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Biochemical Analytical Biochemical Analytical ProcedureProcedure

The major steps involved in are:-

- REQUESTING

- PERFORMING

- EVALUATING

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Biochemical Tests Are Biochemical Tests Are Required ForRequired For

a- Screeningb- Diagnosingc- Monitoring or Managementd- To establish prognosis

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ACTUAL ANALYTICAL ACTUAL ANALYTICAL PROCEDUREPROCEDURE

A- PRE-INSTRUMENTAL PHASE 1- Selection of Patients 2- Prepration of Patients 3-Collection of Sample 4- Transportation of Sample B- INSTRUMENTAL PHASE 1-Methodology 2-Calculation of Values. C- POST INSTRUMENTAL PHASE 1-Reporting 2-Evaluating the reports with Patients clinical stage.

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EXPRESSING THE EXPRESSING THE RESULTSRESULTS

As- Qualitative-Grades

(using signs- +, ++, +++) - Quantitative-Number /Units

eg.-Serum Sodium=132m mol/L

-Blood Sugar =75mg/dl

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INTERNATIONAL VALUESINTERNATIONAL VALUES

1- International Union of Pure & applied chemistry.(IUPAC)

2- International Federation of clinical chemistry.(IFCC)

3- Systemic International units.(SI)These units help us to communicate the

results to larger scientific community with clearity.

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PRE-INSTRUMENTAL PRE-INSTRUMENTAL VARIATIONSVARIATIONS

A- Variations Due To Prepration of Patients

a- Exercise statland

b- Previous Diet

c-Drug Intake

d-Tobacco Smoking

e-Posture

f-Tourniquet Pressure

g-Stress

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EXERCISEEXERCISE A- TRANSIENT EFFECT-(within an hr.) -These are due to increased metabolic activity eg.-Free Fatty Acids-

-Alanine conc.-180% -Lactate -300% -They are corrected to pre-exercise level soon after the cessation of the

exercise. B- LONGER LASTING EFFECT-of exercise is mainly on the muscle

enzymes eg. Creatinine kinase, aldolase, SGOT, LDH etc. Kings studied – One hour moderate to strenuous exercise can cause changes

even upto 11 hrs.

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1 5 11 19 29 45 55 67

120

0

60

40

20

80100

140

CPK

AST

LDH

AP

hr hr hr hr hr hr hr hr

%Change from base

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PREVIOUS DIETPREVIOUS DIET

Usually biochemical analysis is recommended in fasting stage.(12 hrs.)

Prolonged Fasting- 24hrs. Fasting can lead to an increase in - Serum Bilirubin

- Fatty acid - Valine, Leucine > 72 hrs. fast can lead to decrease in - Plasma –glucose -protein,albumin -pre-alb,transferrin -complement-C3 -

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After Meal- Physiological effects of eating a meal within 2-4 hrs. includes increase in

-Triglycerides -Potassium In vivo changes are dependent upon the type and quantity of food ingested

eg.- 1-High fatty diet:- increases -Intestinal isoenzyme of alkaline phos.sp. In ‘O’-B’ group -Turbidity- LACTESCENT SERUM- Which can interfere in many

assays. 2-High Protein diet;- can cause increase in -Blood Urea,Ammonia, Urate etc. 3-High Ratio Unsaturated: Saturated Fats causes decrease in

CHOLESTEROL.

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Diet rich in Purine:- Increases - Urate conc Banana,Pineapple,Tomato. Because of presense of serotonin can cause increase in Renal 5 Hyd roxyindol acetic acid . (Diagnostic-Carcinoid) Coffee:- causes threefold rise in -Nonesterified free fatty acids. -Cholestrol -Cause a release of catecholamine from the adrenal medulla. Ethenol;-1.-Immediate changes are increase in Lactate,Urate, Acetaldehyde,Acetate 2.-Intermediate changes ( 10-100HRS.) (mainly on serum enzymes)

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0

+30

+20

+10

-10

-2015 hrs 36hrs 60 hrs 100 hrs

AST

ALT

CPK

APGGT

LDH

% change from base

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Long time effect:- Increase in Triglyceride It has been noted that chronic alcoholics have higher

plasma HDL cholesterol conc.than do matched control subjects. Abusers of alcohol show increased value of serum GAMMA GLUTAMYL TRANSFERASE,URATE &MEAN ERYTHROCYTE VOL.

