103 Practical

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Final revision module 103 practical

Specimen collection and processing

Types of human samples:

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

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3- What is the technique for venous puncture?

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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:

a. Patients’ full name.


b. Age
c. Identification number.
d. Tests to be done.
4- Patient reassurance: the venipuncture will be slightly painful, it will be short duration.

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

7- Sterilization: clean the area for 30 seconds with 70 % alcohol wipe.

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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.

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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

Sites of skin puncture:

• The finger for adult patients and older children &


• The heel for infants and younger children.
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• Puncturing the dermis layer of the skin to access the capillary beds which run through
the subcutaneous layer of the skin
Uses:

• Minimize the amount of blood drawn from a patient.


• The 10 or 20 microliters can be used to look for anemia, check blood sugar or even to
evaluate thyroid function.
Mention Tubes used in blood collection

ESR tube (black cover): it contains Na citrate for whole blood collection
to prevent blood coagulation

Coagulation profile tube (blue cover): it contains Na citrate for


measurement of PT, PTT, & Fibrinogen

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plain tube (red cover): for lipid profile

CBC tube (violet = purple cover): it contains EDTA to prevent blood


coagulation.

Blood glucose tube (grey cover): it contains Fluoride that prevent


glucose consumption by inhibiting enolase enzyme action.

Heparin tube (green cover) for ABG (arterial blood gases)

Gel tube (yellow cover): it accelerates blood coagulation for serum samples

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2- Urine sample

What is the best urine sample and why?

The first sample in the morning where urine is concentrated so any pus cells, RBCs, bacteria
& crystals can be easily detected

Types of urine samples:

1- Random urine sample (Pus, RBCs, Crystals & casts).

2- 24 hours urine sample (for protein & creatinine, VMA estimation)

3- Sterile urine sample for culture & sensitivity (Should be collected in a sterile container)

3- Stool sample

 What is the recommendations of stool sample?


It should be collected in a clean container & sent to the lab within 2 hours of collection

 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

What is the Site of puncture of bone marrow?

 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

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What are the uses of CSF?

What is the site of CSF puncture?

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7- Swabs

 A swab is a small piece of cotton wool wrapped around a wooden handle


Uses of swabs:

 Take microbiological cultures.


 They are also used to take DNA samples from the inner cheek.

What is the name of this technique? What are the uses?

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MCQ Questions:

What is the procedure?

a. Arterial puncture

b. Venous puncture

c. Skin puncture

d. CSF puncture

Determine the site of skin puncture in newly born infant ?

a. Fingertip

b. Toe

c. Heel of the foot

d. Abdomen

What is the chemical substance used in this tube? (violet = purple color)

a. Na citrate

b. EDTA

c. Heparin

d. None (a Plain tube)

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What is the function of the substance found in this tube?

a. To prevent blood coagulation in blood sample.EDTA

b. to increase blood coagulation

c. to separate blood cells from fluid in blood sample.

d. to prevent glucose oxidation in blood sample.

Mention one example of lab tests using this tube?

a. CBC Test

b. Coagulation profile test

c. Blood glucose level

d. Arterial Blood Gas (ABG) analysis

For which lab test is the green tube used?

a. Coagulation profile

b. CBC

c. Blood glucose

d. ABG

What is the function of the substance added to the gray tube?

a. Inhibits enolase enzyme ( inhibit glycolysis).

b. Prevents blood coagulation

c. Accelerates blood coagulation

d. Increases glucose consumption

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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

d. Any intervertebrae space

Spectrophotometry = Colorimetry= principle of colorimetry and pipetting

Clinical biochemistry:

 Clinical biochemistry is generally concerned with analysis of body fluids helping to


diagnose and manage diseases.
 These are performed on any kind of body fluid, but mostly on serum or plasma.
Separation of plasma and serum by centrifugation

 Method of separation: Centrifugation (what is this apparatus? Define? What is the


principle of its use? What is the application?)

Principle:

- High speed rotation around an axis.


