103 Practical
103 Practical
103 Practical
1- Blood sample, urine sample, stool sample, semen sample, fluid sample (CSF, ascetic fluid,
amniotic, pleural fluid)
2- What is the primary source of specimen for clinical laboratory? Venous puncture
A- Venous puncture
1
3- What is the technique for venous puncture?
2
1- Equipment preparation: syringe, tourniquet, test tube, alcohol swab
2- Patient condition: The patient asked to fast in some types of analysis:
• 6- 8 hours for fasting blood glucose test, uric acid, ESR
• 12 – 14 hours for lipid profile test (TAG, T.Colesterol, HDL, LDL, VLDL)
3- Information record: The information should be recorded on the specimen labels:
5- Positioning the patient: The patient s sit comfortably in a chair with his arm fully extended
also Samples can be taken while the patient lie comfortably on the back.
6- Identify the vein: The most common vein is the median cubital vein; however, the median
cephalic and median basilic veins are also used) Apply a tourniquet above the site of puncture
3
8- Sample withdrawal: Release tourniquet, remove the needle and place a sterile cotton ball
Vacutainer system
a safety-engineered, multi-sample blood collection needle that offers a simple, effective way to
collect blood while reducing the possibility of needle stick injuries.
Place the blood in prepared tubes (plain tubes or tubes that contain special substances as
anticoagulants). If using anticoagulant mix the tubes ten times by gentle inversion
immediately after collection.
4
B- Arterial Puncture
What is the precautions taken in arterial puncture?
● These syringes are pre-heparinized and minimize air exposure that will alter the blood
gas values.
● What is the first choice of arterial puncture?
● The first choice is the radial artery, because of its small size.
● Alternative sites for access are brachial or femoral arteries.
C- Skin puncture
What is the name of this technique? Mention sites of this technique and two uses?
Skin puncture
ESR tube (black cover): it contains Na citrate for whole blood collection
to prevent blood coagulation
6
plain tube (red cover): for lipid profile
Gel tube (yellow cover): it accelerates blood coagulation for serum samples
7
8
2- Urine sample
The first sample in the morning where urine is concentrated so any pus cells, RBCs, bacteria
& crystals can be easily detected
3- Sterile urine sample for culture & sensitivity (Should be collected in a sterile container)
3- Stool sample
What is the uses of stool sample? It is used to diagnose parasitic & protozoal infections.
What is the diagnosis of Occult blood in stool? It is used to diagnose: Colon cancer,
esophagitis, peptic ulcers, gastritis, inflammatory bowel disease.
4- Sputum sample
The patient asked to give the sample in the morning during fasting.
The sample may be taken for TB examination or for bacterial culture.
5- Bone Marrow aspiration Samples
From Sternum in front of 2nd or 3rd intercostal space or from Anterior Superior iliac
spine.
What are uses of bone marrow aspiration? Diagnosis of blood cancers as leukemia,
aplastic anemia & thrombocytopenia.
Needle used:
6- CSF
Define: Cerebrospinal fluid (CSF): is a clear, colorless, watery fluid that flows in and
around brain and spinal cord.
It helps to protect the brain & spinal cord & to removes waste products
9
What are the uses of CSF?
10
7- Swabs
11
MCQ Questions:
a. Arterial puncture
b. Venous puncture
c. Skin puncture
d. CSF puncture
a. Fingertip
b. Toe
d. Abdomen
What is the chemical substance used in this tube? (violet = purple color)
a. Na citrate
b. EDTA
c. Heparin
12
What is the function of the substance found in this tube?
a. CBC Test
a. Coagulation profile
b. CBC
c. Blood glucose
d. ABG
13
If this sample is CSF aspirate : What is the site for CSF aspiration ?
a. Between L3 and L4
b. Between T3 and T4
c. Below L5
Clinical biochemistry:
Principle:
14
Blood sample in a centrifuge tube, After centrifugation, separation causes:
A] Denser blood cells and platelets descend to the bottom of the centrifuge tube
(pellet).
Plasma:
Plasma is the part of the blood that contains both the serum and clotting factors.
Plasma is obtained by centrifugation before clotting occurs.
Plasma = blood- cellular components
15
Serum:
Serum: is the part of the blood that remains once the clotting factors like fibrin have been
removed.
