Dr.

Nagy Morsy Blood and body fluids Biochemistry

Practical Manual

Blood and body Fluids Biochemistry

SCBC - 228

By

Dr. Nagy Morsy

fahad Ataih ali alhurtom/2542209

Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC - 228
Experiments 1
Determination of hemoglobin concentration of blood

Objectives
Haemiglobincyanide (Cyanmethaemoglobin) Method
The haemiglobincyanide (Cyanmethaemoglobin) method is the internationally recommended method
for determining the hemoglobin concentration of blood.

Principle
Blood is diluted in Drabkin's solution, a solution containing potassium cyanide and potassium ferricyanide.
Potassium ferricyanide oxidizes hemoglobin to methaemoglobin. Methaemoglobin then combines with
potassium cyanide to form Cyanmethaemoglobin (HiCN). The absorbance of the solution is measured in
a spectrophotometer at a wavelength of 540 nm against Drabkin's solution as a blank. The result is
calculated using the formula provided below and it is expressed in
gm/dL.

Test Sample
Venous or capillary blood collected into an EDTA tube. Alternatively, free flowing capillary blood
may be added directly to the diluting fluid and measured.

Procedure
1. Pipette 4ml of Drabkin's solution into a test tube.
2. Pipette 20 μL (0.02 ml) of properly mixed blood.
3. Clean outside of pipette and wash out the blood in the tube containing the diluent.
4. Mix and leave for 5-10 minutes for the reaction to complete.
5. Using Drabkin’s solution as a blank, read the absorbance in the spectrophotometer at
wavelength 540 nm.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Calculation
Use the following formula:

Hb (g/dL) = A X 29.3

Where: A = Reading of absorbance of Hb solution

64500 = Molecular wt. of Hb
44 = Millimolar extinction coefficient

D = Thickness of cuvette = 1
1000 = Conversion factor of mg to gm
DIL = Dilution Factor = 200

Notes on Technique:
· The blood sample must be properly mixed before sampling and allowed to warm
· Care should be taken when handling potassium cyanide.
· Use clean tubes and pipettes.

Comments
The cyanmethaemoglobin method is the reference method for Hb estimation because:
a) all Hb forms except sulphaemoglobin are estimated,
b) highly reliable and stable reagents are available and
c) the method can be easily standardized.

Normal Ranges
Adult males: 13 – 17 g/dL
Adult females: 11.5 – 16.5 g/dL
Newborns and infants: 14 – 22 g/dL

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 Dr. Nagy Morsy Blood and body fluids Biochemistry
Student Name: Academic Number:
‫هذه الورقة تسلم في المعمل‬

Experimental report (1)

Determination of hemoglobin concentration of blood

1. Determine the hemoglobin concentration in a blood sample by Haemiglobincyanide

(Cyanmethaemoglobin) Method where:
A = 0.35
Molecular wt. of Hb = 64500
Millimolar extinction coefficient = 44
Thickness of cuvette = 1
Conversion factor of mg to gm = 1000
DIL = Dilution Factor = 200

Hb(g/dL)} = A × 29.3
Hb= 0.35×29.3
Hb= 10.255 g/dL

2. What are the constituents of Drabkin’s solution?

Potassium Cyanide (KCN) and Potassium Ferricyanide.

3. What is the wave length of measurements in determination of hemoglobin concentration

in a blood sample by Haemiglobincyanide (Cyanmethaemoglobin) method.

540 nm

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC - 228
Experiments 2
Red Blood Cell Count

Objectives
A red blood cell count is typically ordered as part of a complete blood count (CBC). It is used as a
screening test for anemia and polycythemia. Manual Red Blood Cell Count (Using Formal Citrate as a
Diluent)

Principle
A suitable dilution of blood (1/200) is made in formal citrate solution. This diluent lyses the white cells
leaving the red cells intact. The number of RBCs in 1/5th of the RBC square is counted using an improved
Neubauer counting chamber called a hematocytometer. The hematocytometer consists of 9 counting
squares with each square having an area of 1mm2. Every square is filled with 0.1 ml of diluted blood. The
result in expressed as RBCs/ lit of blood.