Tobacco smoking:-results in increase in the % of blood Carboxy Hb

Acute effects of Tobacco smoking include increase in plasma catecholamine and serum cortisol.(Probably due to the nicotine)

Chronic effects are increase in Hb,MCV,TLC

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EFFECT OF DRUGSEFFECT OF DRUGS

Young had studied about 15,000 drugs and found that they have

A.- Physiological effect.- In vivo effects of the drug or its metabolites.

B.- Chemical interference.- In vitro due to some physical or chemical property which interfere with the assay.

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Glucose (A) (B)

ACTH-Corticosteroid Acetaminophen

Epinephrine PAS

Ethacrynic acid Ascorbic acid +/-

Furosemide Dextran

Thiazide Hydralazine

Phenytoin Levo Dopa

Propanonol D Nalidixic Acid

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Urea (A) (B) Alk.antacid Chloral hydrate Antimony salts Chlorobutol Cephaloridin Guanethedine Frusemide Gentamycin Kanamycin Methyl Dopa

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AST/ALT (A) (B)

Cephalothin Ascorbic acid

Gentamycin Erythromycin

Opiate L-Dopa

Oxacillin PAS

INH

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A no. 0f drugs are known to affect the liver by inducing hepatic microsomal enzyme or by producing hepatocellular damage or cholestatic jaundice.eg.

-Synthetic steroid -Sulfonylurea derivatives eg. Chlorpromazine Thiouracil Tolbutamide - Erythromycin -INH - Tetracycline -Aspirin Cholestatic changes are increase in- Alk.Phos.,Bromosulphalein,Bilirubin,AST/ALT -

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Oral contraceptives:-Physiological effects include increase in- -ALT - Ceruloplasmin - Transcortin -Thyroxin binding globulin - Iron - Triglyceride -Gamma Glutamyl Transferase decrease in -Albumin -Zinc These effects are probably related to the estrogen portion of the drug.

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POSTUREPOSTURE

Differ OPD:INDOOR Samples are usually taken in either supine or

sitting position.As patient goes from supine to standing posture there will be an efflux of water and filterable subs.from I/V to interstitial fluid space.Non-filterable subs.like protein and protein bound subs.are increased in blood.There is sig. Change in supine to standing stages.-

STATLAND:- Within 30 mints.there is an increase in:-

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-Sodium 1% -Lipid 9 % -Potassium 4% -Cholestrol 8%

-Calcium 4% -AST 6% -Chloride 2% -ALT 14% -Phosphate 3% -Alk.Phos. 12% -Urea 1% -Acid Phos. 6% -Creatinine 4% Decrease in:- -Protein 8% -Uric acid 4% -Albumin 9% -Iron 7% Change in posture dramatically increase- -Norepinephrine -Aldosterone So posture of patient at the time of taking sample should be mentioned.

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EFFECT OF TOURNIQUET EFFECT OF TOURNIQUET OR FIST EXERCISEOR FIST EXERCISE

Prolonged application of tourniquet or fist exercise cause significant changes in the conc.of many serum constituents eg.

-Lactate -Enzymes -Protein -Protein bound subs.such as Cholestrol Triglyceride Calcium&Iron

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Statland compaired 1mt.:3mt.Tourniquet application and found an increase in

Protein 5% Cholesterol 5% Iron 6% Bilirubin 8% AST 10% STRESS:- Effects Adrenal Hormones.i.e.why Cortisol estimation is done at 8AM&8PM ANXIETY Hyperventilation leads to disturbance in acid base balance.An increase

in - Lactate -Non-esterified fatty acid

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ERRORS DUE TO ERRORS DUE TO PREPRATION OF SPECIMENPREPRATION OF SPECIMEN

-During collection

-Specimen interferance

-During processing of specimen

-During storage

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COLLECTION OF COLLECTION OF SPECIMENSPECIMEN

A.Identification error:-a. Proper Reqesition Form b.Name,CR No.,Bed No.etc. c.Collection time in 24hrs. specimen. d.Correct type of container -Anticoagulant -Preservative B.Site of Blood drawing:- a.Postural b.Tourniquete c.Capillary or venous

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Technician should be certain to avoid sampling from an extremity in which an I/V catheter is delivering parentral soln.

Site of venipuncture must be distal to the I/V needle and the tourniquete must be placed between the I/V needle and the site of venipuncture.

