Professional Documents
Culture Documents
Lab. Practical 1
Lab. Practical 2
Lab. Practical 3
Lab. Practical 4
Measurment of Triglycerides
Lab. Practical 5
Lab. Practical 6
Lab. Practical 7
Lab. Practical 8
Lab. Practical 9
Lab. Practical 10
Lab. Practical 11
Lab. Practical 12
Lab. Practical 13 15
Tutorials
heat &acidity
glucosamine (colored )
C) Enzymatic methods:
1-Hexokinase methods(The reference method).
With pre-deproteinization of sample or without.
Glucose +ATP +HKADP+G6P
G6P +NAD +G6PD 6 P-gluconolactone +NADH+H
(measured at 340)
2- Glucose oxidase methods:
-Trinders (Enz.-Dye Colorimetric ) method:
which is colorimetric either by spectrophotometer or refractrometer
(refractrometeric methods either in a film form [kodak Ectachem] or a strip form
[Dry chemistry] ).
-Kinetic method:
by measuring the increase in absorbance through increase in NADH+H
-Polarigraphic method:
using O2 electrode to detect O2 utilization.
N.B. GOD/POD method can not used for detection of urine glucose because the urine
contains interfering substances for peroxidase (POD) .
- To use this method treatment of the urine sample either by Somogi Nelson filtrate or Ion
Exchange Resin is taken before running . Also, using GOD/POD method in urine with
modification like , Polarigraphic determination with post-reaction elimination of H 2O2 by:
ethanol & catalase or Iodide & molybdate.
3- Glucose Dehydrogenase Method:
Glucose +NAD
GDH
Mutarotase
-D-glucose +H2O+O2
-D-glucose
Glucose oxidase
H2O2+ 4-aminophenazone+phenol
D-gluconic acid+H2O2
Peroxidase
Quinonemine +4 H2O
The intensity of the color formed is proportional to the glucose concentration in the sample.
CLINICAL SIGNIFICANCE
Glucose is a major source of energy for most cells of the body; insulin facilitates glucose entry into the
cells. Diabetes is a disease manifested by hyperglycemia; patients with diabetes demonstrate an
inability to produce insulin. Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.
PREPARATION
lank
1.0
WR (mL)
-Standard (L)
-Sample (L)
14. Mix and incubate for 10 min at 37C or 15-20 min at room temperature (15-25C).
15. Read the absorbance (A) of the samples and standard, against the Blank.
The colour is stable for at least 30 minutes.
CALCULATIONS
Interpretation:
II - Hyperglycemia :
Diabetes Mellitus
Hemochromatosis
Hypokalemia
Stress
Pheochromocytoma
Anesthesia
Pregnancy
Hyperthyroidism
Cushing disease
Hyperpituitarism (gigantism)
Discussion:
*Physiological & Biochemical Background:
Glucose metabolism, Insulin action and other hormonal effects on glucose in
the human body.
*Pathological & Disease Correlation: Diabetes Mellitus, Cushing
disease ,Hyperthyroidism ..etc
Questions:
12345-
On standard oral glucose dose, the response of the body regarding the absorption and
metabolism of glucose said to be tolerant on meeting the normal elevation and return. Whereas
abnormal and improper glucose metabolism is termed glucose intolerance. This used to diagnose
diseases where the glucose metabolism is impaired as in Diabetes mellitus. Oral glucose tolerance test
(OGTT) has been widely used as the golden standard for diagnosing diabetes mellitus in clinically
doubtful cases. Lately, thought, the use of OGTT in primary care has been questioned for several
reasons. It has low reproducibility and is very expensive. However, for the detection of diabetes in
pregnant women, it is still recommended.
Objectives:
It is to practice the OGTT and knowing the uses and interpretation regarding the diagnostic
benefits of this laboratory test.
Indications:
1- Borderline fasting blood sugar for >2 times (~ 110 125mg/dl)
2- Diagnosis of Gestational Diabetes (GDM) at 24 28 weeks of gestation especially for those
have a family history of diabetes.
3- After delivery for those was suffering from GDM.
OGTT (Experiment # 2):
*Patient preparation (Perquisites) ;
Activity--Don't smoke or exercise strenuously for 8 hours before the test or during the
test.
Diet--Eat a high-carbohydrate diet (> 150 g/day) for 3 days, then fast for 10 to 12 hours
before the test. Don't drink coffee or alcohol for 8 hours before the test.
Drugs (medicines)-Inform the person performing the test to omit any medications listed,
as under taking these drugs the test results may differ (contraceptives to be stopped one
cycle before the performance of OGTT).
The test must be performed at daytime (morning).
* General description of test
Test usually takes 3 hours but can last as long as 6 hours (extended OGTT).
Drink water frequently during the test (the only allowed fluid to drink).
The first blood sample and the first urine sample are collected between 7 A.M. and 9
A.M., after you have fasted for 12 hours.
Operator gives a test load of glucose, usually 75 100 gram dextrose / 300 ml water,
lemon flavored . Drink the entire solution in 5 minutes.
Blood and urine samples are collected at 30 min., 60 min., 90 min.,120 min. and 3 hours
and sometimes immediately after drinking oral glucose solution.
Dose of Oral Glucose:
Dextrose:
1 1.75 g/kg. body wt. (for adults0 and not exceeds 100 g.
It is to be dissolved in 250 300 ml lemon flavored water.
Fortical : 113 ml completed to 300 ml water
Lucozade: 350 ml. (ready to use)
*Samples:
Blood samples ; fasting(basal) sample, 30min. after oral glucose load, 60min, 90min,
120min. (in extended OGTT another 2 samples will be taken at 2hour and 3 hours).
Urine samples ; first fasting urine and the hourly collected urine samples.
Calculation: there are different methods to calculate and interpret the glucose levels (mg/dl)in OGTT:
Glucose sample
Fasting
30 min.
60 min.
90 min.
120 min.
2 hour
Calculation of Results
Wilkerson Criteria
> 130 1 point
>190 point
>140 point
>130 1 point
2 3 point Diabetic
- 1 point Suspect
Zero Non diabetic
Fajan-conn criteria
>190 +1
> 165 +1
> 140 +1
3 Diabetic
1 2 Suspect
Zero Non diabetic
Revised Summation
*If of results
(F + 60min. + 90 min. + 120
min.) > 600 mg/dl =
Diabetic
*If of results < 600 = non
diabetic
Results and Diagnosis: Glucose tolerance tests may lead to one of the following diagnoses:
Normal Response
A person is said to have a normal response when the 2-hour glucose level
is less than or equal to 110 mg/dl, or following this normal levels.
Time
Pregnancy
Other Adults
Child
Fasting
<100
<110
<130
<200
<200
<200
120 minutes
<145
<140
<140
Discussion:
Drugs may affect OGTT results
Amphetamines.
Arginine.
Benzodiazepines.
Beta-adrenergic blockers.
Chlorthalidone.
Clofibrate.
Corticosteroids.
Dextrothyroxine.
Diazoxide.
Epinephrine.
Furosemide.
Glucose I.V.
Insulin.
Lithium.
MAO inhibitors.
Nicotinic acid (large doses).
Oral contraceptives (estrogen-progestogen combination).
Oral hypoglycemics.
Phenolphthalein.
Phenothiazines.
Phenytoin.
Thiazide diuretics.
Triamterene.
Other factors that may affect test results
Ethanol.
Caffeine.
Recent infection.
Fever.
Pregnancy.
Acute illness.
People over age 50 tend toward decreasing carbohydrate tolerance, which may cause
conflicting results.
