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this is a simple illusteration of the practical course of clinical biochemistry to all medical students

this is a simple illusteration of the practical course of clinical biochemistry to all medical students
i hope it will be usful to all my students

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clinical Biochemistry _aaser clinical Biochemistry _aaser Presentation Transcript

  • Practical clinical Biochemistry AASER ABDEALAZIM Assistant lecturer of Biochemistry [email_address] [email_address]
  • Introduction to Clinical Biochemistry
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  • 1- Precision and accuracy Precision: reproducibility of the results Accuracy: How closed the measured value is to the actual value
  • 2- sensitivity and specifity Sensitivity: how little of the analyte the method can detect Specifity: how good the assay discriminating the requested test 3- quality assurance Use the quality control samples to assure that the samples measured give satsfactory results with patient specimen 4- reference range Biochemical values are Compared to refernce range from healthy population
  • Total serum proteins 1
  • Precautions before taking blood samples for protein determination
    • Patient should be not fasted
    • Serum samples are more used
    • Preservation in room for a day or at -20 for more one month
    • Avoid hemolysis , increase level due to leakage of RBCs proteins
    • Hyper proteimemia
    • Dehydration (diarrohea, vomition)
    • Increase antibody production
    • Acute hepatitis, typhus, malaria
    • Hypoproteinemia
    • Loss (blood, Urine, GIT)
    • Dec. synthesis (liver diseases, immun. Diff)
    • Dec. intake(mal-nutrition, mal-absorption)
    • Incr. catabolism(trauma, burns)
    Normal serum levels= 6-8.2 g/dl
    • More 100 proteins were determined
    • After birth , level decrease then reach to adult level after 3 years then decrease By age
    Abnormalities of serum Total proteins:
  • Blood glucose and Diagnosis of Diabetes 2
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  • Normal level = 70 – 110 mg/dl
    • Diabetes mellitus
    • Gestational diabetes(24 th - 28 th week)
    • Acromegaly
    • Acute stress (response to trauma, heart attack, stroke)
    • Chronic renal failure
    • Cushing syndrome (excess glucocorticoids)
    • Hyper thyrodism
    • Pancreatitis
    • Pancreatic tumors
    • Excess food intake
    • Drugs (corticosteroids, tricyclic antidepressants, diuretics, epinephrine, estrogen
    • Lithium, phenytoin, salicylates.)
    Blood Glucose I- Hyper glycemia
  • 1) Hypo glycemia in Diabetic patients
    • Patients taking blood glucose lowering medications
    • Excess dose of insulin
    • High dose of sulfonylurea
    • Extra dose of Meglitinides.
    • Incerase the activity or the exercise
    • Excess drinking of alcohols .
    2) Hypoglycemia in non Diabetic patients Reactive hypoglycemia(postprandial, after meal) Fasting (post absorptive) II- Hypo glycemia
  • (1) Reactive hypoglycemia (appears after 3-4 hrs after meals)
    • Some people that were sensitive to normal release of epinephrine
    • Deficiency in Glucagon secretion
    • Gastric surgery (rapid passage of food to intestine).
    (2) Fasting hypoglycemia Diagnosed from blood samples lower than 50 mg/dl
    • Alcohols especially binge drinking(depletion of pyruvate and Oxaloacetate)
    • Critical illness (hepatocellular damage, renal insufficiency , sepsis, starvation )
    • Hormonal deficiency(cortisol, GH, Glucagon, epinepherine )
    • Tumors (insulinoma: tumor of B – cells )
    • Drugs(salicylates in large dose, Pentamedines (pneuomenia treatment),
    • Quinine(malaria ttt) )
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  • Blood sugar estimation
    • Blood sugar
    • Urine sugar
    • 3. Glycated HB
    • 4. Glycated proteins
    Samples: Precautions: Types of samples : Monitoring 1- venous blood 2- capillary blood RBCs level is less than plasma level due to less water contents
    • Separated rapidly
    • Transported to lab. Fastly
    • Cooling of samples
    • Add sod. Fluoride to inhibit glycolysis
    • Fasting
    • Random
    • Timed interval ( glucose tolerance )
    (1) Oral glucose tolerance test (2) Venous glucose tolerance test
  • Oral glucose tolerance test
    • Blood glucose measured after 12 hour fast
    • Patient take 75 gm sugar orally
    • Every hour take blood sample for BG and urine samples for Glucose in urine
    • Draw the curve
    • Indications:
    • Mild or no DM symptoms
