration.

Principle: Hemometer Sahli is rack with three tubes. The lateral tubes filled with a standard
solution of hematin and closed. Middle tube has a graduates and used for research of blood.

Materials and reagents

1. Sahli hemometer with capillary pipette
2. Pipette
3. Glass rod
4. Distil water
5. 0.1N of Hydrochloric acid solution (HCL)
6. Scarifier
7. Methanol
8. Iodine
9. Cotton
10. Researching specimen of blood

Procedure

1. Previously into the middle tube of Sahli place 0.2 ml of 0.1N HCL solution
2. Take the researching blood specimen into the pipette of 0.02 ml and blow out to Sahli’s middle
tube (the hemoglobin converts to muriatic haematin of brown color).
3. Add distil water and mix with glass rod till then color is coincide with standard solutions color.
4. Mark the level of middle tube’s liquid on the scale of hemometer. It wills quantity of hemoglobin
in g%.
In normal, the hemoglobin concentration is:
For men 13-16 g% (130-160 g/l)
For women 12-14 g% (120-140 g/l)
RECOMMENDATIONS FOR WORK
1. Determine the hemoglobin concentration in a blood sample;
2. Compare with reference data

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LAB. W: CALCULATION OF RBCS NUMBER CONCENTRATION
Background
Red blood cells (RBCs) are the non-nucleated formed elements in the blood. Red color of the
RBCs is due to the presence of the coloring pigment called hemoglobin. RBCs play a vital role in
transport of respiratory gases. RBCs are larger in number compared to the other two blood cells,
namely WBCs and platelets. Average lifespan of RBCs is about 120 days. After the lifetime, the
senile (old) RBCs are destroyed in reticuloendothelial system
Normal values of RBCs for humans:
Male – 4.5 – 5.0 x 106/mm3
Female – 4.0 – 4.5 x 106/mm3

Clinical aspects:
I. Physiological variations: Increase in RBCs count
• Age
• Sex
• High altitude
• Muscular exercise
• Emotions
• Environmental temperature
• After meals
Decrease in RBCs count
• High barometric pressure
• During sleep
• Pregnancy
II. Pathological variations
1. Pathological Polycythemia is the abnormal increase in RBCs count above 7x106/mm3
• Primary
• Secondary
2. Anemia is abnormal decrease in RBCs count 3,5x106/mm3

Principle: The number of erythrocytes contained in 1 L of blood is called the erythrocytes number
concentration. This method uses a counting chamber in which erythrocytes are counted under the
microscope. Gorayev’s Hemocytometer consist of big, middle and little squares. The count unit is
little square. Its side equal 1/20 mm and area = 1/20 x 1/20 = 1/400 mm2 x 1/10 mm = 1/4000 mm3
(1/10 mm is height of blood layer)

Materials and reagents:

1. Gorayev’s Hemocytometer with cover glass
2. Test-tubes
3. Capillary tube (Sahli pipette 0.02 mL)
4. Microscope
5. Researching specimen of blood
6. 3% Sodium chloride solution (NaCL)

Procedure:
1. Fill the test-tube till 4 mL of 3% NaCL solution
2. Take capillary blood in capillary tube till 0,02 mL
3. Mixed blood specimen with saline solution and shake

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4. One drop of preparing solution distributes in Gorayev’s Hemocytometer under the cover
glass as shown on the Figure below:

5. Use microscope x10 objective lenses for calculation.

6. Calculate the erythrocytes in placed obliquely five big squares which have by 16 little
squares.

7. Sum up the red blood cells in all the descriptive squares above and using the formula below
calculate:
RBCs = Sum of RBCs x 4000 x 200,
80
where:
- the Sum of RBCs in five big squares,
- 4000 is the volume of chamber part (in the 1 little square)
- 200 is diluting of blood.
- 80 is quantity of little squares in five big squares (5 x 16 = 80)

RECOMMENDATIONS FOR WORK:

1. Count the RBCs number concentration in a blood sample;
2. Compare with reference data

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LAB. W: CALCULATION OF COLOR INDEX

Background
The color index of blood is an indicator of the degree of saturation of erythrocytes with hemoglobin
and reflects the ratio between the number of erythrocytes and hemoglobin in the blood. The color
index is used for differential diagnosis of hypo-, normo- and hyperchromic anemia. Color index -
its normal value in people of any age is 0.85-1.15. If a reduced number of red blood cells is noted
against this background, they talk about normochromia, characteristic of acute or chronic bleeding,
renal pathology leading to a lack of erythropoietin, intravascular hemolysis and aplastic conditions.

Clinical aspects:
The decrease indicates hypochromic anemia, which happens when:
• Iron and vitamin B6 deficiency
• Heavy metal poisoning
• Violation of the synthesis of proteins and other compounds that make up hemoglobin
(thalassemia)
• The general serious condition of the patient.

Hyperchromia, when the Color index is higher than normal, indicates:

• Deficiency of vitamin B12 and folic acid in the body
• Toxic damage to the bone marrow
• Overdose or prolonged treatment with cytostatics, anticonvulsants, antiviral drugs
• Hypothyroidism
• Violation of the liver.

New-born children have high Color index.

Principle: The hemoglobin content inside the erythrocytes, the color index and the average
concentration reflect how much of the key oxygen-carrying protein is inside the erythrocyte. It
should be understood that the key point at which the hemoglobin content decreases or the color
index drops is iron deficiency.