Cushing's disease, hemochromo-cytosis, pheochromocytoma, injury to central nervous system,
tumor of pancreas islet cells, malabsorption, Addison's disease, hypothyroidism, hypopituitarism.
Reduced carbohydrate intake for several days before the test.
Failure to follow dietary and exercise restrictions.
QUESTIONS:
a. What are the indications of OGTT ?
b. What are the prerequisites of OGTT ?
c. Draw a graph of a normal glucose tolerance.
Glycated Hemoglobins
Introduction:
Glycohemoglobin (GHb, glycated hemoglobin, glycosylated hemoglobin) is a generic term for
hemoglobin bound irreversibly (ketoamine form) to glucose. Often, the term is used to mean total
glycated hemoglobin, and sometimes to mean hemoglobin A1c.
Total glycated hemoglobin (Total GHb) refers to all the glycated hemoglobins, including glycated
hemoglobin variants. Total glycated hemoglobin is usually determined by affinity chromatography or
immunassays.
Hemoglobin A1c (HbA1c) is the major subfraction of the glycated normal hemoglobin (HbA1).
Determination of HbA1c is usually achieved by ion-exchange HPLC or gel electrophoresis.
Objectives:
It is to know the importance of glycated hemoglobin as a long term monitoring test which may be used
to help controlling the treatment of diabetes mellitus.
Types of Glycated hemoglobins:
HbA1a1 = Hb + Fructose-1,6-bisphosphate (FBP)
HbA1a2 = Hb + Glucose-6-phosphate
HbA1b
= Hb + Pyruvic acid
HbA1c
HbA1d
Using GHb :
Monitoring blood glucose is a key component of successful diabetes management. With the availability
of self-monitoring and HbA1C testing, laboratory testing for fasting glucose and 2-hour post-75g glucose
load should no longer be used routinely to assess glucose control. Laboratory measurement of glucose,
however, may be useful to verify the accuracy of home glucose monitoring equipment or when there has
been a loss of diabetic control.
HbA1C measurement provides a quantitative and reliable measure of glycemic status and control over an
extended period of time, thereby complementing day-to-day monitoring. HbA 1C levels are a better (and
less expensive) measure of long-term glucose control than repeated fasting and p.c. glucose levels.
Over the life of a red blood cell (which averages 120 days), a fraction of hemoglobin will become
covalently bound to glucose and other sugar molecules. This reaction occurs non-enzymatically and at a
rate which is proportional to the concentration of glucose in the blood. HbA 1C is the largest single
component of these glycated hemoglobins.
N.B. Blood Glucose level reflects the previous few hours glycemic state, glycated Albumin reflects 10
14 days glycemic state, while HbA1c reflects the longest (2-3 months) glycemic state.
Methods:
There are currently four main techniques for determining glycated hemoglobins:
1. Cation-exchange chromatography - separates hemoglobins using HPLC based on net charge
as a result of glycation;
2. Gel electrophoresis;
3. Affinity chromatography - separates total glycated hemoglobins by binding to solid-phase
dihydroxyborate;
4. Immunoassay - based on binding to specific antibodies.
Experiment # 3: Estimation of HbA1c by using affinity chromatography column
Principle:
In a chromatography column, the hemoglobins in a hemolysed sample is bound by different affinity to
dihydroxyborate. Elution of HbA1c is carried out by phosphate buffer, while the other hemoglobins
separate (elute) after by sodium chloride solution.
Procedure:
Calculation: % HbA1c =
A1 X 100
Reference ranges:
Degree of glucose control
Total GHb
Hb A1c
Normal (non-diabetic)
< 7%
< 6%
Near normoglycemic
7 to 8%
6 to 7%
Less than 7%
In good control
8 to 9%
7 to 8%
Actions suggested
9 to 11%
8 to 9%
Not in control
> 11 %
> 9%
The determination of a glycated hemoglobin level may assist in the initial diagnosis of diabetes, or it
may be used to indicate the degree of long-term diabetic control in diabetic patients. The significance of
a low glycated hemoglobin level has not been established.
Annual HbA1c < 1.1 times the upper limit of normal (8.8%), suggesting less likely occurring
complications.
Annual HbA1c > 1.7 times the upper limit of normal (13.5%), suggesting more likely occurring
complications.
Correlation with Mean Blood Glucose Levels
A single fasting blood glucose measurement only gives an indication of the patient's immediate past
(last 1 to 2 hours) condition, and may not represent the true status of blood glucose regulation. In
contrast, the level of glycated hemoglobin is directly related to the average glucose concentration over
the life-span of the hemoglobin in the circulation.
Various formulae have been proposed to demonstrate the correlation between the mean blood glucose
(MBG) and Hemoglobin A1c (HbA1c).
MBG mg/dl = (33.3 X HbA1c) - 86
Or, MBG mg/dl = 10 ( HbA1c +4 )
Discussion:
Causes of elevated HbA1c:
-
Uncontrolled D.M.
HbF
Triglycerides
Lead toxicity
iron anemia
Splenectomy
CRF Hemodialysis
Questions:
-
Lipid Profile
Introduction:
Some beneficial aspects of lipids include the following: energy course, function and structural
components of cell membranes, and precursor compound to many important substances such
as vitamin D and steroid (sex) hormones.
With evidence of a link between elevated lipids and atherosclerosis (also known as
arteriosclerosis or atherothrombosis), there is increase interest from both the medical and lay
community in the battery of tests commonly ordered as a lipid profile. Preparation for having
blood collected for lipid testing should include a 12-14 hour overnight fast.
Objectives:
- The contribution of hypercholesterolemia to coronary heart disease (CHD) risk, including the
importance of elevations in total cholesterol, LDL cholesterol, HDL cholesterol, ratio of total to
HDL cholesterol.
- The classification of dyslipidemias, including who to screen, and how often
- The available diagnostic studies and their use, particularly determinations of HDL, LDL and
total cholesterol, as well as the need to test for other cardiovascular risk factors .
Experiment # 4:Glycerol-Phosphate Oxidase method for Triglycerides
PRINCIPLE OF THE METHOD
Sample triglycerides incubated with lipoproteinlipase (LPL), liberate glycerol and free fatty acids.
Glycerol is converted to glycerol-3-phosphate (G3P) and adenosine-5-diphosphate (ADP) by glycerol
kinase and ATP. Glycerol-3-phosphate (G3P) is then converted by glycerol phosphate dehydrogenase
(GPO) to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2). In the last reaction,
hydrogen peroxide (H2O2) reacts with 4-aminophenazone (4-AP) and p-chlorophenol in presence of
peroxidase (POD) to give a red colored dye:
Principle:
Triglycerides + H2O
Glycerol + ATP
lipase
Glycerol + FFA
glycerol kinase
Glycerol-3-phosphate + O2
Glycerol-3-phosphate + ADP
Glycerol-Phosphate Oxidase
DHAP + H2O2
peroxidase
Quinoneimine
The intensity of the color formed is proportional to the triglycerides concentration in the sample.
CLINICAL SIGNIFICANCE
Triglycerides are fats that provide energy for the cell. Like cholesterol, they are delivered to the bodys
cells by lipoproteins in the blood. A diet with a lot of saturated fats or carbohydrates will raise the
triglyceride levels. The increases in serum triglycerides are relatively non-specific. For example liver
dysfunction resulting from hepatitis, extra hepatic biliary obstruction or cirrhosis, diabetes mellitus is
associated with the increase Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.
PREPARATION
Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes into one bottle of R 1 Buffer.
Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in 10 mL of R 1 Buffer.
Cap and mix gently to dissolve contents.
WR stability: 6 weeks at 2-8C or 1 week at room temperature (15-25C).
