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تحميل الدليل التدريبي

أسئلة شائعة


 

Estimation of Serum Creatinine

 Introduction:

 Kidney functions :  

§         Regulation of water and electrolyte balance.

§         Regulation of acid base balance.

§         Regulation of arterial blood pressure.

§         Excretion of  metabolic waste products and foreign chemicals.

§         Hormonal Function : secretion of erythropoietin and 1,25 DHCC.

§         Metabolic Function : site for gluconeogensis.

 

Functional units :  

§    The nephron is the functional unit of the kidney . Each kidney contains about one million nephrons.

§    The nephron is composed of glomerulus and renal tubules . The nephron performs its function by ultra filtration at  glomerulus and secretion and reabsorption at renal tubules.

 

Renal diseases :

Many diseases  affect renal function. In some, several functions are affected. In others, there is selective impairment of glomerular  function or one or more tubular  function .Most types of renal diseases cause destruction of  complete nephron.

 

Routine kidney function test include the measurement of :

§          Serum creatinine.

§          Creatinine clearance.

§          Serum urea.

 

 Both serum creatinine  and creatinine clearance are used as kidney function tests to :

§         Confirm the diagnosis of renal disease.

§         Give  an idea about the severity of the disease.

§         Follow up the treatment.

 

Serum creatinine ( 55-120 mmol/L  in adult ) :

 

Creatinine is the  end product of creatine catabolism. 98% of the body creatine is present in the muscles where it functions  as store of high energy in the form of creatine phosphate.

About 1-2 % of total  muscle creatine or creatine phosphate  pool is converted daily to creatinine through the spontaneous, non enzymatic  loss of water or phosphate. 

Creatinine in the plasma is filtered freely at the glomerulus and secreted by renal tubules  (10 % of urinary creatinine). However, creatinine is not reabsorbed by the renal tubules. Plasma creatinine is an endogenous  substance not affected by diet. Plasma creatinine  remains  fairly constant throughout  adult life.

 

Creatinine clearance : 

The glomerular filtration rate (GFR) provides a useful index of the number of functioning glomeruli. It gives an estimation of the degree of renal impairment by disease.

Accurate measurement of GRF by clearance tests requires determination of the concentration in plasma and urine of a substance that is:  

§       Freely filtered at glomeruli.

§       Neither reabsorbed nor secreted by tubules.

§       Its concentration in plasma needs to remains constant throughout  the period of urine collection.

§       Better if the substance is present endogenously.

§       Easily measured.

 

Creatinine meets most of these criteria.  Creatinine clearance is usually about 110 ml/min in the 20-40 year old adults . It falls slowly but progressively  to about 70 ml/min in individuals over 8o years of age. In children, the GFR should be related to surface area, when this is done, results are similar to those found in young adults. 

Clearance is the volume of plasma cleared from the substance excreted in urine per minute. It could be calculated from the following equation.

 

Clearance (ml / min )  =     U  x  V

                                               P

 

      U = concentration of creatinine in urine m mol /L  

    V = volume of urine per min

    P = concentration of the substance in serum m mol /L

 

  Serum  creatinine is a better kidney function test than creatinine clearance  because  :

§     Serum creatinine estimation is more accurate.

§     Serum creatinine level is constant throughout adult life.

 

   Creatinine clearance is only recommended in the following conditions:

§         Patients with early ( minor ) renal disease.

§         Assessment of possible kidney donors.

§         Detection of renal toxicity of some nephrotoxic drugs.

 

  Serum Urea ( 2.5 - 6.6 mmol/L in adult):

 

Urea is formed in the liver  from ammonia released from deamination  of amino acids. As a kidney function test, urea is inferior to serum creatinine because:

§         High protein diet increases urea formation.

§         Any condition of increased proteins catabolism (Cushing's syndrome, diabetes mellitus, starvation, thyrotoxicosis ) will increase urea formation.

§         50 % or more of urea filtered at the glomerulus is passively reabsorbed by the renal tubules.

Estimation of Serum Creatinine:

 


Principle:

 

A common procedure for creatinine determination consists of reacting a protein free filtrate of plasma or serum with an alkaline picrate solution giving rise to a red colour (alkaline creatinine picrate) which is measured at 520 nm.

Procedure:

 

 

Test

Standard

Blank

Picric acid

6 ml

6 ml

6 ml

Serum or plasma

0.5 ml

-

-

H2O

1.5 ml

1.5 ml

2.0 ml

Creatinine standard

-

0.5 ml

-

 

§     Mix well and immerse the tube for Test in vigorously boiling water bath for 40 seconds, then cool under tap water and filter (Blank and Standard need not to be filtered).

§     Transfer 4 ml aliquots of the filtrate, standard and blank from each tube to                a corresponding tube.

§     To each tube, add 0.2 ml of 2.5 M NaOH solution and mix.

§     Allow to stand for 20 minutes at room temperature.

 

§     Read the absorbances of test and standard against blank at 520 nm.

 

Calculations:

                                                             A Test

Serum Creatinine (mmol/L) =                                   x conc. of standard (250 mmol/L)

                                                          A  Standard

 

                                                            Urine creatinine level

 Creatinine clearance (ml/min) =                                         x Urine volume  (ml/min).

                                                           Serum creatinine level

Normal adult reference values:

i.         Urinary excretion of creatinine is 0.5-2.0 g  per 24 hours in a normal adult                                

             varying according to muscular weight.

       ii.   Serum creatinine       :   55-120  mmol/L                                            

       iii.  Creatinine clearance  :   90-140 ml/min/1.73 m2          (Males)

                                                 80-125 ml/min/1.73 m2         (Females)

Case study 1

 

A 62-year-old man visited his general practitioner and complained of malaise, tiredness and weight loss over the previous 6 months. His only other complaint was of passing more urine than usual, especially at night, when he had to get up three or four times. He appeared pale, was hypertensive, with a blood pressure of 182/114 mmHg. Urine analysis revealed protein, but no glucose.

 

The results of simple initial investigations were as follows:

 

Laboratory test

Patient's result

Reference range

Sodium

132

135 -145       mmol/L

Potassium

5.6

3.5 - 5.1        mmol/L

Urea

38.2

2.5 - 6.6        mmol/L

Creatinine

635

55 -120         mmol/L

Glucose

5.2

4.0 - 6.0        mmol/L

Calcium

1.88

2.12 - 2.62    mmol/L

Phosphate

2.38

1.8 - 1.4        mmol/L

Hb

9.2

13.5 -18.0          g/dL

 

 

Case study 2

 

A 6-year-old boy developed marked edema over a period of a few days, and his parents had noted that his urine had become frothy. His general practitioner detected proteinuria, and arranged admission to a hospital, where the following results were obtained.

 

The results of simple initial investigations were as follows:

 

Laboratory test

Patient's result

Reference range

Sodium

135

135 - 145        mmol/L

Potassium

4.0

3.5 - 5.1          mmol/L

Urea

3.4

2.5 - 6.6          mmol/L

Creatinine

48

55 - 120           mmol/L

Albumin

14

35 - 50                   g/L

Total protein

34

60 - 80                   g/L

24-hour urine protein

12

0.03 - 0.10                g

 

 

 

 

 

 

 

Case study 3

 

A young man sustained multiple injuries in motorcycle accident. He received blood transfusion and underwent surgery, 24 hour after admission, he had only passed 500 ml of urine. He was clinically dehydrated and his blood pressure was 90/50 mmHg.

 

The results of the clinical chemistry investigations were as follows:

 

Laboratory test

Patient's result

Reference range

Sodium

131

135 - 145       mmol/L

Potassium

5.5

3.5 - 5.1         mmol/L

Urea

20

2.5 - 6.6        mmol/L

Creatinine

115

55 - 120         mmol/L

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Estimation of Serum Uric Acid

 

Introduction:

In human, uric acid (2,6,8-trihydroxypurine) is the major product of the catabolism of purine nucleosides. The clinical importance of purines is related to the disorders characterized by increased plasma urate level.

The latter may arise from:

1-     Increased intake of urate or its precursors (purine-rich foods are liver, kidneys, red meat, and sardines

2-     Increased production of urate in the body.

