1
Biochemical Analytical
Procedure
The major steps involved in are:- REQUESTING
- PERFORMING
- EVALUATING

2
Biochemical Tests Are
Required For
 a-

Screening
 b- Diagnosing
 c- Monitoring or Management
 d- To establish prognos...
ACTUAL ANALYTICAL
PROCEDURE
 A-

PRE-INSTRUMENTAL PHASE
1- Selection of Patients
2- Prepration of Patients
3-Collection o...
EXPRESSING THE
RESULTS
 As

Qualitative-Grades
(using signs- +, ++, +++)
- Quantitative-Number /Units
eg.-Serum Sodium=1...
INTERNATIONAL VALUES
 1-

International Union of Pure & applied
chemistry.(IUPAC)
 2- International Federation of clinic...
PRE-INSTRUMENTAL
VARIATIONS
 A-

Variations Due To Prepration of Patients
a- Exercise
statland
b- Previous Diet
c-Drug In...
EXERCISE
 A

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

-Ala...
9
%Change
from
base

CPK
AST
LDH

140
120
100
80

AP

60
40
20
0

1 hr

5 hr

11 hr

19 hr

29 hr 45 hr55hr67 hr
PREVIOUS DIET
 Usually

biochemical analysis is recommended in
fasting stage.(12 hrs.)
 Prolonged Fasting- 24hrs. Fastin...
After Meal- Physiological effects of eating a meal
within 2-4 hrs. includes increase in
-Triglycerides
-Potassium
 In viv...
 Diet

rich in Purine:- Increases
- Urate conc
 Banana,Pineapple,Tomato. Because of presense of
serotonin can cause incr...
AP
GGT
LDH

% change
from base

AST
ALT
CPK

+30
+20
+10
0
-10
-20
15 hrs

36hrs

60 hrs

100 hrs
13
 Long

time effect:- Increase in Triglyceride

It has been noted that chronic alcoholics
have higher plasma HDL choleste...
EFFECT OF DRUGS
 Young

had studied about 15,000 drugs and
found that they have
 A.- Physiological effect.- In vivo effe...
 Glucose

(A)
ACTH-Corticosteroid
Epinephrine
Ethacrynic acid
Furosemide
Thiazide
Phenytoin
Propanonol
D

(B)
Acetaminoph...
 Urea

(A)

Alk.antacid
Antimony salts
Cephaloridin
Frusemide
Gentamycin
Kanamycin
Methyl Dopa

(B)
Chloral hydrate
Chlor...
 AST/ALT

(A)
Cephalothin
Gentamycin
Opiate
Oxacillin

(B)
Ascorbic acid
Erythromycin
L-Dopa
PAS
INH

18
A

no. 0f drugs are known to affect the liver by
inducing hepatic microsomal enzyme or by
producing hepatocellular damage...
 Oral

contraceptives:-Physiological effects include
increase in-ALT
- Ceruloplasmin
- Transcortin
-Thyroxin binding glob...
POSTURE
 Differ

OPD:INDOOR
 Samples are usually taken in either supine or
sitting position.As patient goes from supine ...
-Sodium
1%
-Lipid
9%
-Potassium 4%
-Cholestrol 8%
-Calcium
4%
-AST
6%
-Chloride
2%
-ALT
14%
-Phosphate 3%
-Alk.Phos. 12%
-...
EFFECT OF TOURNIQUET
OR FIST EXERCISE
 Prolonged

application of tourniquet or fist
exercise cause significant changes in...
 Statland

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

Protein
5%
Cholesterol 5%
Iron
6%
Bilirubi...
ERRORS DUE TO
PREPRATION OF SPECIMEN
-During collection
-Specimen interferance
-During processing of specimen
-During stor...
COLLECTION OF
SPECIMEN
A.Identification error:-a. Proper Reqesition Form
b.Name,CR No.,Bed No.etc.
c.Collection time in 24...
 Technician

should be certain to avoid sampling
from an extremity in which an I/V catheter is
delivering parentral soln....
3.Glass tube,Cork etc.-Inceases
calcium.
4.Container with lead.-Increases Lead
5.Metal of needle:-Interferes with
-Assay o...
 The

majority of chemical measuremants are
performed on specimen obtained from the
e.c.f.ie.usually plasma or serum.Cert...
1% of Haemolysis can change:LDH
+272%
AST
+220%
POTASSIUM +25%
ALT
+55%
GLUCOSE
--5%
INORGANIC PHOS. +9.1%
SODIUM
--1%
CAL...
 Lactescent

Serum:-

Increases:Decreases:Triglyceride
Amylase
Albumin
Urea
Calcium
Urate
Inorganic Phos.
Creatinine
Bili...
EFFECT OF
ANTICOAGULANTS
 A.-Potassium

oxalate or EDTA:-Causes decrease
in
-Calcium
-Amylase
-LDH
 B.-Floride:- Inhibit...
 A.