SAMPLES
Serum or heparinized or EDTA plasma1.
Stability of the sample: 5 days at 2-8C .
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . . . . 505 nm (490-550)
Cuvette: . . . . . . . . . . . . . . . . . . . . . . . . 1 cm light path
Temperature . . . . . . . . . . . . . . . . . . . . 37C / 15-25C
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
Standard Blank
1.0
1.0
10
----
WR (mL)
Standard (L)
Sample (L)
QUESTIONS:
a. Give the different methods for the determination of triglycerides.
b. Write a short note on hypertriglyceridemia.
c. Give the upper cut off value of triglyceride for a diagnosis of
hypertriglyceridemia.
DETERMINATION OF CHOLESTEROL:
INTRODUCTION:
Cholesterol is a waxy substances used in every cell membrane you have and as a base for
several hormones. The recommended daily allowance for dietary cholesterol intake is 300 milligrams.
Most cells have some capacity to synthesize cholesterol. The largest percentage of synthesized
cholesterol is made in the liver. Cholesterol lowering medications prescribed by physicians inhibit the
synthesis of cholesterol by the liver, thereby reducing the level in the blood stream.
OBJECTIVES:
The estimation of cholesterol along with other parameters of lipid profile is necessary for the
classification and diagnosis of lipemias
PRINCIPLE OF THE METHOD
The cholesterol present in the sample originates a coloured complex, according to the following
reaction: The intensity of the color formed is proportional to the cholesterol concentration in the sample
Principle (Experiment #5):
Cholesterol esters + H2O
Cholesterol + O2
H2O2 + 4-AAP + Phenol
Cholesterol esterase
Cholesterol Oxidase
Peroxidase
Cholesterol + FA
Cholesterol-3-one + H2O2
Quinonimine
CLINICAL SIGNIFICANCE
Cholesterol is a fat-like substance that is found in all body cells. The liver makes all of the cholesterol
the body needs to form cell membranes and to make certain hormones. The determination of serum
cholesterol is one of the important tools in the diagnosis an classification of lipemia. High blood
cholesterol is one of the major risk factors for heart disease5,6. Clinical diagnosis should not be made
on a single test result; it should integrate clinical and other laboratory data.
PREPARATION
Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer.
Cap and mix gently to dissolve contents.
(WR) is stable: 4 months at 2-8C or 40 days at 15-25C. Avoid direct sunlight.
SAMPLES
Serum or plasma1,2: Stability of the sample for 7 days at 2-8C or freezing at 20C will keep samples
stable for a few months.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . . 505 nm (500-550)
Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path
Temperature . . . . . . . . . . . . . . . .. . . . .37C /15-25C
2. Adjust the instrument to zero with distilled water.
WR (mL)
Standard (L)
Sample (L)
14. Mix and incubate for 5 min. at 37C or 10 min. at room temperature.
25. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable
for at least 60 minutes.
CALCULATIONS
A (Sample) x 200 (Standard conc.) = mg/dL cholesterol in the sample
A (Standard)
Conversion factor: mg/dL x 0.0258= mmol/L.
*Normal range: Desirable blood cholesterol level < 200 mg/dl
Suspect to vascular and CHD 200 240 mg/dl
High risk group for CHD > 240 mg/dl
Risk evaluation:
Normal
Less than 200 mg/dL
Borderline mg/dL 200-239
High
mg/dL and above 240
LDL Cholesterol
PRINCIPLE OF THE METHOD
Direct determination of serum LDLc (low-density lipoprotein cholesterol) levels without the need for
any pre-treatment or centrifugation steps. The assay takes place in two steps.
1 Elimination of lipoprotein no-LDL
Cholesterol esters + H2O
Cholesterol Oxidase
Cholesterol + O2
H2O2
Cholesterol esterase
catalase
Cholesterol + FA
Cholesterol-3-one + H2O2
2 H2O + O2
2 Measurement of LDLc
Cholesterol esters + H2O
Cholesterol + O2
H2O2 + 4-AAP + Phenol
Cholesterol esterase
Cholesterol Oxidase
Peroxidase
Cholesterol + FA
Cholesterol-3-one + H2O2
Quinonimine + 4 H2O2
The intensity of the color formed is proportional to the LDLc concentration in the sample.
CLINICAL SIGNIFICANCE
The LDLc particle is lipoproteins that transport cholesterol to the cells. Often called bad cholesterol
because high levels are risk factor for coronary heart disease and are associated with obesity, diabetes
and nephrosis 1,5,6. Clinical diagnosis should not be made on a single test result; it should integrate
clinical and other laboratory data.
PREPARATION
- R 1 and R 2: Are ready to use. HDLc/LDLc CAL: Dissolve the contents with 1 mL of distilled water.
Cap vial and mix gently to dissolve contents.
SAMPLES
Serum : After sampling, the test should be performed without delay. Repeated freezing and thawing
should be avoided. Stability of the sample: 7 days at 2-8C .
PROCEDURE
. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . . 600 (590-700) nm
Cuvette: . . . . . . . . . . . . . . . . . . . . .. . .1 cm. light path
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . .37C
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
R1(L)
Standard (L)
Sample (L)
Blank Standard
300
300
------4
------- --------
Sample
300
-----4
100
100
6. Mix and incubate for 5 min. at 37C. 7. Read the absorbance (A), against the Blank.
CALCULATIONS
(A) Sample x Standard.conc. = mg/dL of LDLc in the sample
(A) Standard
Conversion factor: mg/dL x 0.02586
. REFERENCE VALUES
Levels of the risk
Desirable < 100 mg/dL
Medium 130-160 mg/dL
High > 160 mg/dL
HDL cholesterol
PRINCIPLE OF THE METHOD
The very low density (VLDL) and low density (LDL) lipoproteins from serum or plasma are precipitated
by phosphotungstate in the presence of magnesium ions. After removed by centrifugation the clear
supernatant containing high density lipoproteins (HDL) is used for the determination of HDL cholesterol
CLINICAL SIGNIFICANCE
HDL particles carry cholesterol from the cells back to the liver. HDL is known as good cholesterol
because high levels are thought to lower the risk of heart disease. A low HDL cholesterol levels, is
considered a greater heart disease risk. Clinical diagnosis should not be made on a single test result; it
should integrate clinical and other laboratory data.
Procedure :
PTA + B. sample
SAMPLES
Serum or plasma1: Free of hemolysis. Removed from the blood clot as soon as possible.
Stability : HDL Cholesterol is stable for 7 days at 2-8C .
PROCEDURE
Precipitation
11. Pipette into a centrifuge tube:
100 R (L)
1.0 Sample (mL)
12. Mix well; allow to stand for 10 min at room temperature.
23. Centrifuge at 4000 r.p.m. for 20 min or 2 min at 12000 r.p.m..
34. Collect the supernatant and test HDLc.
Test
Following the Cholesterol reagent instructions.
CALCULATIONS
- With Factor:
A505 nm Sample x 320 = mg/Dl HDLc in the sample.
A546 nm Sample x 475 = mg/Dl HDLc in the sample
Calculation of LDL-cholesterol
According to the Friedewald Formula:
LDL cholesterol = Total cholesterol - Triglycerides -HDL cholesterol
5
Questions:
a. Write a short note on Hyperlipidemia.
b. Which one of the two HDL/LDL is more dangerous to health and
give reason.
c. Which diet can cause increase in HDL-Chol ?
Write the equation for HDL-cholesterol calculated from TG, Total Chol.& LDL.