3-     Defects in renal elimination of urate.

Plasma urate level varies with the following physiological factors:

1-     Gender: male > female.

2-     Age: increases gradually with age.

3-     Pregnancy, plasma uric acid concentration falls during the first trimester and until 24 weeks of pregnancy, when concentrations begin to rise and eventually exceed nonpregnant concentrations.

4-     Weight: increases in obese.

5-     Social class: higher in the more affluent (rich) social classes.

6-     Diet: higher in individuals taking a high-protein diet (also high alcohol consumption increases plasma urate level).

7-     Genetic factors.

 

Biochemistry and Physiology:

Uric acid is a weak acid:

§       The first acid dissociation constant (pKa1) of uric acid is 5.57.

§       At pH > pKa1, uric acid exists mainly as urate ion, which is more soluble than uric acid.

§       At pH < pKa1, uric acid is the predominant form.

 

Reference intervals of uric acid:

1- Plasma [uric acid]:

     Using an enzymatic method, the reference interval for uric acid is:

          In men: 0.208-0.428 mmol/L (3.5-7.2 mg/dL).

          In women: 0.155-0.357 mmol/L (2.6-6.0 mg/dL).

2- Urinary [uric acid]:

§  In individuals on a diet containing purines, the urinary [uric acid] is 150-450 mmol/day (250-750 mg/day).

§  In individuals on a purine-free diet, urinary excretion may decrease by 20-25%.

 

Total body pool of uric acid:

§ In normal human, the total body pool of exchangeable urate when consuming      a purine-free diet is:

               - In men: 1200 mg.

               - In women: 600 mg.

§       In patients with gouty arthritis and tissue deposition of urate: the total body pool of urate may reach as high as 18000 to 30000 mg.

 

Uric acid excretion:

 

In humans, approximately 75% of uric acid excreted is lost in the urine; most of the remainder is secreted into the gastrointestinal tract, where it is degraded to allantoin and other compounds by bacterial enzymes.

The role of the kidney in uric acid excretion:

1-     Free glomerular filtration.

2-     Tubular reabsorption: in proximal convoluted tubules, of ~ 98-100% of filtered uric acid.

3-     Tubular secretion: into the lumen of the distal portion of the proximal tubules.

4-     Further tubular reabsorption in the distal tubule.

5-     The net urinary excretion of uric acid is 6-12% of the amount filtered.

 

Clinical significance:

 

I. Hyperuricemia:

Defined as plasma urate level >420 mmol/L (7.0 mg/dL) in men, or >360 mmol/L (6.0 mg/dL) in women.

 

Causes:

1-     Increased formation

          - Primary:

                            - Idiopathic.

                            - Inherited metabolic disorders.

          - Secondary:

                        - Excess dietary purine intake.

- Increased nucleic acid turnover (e.g. cancer, radiotherapy, chemotherapy).

2-     Decreased Excretion:

          - Primary: - Idiopathic.

         

- Secondary:

- Kidney disease (acute or chronic).

- Increased renal reabsorption.

- Reduced renal secretion.

- Lead poisoning.

- Lactate and ketone bodies: (interfere with renal tubular secretion of urate).

- Drugs:  Salicylates (low dose) and thiazide diuretics.

- Down syndrome.

Gout:

§       It occurs when monosodium urate precipitates from supersaturated body fluids into body tissues.

§       Gouty arthritis: associated with urate crystals in joint fluid and deposits of crystals (tophi) in tissue surrounding the joint leading to an intense inflammatory response (mediated by polymorph nuclear leukocytes and macrophages).

§       The big toe joint is the classic site for gout.

§       Gout is characterized by occasional acute attacks and long periods of remission.

§       Kidney disease associated with hyperuricemia may be one of the followings:

          - Gouty nephropathy: with urate deposition in renal parenchyma.

          - Acute intratubular deposition of urate crystals.

          - Urate nephrolithiasis (urate stones).

Management of gout:

A- Acute attacks:

§       Nonsteroidal anti-inflammatory drugs (NSAIDs).

§       Colchicine.

§       Avoid purine-rich food.

§       Avoid drugs that affect urate excretion (thiazide diuretics and salicylates).

B-   Long-term treatment:

§       Uricosuric drugs (e.g. probenicid).

§       Inhibitors of urate synthesis (e.g. allopurinol: an isomer of hypoxanthine that will inhibit xanthine oxidase.

§       High fluid intake and alkalanization of urine especially in patients in whom urate stones seem likely to form.  

 

II. Hypouricemia:

§       Defined as plasma urate level < 120 mmol/L (2.0 mg/dL).

§       It is much less common than hyperuricemia.

Causes: secondary to:

1-     Severe hepatocellular disease (with reduced purine synthesis or xanthine oxidase activity).

2-     Defective renal tubular reabsorption of uric acid:

a. Congenital.

b. Acquired:

- Acute: secondary to injection of radiopaque contrast media.

- Chronic: secondary to exposure to toxic agents.

3- Overtreatment of hyperuricemia with allopurinol or uricosuric drugs and cancer chemotherapy with an agent that inhibits de novo purine synthesis (e.g. 6-mercaptopurine, or azathioprine).

4- Inherited metabolic defect: very rare; e.g. hypouricemia and xanthinuria suggesting xanthine oxidase deficiency.

 

Analytical Methodology:

§       Uricase, phosphotungstic acid (PTA), and high performance liquid chromatography (HPLC)-based methods have been used for measuring uric acid.

§       The uricase methods are more specific than PTA method and have replaced PTA methods in most current instrumentation.

§       HPLC methods are specific and fast and might be recommended for reference use.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Estimation of Serum Uric Acid (Uricase/peroxidase method):

 

Principle:

 

Uric acid (2,6,8 trioxy purine) is the end product of purine catabolism in humans. Other species, however, contained uricase, an enzyme that converts uric acid into        a more water-soluble product, allantoin. Quantitative measurement of serum uric acid provides a clue for diagnosis of hyperuricemia, gout, renal impairment, etc.

 

                     Uricase

Uric acid + O2 + H2O                             allantoin + CO2 + H2O2

 

        Peroxidase

H2O2 + * DHBS* + 4-aminophenazone                         quinoneimine

The absorbance of quinoneimine dye at 546 nm is directly proportional to serum uric acid concentration.

Procedure:

 

 

Test

Standard

Blank

Reagent           

2.5 ml

2.5 ml

2.5 ml

Serum              

0.05 ml

-

-

Standard          

-

0.05 ml

-

H2O                 

-

-

0.05 ml

 - Mix and incubate for 10 minutes at room temp. (20-25 °C).

 - Measure the absorbance of test and standard against blank at 546 nm.

    

 

Calculation:

                                                               A  Test         

Uric acid conc. (μmol/L)   =                             X   conc. of standard (596)

                                                           A  Standard

         

       N.B: Uric acid (mg/dL)   =    Uric acid (μmol/L) divided by 59.6  

 

Normal reference values :

 

Males       :  200- 420 μmol/L (3.5 - 7.0 mg/dL).

Females   :  150- 350 μmol/L (2.5 - 5.8 mg/dL).

------------------------------------------------------------------------------------------------

* DHBS = 3,5 – dichloro –2- hydroxybenzenesulfonic acid.

 

 

 

Case study

 

A 46-year-old male presents to the emergency department with severe right toe pain. The patient was in usual state of health until early in the morning when he woke up with severe pain in his right big toe. The patient denies any trauma to the toe and no previous history of such pain in other joints. One year ago, he had an episode of renal colic.

On examination, he was found to have a temperature of 38.2ºC and in moderate distress secondary to the pain. The right big toe (the first metatarsophalangeal joint) was swollen, warm, red, and extremely tender. The remainder of the exam was normal. Synovial fluid was obtained for microscopic analysis.