- Separates contents of tube according to density.
Application: separation of blood sample to plasma and serum

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 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).

B] The liquid fraction of blood remains above (supernatant).

Compare between plasma and serum?

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

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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

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Colorimetry and spectrophotometry

 Define Colorimetry: The measurement of the intensity of light at selected wavelengths.


 Define Spectrophotometry = principle of spectrophotometry: it is a method to measure
how much a solution absorbs light by measuring the intensity of light beam passing through
(absorbed by) sample solution.
 Substance to be estimated colorimetrically, must be either colored itself or colored through the
addition of reagents.
On biochemical basis explain why is red color seen as red? , why is white color seen as white?
And why is black color seen as black?

 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)

Quantitative estimation of color.

To be estimated colorimetrically, a substance must be either colored or capable of forming


a specific color through the addition of reagents.

Mention the requirements of Calorimetry test?

Spectrophotometer, cuvette, colorimetry kit and micropippet

What is this device? What is the components? What tests can be done by it? What is the
principle of its use?
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Spectrophotometry, components of spectrophotometry:

Colorimetric kit: it contains:

1. Colorimetric reagent
2. Standard
3. Written protocol to follow
What is this kit? What are the components?

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Define Colorimetric reagents have substances that bind to specific analyte (e.g. glucose)
generating a color.

This color intensity is directly proportional to the concentration of the analyte.

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’

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 A micropipette is an accurate device that withdraws and releases the amount of solution to be
applied.

(Test tube) (pippet)

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What is the missing equipment in colorimetry test? What are their types?

pipettes

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Types of pippet:

(I) A pipette can be Fixed or variable. In variable

pipette, volume can be adjusted by rotating the knob

(II) Different pipette sizes are present:

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

What is the type of pippet used in spectrophotometer?

2
1

Answer: 1: micropippet

How to use the pippet?

How to pipette properly: solution withdrawal from tube:

1. Set the required volume on the pipette.


2. Attach the tip surely.
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3. Depress the plunger until you feel resistance.
4. Immerse the tip below the surface of the solution. Make sure the tip is not touching the
bottom or the sides of the tube.
5. Slowly let the plunger go up to its resting position.
6. Wait about one second then pull the pipette tip out of the solution.
7. Make sure there are no bubbles in the tip.
How to pipette properly: solution release into the receiving tube

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.

Complete: each tube contains reagent +……..

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Types of cuvette:

Material Transmission Price


Optical glass 340-2500 nm $
UV Quartz 190-2500 nm $$
IR Quartz 220-3500 nm $$$
sapphire 250-5000 nm $$$$

Preparation of solutions for measurements:

In colorimetric estimations, it is necessary to prepare 3 solutions: blank, test and standard


in 3 different test tubes marked B, T, and S.

1.The test solution: serum + colorimetric reagent.

2.The standard solution: standard solution + colorimetric reagent.

3.Blank solution: distilled water + colorimetric reagent.

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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.

What tests can be done by spectrophotometry?

 The use of spectrophotometers in biochemistry and molecular biology includes:


1- Determination of concentrations of proteins, glucose, TAG, urea, creatinine, AST, ALT
and many others.
2- Measurements of enzyme activity.
3- Following DNA and RNA extraction to detect sample purity. but this is done using a
spectrophotometer, which is a similar technique that detects non-visible wavelengths.
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MCQ Questions:
Identify the device

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.

b. It contains the cellular components

c. Anticoagulants are not needed

d. It contains the clotting factors.

Unlike plasma, which of the following is characteristic to serum?

a. It is formed after clotting of the blood.

b. It contains the cellular components

c. Anticoagulants are needed

d. It contains the clotting factors.

What is meant by standard solution in colorimetric kit?

a. A solution that binds to specific analyte generating a color. (Reagent).

b. A solution used as the zero point (Blank).

c. A solution with a known concentration (standard solution)

d. A solution that contains the patient’s sample

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What's the function of this tool?

a. An accurate device used only for mixing solutions.

b. An accurate device that measures the temperature of a solution

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.