    • Glucose absorbance and utilization
    • Renal threshould
    The curve contains Ascending limb: explains the glucose absorbance Descending limb: explains glucose utilization and insulin response Normally the level of BG after 2 hour equals the 1.5 of FBG
  • Types of curves (1) Sever Diabetic curve (3) Hypoglycemic (6) Flat curve (4) Lag storage curve (5) Renal glucosuria (2) Mild diabetic Urine +++ Urine ++ Urine +
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  • Lipid metabolism disorders Sequelae
    • Coronary heart diseases(CHD)
    • Acute pancreatitis
    • Failure to thrive and weakness
    • Cataract
    Major points for sampling for blood lipid profile
    • Patient should be fasted at least12 hours before sampling
    • Test should not performed during acute illness
    • Test should not performed on hospitalized patients until 2-3 months of illness
    • Blood lipids affected by body posture, drugs, smoking and alcohol
    • Samples should not heparinized
    • Plasma or serum Samples should be separated as soon as possible
    • Body weight should be remain constant at least for 2-3 weeks
    • Abnormal results should be confirmed before change the type of therapy
    3
  • (1) Plasma appearance Clear: TG <200 mg/dl Turbid: TG~300 Cloudy to opaque: TG >600 Creamy layer appeared on the surface of the plasma within 4 hours: CM present (2) Serum Total Cholesterol (N= 130-220 mg/dl) Used to monitoring the CHD Monitoring other lipoprotein disorders
    • Increased in
    • Idopathic hyper cholestrolemia
    • Hyperlipoproteinemia
    • Chronic renal failure
    • Smooking
    • Hypothyrodism
    • Obstructive liver diseases
    • Pregnancy
    • Pancraetic diseases include DM
    • Decreased in
    • Hyperthyrodism
    • Malnutrition
    • Severe liver cell damage
    • Chronic anemia
  • (3) Serum HDL-Cholesterol (N=30-75 mg/dl )
    • Increased in (>60 mg/dl good indication –ve CHD)
    • Vigorous exercise
    • Some familial lipoproteins disorders
    • Mild alcohol intake
    • Estrogen treatment
    • Insulin treatment
    • Increase clearance of TG
    • Decreased in
    • Nephrosis
    • Chronic liver diseases
    • Stress
    • Obesity
    • Smoking
    • Lack of exercise
    • Hyperthyroidism
    • DM
    • Familial LCAT deficiency
    Every 1 mg/dl decrease in HDL increases the risk of CHD by 2-3% Risk factor for CHD = Total cholesterol/HDL-cholesterol Low :3.3-4.4 average: 4.4-7.1 moderte: 7.1-11 high:>11
  • (4) Serum LDL-Cholesterol (N=65-175mg/dl)
    • Increased in
    • Idopathic hyper cholestrolemia
    • Hyperlipoproteinemia
    • Chronic renal failure
    • Smooking
    • Hypothyrodism
    • Obstructive liver diseases
    • Pregnancy
    • Pancraetic diseases include DM
    • Diet reach in choletserol, saturated FA
    • antihypertensive B blocker drugs
    • Decreased in
    • Oral estrogen
    • Sever illness
    • A betalipoproteinemia
    Determination of LDL-cholesterol level Total Cholesterol= HDL+VLDL+LDL (in lab we can mesure TC,HDL,TG but neither LDL nor VLDL ) Major amount of TG present in VLDL= 5 times the amount of C so we can calculate the amount of C in VLDL by TG/5 Friewald formula: LDL-C=(HDL+TG/5)-Total C
  • (5) Serum Triglycerides (N=40-160 mg/dl)
    • Increased in
    • Idopathic hyper cholestrolemia
    • Hyperlipoproteinemia
    • Chronic renal failure
    • Smooking
    • Hypothyrodism
    • Obstructive liver diseases
    • Pregnancy
    • Pancraetic diseases include DM
    • TG should be measured after 12 hours
    • fasting serum levels are 5% higher than plasma
    • While HDL is the same at fasting and after meal
    • TGs are not strong predictors for athersclerosis and CHD
    Precautions :
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  • Kidney function tests 4
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  • Renal function tests Glomerular function Tubular function
    • Urine examination
    • BUN
    • Serum Creatinine
    • Creatinine clearance
    • Urea clearance
    • Urine conc. Test
    • Urine dilution test
    • Plasma electrolytes
    • Maximal tubular reabsorbtion of glucose and secrete PAH
  • Urine examination Physical Chemical Microscopical
    • Colour: amber yellow
    • Odour: aromatic
    • Aspect: clear
    • Specific gravity: 1015-1025
    • Reaction: acidic
    • Volume: 750-2000ml
    • Proteins
    • Glucose
    • Blood
    • Ketone bodies
    • Pigments
    • Amino acids
    • Lipid
    • Cells or casts : RBCs, pus
    • Crystals: urate, Ca oxalate, phosphate, cystine (inborn error)
    • Parasite egg
