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BIOANALYTICAL/CLINICAL ANALYSIS

Prof. B. Kuswandi
University of Jember
(Edieted from G.G Guibault)
CLINICAL BIOMEDICINE
STUDY OF CHEMISTRY OF HUMAN SYSTEM
-WHAT CHEMICALS ARE INVOLVED IN DISEASES
- WHICH ONES AFFECT WELL BEING
- WHY IMPORTANT
2/3 OF ALL ANAL WORK IS IN THIS AREA
1 OF 2 OF ALL PERSONNEL IN THIS AREA
HISTORY OF CLINICAL:
l. OTTO WARBURG IN 1940 NADH ABSORBS AT 340nm
2. 1965 OVERHAUL OF CLINICAL ANALYSIS(NEXT)
3. 1970 SKEGGS - AUTOMATION
HIGH GLUCOSE = DIABETES- IMBALANCE OF
METABOLISM
SUCROSE GLUCOSE GLYCOGEN
IN 1965 NATIONAL INSTITUTES OF HEALTH DID A STUDY
OF THE ABILITY OF ANALYTICAL LABS TO GET THE
CORRECT RESULT
TRUE RESULT WAS NORMAL: 90 mg/dL
30% OF LABS REPORTED HIGH(RESULT:INJECTION OF
INSULIN DEATH)
40% OF LABS REPORTED NORMAL
30% OF LABS BELOW NORMAL(RESULT:INJECTION OF
SUGAR LETHAL TO DIABETIC)
RESULT: BIG SHAKEUP IN LABS LEADING TO BETTER
RESULTS AND A MODERNIZATION OF ANALYSIS.
MOST IMPORTANT TESTS
l. GLUCOSE HIGH DIABETES( IMBALANCE OF
METABOLISM)
2. UREA/CREATININE MALFUNCTIONING OF KIDNEY
3. CHOLESTEROL/HDL/LDL/VLDL MYOCARDIAL
INFARCT(HEART)
4. URIC ACID GOUT
5. ALKALINE PHOSPHATASE DAMAGE TO LIVER
6. AND OF COURSE PHARMACEUTICAL INDUSTRY
BENEFITS BY PROVIDING NEW DRUGS FOR THESE
DEFICIENCIES
BIGGEST DIABETES NEW DRUG RECENTLY
APPROVED FOR TYPE 2 DIABETES = JANUVIA
PURPOSE OF CLINICAL LAB=QUAL AND QUANT
ANALYSIS OF BODY FLUIDS-BLOOD,URINE,SPINAL
FLUID,FECES,etc
UNLIKE ALL OTHER ANALYSIS HERE WE USE LIQUID
NOT MASS(TAKE FEW MICROLITERS BLOOD).USE dL AS
UNIT.IN IRELAND/UK USE mM,ALL OTHERS mg/dL:
Mg per liter/Molecular Weight = mM
CURRENT TYPICAL VALUES IN PLASMA
GLUCOSE,mg% 90 (5.0 mM)
UREA,mg % 16
CREATININE,mg% 1.1
URIC ACID, mg% 4.6
TOTAL CHOLESTEROL 194
POTASSIUM,meq/liter 4.4
ALKALINE PHOSPHATASE,units 3.0
LDH, units 360
ORIGINS OF SPECIES DETERMINED
STAGE 1 TESTS ARE ON ENZYMES OR SUBSTRATES COMING FROM
SPECIFICALLY DAMAGED ORGANS
l. ALKALINE PHOSPHATASE DAMAGED LIVER
HIGH: OBSTRUCTIVE JAUNDICE,PADGETTS DISEASE, RICKETS
2. CHOLINESTERASE DAMAGE TO NERVE ENDINGS
HIGH: NEPHROTIC SYNDROME
LOW: PESTICIDE POISONING, ANEMIA, MALNUTRITION
3. LACTATE DEHYDROGENASE(LDH)
MYOCARDIAL INFARCTION, LEUKEMIA
4. GLUCOSE DIABETES(TYPES 1 AND 2)
5. UREA/CREATININE DISEASES OF KIDNEY
QUALITY CONTROL AND VALIDATION
VERY IMPORTANT IN CLINICAL LABS WHERE LIFE/DEATH
DECISIONS ARE MADE
Reference: N. TIETZ-FUNDAMENTALS OF CLINICAL
CHEMISTRY
A. MUST CALIBRATE INSTRUMENT TWICE DAILY WITH
STANDARD BLOOD SAMPLE WITH KNOW VALIDATED
VALUES
B. MUST USE REAL BLOOD SAMPLES:
l. POOLED SERUM
2. FREEZE DRIED SERUM CONTROLS
a. DADE MONITROL: NORMALS FOR ALL
SUBSTRATES(eg GLUCOSE),METALS, ENZYMES
b. LEDERLE LABS LEDERNORM(MOST TESTS)
NORMAL VS ABNORMAL
WHAT IS NORMAL = AVERAGE PERSON(?)
-ACCEPTED AS ABOUT 80% OF POPULATION
-RANGE IN WHICH 95% FIT(1 in 20 OUTSIDE)
- 80 to 95% OF POPULATION ARE GREY AREA
- 5% CONSIDERED ABNORMAL(See TIETZ)
DADE FOR EXAMPLE CAREFULLY SCREENS DONORS FOR
DISEASES, MEDICAL HISTORY.
-PICKES SEVERAL HUNDRED,COLLECTS BLOOD=FORMS A
POOL. FREEZE DRY BLOOD, ASSAY AND REPORT ALL
VALUES(SEPARATE FOR EVERY DIFFERENT
INSTRUMENT).ONLY ABOUT 5% TAKEN AS
REPRESENTATIVE FOR ANALYSIS.