It is the yellow watery part of blood that is left after blood has been allowed to clot and all
blood cells have been removed
Plasma – clotting factors = Serum
16
Colorimetry and spectrophotometry
Red is what we see when the object absorbs all wavelength except the ‘red’ spectrum. Red
wavelength spectrum is reflected and seen by our eyes.
White is what we see when all wavelengths of light are reflected off an object i.e. no
absorption.
Black is what we see when all wavelength of light are absorbed i.e. no reflection.
Colorimetry (spectrophotometry)
What is this device? What is the components? What tests can be done by it? What is the
principle of its use?
17
Spectrophotometry, components of spectrophotometry:
1. Colorimetric reagent
2. Standard
3. Written protocol to follow
What is this kit? What are the components?
18
Define Colorimetric reagents have substances that bind to specific analyte (e.g. glucose)
generating a color.
Define standard solution: has a precisely known concentration e.g. standard solution of
glucose has 100 mg/dl glucose.
Patient’s serum contains the analyte to be measured e.g. glucose of unknown concentration.
To be able to apply solutions to test tubes, you need a ‘micropipette’
19
A micropipette is an accurate device that withdraws and releases the amount of solution to be
applied.
20
What is the missing equipment in colorimetry test? What are their types?
pipettes
21
Types of pippet:
1. 0.5 – 5 ul
2. 1 – 10 ul
3. 10 – 100 ul
4. 100 – 1000 ul
One should always choose the smallest pipette for the required volume.
(III) If the same solution will be added in the same quantity to successive wells in a plate, a
multichannel pipette can be used
2
1
Answer: 1: micropippet
1. Put the pipette in the receiving tube, with the tip below the solution.
2. Depress the plunger to the second stop.
3. Pull the pipette out of the receiving tube.
4. Allow the plunger to return to its rest position.
23
Types of cuvette:
24
Use of blank: blank is used in the beginning to be used as the zero point, starting from which,
color intensity to be measured in standard and test samples.
a. A centrifuge
b. A colorimeter
c. An electrophoresis apparatus
d. A sensitive balance
Unlike serum, which of the following is characteristic to plasma?
a. It is formed after clotting of the blood.
26
What's the function of this tool?
c. an accurate device that withdraws and releases the amount of solution to be applied.
d. An inaccurate device that withdraws and releases the amount of solution to be applied.
a. Multichannel Pipette
b. Micro Pipette
c. Pasteur pipette
27
a. Multichannel Pipette
b. Micro Pipette
c. Pasteur pipette
a. Patients sample
b. Glucose
c. Standard
d. Distilled water
a. Patients sample
b. Glucose
c. Standard
d. Distilled water
28
In colorimetry test, choose the missing component to this tube:
a. Patients sample
b. Glucose
c. Standard
d. Distilled water
Which tube used to apply the sample into the colorimetry device?
Mention the equation to calculate the concentration of a patient’s blood glucose. Where T =
patients sample, S = standard
Absorbance of “T”
a. × Concentration of “S”.
Absorbance of “S
Absorbance of “S”
b. × Concentration of “S”.
Absorbance of “T
Absorbance of “T”
c. Concentration of “S × Absorbance of “S”.
Concentration of “S”
d. × Absorbance of “S”.
Absorbance of “T
29
Calculate the concentration of a patient’s blood glucose provided that the absorbance of the
test= 0.8, the absorbance of the standard= 0.25, and the concentration of the standard is
100mg/dl. Comment on your result if this sample is Random Blood Sugar ?
𝟎.𝟖
a. 𝟎.𝟐𝟓 × 100 = 320 mg/dl which means hyperglycemia
0.8
b. 0.25 × 100 = 320 mg/dl which means normoglycemia
0.25
c. × 100 = 31.25 mg/dl which means hypoglycemia
0.8
0.25
d. × 100 = 31.25 mg/dl which means normoglycemia
0.8
A- Clinical diagnosis: The 3 P’s of DM: Polyuria, polydipsia, polyphagia, Other symptoms
may include: weight loss, delayed wound healing, fatigue, and weakness
B- Laboratory tests:
30
II- Laboratory tests for follow up diabetic patients
After 8-12 hours fasting, a fasting blood and urine samples are obtained.
The patient takes, orally 75 g of glucose dissolved in 300 ml of water.
4 samples are obtained (at ½ hour, 1 hour, 1½ hour, and 2 hours)
The test may be extended for 3 hours especially in case of gestational diabetes.