 

1. What is the likely diagnosis?

2. How would you make a definite diagnosis?

3. What is the possible cause for this patient’s previous episode of renal colic?

4. What are the possible characters in patients most susceptible to this disorder?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Extraction and purification of DNA

A)  DNA isolation and purification:

1.     Sample: nucleated cells, e.g., mononuclear blood cells, biopsy specimen, tissue-culture cells

2.     Principle:

§       Lysis of cells

§       Removal of contaminants (any materials other than DNA, e.g., proteins, debris,..etc)

 

3.      Nucleotides absorb UV light at 260 nm wave length. The purity of DNA is evaluated by determination of A260/A280 ratio of DNA solution:

      260/A280 ratio, 1.7-1.9 is acceptable, lower ratios (<1.6) indicate protein contamination, higher ratios (> 2) indicate RNA contamination.

 

     The concentration of DNA solution is evaluated by measuring its absorbance at 260 nm (A260).

 

For ds DNA 1.0 A260 = 50  μg/ml

For ss  RNA 1.0 A260 = 40  μg/ml

N.B. The UV light is a potent mutagen because of the ability of nucleotides to absorb UV light.

 

B)         Denaturation (melting) of ds DNA:

It is the separation of the 2 strands of ds DNA with loss of hydrogen bonds between the complementary bases and unstacking of the bases, while the nucleotides of each strand are still linked by the phosphodiester bonds. Denaturation of DNA in solution can be achieved by increasing temperature or by decreasing salt conc.

Melting temperature (Tm): The DNA strands separate over a temp. range, the mid-point is the Tm. It is higher for GC-rich DNA.

Hyperchromicity of denaturation: It is the increase in the absorbance at 260 nm by nucleotides, associated with denaturation. It can be used to monitor the degree of denaturation.

Viscosity of DNA solution: The viscosity of DNA solution is lost upon denaturation (DNA solution is viscid due to the presence of hydrogen bonds and the stacking between bases).

Applications: Denaturation is important for analysis of DNA:

1.      Southern blotting: Denaturation is essential to allow hybridization between target DNA sequence and the labeled probe.

2.     PCR reaction: Denaturation is the first step in PCR amplification cycles, including, denaturation, annealing, and polymerization (extension) steps.

A protocol for the extraction and purification of DNA from whole blood sample

(QIAamp DNA blood mini kit, Qiagen)

 

Reagents:

- Qiagen Kit:

Protease stock solution

Lysis buffer: Buffer AL

Wash buffer 1: AW1

Wash buffer 2: AW2

Elution buffer: AE

Spin columns

Collection tubes

- Other reagents:

Absolute ethanol (96 – 100 %)

Microcentrifuge tubes (1.5 ml)

 

Equipments:

          - Automatic pipettes

            - Microcentrifuge

            - Vortex

            - Water bath

            - UV-spectrophotometer

 

Before starting:

1.      Allow samples and reagents to equilibrate at room temperature (15–25°C)

2.      Adjust a water bath to 56 °C.

3.      Ensure that protease, AW1 and AW2 are prepared according to manual   instructions.

4.      If precipitate has formed in buffer (AL), dissolve by incubating at 56 °C.

 

Steps:

1.      Pipette 20 μl Qiagen protease into the bottom of 1.5 ml microcentrifuge tube.

2.      Add 200 μl whole blood sample.

3.      Add 200 μl buffer AL and mix by pulse-vortex for 15 seconds.

4.      Incubate at 56 °C for 10 minutes.

5.      Brief centrifuge to remove drops from the inside of the lid.

6.      Add 200 μl ethanol (96 –100 %), mix by pulse-vortex for 15 seconds and brief centrifuge to remove drops from the inside of the lid.

 

7.      Carefully apply the mixture from step 6 to the QIAamp spin column (in a 2 ml collection tube) without wetting the rim, close the cap.

8.      Centrifuge at 6000 x g (8000 rpm) for 1 minute.

9.      Place the spin column in a clean 2 ml collection tube and discard the tube containing the filtrate.

10.  Carefully open the column and add 500 μl buffer AW1 without wetting the rim, close the cap.

11.  Centrifuge at 6000 x g (8000 rpm) for 1 minute.

12.  Place the spin column in a clean 2 ml collection tube and discard the tube containing the filtrate.

13.  Carefully open the column and add 500 μl buffer AW2 without wetting the rim, close the cap.

14.  Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes.

15.  Place the spin column in a clean 1.5 ml microcentrifuge tube and discard the tube containing the filtrate.

16.  Carefully open the column and add 200 μl elution buffer AE and incubate at room temperature for 5 minutes.

17.  Centrifuge at 6000 ´ g (8000 rpm) for 1 minute.

18.  Save the eluant and discard the column.

 

Determination of Purity, concentration and yield of DNA:

- Purity of DNA solution:

Determine A260/A280 ratio

1.7 – 1.9 is accepted

- Concentration of DNA (μg/ml):

            Measure A260

Calculate the DNA concentration (μg/ml) provided

DNA is 50 μg/ml when A260 = 1.0

- Yield of DNA (μg):

            DNA concentration ´ Total volume of purified DNA

 

 

 

 

 

 

 

 

 

 

Polymerase Chain Reaction ( PCR )

 

Introduction:

 

Polymerase chain reaction (PCR) is an in-vitro molecular biology technique applied for replicating DNA by a special DNA polymerase. It does not require a living organism. The technique allows a small amount of the DNA molecule to be amplified exponentially. It can be extensively modified to perform a wide array of genetic manipulations. PCR is commonly used in medical and biological research labs for            a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, and the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.

 

I. Principle:

 

PCR technique exploits special DNA polymerases to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA in the sample. This leads to an exponential increase in DNA with repeated cycles of amplification.

 

II. Components of PCR reaction (the mixture put in a test tube):

 

1. Target DNA sequence (template).

2. Primer Set (two oligonucleotide primers: forward and backward).

3. DNA polymerase enzyme (e.g. heat stable Taq polymerase).

4. dNTPs.             ­

5. Buffer (including MgCl2) and sterilized water.

 

III. Equipment: Thermal cycler.

 

IV. Steps of PCR:

 

1. Primer construction:

 

It is necessary to know the nucleotide sequence of short segments on each side of the target DNA which are called flanking sequences. Thus, the whole nucleotide sequence of the target DNA is not required to be known for primers designing.

The flanking sequences nucleotides are used to construct two single stranded oligonucleotides 20-35 nuc1eotides long, which are complementary to the flanking sequences.

 

 

 

 

2. One PCR amplification cycle includes three consecutive steps:

- Step 1: Denaturation of DNA at 95°C: DNA to be amplified is heated to        

       separate the double -stranded target DNA into single strands.

- Step 2: Annealing of primers to single-stranded DNA at 55oC: The separated      

    strands are cooled and allowed to anneal to the two primers (one for each    

      strand).

- Step 3: Chain extension at 72oC. DNA polymerase and dNTPs are added to     

    the mixture to initiate the synthesis of two chains of DNA complementary to      

      the original DNA chains.

         

 N.B: Taq polymerase is a heat stable enzyme derived from a bacteria that normally lives at a      high temperature (thermophilic bacteria). So, the enzyme is not denatured after each cycle.

 

3. The PCR cycle is repeated many times. (~ 20 -30 cycles)

 

4. Analyzing of the amplified DNA sequence (PCR product):

     By agarose gel electrophoresis, southern hybridization or sequencing of DNA.

 

V. Advantages of PCR over other amplification techniques:

 

§        PCR is a relatively less time consuming than other amplification techniques as it permits the synthesis of millions of copies of specific nucleotide sequence in a few hours.

§        PCR is less technically difficult.

§        It can be used to amplify trace amounts of DNA.

§        DNA sequences from any source, bacterial, viral, plant or animal are suitable for amplification.

 

VI. Applications of PCR in Medicine:

 

1. Diagnosis of infectious diseases:

 

PCR is applied in detecting DNA or RNA of infecting organisms as bacteria and viruses. In this respect, PCR has the advantage of detecting the occurrence of infection even in the early stages, in which only a small amount of nucleic acid is available in blood. In addition, real time-PCR (RT-PCR), which is a special modification of PCR technique, allows the quantitative determination of viral infection. This developed technique facilitates the decision of proper and adequate medication and the follow up of patients' condition and their response to treatment.