-ABNORMAL- SPIKE WITH PURE REAGENTS.ASSAY
TYPES OF ASSAYS
l. EQUILIBRIUM LET ALL REACT, MEASURE AT EQN
A + B PRODUCTS
MEASURE PRODUCTS BY COLOR, FLUORESCENCE,
ELECTROCHEMICAL METHOD
2. KINETICS
MEASURE RATE OF REACTION AS AFFECTED BY A ABOVE AT
CONSTANT B.
4 X C1
ABSORBANCE 3 X C1
2 X C1
C1
TIME
ASSAY OF ENZYMES
MUST BE DONE BY KINETIC(RATE ) METHOD SINCE THEY
ARE CATALYSTS
ADVANTAGES: THEY ARE HIGHLY SPECIFIC AND SENSITIVE
BASE EQUATIONS E + S(k1/k2) ES (k3) PRODUCTS + E
Vo = Vmax[So]/Km + So where Vmax = k3[Eo]
Km = k2 + k3/k1
ENZYME ACTIVITY = HOW MUCH SUBSTRATE IS CONVERTED
AMBIGUITY ARISES SO USE INTERNATIONAL UNITS
IN OLD SYSTEM WE HAD MANY ASSAYS WITH DIFFERENT
CONDITIONS/IMPOSSIBLE TO CORRELATE
THERE ARE A NUMBER OF IMPORTANT ENZYMES WE WILL
LOOK AT AND ALSO ISOENZYMES.
TO SYSTEMATIZE:
COMMISSION ON ENZYMES IF INTERNATIONAL UNION
OF BIOCHEMISTRY IN 1961 MET IN NEW YORK:
RECOMMENDATIONS OF I.U.B., ELSEVIER, 1965
ONE UNIT = AMOUNT OF ENZYME THAT CATALYZES
REACTION OF ONE MICROMOLE OF SUBSTRATE PER mg
ENZYME PER MINUTE AT STANDARD
CONDITIONS(USUALLY T=30 C,pH OPTIMUM ETC)
RECOMMEND U/mL or U/L at 30 C
REMEMBER: ENZYMES ARE SECONDARY INDICATORS OF
CELL OR TISSUE ACTIVITY- IF UP OR DOWN=
SOMETHING WRONG.BETTER = DIRECT ASSAY OF
TISSUE. THIS IS DIFFICULT:IN SECOND GENERATION
TESTS WE USE (l) IMMUNOCHEMISTRY,eg PSA TEST .
ENZYMES AND DISEASES.
ENZYME ORGAN OF DISEASE SPECIMEN
Acid Phosphatase Prostate Serum
Alkaline Phosphatase Liver, bone Serum/urine
Amylase Pancreas Serum/urine
Lipase Pancreas Serum/urine
Lactate Dehydrogenase(LDH)Liver,Heart Serum/urine
Aspartate transaminase(GOT) Liver,Heart Serum/urine
Alanine transaminase(GPT) Liver,Heart Serum/urine
Creatine Hinase(CK) Heart,Muscle,Brain Serum
Aldolase Copper transport disease(Wilson) Serum
USE OF PROFILING
SEVERAL TESTS USED TO INDICATE A SPECIFIC
FUNCTIONING OF BODY
WHY-COVER ALL POSSIBILITIES(AVOID LAWSUITS)
l. LIVER : LDH, GOT,GPT,ALK PHOSPATASE,GAMMA
GLUTAMYL TRANSPEPTIDASE(GGTP)
2. KIDNEY : UREA, CREATININE, GGTP
3.PANCREAS : LIPASE, AMYLASE,LEUCINE
AMINOPEPTIDASE
4. MUSCLE: CK, LDH
5. BONE: ALKALINE PHOSPHATASE TARTRATE
INHIBITED(ISOENZYME 1)OR UREA INHIBITED(ISO 2)
6. CANCER : ALK AND ACID
PHOSPHATASE,LDH,ASPARAGINASE,LAP
ISOENZYMES=MAJOR IMPROVEMENT in ASSAYS
WHAT ARE THEY:SOME ENZYMES FROM AN INDIVIDUAL
ORGANISM CAN EXIST IS SEVERAL DIFFERENT
FORMS=ISOENZYMES
DIFFERENCES-
l. CHARGE = CAN BE SEPARATED BY ELECTROPHORESIS
2. STABILITY TO HEAT DENATURATION
3. REACTION TO CHEMICAL INHIBITORS(Alk Phosphatase)
4. AFFINITY FOR SUBSTRATES OR COENZYMES
WHICH ONES ARE COMMON:
LDH,CK,ALK AND ACID PHOSPHATASE(PERHAPS ALL
BUT NOT GLUCOSE OXIDASE)
ANALYSIS OF ENZYMES
WHICH ARE MOST IMPORTANT:
A. ALKALINE PHOSPHATASE SIGNIFICANCE:
LIVER DISEASE INDICATOR, OBSTRUCTIVE
JAUNDICE,PADGETTS AND OTHER LIVER DISEASE
INDICATORS
ASSAY: PHENYLPHOSPHATE PHENOL + Pi
PHENOL + DIAZOREAGENT RED COLOR
ACTIVATORS Mg(II) AND Mn(II)
OR p-NITROPHENYLPHOSPHATE p-
NITROPHENOL(410nm)
OR FLUOROMETRIC USING NAPTHOL ASBI
PHOSPHATE GREEN FLUORESCENCE