    • Colour: dark yellow, black, red, brown
    • Odour: rotten apple, putrid
    • Aspect:turbid
    • Specific gravity: high or low
    • Reaction: alkaline
    • Volume: polyurea, oligurea
  • Blood urea nitrogen
    • Urea mainly come from metabolism of proteins
    • It is a save product of ammonia (urea cycle)
    • Secreted in urine (N= 20-40 gm /day)
    • Normal plasma level= 20-40 mg/dl
    • Increased in:
    • Chronic renal failure
    • Decrease renal perfusion (CHF)
    • Renal tract obstruction (stones, tumors)
    • Nephritis
    • Afferent arteriole vasoconstriction
    • High protein diet
    • Gastrointestinal bleeding
    • Dehydration
    • Drugs
    • Aminoglycosides
    • Diurtetics
    • Lithium
    • Corticosteroids
    • Decreased in:
    • Mal nutrition
    • Liver diseases
    • Low protein diet
    • Renal dialysis
    • Over hydration
    • Third trimester of pregnancy
    Almost high BUN > 80 mg/dl indicates impired renal function
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  • Serum creatinine
    • Normal serum = men: 0.8-1.3 mg/dl , women: 0.6-1.0 mg/dl
    • Creatinine is best used to urea for assessment of renal functions
    • Not affected by diet
    • Completely execrated in urine not reabsorbed from tubules
    • Mainly of endogenous origin
    • Increased in :
    • Renal failure
    • Decrease renal perfusion
    • Renal obstruction
    • Skeletal muscle trauma
    • Ketonemia
    • Diabetic ketoacidosis
    • Creatine supplement
    • Drugs
    • hydantion: tranqulizer
    • Cephalosporins ,Aminoglycosides, Diuretics, methyldopa: parkinsonism
    • Cemetidine , trimethoprim
    • Decreased in:
    • Amputations
    • Low muscle mass
    • Pregnancy
  • Creatinine coeffecient The amount of cratinine excreted in urine/ kg BWt/ day N= 1.5 gm /day in male and 1.0 gm/day for female Depends on Body mascle mass Body conditions Creatinine clearance
    • Used for
    • Assess renal glomerular function
    • Drug monitoring
    • Advanced stages of renal failure
    Amount of creatinine filterd = the amount of cratinine execreted GFR x Cr conc. In Plasma = Cr conc. In urine x urine Volume GFR(C) x P = U x V C (Cr clearance) = U x V / P Mg/dl x ml/ min /mg/dl ml / min N= 90 ml / min
  • Creatinine clearance
    • Increased :
    • Pregnancy
    • Exercise
    • Decreased:
    • Renal insufficiency
    • Advanced age: decreased by 1 ml / min after 30 years
    • Inadequate urine specimens
    • Medications:
    • -Cimetidine
    • -Procainamide : anthesia
    • -Antibiotics
    • -Quinidinine
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  • Tubular function (1) Urine concentration test (water deprivation test)
    • Uses:
    • Used for testing the renal concentration ability in response to water deprivation
    • Can assess :
    • Normal ADH
    • Normal response of DCT to ADH.
    • Contra indication:
    • High blood urea
    • Pateints with clinical signs of RF
    • Test:
    • Allow pateint to take water after 6 pm but no food.
    • Come to lab at 7am,
    • Take urine samples at 7(zero time), 8, 9 am
    • Results
    • Any of these samples its Sp Gr should be more 1026 indicates good renal function
    • With in 1020 minor renal failure
    • 1010-1015 : severe renal damage
  • (2) Vasopressin test
    • Test
    • At 8 pm patient take 5 units of vasopressin tanate.S/C
    • Collect urine at 7, 8, 9 am
    • Sp gr should be more 1020 : good renal function
    (3) Urine dilution test(water load test)
    • Water not allowed to patient over night
    • Come at mooring to lab
    • Take 1000 ml water to drink completely
    • Collect 4 urine samples one hour interval after drinking
    • Results :
    • Normal urine led down in 4 hrs is 700 ml at least
    • Sp gr. Of one of the 4 at least = 1004
    • Severe damaged kidneys secrets urine with sp gr not less than 1010 and volume not mor than 400 ml
    • Contra indication
    • Odema
    • Hyponatremia
    • Renal failure with water intoxication
  • (4) Tubular maximum of glucose reabsorption and PAH secretion TmG =GFR x Pg – Ug x V Amount of glucose reabsorbed= amount of glucose filterd – amount of glucose secreted N = 350±75 minutes Tm PAH = UPAH x V – GFR x P PAH Para amino hippuric acid (PAH) actively excreted = PAH filtered- PAH secreted
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  • Liver Functions Tests Liver is the master organ in the body, it serve all other organs
    • Functions:
    • Metabolism(lipid, proteins, carbohydrates)
    • Storage (glycogen, vitamins, vitamin B12)
    • Excretory function (bilirubin, cholesterol).