 

 

 

Detection of infectious diseases by PCR is nowadays recommended to be performed as a routine for blood of donors in blood transfusion. This can help to limit risk of transmitting infected blood with viruses (as hepatitis B, HIV, etc) from carriers who may show negative results by ordinary serological methods which depend on antibodies detection in serum. Appearance of antibodies in serum may take a long time to develop.

 

Examples of infectious organisms detected by PCR

§        Bacterial infections: Tuberculosis (TB).

§        Viral infections: hepatitis B and C virus.

§        Human Immunodeficiency Virus (HIV) which causes AIDS.

 

2. Detection of mutations in single genes:

Some single gene mutations may cause inborn errors of metabolism. Accurate and early diagnosis even before appearance of clinical signs of such disorders may help to avoid serious consequences as it permits treatment to be applied in proper time.

 

A set of primers is designed to amplify the single gene of interest. Amplified gene can be further studied by sequencing in order to identify the presence of mutations of any type. Sometimes, it is required to design many sets of primers for different locations on the same gene. This technique is called multiplex-PCR.

 

Examples of application of PCR in single gene mutation:

 

Cystic fibrosis:        

 

It is an autosomal recessive genetic disease. The clinical disorder results from mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The most common mutation is three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein.

PCR helps to verify the CFTR gene for the presence of mutation. As the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size the PCR products obtained by amplifying the portion of the DNA.

 

Duchenne muscular dystrophy (DMD):

 

DMD is among the most common human genetic diseases. This gene is greater than 2 million base pairs in size and contains at least 70 exons separated by an average intron size of 35 Kb. Intragenic deletion mutations account for up to 60% of all cases of DMD. In order to detect deletions in this large gene by PCR, at least nine separate regions of the dystrophin gene are amplified at the same time using    a single PCR procedure called multiplex PCR. To fulfill technique requirements, several sets of PCR primers are designed and applied in equimolar concentrations to permit specific and simultaneous amplification of suspected loci on the gene.

 

3. Forensic applications of PCR:

 

DNA isolated from a single human hair, a tiny spot of blood or a semen sample is sufficient to check whet4er the sample belongs to a specific individual or not. Accordingly, evidences from crime scenes could be analyzed in order to verify whether a suspect (if available) is guilty or not guilty.

 

4. Molecular Genetics and Molecular Biology research applications of PCR.

 

The study of segments of DNA may require their amplification by PCR. Amplified DNA can be analyzed by sequencing. In this respect, PCR technique as a relatively rapid technique of DNA amplification, facilities the human genome project which achieved to sequence all DNA in human cells.

 

In addition, scientists try to understand the physiological function of each domain of various proteins as enzymes. This could be easily performed by exploiting the cDNA coding these proteins. cDNA of a protein of interest may be amplified by PCR. Then, amplified cDNA is cloned in expression vectors. Cloned vectors are introduced to expression prokaryotic or eukaryotic host cells in order to synthesize the protein of interest.

 

Many studies of genes may require inducing mutagenesis in a specific single or multiple loci on the DNA coding the protein by using PCR. Then, the mutated gene is further amplified by PCR and cloned in expression vectors. Expressed mutated protein is studied for the biological importance of the knocked out region of DNA.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Estimation of Serum Iron

Introduction:

Sources:

The liver, kidney, spleen, heart meat and egg yolk are very good sources of iron. Molasses, dates, legumes, vegetables and whole cereals are also good sources.

 

Absorption:

On an average diet containing 10-15 mg of iron, only about 5 -15% of the iron is absorbed mostly from the duodenum where the pH is relatively low. Little iron may be absorbed from the lower small intestine.

The absorption of iron is affected by:

 

a) Amount of iron ingested:

The greater the amount of iron ingested in the diet the greater is the amount absorbed.

 

b) State of iron: 

Iron is liberated from the organic complex, e.g. ferritin, forming inorganic salt. This is helped by gastric HCl. Ferric iron (Fe3+) is converted to the ferrous state (Fe2+) before absorption. This is helped by reducing substances in food, as ascorbic acid and cysteine. Heme is better absorbed than other forms of iron making meat an excellent source of iron.

 

c) Solubility of iron:

The absorption of iron is reduced by factors that decrease its solubility. These include high pH, excess phosphate, oxalate, phytate and unabsorbed fatty acids. This makes the iron of animal origin more absorbed than that of plant origin.

 

d) Copper: 

     The iron absorbed from the intestine enters the blood in the ferrous state. In the plasma, it becomes oxidized rapidly to the ferric state to be carried by transferrin. Oxidation is catalyzed by ceruloplasmin (a copper-containing protein) which is important for the absorption of iron from the intestine and for the mobilization of iron from tissue stores. Copper deficiency leads to anemia.

 

e) Body needs:

Both ferritin and transferrin are present in the enterocytes and are believed together to regulate iron absorption. Iron absorption is reduced when body stores of iron are already increased. Conversely, with iron deficiency, iron absorption is increased. Iron absorption is also increased when erythropoiesis is increased.

 

Distribution and functions:

-       The total iron in the body is about 4 g.

-      It is present in the body in 2 forms:

 

a) Functional forms (75%):

 - These are mostly in the form of hemoproteins. They are responsible  for cellular respiration .

 - They include:

1. Hemoglobin (67%): This is the main form of iron in the body. It is a hemoprotein that carries oxygen from the lungs to the tissues and helps the carriage of carbon dioxide in the opposite direction. 

2.  Myoglobin (7.5%): This is a hemoprotein found in the muscles. It acts as temporary carrier of oxygen.

 3. Respiratory enzymes (0.5%): These are mostly hemoproteins, which include cytochromes, cytochrome oxidase (electron carriers in the respiratory chain), catalase, peroxidase (important in the detoxication of hydrogen peroxide) and tryptophan pyrrolase (important in tryptophan metabolism). In addition, some flavoprotein enzymes contain non-heme iron  e.g. NADH and succinate  dehydrogenases.

 

 b) Nonfunctional forms (25%):

Free iron is very toxic and protein binding allows iron to be transported and stored in a non-toxic form. The nonfunctional forms of iron are the transport and storage forms. They are non-heme metalloproteins.

 

1. Transferrin (0.1%): This is the transport form of iron in the blood plasma.

 

2. Ferritin and Hemosiderin (24.9%):

- Ferritin:

This is the chief storage form of iron in the tissues. It is present in the liver, kidneys, spleen, bone marrow and intestinal mucosal epithelium. It is a spherical molecule consisting of an apoferritin protein shell surrounding an interior ferric oxhydroxide (Fe OOH) crystalline core. It contains about 23% iron.

-  Hemosiderin:

This is present in iron stores when the body contains excess iron. It is similar to ferritin in structure, but contains about 35% iron.

 

                     Normal ratio of Ferritin  :  Hemosiderin is 1.1  :  1

 

Blood Iron:

 

a) In the red blood cells:

The erythrocytes contain hemoglobin which contains 3.4 mg of iron per gram. There is 15 g of Hb i.e. 50mg of iron per 100 ml of blood

 

b) In the plasma: 

1.Transferrin: The plasma iron concentration is 11.5 – 30 µmol/L for adult male. Iron is carried by a glycoprotein transferrin (2 - 4 g/L) which carries 2 atoms of ferric iron (Fe3+) per molecule. It is synthesized in the liver and runs with the beta globulin in the electrophoresis. 

Transferrin may carry up to 250-400 µg of iron per dl. plasma. This is known as the total binding capacity (TIBC). This means that, on average, only about 30% of TIBC is saturated. In iron deficiency anemia, the plasma iron decreases while the TIBC tend to increase. In liver diseases, both the plasma iron and TIBC tend to decrease.

Measurement of plasma-soluble transferrin receptor concentration is                      a recent addition to the tests available for the investigation of iron status.  The plasma concentration increases to two to three times normal in presence of anemia, but this rise occurs only after the iron stores became functionally depleted.

2. Ferritin: Plasma contains very low concentration of ferritin (20-250 µg/L), which is a very good index of iron storage. It decreases in iron deficiency anemia and increases in hemosiderosis.

 

Excretion:

a) Feces (90 – 95 %):

Fecal iron is mostly unabsorbed iron. Only minute amount of iron may be excreted in the bile after absorption or in sloughed intestinal cells.