Hematocytometer
Procedure
1. Pipette 4 mL (3980 μL) of RBC diluents in a tube.
2. Pipette 20 μL of well mixed blood into the tube containing the diluents.
3. Clean outside of pipette and wash out the blood in the tube containing the diluent (dil 1/200).
4. Mix the contents of the tube and fill the counting chamber with the diluted blood (0.1 ml of
diluted blood in each square).
5. Leave on bench for 2-5 minutes for the cells to settle.
6. Count the RBCs under the microscope using the (x40) objective lens and lowering the
condenser. The RBCs are counted in 1/5th of the RBC square i.e. 0.2 mm2.

Note:
1 µL = mm3

Calculations
N = the number of cells in 0.02 mL of diluted blood (the number of cells in five squares)

Number of cells in 1 µL of whole blood = N x 50 x 200 = N x 10000

Normal Ranges High RBC count is called polycythemia

• Men: 4500000 - 6 000000/ mL
• Women: 4200000 – 5000000 / mL Low RBC count is called anemia

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Figure: Illustration showing different parts of the counting chamber.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Student Name: Academic Number:

‫هذه الورقة تسلم في المعمل‬

Experimental report (2)

Red Blood Cell Count

1. Calculate the number of red blood cells in the following red square and what is the RBCs
number if the other 4 squares are the same? What is the diagnosis of this patient?

2. What is the apparatus used to count red blood cells in a blood sample?

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC - 228
Experiments 3
White Blood Cell Count

Introduction:
• The WBC or leukocyte count method estimates white blood cells per microlitres of
your blood. By enumerating the total number or concentration of leukocytes, you
could determine the condition of your immune health. The average WBC count is
between 4000 to 11000 cells/µL of blood.
•
• An increase in the concentration of leukocytes leads to a medical condition called
“Leukocytosis”. Oppositely, a decrease in the concentration of leukocytes
eventually leads to “Leukopenia”. Therefore, white blood cells make a significant
difference to health, as immunity depends on it.

• The total WBCs in the blood smear is called WBC count. It involves counting
WBCs manually using a haemocytometer and automated WBC counters using
impedance, quantitative buffy coat, and flow cytometry techniques. Here, we will
discuss the manual method to count white cells.

The principle of the WBC count:

• The WBC count method depends upon the dilution of either anticoagulated blood
or capillary blood, using WBC diluent. Then the cover glass is mounted over the
diluted blood smear in the central ruled area of the Neubauer’s chamber.

• Finally, the prepared slide is examined under the light microscope to count the total
number of WBCs within the four large corner squares of the haemocytometer. This
post describes the meaning and types of WBCs. You will also get to know the
requirements, procedure (Thoma pipette method and tube method), criteria to
count WBCs under the microscope and the calculation of WBCs.

Meaning of WBC:
WBCs, leukocytes or white blood cells are the immune cells contributing 1% of your
blood. Hematopoietic stem cells of the bone marrow form white blood cells that flow
through the bloodstream. Spleen, liver and kidneys regulate the WBC’s production. The
blood and lymphatic system carry the leukocytes.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Unlike RBCs, white blood cells possess nuclei and lack haemoglobin. Based on the
presence of granules, there are five types of WBCs.

1. Neutrophils have a segmented nucleus and contain light pink coloured granules
in the cytoplasm. These play a crucial role to phagocytose bacteria and
promoting inflammation.
2. Basophils have a bilobed nucleus and dark blue or purple coloured granules in
the cytoplasm. They mediate allergic responses.
3. Eosinophils have a bilobed nucleus and contain dark pink or red coloured
granules in the cytoplasm. These are active against parasites and helminths.
4. Lymphocytes have a large spherical nucleus with agranulated cytoplasm. These
include effector and regulatory cells (like T, B and NK cells) that trigger an
immune response.
5. Monocytes have a large indented nucleus with agranulated cytoplasm. These
play a significant role to form macrophages and other antigen-presenting centres.

Requirements of WBC Count:

To manually count the white blood cells, you need the following devices and reagents:

Materials required to collect the capillary blood:

Cotton, lancet, ethyl alcohol and glass slide are the materials needed to collect the
blood sample. We will discuss the process of collecting blood further in this post.