Name the type of pipette?

a. Multichannel Pipette

b. Micro Pipette

c. Pasteur pipette

d. Pipette filler bulb

Name the type of pipette? How to use it ?

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a. Multichannel Pipette

b. Micro Pipette

c. Pasteur pipette

d. Pipette filler bulb

In colorimetry test, choose the missing component to this tube:

a. Patients sample

b. Glucose

c. Standard

d. Distilled water

In colorimetry test, choose the missing component to this tube:

a. Patients sample

b. Glucose

c. Standard

d. Distilled water
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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?

a-cuvette b-test tube c-cuvette d-test tube

Mention the equation to calculate the concentration of a patient’s blood glucose. Where T =
patients sample, S = standard

Concentration of test (T) =

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

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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

Laboratory diagnosis of diabetes mellitus

Diagnosis of Diabetes Mellitus

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:

1- Fasting and 2-hour post-glucose or postprandial (PP) plasma glucose levels


Fasting is defined as no caloric intake for at least 8 hours
Individuals with impaired glucose tolerance (IGT) are considered “pre- diabetic” and are at increased
risk for developing Type II DM.
Normal IGT = Pre diabetic Diabetes
FPG (mg/dL) < 100 100-125 ≥ 126
2-hour PG (mg/dL) < 140 mg ≥ 140 - < 200 ≥ 200

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II- Laboratory tests for follow up diabetic patients

I- Measurement of Glycated-Hb (HbA1C):


 HbA1c results from Non-enzymatic Glycation of hemoglobin (glucose binds to N-
terminal valine of β-chain of Hb)
 HbA1c reflects the mean blood glucose level over the preceding 8-12 weeks (life span of
RBCs is ~120 d)
 HbA1c is the investigation of choice to monitor therapy and provides valuable information
for assessing diabetic control and follow up of diabetes mellitus
 4- 5.7% →Normal
 5.8 – 6.4 pre-diabetic
 ≥ 6.5% → Diabetic
 6.5 -7% → Target for Diabetic
 ≥ 8% → Uncontrolled DM
II- Measurement of Plasma Fructosamine
 Fructosamine is formed when carbonyl group of glucose reacts with an amino group of
a protein such as albumin
 Fructosamine reflects the mean blood glucose level over the preceding 1-2 weeks (serum
albumin has a much shorter half-life than hemoglobin)
 Normal values vary in relation to the serum albumin concentration and are 1.5–2.4
mmol/L
 When Measurement of HbA1C is not appropriate?= not indicated?= fructoseamine is better
1- Change in diet or treatment within 6 weeks
2- Abnormalities of red blood cell (blood loss or hemolytic anemia, or hemoglobinopathy such
as sickle-cell anemia)
3- Pregnancy

III- Oral Glucose Tolerance Test (OGTT)

 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.

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 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

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Renal glucosuria

Additional Tests include:


1. Random blood Sugar
2. Self-monitoring of blood glucose
3. Urinalysis
4. Lipid profile
5. C-peptide measurement
6. Measurement of islet cell antibodies
1. Random blood Sugar
 Random is defined as without regard to time since the last meal.
 FPG is better than random blood glucose
 It is required only during emergency.
 A random plasma glucose concentration >200 mg/dL accompanied by classic symptoms of
DM is sufficient for the diagnosis of DM.
2. Self-monitoring of blood glucose
 Capillary blood glucose measurements performed by patients themselves, as outpatients, are
extremely useful. Also, In type 1 patients in whom “tight” metabolic control is attempted

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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)

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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.
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 Many individuals with new onset type 1 DM retain some C-peptide production

6. Measurement of islet cell antibodies


 Antibodies to insulin, islet cells, or Glutamic acid decarboxylase (GAD) may be present
in type 1 and absent in type 2 diabetes mellitus.