CHANGES DURING
STORAGE

Evaporation:-Of water from serum
-Results in higher conc.of all
analytes
-Causes increase in...
 C.-Open

Sample:-I.Will lead to evaporation of
CO2 which increases pH to 8.5 in 2hrs.which
causes decrease in Acid phosp...
ERRORS DURING
METHADOLOGY
 -Errors

in Pipetting
 -In instrumentation
 -In preparation of Reagents
 -Caliberation&Calc...
 Idealy

all measurements should be performed
within 1hr.after collection. Prolonged contact of
serum with cell clot beyo...
QUALITY CONTROL
 Quality

control is must in the lab.
-Interlaboratory
-Intralaboratory
 Interlaboratory:-Standards
-Ref...
EVALUATING RESULTS
 Before

evaluating results we should check
-Reliability of Method
-Specificity&Senstivity
-Random Ana...
CONCLUSION
 Total

variation in results of patients can be
grouped:
- Analytical Variation
-Prepration of subjects
-Intra...
 One

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

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Svinbio tests

  1. 1. 1
  2. 2. Biochemical Analytical Procedure The major steps involved in are:- REQUESTING - PERFORMING - EVALUATING 2
  3. 3. Biochemical Tests Are Required For  a- Screening  b- Diagnosing  c- Monitoring or Management  d- To establish prognosis 3
  4. 4. ACTUAL ANALYTICAL PROCEDURE  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. 4
  5. 5. EXPRESSING THE RESULTS  As Qualitative-Grades (using signs- +, ++, +++) - Quantitative-Number /Units eg.-Serum Sodium=132m mol/L -Blood Sugar =75mg/dl 5
  6. 6. INTERNATIONAL 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. 6
  7. 7. PRE-INSTRUMENTAL VARIATIONS  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 7
  8. 8. EXERCISE  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. 8
  9. 9. 9 %Change from base CPK AST LDH 140 120 100 80 AP 60 40 20 0 1 hr 5 hr 11 hr 19 hr 29 hr 45 hr55hr67 hr
  10. 10. PREVIOUS 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 10
  11. 11. 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 SERUMWhich 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. 11
  12. 12.  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) 12
  13. 13. AP GGT LDH % change from base AST ALT CPK +30 +20 +10 0 -10 -20 15 hrs 36hrs 60 hrs 100 hrs 13
  14. 14.  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 14
  15. 15. EFFECT 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. 15
  16. 16.  Glucose (A) ACTH-Corticosteroid Epinephrine Ethacrynic acid Furosemide Thiazide Phenytoin Propanonol D (B) Acetaminophen PAS Ascorbic acid +/Dextran Hydralazine Levo Dopa Nalidixic Acid 16
  17. 17.  Urea (A) Alk.antacid Antimony salts Cephaloridin Frusemide Gentamycin Kanamycin Methyl Dopa (B) Chloral hydrate Chlorobutol Guanethedine 17
  18. 18.  AST/ALT (A) Cephalothin Gentamycin Opiate Oxacillin (B) Ascorbic acid Erythromycin L-Dopa PAS INH 18
  19. 19. 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 19
  20. 20.  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. 20
  21. 21. POSTURE  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:21
  22. 22. -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. 22
  23. 23. EFFECT OF TOURNIQUET OR 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 23
  24. 24.  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 24
  25. 25. ERRORS DUE TO PREPRATION OF SPECIMEN -During collection -Specimen interferance -During processing of specimen -During storage 25
  26. 26. COLLECTION OF SPECIMEN 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 26
  27. 27.  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. 27
  28. 28. 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 28
  29. 29.  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. 29
  30. 30. 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
  31. 31.  Lactescent Serum:- Increases:Decreases:Triglyceride Amylase Albumin Urea Calcium Urate Inorganic Phos. Creatinine Bilirubin Protein  Can be eliminated by using serum blank or ULTRACENTRIFUGATION 31
  32. 32. EFFECT OF ANTICOAGULANTS  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 32
  33. 33.  A. CHANGES DURING STORAGE 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
  34. 34.  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. 34
  35. 35. ERRORS DURING METHADOLOGY  -Errors in Pipetting  -In instrumentation  -In preparation of Reagents  -Caliberation&Calculation 35
  36. 36.  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. 36
  37. 37. QUALITY CONTROL  Quality control is must in the lab. -Interlaboratory -Intralaboratory  Interlaboratory:-Standards -Referance samples-Low -High 37
  38. 38. EVALUATING RESULTS  Before evaluating results we should check -Reliability of Method -Specificity&Senstivity -Random Analytical Variation -Dynamic Range -Interferance 38
  39. 39. CONCLUSION  Total variation in results of patients can be grouped: - Analytical Variation -Prepration of subjects -Intra Individual Physiological -Inter Individual Biological variations of Mean Values. 39
  40. 40.  One has to appreciate and keep in mind these expected non pathological source of variation in Diagnosing and management of Disease. 40
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