Diacetyl-Urea
Berthlots
Urease
CO2+ NH3
( HgI2
+KI)
Kinetic
Multienzymatic
method
NH3
Conductivity
difference between
non-ionized urea and
ionized ammonia
pH indicator
dye (dry
chemistry)
Ammonia
detecting
electrode
Urease
CO2+ NH3
Indophenol
PREPARATION
- Working reagent (WR): Dissolve one tablet R 3 Enzymes in one bottle of R 1 Buffer. Cap and mix
gently to dissolve contents.
Stability: 4 weeks in the refrigerator (2-8C) or 7 days at room temperature (15-25C).
- R 2 NaClO is ready to use
:
SAMPLES
1- Serum or heparinized plasma: Do not use ammonium salts or fluoride as anticoagulants.
2- Urine: Dilute sample 1/50 in distilled water. Mix. Multiply results by 50 (dilution factor). Preserve
urine samples at pH < 4.
Urea is stable at 2-8C for 5 days;
PROCEDURE
11. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . . . .. . . . . . 580 nm
Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path
Temperature. . . . . . . . . . . . . . . . . .. . . 37C / 15-25C
12. Adjust the instrument to zero with distilled water.
13. Pipette into a cuvette:
Blank
1.0
WR (mL)
-Standard (L)
-Sample (L)
nk
14. Mix and incubate 5 min at 37C or 10 min at room temperature (15-25C).
25. Pipette:
R 2 (mL)
16. Mix and incubate 5 min at 37C or 10 min at room temperature (15-25C).
27. Read the absorbance (A) of the samples and calibrator, against the Blank. The colour is
stable for at least 30 minutes at 15-25C.
CALCULATIONS
(A) Sample x 50 (Standard conc.) = mg/dL urea in the sample
(A) Standard
10 mg/L urea BUN divided by 0.466 = 21 mg/L urea = 0.36 mmol/L urea.
Conversion factor: mg/dL x 0.1665 = mmol/L.
REFERENCE VALUES1
Serum : 15- 45 mg/dL (2.49-7.49 mmol/L)
Urine : 20 - 35 gr/24 h.
Blood Urea Nitrogen (BUN) 8 25 mg/dl
Interpretation:
Interpretation of the BUN is usually straightforward, though there are a few things to remember.
#Increased BUN is, by definition, azotemia. It is due either to increased protein catabolism or impaired
kidney function.
*Increased protein catabolism results from:
N.B. In acute renal failure, BUN increases around 20 mg/dL each day (*estimates
vary; range of increase is 10-50 mg/dL daily).
#Decreased BUN
Discussion:
-Physiological & Biochemical Background:
-Pathological & Disease Correlation:
Questions:
-
purplish rose
Deaminase
- Creatinine
- Creatine
Creatine kinase
- Creatine-p +ATP
- ADP + P-enol pyrovate (PEP)
Creatine
Creatine -p
Creatine +ADP
ATP +Pyruvate
LDH
- Pyruvate + NADH+H+
Lactate + NAD
(with diminished absorbance at 340 nm)
CLINICAL SIGNIFICANCE
Creatinine is the result of the degradation of the creatine, component of muscles, it can be transformed
into ATP, that is a source of high energy for the cells. The creatinine production depends on the
modification of the muscular mass, and it varies little and the levels usually are very stable. Is excreted
by the kidneys. With progressive renal insufficiency there is retention in blood of urea, creatinine and
uric acid. Elevate creatinine level may be indicative of renal insufficiency1,4,5. Clinical diagnosis should
not be made on a single test result; it should integrate clinical and other laboratory data.
PREPARATION
Working reagent (WR):
Mix equal volumes of R 1 Picric Reagent and R 2 Alkaline reagent.
The working reagent is stable for 10 days at 15-25C.
Signs of reagent deterioration:
1- Presence of particles and turbidity.
2- Blank absorbance (A) at 492 nm 1.80.
SAMPLES
1- Serum or heparinized plasma.
Creatinine stability: 24 hours at 2-8C.
1- Urine: Dilute sample 1/50 with distilled water. Mix. Multiply results by 50 (dilution factor);
Creatinine stability: 7 days at 2-8C.
PROCEDURE
11. Assay conditions:
Wavelength: . . . . . . . . . . . . . . . . . 492 nm (490-510)
Cuvette: . . . . . . . . . . . . . . . . . . . . . . . . 1 cm. light path
Temperature. . . . . . . . . . . . . . . . . . . . . 37C / 15-25C
12. Adjust the instrument to zero with distilled water.
23. Pipette into a cuvette:
ard Blank
1.0
WR (mL)
-Standard (L)
-Sample (L)
1
4. Mix and start stopwatch.
25. Read the absorbance (A1) after 30 seconds and after 90 seconds (A2) of the sample addition.
36. Calculate: A= A2 A1.
CALCULATIONS
A Sample A Blank
x 2 (Standard conc.) = mg/dL of creatinine in the sample
A Standard ABlank
Conversion factor: mg/dL x 88.4 = mol/L. l
REFERENCE VALUES
plasma
1.8 123.7) mol/L 1,4 - 0,7
3.0 97.2 ) mol/L 1,1 - 0,6
5 mg/Kg/24 h
= 88 177 mol/Kg/24 h 20 - 10
= 71 177 mol/Kg/24 h 18 8
Male
Female
Male
Female
Tutorial
Creatinine clearance : is widely used to approximate glomerular filtration. You need a timed urine
sample and a blood sample.
The clearance of a substance is the volume of plasma "cleared" of that substance per unit time.
Clearance =
In deciding how to "time" your collection, remember that you don't really need to collect urine for
a full 24 hours. One group got more reliable results by a controlled collection over 4 hours,
monitoring body position (kept them lying down) and hydration with body surface area
measurement.
Creatinine clearance is not a perfect measure of GFR, because some is not filtered and some is
secreted into the proximal tubule. These fractions tend to cancel each other out in health, but
when GFR drops below 30 mL/min, tubular secretion approaches or even exceeds the amount
filtered at the glomerulus.
*Also, lots and lots of red meat (protein and creatinine-rich) can lead to overestimates (maybe
30%) in GFR in renal failure patients.
Reference range for creatinine clearance is 90-120 mL/min for young adults; values tend to fall
by around 0.5 mL/year over age 20, worse for hypertensives .
*Formulas to adjust "normal" for body surface area have been devised, etc. For kids, a
height/creatinine ratio of 2.1 or less is normal. GFR for adults can be estimated by various
formulas; try 1.12 x Creatinine Clearence - 20.6.
Another formula to correct the clearance according to body surface area is {Corrected
Cr.Cl. = Cr.Cl. X (1.7/ body surface area)}
*Whether or not "corrections" are applied, creatinine clearance is a pretty good estimate of
glomerular filtration rate except at very low values, when tubular secretion of creatinine
become proportionately greater.
OBJECTIVES:
METHODS:
Quinoneimine+ 4H2O
The intensity of the red color formed is proportional to the uric acid concentration in the sample.
CLINICAL SIGNIFICANCE
Uric acid and its salts are end products of the purine metabolism. With progressive renal insufficiency,
there is retention in blood of urea, creatinine and uric acid. Elevate uric acid level may be indicative of
renal insufficiency and is commonly associated with gout Clinical diagnosis should not be made on a
single test result; it should integrate clinical and other laboratory data.
PREPARATION
Working reagent (WR): Dissolve the contents of one vial R 2 Enzymes in one bottle R 1 Buffer. Cap
and mix gently to dissolve contents. (WR) is stable after reconstitution 1 month at 2-8C or 10 days at
room temperature.
SAMPLES Serum or plasma: Stability 3-5 days at 2-8C or 6 months at 20C.