Estimation of blood glucose using a spectrophotometer

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

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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

1. Total cholesterol: measurement of cholesterol in all lipoprotein particles


Total cholesterol = LDL + HDL + (1/5 triglycerides)
2. LDL-C: it is bad cholesterol, because it deposits cholesterol in blood vessels and cause
atherosclerosis, LDL-C is calculated using the results of total cholesterol, HDL-C and
triglycerides
3. HDL-C: it is good cholesterol as it removes cholesterol and carries it to liver, it measures the
cholesterol in HDL particles measures the cholesterol in HDL particles (function: reverse
cholesterol transport , anti inflammatory, antioxidant , atheroprotective, reservoirs for apo
C and apo E)
4. VLDL-C: calculated from (triglycerides/5)
5. NON-HDL cholesterol:= Total cholesterol – HDL cholesterol (it contains all bad cholesterol),
optimal level is less than 130 mg/dl
Calculate HDL- C if you know that: total cholesterol : 347.2, triglycerides: 446, LDL: 228
Total cholesterol = LDL + HDL+ (1/5 triglycerides)
347.2= 228+ HDL+ 89.2
HDL-C = 30 mg/dl
When lipid profile is requested?
1- As part of routine cardiovascular health check every 5 yrs (cholesterol only  if increased
 full lipid profile.
2- Patients at high risk of cardiovascular disease (tested more frequently)
3- Medical condition which can be due to abnormal lipids ( e.g. pancreatitis)
4- Monitor response to prescribed lipid-modifying treatments, such as statins or lifestyle
modification as diet and exercise.
5- High risk children  first lipid profile between 2 and 10 years.
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What is the risk factors of CVS diseases?

 +45 male, + 50 female, smoking, obesity, unhealthy food, sedentary life, hypertension,
diabetic, prediabetic, family history of CVS disease.

How to prepare patient with lipid profile test?

• Fasting for 9-12 (12-14) hours before the test


-do not eat or drink anything.

-only water is allowed.

• Blood sample by inserting a needle in the vein of the arm

• Sometimes a drop of blood from fingertip  portable testing device (health fair)

What are the abnormal lipid profile results?

Optimal Borderline High risk


LDL cholesterol <100 mg/dl 100-159 mg/dl >160mg/dl

HDL cholesterol >60 mg/dl 35-45 mg/dl <35 mg/dl

Triglycerides = TG <150 mg/dl 150-199 mg/dl >200 mg/dl


Total cholesterol <200 mg/dl 200-239 mg/dl >240 mg/dl

Chol/HDL ratio <4 5 >6

LDL/ HDL ratio <2.5 2.5-3.3 >3.3

Why LDL is risk factor?

 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

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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

Disorders of Plasma Lipoproteins (Dyslipoproteinemias)


A- Hyperlipoproteinemia
1. Primary hyperlipoproteinemia
- Familial deficiency of plasma lipoprotein lipase: This produces increase in the plasma levels
of chylomicrons and VLDL.
- Familial Hypercholesterolemia: It is due to defect in LDL receptors in liver and other tissues,
which produces marked increase in LDL.
- Familial Dysbetahyperlipoproteinemia: There is increase in chylomicrons remnants due to
defect in apo-E receptors liver cells.
- Familial Hypertriacylglycerolemia: It is due to overproduction of VLDL which is usually
associated with coronary heart diseases, type II diabetes mellitus, obesity and alcoholism.
2. Secondary Hyperlipoproteinemia
Secondary hyperlipoproteinemia is due to other diseases e.g. diabetes mellitus ,
obstructive jaundice, hypothyroidism, nephrotic syndrome and obesity.

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).

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Atherosclerosis

 It a narrowing and hardening of the arteries.