31
Normal OGTT: There is an increase in plasma glucose levels after 30 and 60 minutes
from the glucose load due to the absorption of glucose
Followed by a drop due to increased uptake and utilization of glucose under the effect of the
stimulated insulin secretion.
No glucosuria occurs during the test
Abormal OGTT:
1. Diabetic curve: The curve points obey the plasma glucose levels of DM + glucosuria +
delayed decline of plasma glucose levels to the fasting levels.
2. Impaired glucose tolerance : The curve points obey the plasma glucose levels of
impaired glucose tolerance
3. Renal glucosuria: Plasma glucose is below 180 mg/dL all-through GIT, however glucosuria
is evident. The curve looks normal.
The green is for a normal patient while the red curve is for a diabetic patient
32
Renal glucosuria
33
3. Urine Analysis:
A. Glucosuria:
Normally glucose does not appear in urine until the plasma glucose rises above 180 mg/dl or
more.
In certain individuals due to low renal threshold glucose may be present despite normal blood
glucose levels.
Conversely renal threshold increases with age so many diabetics may not have glycosuria despite
high blood sugar levels.
A specific and convenient method to detect glucosuria is the urine strip impregnated with glucose
oxidase.
B. Ketonuria:
Qualitative detection of ketone bodies can be accomplished by nitroprusside tests (Acetest or
Ketostix).
Ketone bodies may be present in:
1. Normal subjects with prolonged fasting (Ketogenic diet).
2. Severe uncontrolled DM (DKA)
34
C. Albumin in urine
Normal (2.5-25 mg/day)
Microalbuminuria (>25-<250mg/day)
Macroalbuminuria ( > 250 mg/day)
Microalbuminuria in type I diabetic patients is a signal of early reversible renal damage.
Despite that, microalbuminuria is a common finding (even at diagnosis) in type 2 diabetes
mellitus and is a risk factor for macro vascular (especially coronary heart disease).
D. Albumin to creatinine ratio.
4. Lipid profile
Most obese patients with type 2 diabetes have diabetic dyslipidemia due to insulin
resistance.
It mostly reveals:
1. High total cholesterol
2. High serum triglyceride level (300–400 mg/dL)
3. High LDL cholesterol
4. Low HDL cholesterol (less than 30 mg/dL)
5. C-peptide measurement
It is a good index of endogenous insulin secretion especially in diabetics on exogenous
insulin.
Serum insulin or C-peptide measurements do not always distinguish type 1 from type 2
DM
A low C-peptide level confirms a patient’s need for insulin.
35
Many individuals with new onset type 1 DM retain some C-peptide production
Calculate the concentration of a patient’s blood glucose provided that the absorbance of the
test= 0.8, the absorbance of the standard= 0.25, and the concentration of the standard is
100mg/dl. Comment on your result if this sample is RBS
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝑻𝒆𝒔𝒕
× 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝟎.𝟖
× 𝟏𝟎𝟎= 320 mg/dl (high)
𝟎.𝟐𝟓
36
Lipid profile
Lipids are transported in blood in the form of lipoprotein:
For TAG transport: chylomicron (TG of dietary origin), VLDL (TG of endogenous hepatic
synthesis)
For cholesterol transport: LDL (mainly free cholesterol), HDL (mainly chelosterolester)
What is lipid profile tests:
A group of blood tests ordered to determine the risk of CAD
1. TOTAL CHOLESTEROL
2. HDL-CHOLESTEROL (Good)
3. LDL CHOLESTEROL (bad)
4. TRIGLYCERIDE
Extended profile includes:
1. VLDL CHOLESTEROL
2. NON-HDL CHOLESTEROL
Some reports include:
1. CHOL / HDL RATIO
Or Risk score
Colorimetry equation
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝑻𝒆𝒔𝒕
× 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅
+45 male, + 50 female, smoking, obesity, unhealthy food, sedentary life, hypertension,
diabetic, prediabetic, family history of CVS disease.
• Sometimes a drop of blood from fingertip portable testing device (health fair)
LDL cholesterol is often called the “bad” cholesterol because it collects in the walls of blood
vessels, raising the chances of health problems by narrowing and hardening of the arteries
This condition called atherosclerosis to be discussed in the next section
38
Clinical correlation:
Hypercholesterolemia
Hypercholesterolemia means a plasma cholesterol level higher than 200 mg/dL.
causes:
1-Dietary causes: Diet rich in saturated fat, carbohydrates and cholesterol.