- Urine (24 h)1: Stability 4 days at 15-25C, pH >8. Dilute sample 1/50 in distilled water. Mix. Multiply
results by 50 (dilution factor);
If urine is cloudy; warm the specimen to 60C for 10 min to dissolve precipitated urates and uric acid. Do
not refrigerate.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . . . . . . . .520 nm (490-550)
Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . . 1 cm light path
Temperature . . . . . . . . . . . . . . . . . . . . . . 37C / 15-25C
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
WR (ml)
Standard (L)
Sample (L)
Blank
1.0
-------------
Standard
1.0
25
---------
Sample
1.0
-------25
DISCUSSION:
Causes of elevated uric acidemia
QUESTIONS:
1.Give the principle for the determination of serum uric acid by uricase
method.
2. Write a short note on Uric acid metabolism.
Values less than 1% indicate prerenal azotemia; values over 2% indicate acute tubular
necrosis.
Several other factors can complicate the picture in such patients.
Diuretics will increase the excretion of filtered sodium, while secondary hyperaldosteronism (as
in cirrhosis) will decrease sodium excretion.
In acute tubular necrosis due to myoglobinuria, sodium excretion is low (the tubules are
plugged, not damaged.)
*Tip: If you obtain urine by squeezing a diaper or the absorptive balls you placed into the diaper, your
estimate of urine creatinine will be low because these things absorb creatinine .
Urine Protein/creatinine ratio
Urine protein/creatinine ratio (UP/UCr) is used to calculate urine protein loss into the urine
without a need for 24 hour urine collection. Compared to conventional 24 hour- urinary protein
value, UP/UCr is less time consuming and less accurate.
Generally with proteinuria, UP/UCr is greater than 1.0.
especially subtle industrial poisoning, acute pyelonephritis, early acute tubular necrosis, and
early transplant rejection. (These functions are now largely taken over by beta-2 microglobulin).
7. Adenosine Deaminase Binding Protein is an enzyme from the brush borders of the
proximal tubule. Like NAG, its presence in urine indicates tubular disease.
8. Alkaline phosphatase in urine comes from the proximal tubular brush border .
9. Beta-2 microglobulin (beta-2-m) is the short chain of the HLA class I proteins. In health, it is
freely filtered by the glomerulus, and fully reabsorbed by the proximal tubule.
Serum beta-2-m has been suggested as a measure of glomerular filtration rate, similar
to creatinine. Obviously this isn't a good idea for patients with tissue necrosis,
lymphomas, etc.
Urine beta-2-m has found widespread acceptance as an research tool. It appears if
levels in the serum and glomerular filtrate exceed what the proximal tubule can
reabsorb (more than 4.5 mg/L) or if there is renal tubular disease. It is very sensitive as
an indicator of the latter.
10. Tubular functions: Urinary amino acids and maximum concentrating ability are
sensitive screens for tubular damage. Lithium clearance is a researcher's way of estimating
delivery to the distal tubule.
11. Isotope scans exist to compare the function of the kidneys. These may prove a valuable
supplement to the intravenous pyelogram. More recently, the 12. color Doppler sonogram,
which is cheap and portable, has proved even more useful than these scans in transplant
patients. Most recent of all, there's a Tc99 scanner that monitors glomerular filtration minute by
minute, suitable for the intensive care .
13. Positron emission tomography is the latest way of measuring renal blood flow.
Serum osmolality: 282 - 295 mOsm/kg water; a serum osmolality of 285 mOsm correlates with
a urine specific gravity of 1.010
Urine osmolality: extreme range of 50 - 1400 mOsm/kg water, but average is about 500 - 800
mOsm. After an overnight fast, the urine osmolality should be at least 3 times the serum
osmolality
Increased serum and urine osmolality (hyperosmolality) levels are seen in:
Renal disease
Congestive heart failure
Addison's disease
Dehydration
Diabetes insipidus
Hypercalcemia
Diabetes mellitus/hyperglycemia
Hypernatremia
Alcohol ingestion
Mannitol therapy
Azotemia
Decreased serum and urine osmolality (hypoosmolality) levels are seen in:
SIADH
Excessive water replacement/overhydration/water intoxication
Panic values for serum osmolality are values of less than 240 mOsm or greater than 321 mOsm. A
serum of osmolality of 384 mOsm produces stupor. If the serum osmolality rises over 400 mOsm, the
patient may have grand mal seizures. Values greater than 420 mOsm are fatal.
When the serum osmolality is normal or increased, the kidneys are conserving water. As the serum
osmolality rises, the urine osmolality should also rise. The higher the number of millosmoles in the urine,
the more concentrated the urine; this is the expected physiological response to dehydration
This table shows the relationship between serum and urine osmolality and the clinical significance of
laboratory values.
Serum Osmolality
Normal values:
282-295mOsm
Normal or increased
Decreased
Normal
Increased or normal
Urine Osmolality
Normal values:
500-800mOsm
Increased
Decreased
Decreased
Decreased (with no increase in
fluid intake)
Decreased
Increased
Clinical Significance
Fluid volume deficit
Fluid volume excess
Increased fluid intake or diuretics
Kidneys unable to concentrate
urine or lack of ADH (diabetes
insipidus)
SIADH
Blank
1.0
R (mL)
-Standard (L)
-Sample (L)
14. Mix and incubate for 10 min at room temperature (15-25C).
25. Read the absorbance (A) of the samples and Standard, against the Blank.
The colour is stable 1 hour at room temperature.
CALCULATIONS
(A) Sample x 5 (Standard conc.) = g/dL albumin in the sample
(A) Standard
Conversion factor: g/dL x 144.9 = mol/L
REFERENCE VALUES
3.5 to 5.0 g/dL.
Introduction:
A Total Protein can be done on either a fasting or non fasting specimen. It is usually done as a general
screening assay since it is composed of two major fractions (albumin and globulin). Elevations or
decreases in a total protein must be investigated to find out which of the two components is causing the
problem. Since many of the next level tests may be reported as percentages or ratios, it is necessary to
have the total protein rerun at the time these tests are performed. Overall, a general reference range is
5.0 - 8.0 gram/dL.. Since this is a stable assay, the range of variation is quite small. Acceptable variation
is 1.0
If both the albumin and globulin are elevated, one possibility is dehydration or a slow down of blood
flow. If both are decreased, the most common culprit is liver function. Since both albumin and globulin
can be assayed individually, they are sometimes reported as an "AG ratio". (See albumin and globulin
for specifics.)
Patients with Waldenstrms macroglobulinemia may have total proteins above 8.5. They should
consider having tests performed on urine specimens as this will lessen the clotting problem found in the
specimen but still provide adequate answers to the physician.
1-Ultraviolet absorption method
2-Specific gravity methods for T.P.
3-Refractrometry.
4-Kjeldahl nitrogen detection method
5-CuSO4 (Cu-Pr complex) Methods
a)Phillips
or b)Lowry &Hunter
a)Titration or
a)Lowry
or
b) kinetic
b) Biuret
Normal (Reference)Ranges:
- Ammonia (Plasma on Heparin)= 15-51 ug/dl
- T.P Premature babies = begin from 3.6 g/dl
Newborns
= 4.6 -5.7 g/dl
7months -1yr.
= 5.1 -7.3 g/dl
1-2yrs.
= 5.7 -7.5 g/dl
Adults
= 6.0 - 8.0 g/dl
- Exercise &Ambulatory 0.5 g/dl to T.P (by extravasation of proteins)
1- Ultraviolet Absorption:
270 290 nm
200 -225 nm
*Used for Solutions rather than serum.
*Using Quartz Cuvette (with no scratches)
On using serum , Dilute 1:1000 with NaCl 0.15 mol/L.