Materials required to dilute blood:

WBC Pipette: It is also called Thoma-pipette. WBC pipette is an apparatus used to
prepare the TLC (total leukocyte count) solution. It gives a dilution of 1:20. The stem of
the WBC pipette has markings 0.5 to 1.0. A white bead is present in the swelled central
portion or bulb of the WBC pipette. The short arm after the bulb is marked with the
endpoint 11. There is a rubber tube with a mouthpiece, through which you can suck the
blood.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Micropipette:
It is required to aspirate or dispense the blood sample and WBC diluent of the desired
volume into the sterile test tube. In the bulk dilution method, a micropipette is an
apparatus used to dilute white cells with the WBC diluent.

WBC Diluting Fluid: Turk’s solution dilutes the blood specimen, and it has the following
components:
• Glacial acetic acid: 2 ml
• Gentian violet: 1 ml
• Distilled water: 97 ml
Gentian acetic acid causes lysis of erythrocytes. Gentian violet stains the WBC’s
nuclei. Distilled water is used to dilute the contents of the Turk’s solution.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Haemocytometer:
It was pioneered by Louis Charles Malassez to count blood cells. Haemocytometer is a
thick microscope slide with three lateral platforms. It possesses four vertical troughs and
one horizontal trough. The central platform has an etched grid at the centre.
Haemocytometer has an H-shaped volume chamber. Raised edges of the
haemocytometer hold the thick cover glass at the height of 0.1 mm from the ruled grid.
The improved Neubauer’s slide has nine large squares in a ruled grid, among which
four large squares at the corners are examined to calculate WBCs per microliter of a
blood sample.

Cover Glass:
It is a specialized coverslip that is thicker than ordinary coverslips.
• Thick: 0.4 mm
• Width: 26 mm
• Length: 20 mm

The special cover glass of the haemocytometer slide is placed 1 mm above the blood
specimen loaded into the counting grid.

The Procedure of Total Leukocyte Count:

The Thoma pipette technique and tube method are commonly used to count the white
blood cells manually. Both use the same dilution of the blood specimen. Let us
summarize the steps of both methods.

Collect Blood Sample:

You can use capillary blood directly by pricking the fingertip. First, rub the tip of your
middle finger with cotton saturated with ethyl alcohol. Then, prick the tip of your finger
by using a sterile lancet. Put 2-3 drops of your blood over the centre of a sterilized glass
slide. The procedure of collecting a blood sample is similar in both the Thoma pipette
method and the tube method.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Dilution of Blood Specimen

Thoma pipette method: Suck blood up to the 0.5 mark through the mouthpiece attached
to the end of a rubber tube. Afterwards, you need to suck the prepared Turk’s solution
(WBC diluting fluid) up to an endpoint 11 in a similar way. Then, horizontally rotate the
WBC pipette using your palms in order to mix the contents.
Tube method: Dilution of blood specimen in the tube method is quite different, as it uses
a test tube to mix the blood sample with WBC diluent instead of using a WBC pipette.
The dilution factor is the same (1:20) as the Thoma pipette method. You need to add
0.02 ml of blood into the sterile test tube using a micropipette. Similarly, dispense 0.38
ml of WBC diluting fluid into a test tube. Then, vigorously shake the test tube to mix the
contents.

Sample Loading and Slide Preparation:

Thoma pipette method: Before loading the sample into the central ruled portion of the
haemocytometer slide, you must discard a few drops from the WBC pipette. Place a
thick cover glass over the central grid with a space leaving about 1 mm. Then, carefully
load the sample from both the edges of a cover glass. Through capillary action, the
sample reaches the central ruled area of the haemocytometer. Finally, allow the cells to

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Dr. Nagy Morsy Blood and body fluids Biochemistry
settle for at least 2 minutes. In the diagram, the areas highlighted in blue are the WBC
counting regions that are denoted by ‘W’.

Tube method:
Charge the diluted blood specimen to fill the central ruled portion of the
haemocytometer using a micropipette. Else, you need to follow the same steps of the
Thoma pipette method to prepare the slide for microscopic observation.

How to count WBCs?