 

b) Urine and sweat (5 –10 %)

The daily loss of iron in the urine and sweat amounts to about 0.5 – 1 mg. Failure of the kidneys to excrete iron is probably due to the strong binding of the plasma iron to transferrin, which is not filterable by the glomeruli.

 

c) Menstruation and milk (5 -10 %) 

About 15 - 30 mg of iron (in the form of Hb) are lost in menstruation per month which is equivalent to 0.5 - 1 mg/day. Breast - feeding accounts for the loss of about 0.5 - 1 mg of iron per day.

 

Toxicity:

 

The body is unable to excrete large load of iron. Iron differs from most other minerals in that its quantity in the body is controlled by regulating its absorption rather than its excretion. Some people can absorb large amounts of iron, from the intestines (20 - 45% of intake), leading to accumulation of excessive amounts of iron in tissues, a condition known as hemosiderosis (hemosiderin accumulates in the tissues) which is an autosomal recessive disease. Hemochromatosis means hemosiderosis with injury to involved tissues as manifested by cellular degeneration and fibrosis. Clinical manifestations include bronzed pigmentation of skin, liver cirrhosis and pancreatic fibrosis leading to diabetes mellitus. Serum iron is elevated and transferrin becomes 70 - 90% saturated with iron. A similar condition is observed in patients with aplastic or hemolytic anemia who have received repeated blood transfusions.

 

 

Requirement:

The RDA is 10 mg/day. This is to be increased to 15 mg for females below the age of 50 years and during lactation and to 30 mg during pregnancy. Iron deficiency leads to microcytic hypochromic anemia.

Causes of Iron deficiency and overload

Iron  deficiency

Iron  overload

- Decreased intake

   ( poor diet, malabsorption)

- Repeated blood transfusions

- Increased requirements (adolescence, pregnancy, menstruating females)

 

- Excessive iron losses (menorrhagia, G.I.T. losses, genitor-urinary losses)

- Excessive absorption (hemochromatosis)

__________________________________________________

 

Estimation of Serum Iron:

 

 

 

 


Principle :

 

Ferric iron is dissociated from its carrier protein, transferrin, in an acid medium and simultaneously reduced to the ferrous form. The ferrous iron is then complexed with the chromogen, a sensitive iron indicator, to produce a blue color which absorbs maximally at 595 nm.

Procedure:

 

 

Test

Standard

Blank

Buffer

2 ml

2 ml

2 ml

Reductant

0.1 ml

0.1 ml

0.1 ml

Serum

0.5 ml

-

-

Standard

-

0.5 ml

-

Water

-

-

0.5 ml

Chromogen

0.1 ml

0.1 ml

0.1 ml

    - Mix and incubate for 20 minutes at 20-25 o C.

- Read the absorbance (A) of the test and standard against the blank at 595 nm.

 

  Calculation:

                  

              Serum iron conc.   =    A sample     x  conc. of standard (36 mmol/L)

     A standard                       

  Normal reference values:

          Children      :   9     – 22  m mol/L

          Adult male  :  11.5 – 30   m mol/L

          Adult female:  9    – 30   m mol/L

 

Case study 1

 

A 45-year-old man presented with weight loss, lassitude and weakness. His skin was noticeably bronzed, although it was winter and he had not been out of the country.     On examination, he was found to have hepatosplenomegaly.

 

 

Investigations

Patient's result

Reference range

Urine

positive for glucose

 

Blood glucose (fasting)

10 

4.0 – 5.8            mmol/L

Serum iron

70 

11.5 – 30           mmol/L

Total iron binding capacity

80 

45 – 80              mmol/L

Ferritin

5000

20 – 250                mg/L

__________________________________________________

 

 

Case study 2

 

Two sisters, whose mother had recently received a diagnosis of hemochromatosis, were referred to a hematologist for assessment of their iron status and genetic testing. Both were well and asymptomatic. Initial biochemical findings were as follows.

 

  Investigations

Patient 1

Patient 2

Reference range

Ferritin

1620

3340

20 – 250               mg/L

Iron

39 

53

9.0 – 30            mmol/L

Transferrin

2.0 

2.1

2.0 – 4.0                 g/L

Transferrin saturation

74 

78

14 – 51                     %

__________________________________________________

 

 

Case study 3

 

A 3-year-old boy was referred for the investigation of failure to thrive: he was short and underweight compared to his age. The boy had frequent diarrhea and didn't appear to enjoy his food. On examination, he was anemic and there was obvious wasting of the muscles of the limbs and shoulder girdle.

 

  Investigations

Patient's result

Reference range

Hemoglobin

97

110 – 140                g/L

Serum albumin

30

35 – 50                    g/L

Serum Iron

6

9 – 22                mmol/L

Serum TIBC

84

45 – 80              mmol/L

Serum ferritin

2

7 – 140                  mg/L

 

A duodenal biopsy showed total villous atrophy.

 

 

Estimation of Plasma Total Proteins 

 

Introduction:

Plasma contains over 300 proteins. Many of these have a specific biochemical role; organic disease may result when their concentration in plasma is reduced. Other plasma proteins, including most enzymes and tumour markers, have no known function in blood, and arise as a result of cell death or tissue damage.

In this practical, the functions and diagnostic roles of a number of the clinically more important plasma proteins are described.

 

Functions of plasma proteins:

Many proteins in plasma have a specific role to play and carry out a wide range of functions, including:

 

§     Maintenance of colloid osmotic pressure – mainly a function of albumin.

§     Transport functions, carried out by various carrier proteins.

§     Defence reactions – functions that depend on:

- Immunoglobulins, synthesized in the lymphoreticular system.

       - The complement system.

§     Coagulation and fibrinolysis. This involves some of the proteins circulating in plasma, and others liberated from damaged cells or tissues (e.g. platelets).

 

Some diseases may affect most plasma proteins, e.g. if there is malnutrition or loss of blood. In other diseases, only certain specific proteins are affected.

 

Methods of investigating plasma proteins:

Chemical and immunological methods are available that can quantify the concentration of a specific plasma protein with a high degree of specificity. Less commonly, electrophoresis is used to provide a semiquantitative estimate of the pattern of serum proteins. Electrophoresis separates the proteins into five broad fractions – albumin, a1-gobulins, a2-globulins, b-globulins and g-globulins ( Fig. 1 ), each of the globulin fractions consists of a mixture of several proteins. Serum proteins electrophoresis has limited diagnostic value, and the measurement of specific protein using immunoassay methods is most commonly used.

 

Electrophoretic pattern of normal serum proteins

+                                                                                      _  

                            G   l  o  b  u  l  i  n  s

                          Alb.      a1          a2      b                g

 

 

 

 

 

 

 

 

 

 

 

( Figure 1 )

 

 

 

Plasma proteins and disease:

A summary of the proteins commonly measured in clinical practice is given in table 1. Most diseases that alter plasma proteins concentrations do so by affecting their volume of distribution or their rates of synthesis, catabolism or excretion. In some patients, more than one of these factors may be operating.

 

Individual plasma proteins

 

Protein

Electrophoretic

bands

Principal function

Clinical significance

Albumin

35 – 50 g/L

- Liver

Albumin

 

- Regulates blood oncotic pressure

 -Transport protein ( Calcium, free fatty acids, bilirubin, therapeutic drugs and thyroid hormones)

  - Decreases  in chronic damage of liver, nephrotic syndrome, burns and malignancy

 

  - Increases  in dehydration

a1- antitrypsin

- Liver

- Macrophages

a1-Globulin

 - Proteases inhibitor

    (Trypsin & elastase)

- Decreases in pulmonary emphysema and liver cirrhosis

Prothrombin

- Liver

-     Blood clotting

 

  - Coagulation screening and also as a liver function test

a1- fetoprotein

 - Liver

 

 - It may play an immunoregulatory role during pregnancy

- Tumor marker for hepatoma

- Neural tube defect

Ceruloplasmin

- Liver

a2-Globulin

 

 

- Copper transport

- Has ferroxidase activity

 

- Decreases in Wilson disease

Haptoglobin

- Liver

 

- It binds Hb to form

  haptoglobin – Hb complex

   this is rapidly broken- down in the lympho-reticular system (conserve body iron).