This method depends on Tryptophan &Tyrosine content of the protein.
*Interference by free tyrosine,tryptophan, bil.&U.A.
4-Kjeldahl Method:
*The reference method
*using protein free filtrate.
*Depend on estimation of protein nitrogen.
Protein
H2SO4+Catalyst
+Na-Molybdate
NH4+
Alkaline PH
NH3
HCl(standard Sol.)
( either)
NADPH +2oxoglutarate
(Titration)
Neutral PH
(Nisselerization)
NADP + glutamate
(Monitor abs.change at 340 nm)
Folin(Fenol)+
Cio-Calteau(PTA+Ph-Molbdic a.)
Molybdinum blue +
Tungesten blue(at 650- 750nm)
LOWRYs Method
Sensitivity:
Specificity:
Less specific
Drug Interference
(salicylates,sulfa&tetracyclines)
K&NaTartarate(=color stabilizer)
BIURETs Method
good for Pr. 2-12 g
specific
One reagent
Dependence on Tryptophan&Tyrosine
e.g Alb.=0.2 % Tryptophan by wt.
Glob.=2% Tryptophan by wt.
Blank
1.0
-----------
Standard
1.0
25
---------
Sample
1.0
------25
Bilirubin estimation
Introduction:
Bilirubin is the end product of red cell lysis and recycling of hemoglobin which is performed
in the liver. The test quantifies two different forms of bilirubin, one is the final product while
the other is an intermediate form.
The build up of bilirubin in the blood stream is called jaundice and is a general sign of liver
disease. Many medications, gall bladder disease as well as viruses such as infectious
mononucleosis and hepatitis will have jaundice. Many infants are born with less than fully
mature livers. As a consequence, for the first several days, they may show "neonatal jaundice"
which is a build up of bilirubin in the blood stream. This should go away as the liver matures.
Bilirubin determinations are used to study liver function and red cell metabolism
CLINICAL SIGNIFICANCE
Bilirubin is a breakdown product of hemoglobin. It is transported from the spleen to the liver and
excreted into bile. Hyperbilirubinemia results from the increase of bilirubin concentrations in plasma.
Causes of hyperbilirubinemia: Total bilirubin (T): Increase hemolysis, genetic errors, neonatal jaundice,
ineffective erythrpoiesis, and drugs. Direct bilirubin (D): Hepatic cholestasis, genetic errors,
hepatocellular damage1,5,6. Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.
MELLOY &EVELYN M.
*Accelerator: Methanol or Urea
*PH
: Neutral
*W.L
: 660
*Color
: Red purple
JENDRASSIL-GROF M.
Caffeine &Na.Benzoate
Alkaline(PH=12)
560 nm
red
Coloumetric Reaction:
*Bil.Glucuronides + DSA Azobilirubin +H2O+Co2+Hcl
(Direct Bilirubin)
*Bil.Glucuronides +DSA +Accelerator Azobilirubin
+H2O+Co2+Hcl(Total Bilirubin)
6-Bilirubin Oxidase Method :
*Specific for Bilirubin only.
*Bil. +Bil. Oxidase Biliverdin (measured at 405 nm)
ect
0
00
Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per
minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).
REFERENCE VALUES
25C
L 4-18 U/L Women
L 6-28 U/L Men
L-Lactate + NAD
CLINICAL SIGNIFICANCE
The AST is a cellular enzyme, is found in highest concentration in heart muscle, the cells of the liver, the
cells of the skeletal muscle and in smaller amounts in other weaves.
Although an elevated level of AST in the serum is not specific of the hepatic disease, is used mainly to
diagnostic and to verify the course of this disease with other enzymes like ALT and ALP.
Also it is used to control the patients after myocardial infarction, in skeletal muscle disease and other
Clinical diagnosis should not be made on a single test result; it should integrate clinical and other
laboratory data.
PREPARATION
Working reagent (WR):
Dissolve one tablet of R.2 Substrate with one vial of R1 Buffer.
Cap and mix gently to dissolve contents.
Stability: 21 days at 2-8C or 72 hours at room temperature (15-25C).
Signs of reagent deterioration:
1- Presence of particles and turbidity.
2- Blank absorbance (A) at 340 nm < 1.00.
SAMPLES
Serum or plasma: Stability 7 days at 2-8C..
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 nm
Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm. light path
Constant temperature . . . . . . . . . . . . . . .25C /30C / 37C
2. Adjust the instrument to zero with distilled water or air.
3. Pipette into a cuvette:
Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per
minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).
REFERENCE VALUES
25C
Men up to
19 U/L
Women up to 16 U/L
30C
26 U/L
22 U/L
37C
38 U/L
31 U/L
hyperparathyroidism. Clinical diagnosis should not be made on a single test result; it should integrate
clinical and other laboratory data.
PREPARATION
All the reagents are ready to use. To prepare monoreagent, mix according to this proportion: 50 vol. of
R1 and 1 vol. of R2.
SAMPLES Serum or plasma: Separated from cells as rapidly as possible. Blood anticoagulants with oxalate or
EDTA are not acceptable since these chemicals will strongly chelate calcium.
- Urine: Collect 24 hour urine specimen in calcium free containers. The collecting bottles should contain
10 ml of diluted Nitric acid (50% v/v). Record the volume.
Dilute a sample 1/2 in distilled water. Mix. Multiply results by 2 (dilution factor). Stability of the samples:
Calcium is stable 10 days at 2-8C.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . . 570 nm (550-590)
Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm. light path
Temperature . . . . . . . . . . . . . . . . . . . 37C / 15-25C
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
R1 (mL)
R2 (2 drop)
Standard (L)
Sample (L)
Blank
2.0
1
---------
Standard
2.0
1
20
------
Sample
2.0
1
----20
4. Mix and incubate for 5 min at 37C / 15-25C. 5. Read the absorbance (A) of the samples and
calibrator, against the Blank.
The color is stable for at least 40 minutes.
CALCULATIONS
Serum and plasma (A) Sample x 10 (Standard conc.) = mg/dL calcium
(A) Standard
Conversion factor: mg/dL x 0.25= mmol/L.
REFERENCE VALUES
Serum or plasma:
Adults
8.5-10.5 mg /dL = 2.1-2.6 mmol/L
Children
10 -12 mg/dL = 2.5 - 3 mmol/L
Newborns
8 -13 mg/dL = 2 - 3.25 mmol/L
RESULTS:
CLINICAL SIGNIFICANCE:
* HYPOCALCEMIA
1. Vitamin D deficiency
2. Hypoparathyroidism
3. Alkalosis (Alkalemia)
* HYPERCALCEMIA
1. Hyperparathyroidism
2. Malignancy of bone
3. Thyrotoxicosis
4. Vitamin D intoxication
5. Idiopathic
DISCUSSION:
TETANY
PRECAUTIONS:
1. Avoid venous stasis (Increase protein & calcium)
2. Do not use contaminated glass ware (Increase calcium)
3. Lipemic Samples (Prepare blank 0.05 ml sample + 2.5 D.W)
QUESTIONS:
1. What is HYPOCALCEMIA? Write a short note on Tetany.
2. Give the principle for the determination of serum calcium by colorimetric method.
3. Enumerate different methods for the determination of serum calcium.
Standard
1.5
50
--
Blank
1.5
WR (mL)
- Standard (L)
-- Sample (L)
4. Mix and incubate for 10 min at 37C or 30 min at room temperature (15-30C).
5- Read the absorbance (A) of the samples and calibrator, against the Blank.
The colour is stable for at least 2 hours.
CALCULATIONS
Serum
(A) Sample x
(A) Calibrator
Conversion factor: mg/dL x 0.323 = mmol/L.