First, you need to focus on the rulings of the central grid under the 10X objective. Then,
focus on the four large squares at the corners by adjusting coarse and fine adjustment
knobs. Then, manually count the leukocytes within four corner squares of the ruled grid
via hand tally.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Calculation of WBCs:
After counting white cells in four large corner squares, we can put the values in the
given formula:

• Numbers of WBCs counted = ‘N’

• Dilution factor= 20
• Depth = 10
• Area= 4 mm2

Hence, WBCs/µL= N X 10 X 20 / 4 = N X 50 cells/µL

Suppose the number of cells in the first and second grid is 160 and 200, respectively.
To find out the total number of WBCs, you need to sum up the above two numbers and
multiply them by 50. Thus, we could get the total leukocyte count by putting the final
value in the equation.
WBCs/µL= 360 X 50 cells/µL = 1800 cells/µL

Conclusion:
Therefore, we can conclude that the WBC count method is a form of blood test through
which we could estimate the concentration of leukocytes. Thus, TLC is a quantitative
measurement of white blood cells manually through a wet mount.

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Student Name: Academic Number:

‫هذه الورقة تسلم في المعمل‬

Experimental report (3)

White Blood Cell Count

1. Calculate the number of white blood cells in the following:

Your sample is diluted 1:20 with a solution containing crystal violet, which
makes the colorless WBCs clearly visible and acetic acid to lyse (break apart)
the RBCs so that they are no longer visible.

After counting white cells in four large corner squares, we can put the values in the given formula:

• Numbers of WBCs counted = ‘N’

• Dilution factor= 20
• Depth = 10
• Area= 4 mm2

2. What is the apparatus used to count red blood cells in a blood sample?

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC – 322
Experiments 4
Physical examination of Urine

Color:
Normal urine color has a wide range of variation ranging from pale yellow, straw, light yellow,
yellow, dark yellow amber due to urochrome pigment (it’s an end product of endogen
metabolism), trace of urobilin and uroerythrin.

The color is affected By :

Concentration of urine, pH, Metabolic activity, Diet intake and Some Drugs

1. Colorless or pale yellow

 High fluid intake
 Reduction in perspiration.
 Using of diuretic.
 Nervousness
 Alcohol ingestion
 Diabetes Mellitus.

2. Dark yellow, Amber, orange

 Low fluid intake.
 Excessive sweating
 Carrots or vitamin (A)
 Dehydration (burns, fever).
 Pyridium and nitrofurantoin (drugs).

3. Brownish yellow
 Bilirubin on shaking yellow foam will appear.
 Urobilin on shaking the foam has no color.

4. Yellow – green
 Bilirubin oxd. Biliverdin (greenish).
 Which give a yellow foam & (- ve) test for bilirubin

5. Blue – Green
 Pseudomonas Infection

6. Pink – Red

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Dr. Nagy Morsy Blood and body fluids Biochemistry
Due to the presence of fresh blood or Hb, fresh blood will give smoky color while Hb gives clear
reddish urine.

Both may be due to

 Trauma
 Calculi
 Urinary tract infection
 Menstrual contamination.

6. Dark brown
 Met hemoglobin if bloody sample long standed, Hb will be
oxidized.
 Melanin
7. Black Urine
 Alkaptonuria, a disease of tyrosine metabolism.
 Melanoma (skin cancer).

2. Clarity (Transparency)
Normal urine clear or transparent, any turbidity will indicate.
 WBCs (pus).
 RBCs
 Epithelial cells
 Bacteria
 Casts (cell, protein or fat aggregation)
 Crystals
 Lymph
 Semen.
 Salts (calcium phosoahate)

3. Odor
 Fresh normal urine has a faint aromatic odor due to the presence of some volatile acids.
 In some pathological conditions, certain metabolites may be produced to give a specific
odor such as:
 Fruity odor is due to Diabetic urine acetone.
 Ammoniac odor urine standing long time
 Offensive odor Bacterial action of pus (UTI).
 Apple odor Asparagus
 Mousy odor Phenylalanine (phenyl keto urea)

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Dr. Nagy Morsy Blood and body fluids Biochemistry

4. Measurement of specific gravity (spg)

1. Urinometer:
 Consists of a weighted float a hatched to a scale that has been calibrated in terms of
urine spg. (1.00 – 1.040)
 The weighted float displaces a volume of liquid equal to its weight and has been
designed to sink to a level of 1.000 in distilled water.
 The additional mass by the dissolved substances in urine causes the float to displace a
volume of urine smaller than of D.W. , the level to which the urinometer sinks represent
the specimen spg.

Disadvantages of urinometer:
• The minimum amount of urine to be measured is about 15 ml.
• If the urine is so turbid it is difficult to read the result.