- Decreases in hemolytic anemia

a2-Macroglobulin

 

- Liver

- Macrophages

- Protease inhibitor, carrier for cytokines

- Increases in nephrotic syndrome

C- Reactive protein

- Liver

b-Globulin

 

- Body's defense mechanism.

- Activates classic pathway of complement.

- Increases during acute phase protein.

- Non-specific test that may  be used instead of ESR

Transferrin

200 – 400 mg/dL

- Liver

- Transport iron (Fe3+)

- Increases in iron deficiency anemia.

- Decreases in malnutrition, liver disease, inflammation and malignancy (negative acute phase protein)

 

 

Individual plasma proteins…(contd)

 

Protein

Electrophoretic

bands

Principal function

Clinical significance

b2- microglobulin

0.1 - 0.2 mg/dL

-   Myeloid and lymphoid cells

 

b-Globulin

 

-   Forms part of the human leukocyte antigen (HLA) system and is a surface constituent of most cells

 

- Increases in renal failure and  malignancy (used as tumor marker for leukemia, lymphomas and multiple myeloma)

Immunoglobulins

(A,G,M,D and E )

    - Synthesized by

       plasma cells

g-Globulin

 

Body 's defense mechanism

 

 

-      Increases in liver diseases,   infection, allergy,

autoimmune diseases, paraproteinaemias, .. etc.

- Decreases in agammaglobulinemia,

  hypogammaglobulinemia and nephrotic syndrome.

 

 

     Prealbumin

      - Liver

 

Migrates faster than albumin

-   Binds thyroxin

-   Complexes with retinol binding protein ® transport vitamin A in blood

 

 

- Decreases in malnutrition, liver disease, nephrotic syndrome, burns and malignancy (negative acute phase protein).

 

 

Causes of Paraproteinemia:

 

1. Multiple myeloma.

2. Waldenstrom macroglobulinemia.

3. Malignant lymphoproliferative disease.

4. Benign paraproteinemia.

 

 

 

 

 

 

 

 

 

 

 

 

Estimation of Plasma Total Proteins (Biuret’s method):

 

 

Procedure:

 

 

 

Test

Standard

Blank

Plasma

0.1 ml

-

-

Standard

-

0.1 ml

-

Dist. H2O

-

-

0.1 ml

Biuret’s reagent

5 ml

5 ml

5 ml

                                               

- Mix, leave at room temperature for 15 minutes.

- Read the absorbance (A) of Test and Standard against Blank at 546 nm.

 

  Calculation:

                                                               A Test

 Plasma protein conc.(g/L)  =                           X conc. of standard (60 g/L)

                                                              A Standard

 

 

Normal reference value:  60 - 80 g/L

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Case study 1

 

A 70-year-old man complained to his doctor of back pain that he had for several months, and of feeling generally unwell. He appeared pale and he was tender over the lumbar spine. His urine contained protein (1 g/L) and his erythrocyte sedimentation rate (ESR) was very high (90 mm in the first hour).

The following abnormalities were reported:

 

Plasma analysis

Patient's result

Reference range

Albumin

32

35 – 50                     g/L

Calcium

2.72

2.12 – 2.62        mmol/L

ALP

90

40 – 125                  U/L

Creatinine

180

55 – 120            μmol/L

Total protein

84

63 – 83                    g/L

IgA

<0.4

0.5 – 4.0                  g/L

IgG

37

5.0 – 13                    g/L

IgM

<0.2

0.3 – 2.5                   g/L

 

How would you interpret these results, and what further chemical investigations would you request in this patient?

__________________________________________________

 

Case study 2

 

A 50-year-old lecturer presented to his doctor, complaining of tiredness, abdominal discomfort and poor appetite. He had worked in Africa in the past, where he had contracted Hepatitis B and had become a carrier. On examination, he was jaundiced and his liver was enlarged. Urine was positive for both bilirubin and urobilinogen.

The following results were found:

 

Plasma analysis

Patient's result

Reference range

Albumin

34

35 – 50                     g/L

ALP

400

40 – 125                  U/L

ALT

150

10 - 40                     U/L

Total bilirubin

60

2 – 17                μmol /L

GGT

150

10 – 55                     U/L

 a1- fetoprotein

3000

Not detectable        kU/L

 

 

 

 

 

Case study 3

 

Immediately following the birth of a baby girl, the attending physician requested            a protein electrophoretic examination of the mother's serum. This was done on            a sample that was obtained upon the mother's admission to the hospital the previous day.

An Electrophoretic examination was also performed on the cord-blood specimen.

Laboratory reports were as follows:

 

Investigations

Mother's

Serum

Cord-blood

serum

Adult

Reference values

Albumin

42

35

35 – 50          g/L

a1 - globulins

3

0.0

1 – 4              g/L

a2 - globulins

7

4

3 – 8              g/L

b - globulins

9

7

6 – 11            g/L

g - globulins

12

10

5 – 17            g/L

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hemoglobin Electrophoresis

 

Introduction:

The major hemoglobin in the erythrocytes of the normal adult is Hb A (97-98%) and there are small amounts of Hb A2 (2 – 3 %) and Hb F "Fetal Hb" (0 – 2 %).

 

     Hemoglobinopathies:

     They are divided into three groups:

      1.  Production of abnormal protein molecules i.e. Sickle cell disease (HbS).

      2.  Reduction in the amount of normal protein synthesis i.e. Thalassemia.

      3.  Developmental abnormalities (hereditary persistence of Hb F).

 

Hemoglobin electrophoresis:

§     It is generally considered the best method for separating and identifying the hemoglobin present in a blood sample.

 

§     Electrophoresis is the migration of charged particles to the opposite electrode in an electrical field.

 

§     The rate of migration depends on :

o     The net electrical charges of the molecule (particle).

o     Size and shape of the molecule.

o     Electrical field strength.

o     Properties of the supporting medium.

o     The temperature of operation.

 

§     Equipments and reagents needed:

o    Charged particles to be separated.

o    An electrical field.

o    A supporting medium on which the migration will occur.

o    Buffer:

           It is tris-boric acid- EDTA buffer with pH 8.6. The buffer is important for:

- Carrying the applied current.

- Keeping the pH at which electrophoresis is carried out.

o    Supporting medium:  cellulose acetate sheet.

o    Hemoglobin stain: Ponceau S. It is used to fix, visualize and locate the hemoglobins separated.

- Electrophoresis apparatus:

The apparatus is formed of two buffer boxes (1) which contain the buffer used in the process. In each buffer box is an electrode (2) of either platinum or carbon. The electrophoresis supporting medium (3) on which electrophoresis takes place is in contact with buffer by means of wicks (4).

The whole apparatus may be covered (5) to minimize evaporation and protect the system. The direct current power supply may be either constant current or constant voltage or both.

 

 

Procedure of hemoglobin electrophoresis:

§     Wet the cellulose acetate sheet in the buffer.

§     Put the cellulose acetate sheet in the apparatus and apply the blood sample.

§     Put the power supply on for a certain time.

§     Stain the sheet in Ponceau S.

§     Wash out the excess dye, clean the sheet background.

§     Read the sheet by densitometer.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Case study

 

 A newly engaged couple decided to do premarital screening before getting married. They went to the hospital to perform the required investigations. The investigations included complete blood count and hemoglobin electrophoresis. On receiving the results, they were asked to see the consultant. The consultant took the family history for each and assured that each one of them was within normal. He added that on examining their investigations their electrophoresis results point out that they might face troubles with their offsprings if they got marry. He added that care should be taken with anesthesia, pregnancy and high altitude.

 

 

-

 

 

 

+

 

 

C

S

F

A

 

 

 

Control

 

 

 

 

Male

 

 

 

 

Female

 

The figure shows the electrophoresis results

 

 

1.      What is the principal of electrophoresis?

2.      Comment on the electrophoresis results?

3.      What did the couple suffer from?

4.      Mention the genetic abnormality underlying this case?

5.      Why were they prepared to expect certain troubles with their offsprings?

 

 

 

 

 

 

 

 

 

 

 

Glucose-6-Phosphate Dehydrogenase:

Introduction:

§     Glucose-6-phosphate dehydrogenase (G6PD) functions to reduce nicotinamide adenine dinucleotide phosphate (NADP) while oxidizing glucose-6-phosphate.