REFERENCE VALUES
Serum:
Children : 4.0 - 7.0 mg/dL = (1.3 - 2.2 mmol/L)
Adults : 2.5 - 5.0 mg/dL = (O.8 - 1.8 mmo/lL)
p-Nitrophenol + Phosphate
1.2
(L)Sample
20
Adults
60 - 170 U/L
Factors affecting ALP activities in a normal population include exercise, periods of repaid growth in
children and pregnancy.
C
WR (mL)
Sample (L)
14. Mix, incubate for 2 minutes.
25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1
minute intervals thereafter for 3 minutes.
36. Calculate the difference between absorbances and the average absorbance differences per
minute (A/min).
CALCULATIONS
A / min x 4127 = U/L CK
A / min x 8095 = U/L CK
Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per
minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).
REFERENCE VALUES
25C
30C
Men, up to
80 U/L 130 U/L
Women, up to 70 U/L 110 U/L
37C
195 U/L
170 U/L
1. WR (mL)
0
40 Sample (L)
Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per
minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).
Percentage of CK-MB activity in sample:
CK-MB Activity
x 100
% CK-MB Activity =
CK total Activity
REFERENCE VALUES
Heart infarct probability is high at the following conditions:
25C
30C
37C
CK-MB > 10 U/L
> 15 U/L
> 24 U/L
CK-MB activity is between 6 and 25% of total CK activity.
Appendix (1)
Collective Knowledge of Most Common Lab.Tests
Blood Tests
Glucose: Glucose is the primary blood sugar test and indicates blood sugar level at the time
blood was drawn. High values are seen in diabetics. In addition to pancreatic functions, Glucose
may be altered by diet and medication. Normal fasting value is 70-110.
Fructosamine: Indicates blood sugar levels over the past one to three weeks.
HGB A1C (Glycohemoglobin): Indicates blood sugar activity for the past three months.
BUN: BUN stands for Blood Urea Nitrogen and is a waste product which should be removed
from the blood by the kidneys. This test measures kidney function. Normal range is 6-20.
Creatinine: Creatinine is a waste product which should be removed from the blood by the
kidneys. This test measures kidney function. Normal range is 0.5-1.2.
ASAT/ALT: Material found in the liver cells and muscle (heart) cells. Damage to these cells will
increase values. Normal range is 10-60.
LDH: LDH is a material found in blood cells and liver cells. Breakdown of the blood cells as in
heart disease or liver damage may increase values. Normal range is 91-180.
Alkaline Phosphorus: A material found in the blood related to liver and bone. Normal range for
adult males is 20-125; normal range for adult females is 42-124.
SGOT, SGPT: Two measures of liver function; occasionally affected by muscle injury.
GGTP: The earliest liver function to become abnormal.
Total Bilirubin: The level of pigment in the blood. Elevations can be associated with liver disease
or breakdown or red blood cells. Slight increases are sometimes seen without significance.
Some people normally have isolated elevations of bilirubin called Gilbert's disease. Normal
range is 1.0-1.2.
Total Protein: This is a combination of albumin and globulin, which are proteins. Abnormal
values occur in liver disease and poor nutrition. Normal range is 6.7-8.0.
Globulin: Globulin helps to combat infection on a normal level. It is the total protein value minus
albumin value. Normal range is 2.3-4.0.
A/G Ratio: Albumin value divided by the globulin value. Normal range is 0.8-2.4.
Calcium: The most abundant mineral found in the human body. Abnormalities are found in loss
of bone, kidney disease and lack of Vitamin D. Normal range is 8.5-10.5.
Phosphorous: Related to bone activity and usually follows exact opposite of calcium. Normal
range is 2.5-4.6.
Uric Acid: A material which, if in excess, can deposit stones in the kidney or in the joints and
cause gout. Normal range for males is 4.0-7.0; normal range for females is 2.0-6.0.
Cholesterol: A blood fat related in part to eating animal fats such as eggs, cheese, cream, liver,
pork, beef, etc. Increased values may indicate a tendency to have hardening of the arteries.
Values of 180 or less are associated with least risk of heart disease; in addition to diet and
exercise.
Lipoproteins: Proteins combined with lipids that serve as carriers of cholesterol. LDL ("Bad"
Cholesterol); HDL ("Good" Cholesterol). The higher the value, the less likely that cholesterol
deposits are in the blood stream and the less likely the chance of coronary heart disease.
Cholesterol/HDL ratio measures the coronary risk factors.
Triglycerides: A blood fat related to calories and starch (sweets) in the diet. High levels can
impair circulation and lead to hardening of the arteries. Alcohol also will increase the value. Fast
overnight test for accurate test results. Normal range for males is 40-160; normal range for
feales is 35-135.
Magnesium: An element absorbed in the intestine. Abnormal levels are found in pancreatitis,
alcoholism and Addison's disease. Normal range is 1.8-2.4.
Socium: A body salt, also termed electrolyte. Kidney disease and some diseases of the adrenal
gland and dehydration can cause abnormal results. Normal range is 135-145.
Potassium: A body salt or electrolyte found mostly inside of cells. "Water pills" may lower
potassium and increase kidney damage. Normal range is 3.6-5.0.
Chloride: A body salt/electrolyte, it usually follows the same pattern as sodium. Normal range is
101-111.
Co2: Buffer system which assists in the transport of carbon dioxide from the tissue to the lungs.
Normal range is 21-31.
HIV antibody: Presence of antibody is associated with having been infected by the virus known
to cause AIDS (Acquired Immune Deficiency Syndrome).
PSA: Abnormal levels in the serum are associated with clinical abnormalities of the prostate,
including prostate cancer. Because PSA is found in normal, malignant and benign prostatic
tissue, clinical discrimination is based upon its serum level.
Appendix (2)
Common Blood Profiles
Reference values for the more commonly employed laboratory tests are given in the following table. The
reference values are in the units currently often used and in the International System (SI) of Units.