    • Detoxification(phenbarbiton, amonia, steroid hormones, benzoic)
    • Hematological function(blood formation, blood volume, blood coagulation)
    Major tests used to diagnose liver functions
    • Plasma enzymes
    • ALT
    • AST
    • GGT
    • ALP
    • LDH
    • 5` nucleosidase
    • Plasma proteins
    • Albumin (cirrhosis)
    • ( α , ᵦ , ɣ) globulins(cholestasis)
    • Immunoglobulins(Igs)
    • IgG: chronic active hepatitis
    • IgA: portal cirhosis
    • IgM: biliray cirrhosis, viral hepatitis
    • Serology
    • Anti mitoch. Ab.(CAH, biliray cirrhosis
    • Anti nuclear: autoimmune hepatitis
    • Anti smooth muscle: CAH
    CAH: chronic active hepatitis 5
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  • Major changes in plasma enzymes and its indication in liver diseases (1) Serum AST (N= up to 40 U/ ml)
    • It present in high conc. In
    • Heart
    • Liver
    • Muscle
    • Kidenys
    Causes of AST increase Physiological increase In newborn its increase = 1.5 of normal level
    • Moderate increase
    • Liver cirhosis
    • Cholestatic jaundice
    • Liver infilteration
    • Skeletal muscle disease
    • After trauma or surgery
    • Marked increase (10-100 times )
    • Myocardial infraction
    • Viral hepatitis
    • Toxic liver cirhosis
    • Circulatory failure due to
    • Shock
    • Hypoxia
    Artefacual increase Due to hemolysis of blood in lab. Led to its release
  • Causes of ALT increase (2) Serum ALT (N= up to 35 U/ml)
    • It presents in high conc. In
    • Liver
    • Skeletal muscles
    • Kidneys
    • Heart
    • Marked increase(10- 100 times)
    • Viral hepatitis
    • Toxic liver hepatitis
    • Circulatory failure
    • Moderate increase
    • Liver cirhosis
    • Cholestatic jaundice
    • Liver congestion
    • Secondary to cardiac failure
    • Extensive trauma
  • (3) Serum Alkaline Phosphatase ALP
    • Presents in high conc. In
    • Bone
    • Liver
    • Kidneys
    • Lactating mammary glands
    • Intestinal wall
    • Placenta
    Causes of ALP increase
    • Physiological
    • Childern until puberty 2.5 times of adult level
    • Pregnancy
    • Bone diseases
    • Osteomalcia
    • Rickets
    • Bone carcinoma
    • Healing stage of bone fractures
    • Liver diseases
    • Cholestatic jaundice
    • Hepatitis
    • Cirhosis
    • Tumors
    • Infilteration
  • (4) Serum Lactate Dehydrogenase LDH
    • Presents in
    • Heart
    • Skeletal muscles
    • Liver
    • Kidneys
    • Brain
    • Malignant tissues
    Causes of serum LDH increase
    • Marked increase
    • Myocardial infarction
    • Hematological diseases
    • Leukemia
    • Shock
    • Pernicious anemia
    • Moderate increase
    • Viral hepatitis
    • Skeletal muscle diseases
    • Pulmonary embolism
    • Infections
    Artefacual increase Hemolysis of samples
    • LDH isoenzymes
    • LDH1:heart, erythrocytes, blast, kidneys
    • LDH2: heart
    • LDH3:intermediate
    • LDH4:liver
    • LDH5:liver
  • (5) Gamma Glutamyle transpeptidase GGT
    • Presents in
    • Liver
    • Kidneys
    • Pancreas
    Causes of GGT increase
    • Liver diseases
    • Cirrhosis
    • Metastatic cancer
    • Hepatic infiltration
    • Cholestasis
    Chronic alcoholism Patient with anticonvulsant therapy
  • Impaired the secretion of bile Then accumulated in the plasma
    • Secondary to a disaess
    • By abscess
    • Parasitic emboli
    • As bilhariziasis cause destruction of cells
    Destruction of cell Acute as viral infection Chronic : Loss of function Major pathological changes in liver Liver cell damage Cholestasis Infiltration of liver
  • (1) Liver cell damage
    • Causes:
    • Viral infection
    • Toxins (alcohole, paracetamol, acetaminophen)
    • Hypoxia and congestion(CHF).