 LDL cholesterol  “bad” cholesterol  it collects in the walls of blood vessels 
narrowing and hardening of the arteries  atherosclerosis
Mechanism:
 LDL is taken up by macrophage and arterial smooth muscle by scavenger receptors or
non-receptor pinocytosis.
 This occurs when plasma LDL level increased and modified into oxidized and glycated
LDL by oxidants (ROS) and hyperglycemia (as in diabetes mellitus).
 The macrophages become overloaded with cholesterol causing its transformation into
foam cells.
 Accumulated foam cell in arterial wall stimulate:
- Release of growth factors
- Proliferation of smooth muscle
- Formation of plaque (atheroma).
- Narrowing of blood vessels and thus, predispose to thrombosis.
 Complications = Consequences:
1-Cardiovascular disease (hypertension, myocardial infarction) 2-Peripheral artery
disease 3-Stroke

Myocardial Infarction

Mechanism of myocardial infarction:


It is caused by Atheromatous obstruction or Severe spasm in a coronary  artery Block blood
flow to area of heart muscle  Lack of Oxygen and Fuel from nutrients No ATP  damage of
cell membrane  leak of enzymes into blood stream
Risk factors of myocardial infarction: hypertension, hypercholesterolemia,
hypertriacylglycerelemia, obesity, diabetes, smoking, stress, sedentary life, unhealthy food, family
history
The most common clinical symptoms: anxiety, nausea, chest discomfort, short breath, fatigue,
dizziness, chest pain Begins in the chest and spreads to shoulders, arms, elbows, back, neck or
jaws. Rapid irregular pulse, sweating, silent myocardial infarction

Diagnosis of Myocardial Infarction (MI)

Enzyme Time of rise Peak level time Fall to normal


CK-MB 3-4 hrs 24 hrs 36 hrs
Troponin I & T 3-12 hrs 24 hrs 5-10 days (I)
2 weeks (T)
AST 12 hrs 24-48 hrs 5th day
LDH 12 hrs 48-72 hrs At least one week

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1- Creatine Kinase-MB Fraction:

 Part of total CK  Specific for cardiac muscle


 Less sensitive than Troponins
 Useful for diagnosis of reinfarction or extension of infarction (as it begins to fall after 1 day)
subsequent elevation  new event
 2 isoforms 1 & 2 (separated by electrophoresis)

- Ratio isoform 2 : Isoform1 information about Myocardial injury


- Ratio equals to or >1.5  Excellent indicator for early myocardial infarction
- Isoform 2  demonstrates elevation before elevation of total CK-MB

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.

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 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

What other investigations for MI?

 Electrocardiogram( ECG)
 Stress test
 Echocardiogram
 Cardiac catheterization

Life style modifications to maintain normal lipid profile = maintain healthy cholesterol level:

1- Quit smoking and tobacco using


2- Exercise regularly for an hour
3- Avoid high fat and high sodium diet
4- Consume vegetables, fruits, legumes and whole grain food
5- Minimize stress and maintain ideal body weight
6- Routine lipid profile tests

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Enumerate the serum markers for diagnosis of myocardial infarction

Mention the most specific of them

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 )

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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:

a. Green curve is normal, but red curve is a diabetic patient.


b. Green curve is normal, but red curve is a prediabetic patient.
c. Green curve is prediabetic, but red curve is a diabetic patient
d. Green curve is urine level of glucose, but red curve is blood level of glucose of a diabetic
patient
A 56-year-old obese female was admitted for routine check up. Physical examination and
ECG were normal. Laboratory investigations showed:
- fasting plasma glucose: 120 mg/dl,

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- 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

a. endogenous insulin secretion


b. exogenous insulin intake.
c. insulin resistance
d. protein synthesis

kidney function test


Creatinine in blood
Synthesis of Creatine and Creatinine
• Creatine is formed from glycine, arginine and methionine.
• Site of synthesis: liver and kidney
• The uptake & retention of creatine by muscles is increased by androgens
• In muscle Creatine phosphate is the storage form of high energy phosphate
The normal plasma or serum levels are as follows:
• Creatine 0.2 to 0.9 mg/dL
• Creatinine 0.5 to 1.2 mg/dL
• Constant amounts of creatinine are formed daily by non-enzymatic reactions in muscles.
• Plasma creatinine level increases in cases of kidney diseases and it is a good index for renal
functions as its level is not affected by diet.
• Excretion of Creatine and Creatinine
• About 2% of body creatine is converted to creatinine.
• The amount of creatinine excreted for each individual is nearly constant (1-2 g/day)
• it is related to muscle mass.
• This is expressed as creatinine coefficient, which is the milligrams of creatinine excreted in
24 hours per kilogram of body weight
• Creatinine Coefficient = 20 – 30 in men (more muscular) = 10 – 20 in women (less muscular)

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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

Uses of urine test, Why it's done?