5. PH
 One of the important functions of the kidneys is pH regulation, the glomerular filtrate of
blood plasma is usually acidified by renal tubules and collecting ducts from a pH of 7.4
to about 6 in the final urine to keep blood pH about 7.4.
 Urine pH must vary to compensate for diet and products of metabolism, this function
takes place in the distal convoluted tubule with the secretion of both H+ & NH3+ and
reabsorption of bicarbonate .
 In cases of alkalosis, urinary pH will be alkaline by stop H+ excretion.
 Normal urine pH is (4.6 – 8.0) as average (6.0)

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Student Name: Academic Number:

‫هذه الورقة تسلم في المعمل‬

Experimental report (4)

Physical examination of Urine

1. Write the physical Examination of the following urine sample.

Test Observation Result

Color Pale yellow
Oder Fruity
Clarity Turbid
pH 8.5

1. What is the expected diagnosis for turbid urine?

2. What is the expected diagnosis for Black Urine?

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC – 322
Experiments 5
Tests for protein in urine
1. Principle of dipstick
 The dipstick is impregnated with citrate buffer (PH = 3.0) containing Bromphenol blue
which is yellow at pH 3.0 and blue at pH 4.2, at pH (3) the indicator is mostly unionized.

 If protein is present in the urine into which it is dipped, the ionized fraction (IN) binds to
the protein, thereby causing more dye to ionize until equilibrium is reached; hence, the
impregnated strip has less yellow and bluer color as the protein concentration increases.
This reaction is seen visually as a change from yellow to green (a mixture of yellow dye
plus blue dye appears green). The color is compared with that of the protein content
from (30 – 1000mg /dL).
 At a constant pH, the development of any green color is due to the presence of protein.
 Colors range from yellow for "Negative" through yellow-green and green to green-blue
for "Positive" reactions.
 Trace positive results (which represent a slightly hazy appearance in urine) are
equivalent to 10 mg/100 ml or about 150 mg/24 hours (the upper limit of normal)
 1+ corresponds to about 200-500 mg/24 hours
 2+ to 0.5-1.5 gm/24 hours
 3+ to 2-5 gm/24 hours
 4+ represents 7 gm/24 hours or greater.

Notes:
False (+ve) may be due to increased urine pH

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Dr. Nagy Morsy Blood and body fluids Biochemistry

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Student Name: Academic Number: ‫هذه الورقة تسلم في المعمل‬

Experimental report (5)

Tests for protein in urine

1. What is the expected diagnosis for the following results of protein dipstick
examinations?

Test Observation Results

PH 8.5
Blood Small
Protein +

Conclusion:
……………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Blood and body Fluids Biochemistry

SCBC - 322
Experiments 6
Tests for glucose in urine
Principles:
 Under normal conditions, almost all of glucose filtered by glomerulus is reabsorbed in
the proximal convoluted tubule, by an active process to maintain the plasma
concentration of glucose.
 Less than 0.1% of glucose normally filtered by the glomerulus appears in urine (< 130
mg/24 hr).
 If the blood glucose concentration is increased, reabsorption of glucose ceases &
glucose appears in urine.
 Glycosuria (excess sugar in urine) generally means diabetes mellitus
 The threshold of glucose is 180 mg / dl.

Threshold substances
Substances that are completely absorbed by the tubules when their plasma concentration is
normal and not completely absorbed by the tubules if their plasma concentration exceeds
normal levels.

Glycosuria may be due to

 Reabsorption defect
 Increase Blood glucose, in the following cases:
 Diabetes mellitus
 Alimentary glycosuria (transitory), after meal.
 Stress in which elevation of epinephrine leads to increase glycogenolysis, and cortisol
increase gluconeogenesis.
 Pancreatic disease affect insulin-secreting gland.
 Decrease reabsorption ability.

Tests for sugar :

1- Benedicts test ”

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Dr. Nagy Morsy Blood and body fluids Biochemistry

2- reagent strip

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Dr. Nagy Morsy Blood and body fluids Biochemistry

Student Name: Academic Number: ‫هذه الورقة تسلم في المعمل‬

Experimental report (6)

1. What is the expected diagnosis for the following results of protein dipstick
examinations?

Test Observation Results

PH
Blood
Protein
Glucose
Bilirubin

Conclusion:
……………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………
……………………………………………………………………………………………………………………………………………………
…………………………………………………………………………………………………………………………………………………….

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