Transcript BIOANALYTICAL/CLINICAL ANALYSIS

CM3107
BIOANALYTICAL/CLINICAL ANALYSIS
Prof. George G. Guilbault
Room 205, Robert Kane Building
University College Cork, Ireland
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%
URIC ACID, mg%
1.1
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
Acid Phosphatase
SPECIMEN
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)
Aldolase
Heart,Muscle,Brain
Serum
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
DIFFERENCESl. 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  pNITROPHENOL(410nm)
OR FLUOROMETRIC USING NAPTHOL ASBI
PHOSPHATE GREEN FLUORESCENCE