 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.

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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).

Acidic urine Alkaline urine


excessive ingestion of meat and Vegetarians.
certain fruits (cranberries)
ketosis (diabetes mellitus & Urinary tract infection.
starvation)
severe diarrhea, metabolic and Respiratory and metabolic alkalosis
respiratory acidosis
Vomiting
Ammonia producing and urea
splitting bacteria

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6- Specific gravity

Measurement of Specific Gravity:


 Normal specific gravity: 1015-1025
 It is measured by urinometer.
 Causes of high specific gravity: restricted water, dehydration, diabetes mellitus,
proteinuria, adrenal insufficiency.
 Causes of low specific gravity: polyuria, high fluid intake, diabetes insipidus,
hypothermia.
Measurement of Specific Gravity:
 Fill a proper cylinder with the sample of urine provided. Remove any froth on the
surface of urine by filter paper. Take the temperature of urine by means of a
thermometer. Float the urinometer in the urine and read, record the observed
reading.
 Correct the specific gravity value for temperature higher or lower than 15 °C on the
basis of one unit specific gravity for each 3 degrees of temperature.

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Conditions of Proteinuria:
● Nephrosis /Nephritis
● Tuberculosis
● Neoplasm
● Diabetic nephropathy
● Bence-Jones proteins in multiple myeloma
● Congestive heart failure
● Hypertension
● Strenuous exercise
● Pregnancy

Causes of Glucosuria are

a- Renal glycosuria
b- Diabetes mellitus
c- Alimentary glucosuria
d- Hyperthyroidism, hyperpituitarism and hyperadrenalism.
e- Stress, severe infections, increased intracranial pressure

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 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

Test for ketone bodies: Rothera's test

permanganate = violet color

Causes of ketnouria:

 Prolonged vomitting
 Starvation
 Diabetic ketoacidosis

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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.

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dry chemistry

 What is this test? How can be done?


 A dipstick — a thin, plastic stick with strips of chemicals on it — is placed in the urine.
The chemical strips change color if certain substances are present or if their levels are
above typical levels.

Procedure

1. Check the expiry date of the urinalysis dipstick.

2. Remove a dipstick from the container whilst avoiding touching the reagent squares.

3. Replace the container lid to prevent oxidisation of the remaining dipsticks.

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.

6. Lay the dipstick flat on a paper towel.


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7. Use the urinalysis guide on the side of the testing strip container to interpret the findings.
Different reagent squares on the strip need to be interpreted at different times, so ensure
you interpret the correct test at the appropriate time interval (e.g. 60 seconds for protein).

8. Once you have interpreted all of the tests, discard the strip into the clinical waste bin
along with your PPE.

9. Wash your hands.

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Mention the causes of positive (glucose-protein-ketone-bilirubin-blood-nitrate) result

Blood: renal stones, cancer

Nitrates: urinary tract infection

MCQ Questions:

When using the dipstick strip, the following must be considered. Mark A for true and B for
false in your bubble sheet:

1. The dipstick should be immersed in urine sample for 10 minutes. (F)

2. The dipstick should be held vertically. (F)

3. The dipstick should be held horizontally to avoid cross-contamination of the reagent


squares. (T)

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
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What are the causes of red color in the urine sample?

a. Normal finding

b. Blood Excretion (hematuria)

c. Hemoglobin excretion (Haemoglobinuria)

d. B & C

Urine may be turbid due to presence of:

a. Bacteria

b. Blood

c. Protein

d. All of the above

Which sample is preferred for a urinary dipstick test?

a. Random Sample

b. Morning, midstream sample

c. Morning, first stream sample

d. supra-pubic aspired sample

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