    • Secondary to bibliray obestruction.
    • Biochemical effect:
    • Release of intra cellular constituents into blood.
    • High SGOT (AST)and SGPT(ALT).
    • Massive destruction: sudden fall after high elevation
    • Chronic destruction: high level for long time
    Means liver viral hepatitis Means excess damage, cirrhosis, hypoxia and tumors ALT than AST AST than ALT
  • (2) Cholestasis With jaundice(Cholestatic jaundice) With out jaundice Extra hepatic Intra hepatic
    • Gall stones
    • Carcinoma (obstruction of bile duct)
    • Some forms of Viral hepatitis
    • Biliary cirrhosis
    • Drugs : phenothiazine.
    • Plasma bilirubin increased to be 50mg/dl
    • ALP increases
    • ALT, AST increases
    • Increase GGT
    • Increase 5` nucleotidase
    • Obstruction to only part of biliary system
    • Cholengitis
    • Primary biliary cirrhosis
    Biochemical changes
  • (3) Liver infiltration Carcinoma from lung or stomach Abcess Amyloidosis Tuberculosis Biochemical changes Increase ALP Increase GGT Bilirubin normal ALT and AST normal or slight raised (1) (2) (4) (3)
    • Increse synthesis in sinusoids
    • Regurge to circulation
    • But it highest in Cholestasis
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  • To perform some techniques as ELIZA, western blott, immunolocalization.we should have Antibodies A. Bodies Polyclonal Monoclonal Produced in rabbit or goat Protein specific Goat Collect serum contains Abs
    • Non specific
    • Risk of contamination
    • Antibodies to different epitops and segments
    • Environmental protein reaction
    Specific protein Spleen Antibodies producing cells 6 Remarks on ELIZA technique
  • Monoclonal antibodies production After collection of spleen and obtaining the antibodies producing cells Only selects cell produce your a b Grow alone on culture media High number
    • Specific
    • No cross reaction
    • May be specific to single epitope
    BUT! It can not identify the protein if it has been modified (phosph. Acetyl., glycosyl.) Hybridoma cell Spleen Cells Myeloma Cells Cell culture Cell culture
    • One of tech. based on Ab and Ag reaction
    • Used to detect particular protein in a sample
    Technique of sandwish (captured)
    • Plastic plate
    • Primary antibody
    • Protein sample(antigen)
    • Secondary antibody linked to enzyme
    • Horse raddish peroxidase(HRP)
    • Alkaline phosphatase (ALP)
    • 5. When the substrate present color will developed
    The color intensity was proportional with the amount of enzyme which proportional with the protein conc. ELIZA technique
  • Uric acid Sources: Excretion = 500 mg /day Normal blood level=2-7 mg/dl
    • It form crystals at acidic PH (DCT)
    • dissolved in more alkaline media (PCT)
    End product of purine catabolism High protein diets(meat)
    • deposited in soft tissues( gout)
    • Kidney stones
    Elevated level called hyperuricemia
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  • Disorders in uric acid level in blood
    • Wilson’s disease(accumulation of cupper in liver, kidneys, cornea leticular nucleolus of brain)
    • Proximal renal tubular acidosis
    • Folic acid anemia
    • Burns
    • Pregnancy
    • Drugs as (Allopurinol, azathioprine , Imuran, Benemid, sulfam)
    • Gout
    • Alcoholism
    • Leukemias, multiple myeloma, metastatic cancer
    • Severe diabetes mellitus
    • Renal failure
    • Chronic heart failure
    • Hemolytic- megalobalstic anemia
    • Lead poisoning
    • Polycythemia
    • Glomerulonephritis
    • Hyperlipoproteinemia
    • Severe eclampsia
    • Strenuous exercise
    • Drugs (ascorbic acid, lasix, aldomet, theophyline, phenothiazine, prolonged use of aspirin)
  • Primary Hyperuricemia
    • Increase the activity of PRPP synthetase(phosphoribosyle pyrophosphate)
    • Decrease the activity of hypothanthine guanine phosphorybosyl transferase (HGRPase)
    • Dec. activity of glucose – 6- phosphatase inc. G6P inc. HMP ribose purines uric acid
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