Choice of capillary or venous blood is not usually of clinical importance except in the case of GTT where capillary conc. is 10-30% higher

C.Contamination of specimen:- 1.Residual detergent:-May contaminate the sample with

Inorganic phosphate 2.Plasticizers:-(I/Vset,Tube,Rubber cork)create spurious

peaks in gas liquid chromatography.

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3.Glass tube,Cork etc.-Inceases calcium. 4.Container with lead.-Increases Lead 5.Metal of needle:-Interferes with -Assay of coagulation -Platelets. Haemolysis;- -While venipuncture -When blood collected in vaccum tubes -While mixing -Greater bore needle has more chances

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The majority of chemical measuremants are performed on specimen obtained from the e.c.f.ie.usually plasma or serum.Certain analytes are present in the formed elements of the blood in conc. many times higher or lower than in the surrounding plasma and therefore lysis of the cells will contaminatethe plasma or serum to a measureable amount.

Direct interference of Hb.increases:- -Bilirubin -Protein -Potassium etc. Haemolysis can occur before venipuncture or during the

analytical procedure.

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1% of Haemolysis can change:- LDH +272% AST +220% POTASSIUM +25% ALT +55% GLUCOSE --5% INORGANIC PHOS. +9.1% SODIUM --1% CALCIUM +2.9% ICTRIC SERUM:-Jaundice of 2.5mg./dl will

interfere with estimation of -Cholesterol -Glucose -Albumin Can be eliminated by using appropriate blanking

procedure or dual wavelength.30

Lactescent Serum:-

Increases:- Decreases:-

Triglyceride Amylase

Albumin Urea

Calcium Urate

Inorganic Phos. Creatinine

Bilirubin

Protein Can be eliminated by using serum blank or

ULTRACENTRIFUGATION

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EFFECT OF EFFECT OF ANTICOAGULANTSANTICOAGULANTS

A.-Potassium oxalate or EDTA:-Causes decrease in -Calcium

-Amylase -LDH B.-Floride:- Inhibits:- -Glucose oxidase -Acid phosphatase -Amylase So preferrably serum is used for biochemical tests

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CHANGES DURING CHANGES DURING STORAGESTORAGE

A. Evaporation:-Of water from serum -Results in higher conc.of all analytes -Causes increase in activity of enzymes 10mts.storage will cause 5% increase in osmolality at 28

degree C Temp.with 25% Humidity. B.Refrigeration:-Of amniotic fluid increases - Lecithin: Sphingomylin -Phospholipase decreases 33

C.-Open Sample:-I.Will lead to evaporation of CO2 which increases pH to 8.5 in 2hrs.which causes decrease in Acid phosphatase.

2.Glycolysis- Decreases Glucose. 3.Proteolytic&Hydrolytic processes increase conc. Of Ammonia. 4.Changes in Erythrocyte Permeability increases - Potassium -Phosphate & Magnesium D.-Exposure to light:-Decreases -Bilirubin -Delta aminolevulinic acid -Porphyrins -Porphobilinogen. -

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ERRORS DURING ERRORS DURING METHADOLOGYMETHADOLOGY

-Errors in Pipetting-In instrumentation-In preparation of Reagents-Caliberation&Calculation

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Idealy all measurements should be performed within 1hr.after collection. Prolonged contact of serum with cell clot beyond two hrs.can cause sig. Changes in certain constituents.Such as Glucose,Potassium,Phosphorus,Creatinine,AST &ALT(Rehak).

Clinically useful data generated by the lab must be reported promptly and accurately to optimize patient management.Delay in reporting can make data useless.

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QUALITY CONTROLQUALITY CONTROL

Quality control is must in the lab. -Interlaboratory -Intralaboratory Interlaboratory:- -Standards -Referance samples-Low -High

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EVALUATING RESULTSEVALUATING RESULTS

Before evaluating results we should check

-Reliability of Method

-Specificity&Senstivity

-Random Analytical Variation

-Dynamic Range

-Interferance

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CONCLUSIONCONCLUSION

Total variation in results of patients can be grouped:

- Analytical Variation

-Prepration of subjects

-Intra Individual Physiological

-Inter Individual Biological variations of Mean Values.

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One has to appreciate and keep in mind these expected non pathological source of variation in Diagnosing and management of Disease.

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