§     It is the only source for production of NADPH in red cells.

§     As NADPH is needed for the production of reduced glutathione, a deficiency renders the red cells susceptible to oxidant stress.

§      G6PD deficiency is an X- linked disease.

§      More than 400 variants due to point mutations of the G6PD gene have been characterized, with only some of these mutations cause clinical manifestations.

§     G6PD deficiency classification ranges from class I with the severe clinical symptoms and residual enzyme activity < 2% to class IV with no clinical symptoms and 60-150% residual activity.

§     G6PDA- is the prototype of the moderate form of the disease (class III) while G6PD Mediterranean is the prototype of a more severe class II.

§     There are number of agents which may precipitate clinical manifestations as:  

o       Infections and acute illness as diabetic ketoacidosis.

o       Drugs as antimalarials (e.g., primaquin), antibacterial (e.g. suphonamides, co-trimoxazole), analgesics (e.g., aspirin, phenacetin), miscellaneous (e.g., vitamin K analogues and probenecid).

o       Ingestion of fava beans.       

§     In case of an attack precipitated by one of the previous factors, there is rapidly developing intravascular hemolysis with hemoglobinuria.

§     The anemia may be self-limiting as new young cells are made with near normal enzyme levels.

§     Other clinical feature includes neonatal jaundice while in class I mutations are often associated with chronic nonspherocytic anemia.

 

Diagnosis: 

§        Between crises, the blood count is normal.  The enzyme deficiency is detected  by a number of screening tests as fluorescent screening test or by direct enzyme assay in red cells.

 

§        Because of the higher enzyme levels in young red cells, red cell enzyme assay may give ‘false’ normal levels in the phase of acute hemolysis with reticulocytosis. Subsequent assay after the acute phase reveals low G6PD level.

 

 

Fluorescence Screening Test for G6P-DH Deficiency:

 

Principle:

        Glucose-6-P + NADP   G6P-DH   Gluconate-6-P + NADPH + H+

 

The production of NADPH causes fluorescence under long wave UV light.

If G6P-DH is not present or markedly deficient, no fluorescence will be observed.

Sample:

Whole blood (Heparin, citrate, oxalate or EDTA are suitable anticoagulants) stable for up to 1 day. Use 10 µl of blood for the assay.

Procedure:

§         Add 10 µl of blood directly into 200 µl of the working reagent. Mix and incubate for 10 minutes at room temperature (25°C).

§         Take 10 µl of the mixture and apply on the filter paper provided and leave to dry (about 20 minutes).

§         When the filter paper is completely dry, view under the long wave UV-lamp in              a darkened room.

Interpretation of results:

§         Samples with normal or slightly deficient G6P-DH activity will show fluorescence.

§         Samples with no fluorescence suggest complete lack or marked deficiency of G6P-DH.

§         This is a qualitative method, when in doubt; a quantitative test has to be followed for confirmation.

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Case study

 

 

 A 10-year-old male patient arrived to the hospital with obvious pallor, general discomfort and a complaint of passing dark-colored urine. On examination there was a tinge of jaundice apparent in his eyes, pallor and low grade fever. History taking revealed recent acute tonsillitis for which he received co-trimoxazole. His mother added that his maternal grandfather has a history of a similar condition.

 

1.      Comment in the previous case

2.      What is the main problem leading to this case?

3.      What is the genetic defect underlying this case?

4.      What are the biochemical investigations would you like to order?

5.       What other investigations would you add?

Urine Analysis

 

Introduction:

Urine is a fluid obtained from the blood through the renal glomeruli with considerable changes before it is excreted as urine.

The first step in urine formation is by ultrafiltration at the glomeruli which is about 170 – 200 L/24 hrs. During the passage of the ultrafiltrate through the renal tubules, reabsorption of solutes and water in various regions reduces the total urine volume to less than 1% of the ultrafiltrate. There is also active secretion at the renal tubules. Collection of urine specimen depends on the test required either random sample or 24- hours sample. Urine specimen tends to deteriorate unless the correct preservative is added (toluene, chloroform, thymol and formalin) or the specimen is refrigerated throughout the collection period.

Urine samples are usually examined for the main items:

                <   Physical examination.

                <   Biochemical examination.

                <   Serological examination.

                <   Microscopic examination.

 

Physical examination:

1. Volume:  Normal urine volume: 0.4 -2.0 L/day.   

§     Increased volume (Polyuria) > 2.0 L/day:

- Physiological: Excessive water and fluid intake.

- Pathological: a. Diabetes mellitus. 

                   b. Diabetes insipidus.

                   c. Chronic renal failure.

 

§     Decreased volume (Oliguria) < 0.4 L/day:

   a. Dehydration.

                                  b. Acute renal failure.

 

2. Appearance:        a. Normal fresh urine is clear.

                                 b. Cloudy (turbid) urine is due to abnormal constituents                                (pus cells, bacteria, salt or epithelial cells).

 

3. Color:

 a. Normal color: is pale yellow or amber yellow (due to urochrome and urobilin pigments).

          b. Colorless urine: very diluted urine due to:

                                                                <   Physiological causes: excessive fluid intake in normal person.

                                                                <   Pathological causes: uncontrolled diabetes mellitus, diabetes insipidus and chronic renal failure.

 

           c. Orange urine: is due to:

                                                                <   Ingestion of large amount of carotenoids (vitamin A).

                                                                <   Concentrated urine (hot weather, high fever, dehydration..etc). 

 d. Yellow - green urine: is due to bilirubin or biliverdin (jaundice).

           e. Red urine: is due to:

§   Ingestion of root beets.

§   Some drugs (rifampin for treatment of T.B., carmurit…etc).

§   Blood or hemoglobin.

§   Porphyrias.

            f. Dark brown or black urine: is due to:

§   Methemoglobin.

§   Homogentisic acid in alkaptonuria.

§   Melanin (melanoma).

§   Malignant malaria (black water fever).

                g. Smoky urine: is due to presence of RBCs in acute glomerulonephritis.

4. Odor:

     a. Urineferous odor: normal odor of fresh voided urine (due to presence of aromatic acids).

     b. Fruity odor: is due to acetone (diabetic ketoacidosis).

     c. Ammoniacal odor: is due to release of ammonia as a result of the bacterial urease enzyme in the contaminated and long standing exposed urine sample.

     d. Mousy odor: is due to PKU.

     e. Burnt sugar odor: is due to maple syrup urine disease.

 5. Deposits: 

a. Normally, the urine contains no deposits.

b. Deposits are mainly due to:

§  Crystals, salts or cells.

§  Blood clots, necrotic tissues and urinary stones.

 

 6. Osmolality:

It is the number of solute particles per unit weight of water (mmol/kg of water). Measurement of urine osmolality is of value in the investigation of:

§  Polyuria:  - with increased osmolality ® Diabetes mellitus.

                                    - with decreased osmolality ® Diabetes insipidus.

§  Oliguria :  - with increased osmolality ® Acute renal failure.

                                              - with decreased osmolality ® Hypovolemia.

7. Reaction (pH):

   a. Normal range: 4.6 - 7.0 (the average pH is about 6.0).

   b. Acidic urine: is due to ketosis (diabetes mellitus & starvation), severe diarrhea, metabolic and respiratory acidosis, excessive ingestion of meat   and certain fruits (cranberries).

c. Alkaline urine: is due to: 

- Respiratory and metabolic alkalosis.

- Urinary tract infection.

- Vegetarians.

 

Chemical examination:

 

Normal composition of urine:

 Urine is a fluid composed of water (95 %) and inorganic and organic solids (5%)

that include:

 

A) Chief inorganic solids include: - Sodium       - Potassium       - Chlorides.

       In addition, smaller amounts of calcium, magnesium, sulfate and phosphates,   and traces of iron, copper, zinc and iodine.

B) Chief organic solids are:

    1. Non-protein nitrogen (NPN) compounds.  

    2. Organic acids   

    3. Sugars.

In addition, traces of proteins, vitamins, hormones and pigments are present in    the urine.