2- Obesity.
3-Diabetes mellitus
4-Hypothyroidism
5-Obstructive jaundice
6-Familial hyperliopoproteinemias
N.B. Statins are a group of drugs that act through competitive inhibition of HMG-CoA reductase
(structural analogs to HMG-CoA) and help to lower plasma cholesterol.
Hypocholesterolemia
Hypocholesterolemia means a plasma cholesterol level less than 120 mg/dL.
Causes:
1- Dietary causes: Prolonged fasting or starvation decreasing the activity of HMG-CoA reductase.
A diet low in cholesterol, carbohydrate, saturated fats, and rich in unsaturated fats decreases plasma
cholesterol concentration.
2-Liver disease
3-Hyperthyroidism
B- Hypolipoproteinemia
1-Abetalipoproteinemia
It is due to failure of synthesis of apo-B, and defective formation of chylomicrons, VLDL and LDL.
2-Deficiency of LCAT
There is marked decrease in cholesterol esters and HDL (the main site of esterification of
cholesterol).
39
Atherosclerosis
Myocardial Infarction
40
1- Creatine Kinase-MB Fraction:
2- Troponins:
Troponin I and T are structural components of cardiac muscle.
They are released into the bloodstream with myocardial injury.
They are highly specific for myocardial injury, more than CK-MB.
Rate of rise for early infarction may not be as dramatic as for CK-MB
More difficult to use it for diagnosis of reinfarction or extension of infarction because of
continued elevation for long time.
Troponin T is elevated in skeletal myopathies and renal failure So it lacks some specificity.
3- Myoglobin:
Myoglobin is a protein found in skeletal and cardiac muscle which binds oxygen.
It is a very sensitive indicator of muscle injury.
However, it is not specific for cardiac muscle, and can be elevated with any form of injury to
skeletal muscle.
41
More difficult to use it for diagnosis of reinfarction or extension of infarction because of
continued elevation for long time
Negative Myoglobin rise Rule out myocardial infarction (elevated before CK-MB)
Myoglobin rise Help to determine the size of infarction
Electrocardiogram( ECG)
Stress test
Echocardiogram
Cardiac catheterization
Life style modifications to maintain normal lipid profile = maintain healthy cholesterol level:
42
Enumerate the serum markers for diagnosis of myocardial infarction
1- Troponin
2- CK-MB
3- Myoglobin
MCQ Questions:
lipid profile test requested in these cases:
a. As part of routine cardiovascular health check at age of 40.
b. Patients at high risk of cardiovascular disease.
c. Medical condition which can be due to abnormal lipids ( e.g. pancreatitis)
d. Monitor response to prescribed lipid-modifying treatments, such as statins
e. All of the above
How to Prepare the patient for Lipid Profile Test?
a. Fasting for 6-8 hours before the test
b. Fasting for 12-14 hours before the test
c. Fasting for 12-24 hours before the test
d. No fasting is required
How to Prepare the patient for Lipid Profile Test? True or false
1. Avoid eating fatty foods (T )
2. Avoid alcohol. (T )
43
The ratio of LDL/HDL that denotes high risk of atherosclerosis and cardiovascular disease is:
a. < 2.5
b. 2.5-3
c. < 2
d. >3.3
The following is not among Atherosclerosis consequences
a. Hypertension
b. Stroke
c. Myocardial infarction
d. Fatty liver
A patient suffers severe chest pain 3 hours ago most probably a case of myocardial infarction
which of the following test is suitable for the diagnosis:
a. Creatinine Kinase
b. Lactate dehydrogenase
c. Alanine transaminase
d. Pyruvate dehydrogenase
The earliest serum marker elevated in blood in case of myocardial infarction is:
a. Troponin I &T.
b. Lactate dehydrogenase.
c. Alanine transaminase.
d. Myoglobin.
Regarding OGTT, Comment on the green and the red curves? The interpretation of this OGTT
curve is:
44
- 2-hours post prandial plasma glucose: 176 mg/dl.
This woman is considered :
a. Having normal blood glucose levels
b. Diagnosed with Diabetes Mellitus type II
c. Diagnosed with Diabetes Mellitus type I
d. Diagnosed with Prediabetes
Comment on this test? C- peptide test is a good index of
45
Urine analysis and renal function test
What is urine: it is an ultrafiltrate of plasma from which glucose, amino acids, water and other
substances essential to body metabolism have been reabsorbed.