Procedure:
1. Estimate erythrocyte number concentration
2. Determine Hemoglobin quantity
3. According to the following formula calculate Color index:

RECOMMENDATIONS FOR WORK:

1. Use your previous data of Hb and RBCs count and calculate Color Index;
2. Compare with reference data

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LAB. W: ESTIMATION OF ESR

Background
Sed rate, or erythrocyte sedimentation rate (ESR), is a blood test that can reveal inflammatory
activity in the body.
When the blood specimen is placed in a tall, thin tube, red blood cells (erythrocytes) gradually
settle to the bottom. Inflammation can cause the cells to clump. Because these clumps are denser
than individual cells, they settle to the bottom more quickly.
The Sed rate test measures the distance red blood cells fall in a test tube in one hour. The farther
the red blood cells have descended, the greater the inflammatory response of immune system.

Clinical implication.
Normal values of ESRs by age group:
Age group ESR (mm/h) Reference
Adults (< 50 years) Manual of basic techniques (for a health
Men <15 laboratory) 2nd edition (WHO) Geneva, 2003
Women <20
Adults (> 50 years)
Men ≥20
Women ≥30

Clinical aspects:
Any disease that produces plasma protein changes will increase the ESR. These include acute and
chronic infections, myocardial infarctions and rheumatoid arthritis. The ESR is also increased in
patients suffering from anemia.

Principle: Blood with an anticoagulant is placed in a long graduated tube held in a vertical position.
The erythrocytes settle to the bottom, leaving a layer of plasma above. The height of the column
of plasma after 1 hour indicates the sedimentation rate of the erythrocytes (ESR).

Materials and reagents:

1. ESR tube
2. Panchenkov stand (or Westergren stand)
3. Test- tubes
4. Researching specimen of blood
5. Timer
6. 5% Sodium citrate solution

Procedure:
1. Previously, wash inside of ESR tube with Sodium citrate solution.
2. Place 5% Sodium citrate solution into the ESR tube till “P” mark and blow out into the test tube.
3. Collect capillary blood specimen into the ESR tube till “K” mark.
4. Add the taking blood specimen to the test-tube containing anticoagulant. Shake gently.
5. Take the citrated blood into the ESR tube till “K” mark.
6. Place the tube in the Panchenkov stand (as shown on the below figure). Leave then to stand at
room temperature for 1 hour.

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RECOMMENDATIONS FOR WORK:

1. Check the height of the column of plasma in mm graduations starting from the “K” mark at
the top of the tube. The result is expressed in millimeter per hour (mm/h).

LAB. W: DETERMINATION OF HEMATOCRIT (packet cell volume – PVC)

Background.
Hematocrit (Packed Red Cell Volume). The hematocrit is the fraction of the blood composed of
red blood cells, as determined by centrifuging blood in a “hematocrit tube” until the cells become
tightly packed in the bottom of the tube. It is impossible to completely pack the red cells together;
therefore, about 3 to 4% of the plasma remains entrapped among the cells, and the true hematocrit
is only about 96% of the measured hematocrit. In men, the measured hematocrit is normally about
0.40, and in women, it is about 0.36.
Significance of determining PCV in
• Diagnosis and treatment of anemia
• Diagnosis and treatment of polycythemia
• Determination of extent of dehydration and recovery from dehydration after treatment
• Decision of blood transfusion

Principle: Calculated hematocrit is determined by multiplying the red cell count by the mean cell
volume. The hematocrit is slightly more accurate as the PCV includes small amounts of blood
plasma trapped between the red cells (Figure 2-1). An estimated hematocrit as a percentage may
be derived by tripling the hemoglobin concentration in g/dL and dropping the units.

Figure 2-1. Diagram of packed cell volume

The packed cell volume (PCV) can be determined by centrifuging heparinized blood in a capillary
tube (also known as a micro hematocrit tube) at 10,000 RPM for five minutes. This separates the
blood into layers. The volume of packed red blood cells divided by the total volume of the blood
sample gives the PCV. Since a tube is used, this can be calculated by measuring the lengths of the
layers.
Another way of measuring hematocrit levels is by optical methods such as spectrophotometry.

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One of the theoretical method is calculation:

Hemoglobin concentration (g/L

PCV= × 100%
RBCs number concentration (first three numbers of RBCs count without comma

Purpose: determine and interpret of PCV.

Materials and reagents:

• Blood samples with different values of hemoglobin concentration in g/L
• Blood samples with different values of RBCs number concentration in x1012/L million in
3
1 mm
• Centrifuge
• Calculator

Procedure:
1. According to the above formula calculate PCV.

RECOMMENDATIONS FOR WORK

1. Compare results with table of normal values.
2. Interpret results and do conclusion.

Clinical implication.
Normal values
The normal values of PCV
In males = 40-45%
In females = 38-42%

Clinical aspects: In severe anemia, the hematocrit may fall as low as 0.10, a value that is barely
sufficient to sustain life. Conversely, there are some conditions in which there is excessive
production of red blood cells, resulting in polycythemia. In these conditions, the hematocrit can
rise to 0.65 (Figure 2-2).

Fig. 2-2. Hematocrit in different states

PCV increases in:
• Polycythemia
• Dehydration

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• Dengue shock syndrome: Dengue fever (tropical disease caused by flavivirus transmitted
by mosquito Aedes aegypti) of grade III or IV severity.

PCV decreases in:

• Anemia
• Cirrhosis of liver
• Pregnancy
• Hemorrhage due to ectopic pregnancy (pregnancy due to implantation of fertilized ovum
in tissues other than uterine wall), which is characterized by vaginal bleeding

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Age___________ Weight___________ Height___________

a) Male b) Female

Your How much

1 Sahli method (measuring of Hb)

2 Calculation of RBCs number

concentration

3 Calculation of color index

4 Estimation of ESR

5 Determination of Hematocrit

Explain your choice:

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