Abnormal constituents of urine:

I. Proteinuria:

§        Normally less than 200 mg protein is excreted in the urine daily; more than  this level leads to a condition called " proteinuria".

§        Proteinuria is either glomerular or tubular.

§        Glomerular proteinuria is due to increased glomerular permeability leading to filtration of high molecular weight proteins (e.g. glomerulonephritis).

§        Tubular proteinuria occurs as a result of decreased reabsorption with normal glomerular permeability leading to excretion of low molecular weight proteins     (e.g. chronic nephritis).

 

Proteinuria is classified into prerenal, renal and postrenal.

         1. Pre-renal proteinuria:

§     Bence-Jones protein:

- This abnormal gamma globulin (light chains only) is synthesized by malignant plasma cells (multiple myeloma).

-  It precipitates at 60oC, redissolves on boiling and reprecipitates on cooling to 60oC.

 

2. Renal proteinuria: 

§     After prolonged standing (orthostatic).

§      Severe muscular exercise.

§     Congestive heart failure, hypertension, fever, stress.

§     Gestational (in the 3rd trimester of pregnancy).

§     Glomerulonephritis.

§     Diabetic nephropathy.

§     Renal ischemia or neoplasia.

§     Nephrotic syndrome.

§     Nephrotoxins (aminoglycosides; gentamycin, streptomycin…etc).

§     Overflow proteinuria (hemoglobin due to intravascular hemorrhage, microglobulin in leukemia and lymphoma, and myoglobin in rhabdomyolysis).

         3. Post-renal proteinuria:

§     Lower urinary tract infection, tumors or stones.

 

II. Glycosuria (presence of detectable amount of  any sugar in urine):

      Includes the following:

         1. Glucosuria: (presence of detectable amount of glucose in urine).

§     Uncontrolled DM: The concentration of glucose in the plasma exceeds    the renal threshold.

§    Renal glucosuria: Normal plasma glucose concentration with proximal tubular malfunction leads to decreased renal threshold (gestational diabetes and Fanconi's syndrome).

 

       2. Fructosuria: (Presence of fructose in urine)

§     Alimentary: due to increased fructose intake.                                                   

§     Metabolic: deficiency of fructokinase or aldolase B enzyme in the liver.

 

       3. Galactosuria: (Presence of galactose in urine)

§     Alimentary: increased galactose intake.

§     Metabolic: deficiency of galactokinase or galactose -1-phosphate uridyl transferase in the liver.  

 

III. Ketonuria:  (Presence of ketones "Acetone, acetoacetic acid and                                                                      β- Hydroxybutyric acid" in urine)

§     Diabetic ketoacidosis.

§      Glycogen storage diseases.

§      Starvation.

§      Prolonged vomiting.

§      Unbalanced diet: high fat and low carbohydrate diet.

 

 IV. Nitrite:

§     Positive nitrite test is significant of bacteruria in urine.

 

  V. Choluria:

        a) Bilirubin / bile salts: in cases of

§     Hepatocellular damage.

§      Obstruction of bile duct: extrahepatic (stone) or intrahepatic (tumors).

 

b) Urobilinogen:

§     Normally, present in trace amounts in urine.

§     Markedly increased in: 

- Hemolytic anemia.

- Hepatocellular damage.

 

  VI. Blood:

            a) Hematuria (Presence of detectable amount of blood in urine):

§     Acute and chronic glomerulonephritis.

§      Local disorders of kidney and genito-urinary tract (trauma, cystitis,      renal calculi, tumors …..etc).

§      Bleeding disorders (hemophilia).

 b) Hemoglobinuria (Presence of hemolysed blood in urine):

§     Hemoglobinopathies (sickle cell anemia and thalassemia).

§     Transfusion reaction (blood incompatibility).

§     Malaria (plasmodium falciparum)

    VII. Chyluria (Presence of lymph/ fat in urine):

-       The urine acquires a milky appearance which disappears on shaking with ether.  

-       It is due to abnormal connection between the intestinal lymphatic system and urinary tract, this may be congenital or acquired filariasis.

 

 

 

 

 

 

 

 

 

 

 

 

 

Urine Analysis (using dipstick):

 

Principle:

 

 Dipsticks are plastic strips impregnated with chemical reagents which react with specific substances in the urine to produce color-coded visual results. They provide quick determination of pH, protein, glucose, ketones, bilirubin, hemoglobin, nitrite, leukocytes and specific gravity. The depth of color produced relates to the concentration of the substance in urine.

 Color controls are provided against which the actual color produced by the urine sample can be compared .The reaction times of the impregnated chemicals are standardized.

 

  Procedure:

1-       Dip the dipstick in the urine sample provided then remove it immediately.

2-       Remove the excess urine.

3-       Read the color produced within 60 seconds.

4-       Match the color changes to the control charts.

5-       Give a full report about:

§         Physical examination.

§         Chemical examination.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Case study 1

A 49-year-old woman with history of diabetes mellitus came to the hospital with fever, weakness and dysuria for the last three weeks.

The results of her laboratory tests were as follows:

 

Laboratory test

Patient's result

Reference range

Serum:

Fasting blood glucose

7.5

3.9 – 5.8          mmol/L

Creatinine

75

55 – 120          μmol/L

Urea

3.7

2.5 – 6.4          mmol/L

Sodium

140

135 –145        mmol/L

Potassium

3.9

3.5 – 5.1        mmol/L

Urine:

Color

Yellow/ turbid

 

pH

7.5

 

Protein

- ve

 

Glucose

+ ve

 

Ketones

- ve

 

Nitrite

+ ve

 

RBCs

< 5 / HPF

 

WBCs

> 20 / HPF

 

MSU culture

E.coli  > 100

 

 

 

 

 

 

 

 

 

 

 

Case study 2

A 6-year-old boy, developed marked edema over a period of few days and his parents had noted that his urine had become frothy. His general practitioner detected proteinuria on doing urine test and arranged admission to the hospital where the results of chemical investigations were as follows:

 

Laboratory test

Patient's result

Reference range

Serum:

Creatinine

58

55 – 120          μmol/L

Urea

3.4

2.5 – 6.4         mmol/L

Sodium

136

135 –145        mmol/L

Potassium

4.0

3.5 – 5.1         mmol/L

Total protein

34

60 – 80                  g/L

Albumin

14

35 – 50                  g/L

Cholesterol

11

3.2 – 5.2         mmol/L

Triglycerides

15

0.5 – 2.27       mmol/L

Urine:

24 hrs urine protein

12

0.03 – 0.10              g

 

 

 

 

__________________________________________________

 

Case study 3

A 12-year old girl was found by her mother in a drowsy and cooperative state. When the general practitioner arrived, the mother told him that her daughter seemed to be unusually thirsty for the last 1-2 months, and she thought that the girl had lost weight. When the doctor tested the patient's urine by dipstick, he found both glucose and ketone bodies. The girl was immediately transferred to the hospital for further investigations.

 

1.         What is the probable diagnosis?

2.        How would you confirm the diagnosis?

3.        What are the other investigations needed for the proper management of the patient?

 

 

 

 

Medical Biochemistry Department

College of Medicine

MBC 142 - Practical

Urine analysis

 

                الاسم                                                       الرقم الجامعي

       1.         …………………………………………………    ……………….…………………………………………

       2.         …………………………………………………    …………….…………………………………………….

       3.         ……………………………………………    …………….………………………………………….…

       4.         ……………………………………………    …………….………………………………………….…

 

 

   Examination:

 

Volume             ……………………………………………………………………….   

 

   I. Physical Properties :

   

               1.  Appearance        …………………………………………………………………….

               2.  Color                  …………………………………………………………………….

               3.  Odor                  …………………………………………………………………….

               4.  Deposits             …………………………………………………………………….

               5.  Reaction (pH)     …………………………………………………………………….

 

 II. Chemical Properties: 

 

Item

Observation

Comment

Protein

 

 

Glucose

 

 

Ketones

 

 

Nitrite

 

 

Bilirubin

 

 

Urobilinogen

 

 

Blood

 

 

 

 

 

 
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