Urine carries waste products and excess water out of the body
To check overall health. routine medical exam, pre-surgery preparation. to screen for a variety
of disorders, such as diabetes, kidney disease or liver disease, during admission to a hospital.
Urine pregnancy test: detect the hormone human chorionic gonadotrophin (hCG), which starts
to be produced around 6 days after fertilization.
Urine drug test : analyzes urine for the presence of certain illegal drugs and prescription
medications.
To diagnose a medical condition. if there is abdominal pain, back pain, frequent or painful
urination, blood in urine, or other urinary problems. A urinalysis can help diagnose the cause of
these signs and symptoms.
To monitor a medical condition. If diagnosis with a medical condition was done, such as
kidney disease or a urinary tract infection, testing urine regularly to monitor condition and
treatment.
Urine Analysis
It included physical exam and chemical exam
Physical examination of urine
1- Urine color:
Normal urine is amber yellow due to the presence of a number of pigments principally
urochrome + urobilin + uroerythrin.
The color may vary from pale yellow to dark yellow according to concentration.
46
Abnormal color of urine:
Nearly colorless : (Diabetes Insipidus, diluted urine and chronic nephritis).
Dark = deep yellow : (Concentrated Urine due to dehydration and fever).
Red color : (Blood hemoglobinuria and hematuria).
Brownish color: (Bile pigments Excretion in certain types of jaundice).
Black: alkaptonuria
2- Urine odor:
Aromatic odor Volatile acids
Ammoniacal odor Long standing sample and
bacterial contamination
Foul odor Urinary tract infection
Mousy smell phenylketonuria
Fruity odor ketonuria (Diabetic ketosis) e.g.
Acetone
3- Appearance:
Normal fresh urine is clear.
On standing urine samples develop slight turbidity due to precipitation of phosphates.
Pathological turbidity in urine may indicate the presence of blood, pus, urates
4- Aspect:
Normally, fresh urine is clear.
5- pH of urine:
It reflects ability of the kidney to maintain H+ ion concentration in
extracellular fluid and plasma
Normally, the urine is slightly acidic (5-7).
47
6- Specific gravity
48
Conditions of Proteinuria:
● Nephrosis /Nephritis
● Tuberculosis
● Neoplasm
● Diabetic nephropathy
● Bence-Jones proteins in multiple myeloma
● Congestive heart failure
● Hypertension
● Strenuous exercise
● Pregnancy
a- Renal glycosuria
b- Diabetes mellitus
c- Alimentary glucosuria
d- Hyperthyroidism, hyperpituitarism and hyperadrenalism.
e- Stress, severe infections, increased intracranial pressure
49
Bile salt in urine is abnormal
Bile pigment in urine is normal
Causes of bile salts in urine: extrahepatic (stone), intrahepatic (tumor or hepatitis),
cause bile duct obstruction
Causes of ketnouria:
Prolonged vomitting
Starvation
Diabetic ketoacidosis
50
Wet chemistry Dry chemistry
Wet chemistry is the ordinary Dry chemistry is the use of strips
chemical reaction that occurs when a impregnated with dry reagents to
liquid reagent and a sample are added which the specimen is added.
to a reaction vessel and mixed.
51
dry chemistry
Procedure
2. Remove a dipstick from the container whilst avoiding touching the reagent squares.
4. Insert the dipstick into the urine sample, ensuring all reagent squares are fully immersed.
5. Remove the dipstick immediately and tap off any residual urine using the edge of the
container, making sure to hold the dipstick horizontally to avoid cross-contamination of the
reagent squares.
8. Once you have interpreted all of the tests, discard the strip into the clinical waste bin
along with your PPE.
53
Mention the causes of positive (glucose-protein-ketone-bilirubin-blood-nitrate) result
MCQ Questions:
When using the dipstick strip, the following must be considered. Mark A for true and B for
false in your bubble sheet:
4. The dipstick can be left for a day before the interpretation of the results. (F)
A 17-year-old girl decided to go on a starvation diet. After 1 week of starving herself, what
substance would most likely be found in her urine?
A. Protein
B. Ketones
C. Glucose
D. Blood
54
What are the causes of red color in the urine sample?
a. Normal finding
d. B & C
a. Bacteria
b. Blood
c. Protein
a. Random Sample
55