Test
Current units
Factor
SI units
Diabetic Screen
65-110 mg/dl
Glucose, fasting
71-180mg/dl
0.055
3.57-6.05 mmol/L
0.055
3.9-10.0 mmol/L
<200 mg/dl
0.0259
<5.2 mmol/L
>35 mg/dl
0.0259
>0.9 mmol/L
<150 mg/dl
0.0259
<3.9 mmol/L
<205 mg/dl
0.0113
<2.3 mmol/L
<5.8
<5.8
0.25-1.5 mg/dl
17.1
4.3-25.6 mol/L
0-0.5 mg/dl
17.1
0 - 8 mol/L
0-0.9 mg/dl
17.1
0-14 mol/L
6.5-8.5 gm/dl
10
65-85 gm/L
Glucose , random
Glycosylated hemoglobin
5.5 - 8.5%
( Hba1c )
Heart disease risk factors
(fasting lipids )
Total Cholesterol
HDL cholesterol
LDL cholesterol
Triglyceride
Total cholesterol/HDL ratio
Liver function tests
Total Bilirubin
Direct Bilirubin
Indirect Bilirubin
Total Protein
3.5-4.8 gm/dl
0.154
0.54 - 0.74mmol/L
2.0-3.9 g/dl
10
20-39 g/L
1-2.5
1-2.5
11-50 IU/L
1.67 X 10-8
Albumin
Globulin
Albumin/Globulin ratio
g -Glutamy transpeptidase
(GGT) -Male
12-58 X 10-8 Katal/L
7-35 IU/L
g -Glutamy transpeptidase
(GGT) -Female
Alkaline Phosphatase
45-125 IU/L(up to
1000 IU/L in young
children)
1.67 X 10-8
5-35 IU/L
1.67 X 10-8
5-40 IU/L
1.67 X 10-8
8-25 mg/dl
0.357
2.9-8.9 mmol/L
0.6-1.7 mg/dl
88.4
53-150 mol/L
3.5-8.0 mg/dl
0.059
0.21-0.47 mmol/L
3.3-4.9 mmol/L
3.3-4.9 mmol/L
135-145 mmol/L
135-145 mmol/L
8.9-10.3 mg/dl
0.25
2.23-2.57 mmol/L
4.5-5.0 mg/dl
0.25
1.12-1.25 mmol/L
2.5-4.5 mg/dl
0.323
0.8-1.5 mmol/L
Alanine aminotransferase
(SGPT / ALT)
Aspartate aminotransferase
(SGOT/ AST)
Current units
Factor
SI units
11-35 mol/L
11-35 mol/L
7.35-7.45
7.35-7.45
80-105 mmHg
0.133
10.6-14.0 kPa
35 - 45 mmHg
0.133
4.7-6.0 kPa
22-31 mmol/L
22-31 mmol/L
50-300 g/dl
0.0186
0.9-5.6 mol/L
0.23-0.58 gm/L
6.7
1.5-3.9 mol/L
95-105 mEq/L
95-105 mmol/L
70-140 g/dl
0.157
11.0-22 mol/L
85-155 g/dl
0.157
13.3-24.3 mol/L
81-175 mg/dl
0.01
0.8-1.75gm/L
12-34 mg/dl
0.01
0.12-0.34 gm/L
2.25
29-326 pmol/L
118-226CH50U/ml
Complement (total, hemolytic)
C3
C4
Creatinine Clearence
60-120 ml/min
Ferritin - Children
13-145 ng/ml
25-240 ng/ml
56-540 pmol/L
12-130 ng/ml
Fibrinogen
150-360 mg/dl
0.01
1.5-3.6 gm/L
Folate - Plasma
1.7-12.6 ng/ml
2.27
3.9-29 nmol/L
153-602 ng/ml
Haptoglobin
100-300 mg/dl
0.01
1.10-3.00 gm/L
Immunoglobulin - IgA
39-358 mg/dl
0.01
0.39-3.58 gm/L
Immunoglobulin - IgM
33-229 mg/dl
0.01
0.33-2.29 gm/L
Immunoglobulin - IgG
679-1537 mg/dl
0.01
6.79-15.37 gm/L
Iron - Male
80-160 g/dl
0.179
14.3-28.6 mol/L
Iron - Female
60-135 g/dl
10.7-24.2 mol/L
250-350 g/dl
44.7-62.6 mol/L
27-292 pmol/L
347-1367 nmol/L
16-57%
16-57%
0.3-1.3 mmol/L
0.3-1.3 mmol/L
1.5-2.1 mEq/L
0.5
0.7-1.1 mmol/L
270-290 mOsm/kg
270-290 mOsm/kg
0.1-0.5 gm/dl
10
1-5 gm/L
0.3-1.2 gm/dl
10
3-12 gm/L
0.7-1.7 gm/dl
10
7-17 gm/L
Magnesium
Osmolality
Protein electrophoresis Alpha- 1- globulin
Protein electrophoresis - Alpha
-2- globulin
Protein electrophoresis - Beta
globulin
0.7-1.7 gm/dl
10
7-17 gm/L
30-95 g/dl
0.035
1.05-3.32 mol/L
200-800 pg/ml
0.739
148-591 pmol/L
Current units
Factor
SI units
1.5-8.1 IU/L
1.67 X 10-8
25-115 IU/L
1.67 X 10-8
Up to 185 IU/L
1.67 X 10-8
Up to 309X10-8 Katal/L
Up to 150 IU/L
1.67 X 10-8
Up to 251X10-8 Katal/L
90-250 IU/L
1.67 X 10-8
4-24 IU/dl
10
40-240 IU/L
2-16 IU/L
1.67 X 10-8
Up to 0.7 IU/L
1.67 X 10-8
Up to 1.2X10-8 Katal/L
Aldolase
Amylase
Creatine kinase - Male
Creatine kinase - Female
Lactic dehydrogenase
Lipase
5' - Nucleotidase
Phosphatase, acid
Common Serum Hormone Values
Test
Current units
Factor
SI units
20-100 pg/ml
0.22
4.4-22 pmol/L
10-160 ng/L
2.77
28-443 mmol/L
8-25 g/dl
0.027
0.22-0.57 mol/L
Up to 20 IU/L
Up to 20 IU/L
Up to 20 IU/L
Up to 20 IU/L
Up to 15 IU/L
Up to 15 IU/L
15-30 IU/L
15-30 IU/L
>40 IU/L
>40 IU/L
Up to 130 pg/ml
Up to 130 ng/L
<5 ng/ml
<5 g/L
<10 mU/L
<10 mU/L
LH - Male
Up to 25 IU/L
Up to 25 IU/L
LH - Female -Follicular
Up to 40 IU/L
Up to 40 IU/L
LH - Female - Luteal
Up to 25 IU/L
Up to 25 IU/L
LH - Female - Midcycle
50-150 IU/L
50-150 IU/L
LH - Female Postmenopausal
>30 IU/L
>30 IU/L
Parathyroid hormone
2-10 U/ml
Progesterone - Male
Up to 100 ng/dl
Progesterone - Female
-Follicular
Up to 150 ng/dl
250-2800 ng/dl
1300-5000 ng/dl
Prolactin - Male
2-12- ng/ml
2-12 ug/L
Prolactin - Female
2-20 ng/ml
2-20 ug/L
0.9-3.3 ng/ml/hr
0.278
0.2-0.9 ng/L.s
280-1000 ng/dl
0.0346
10-35 nmol/L
20 -120 ng/dl
52-280 pg/ml
1.1-6.3 pg/ml
Thyroxine, total ( T4 )
5.0-11.0 ug/dl
12.9
64-142 nmol/L
Thyroxine, free
1.0-2.3 ng/dl
12.9
13-30 pmol/L
T3 resin uptake
35-45%
0.01
100-216 ng/dl
0.0154
1.54-3.23 nmol/L
1.75-4.95
Up to 10 U/ml
Up to 10 mU/L
Triiodothyronine (T3)
T4 index
TSH
Vitamin D, 1,25 dihydroxy
20-76 pg/ml
Vitamin D, 25 hydroxy
10-55 ng/m
1-4 nmol/L
0.00346
0.18-1.0 nmol/L
4-22 X 10- nmol/L
Current units
Factor
SI units
-aminolevulinic acid
1.3-7.0 mg/day
7.6
9.9-53 mol/day
Amylase
0.04-0.30 IU/min
0.04-0.30 IU/min
Calcium
< 250mg/day
0.025
Catecholamines
Up to 135 g/day
Dopamine
90-440 g/day
Epinephrine
< 13 g/day
5.5
Up to 71 nmol/day
Norepinephrine
11-86 g/day
5.9
65-507 nmol/day
Copper
15-50 g/day
0.0157
0.2-0.78 mol/day
Cortisol, free
20-90 g/day
2.76
Creatinine - Male
1.0-2.0 gm/day
0.0088
0.009-0.018 mmol/day
Creatinine - Female
0.6-1.5 gm/day
5Hydroxyindoleacetic acid
1.8-6 mg/day
5.3
9.5-31.8 mol/day
Hydroxyproline, total
25-77 mg/day
7.63
191-588 mol/day
Metanephrine
0.3-1.0 mg/day
Oxalate
Up to 40 mg/day
7.93
Porphyrin-Coproporphyrin
15-125 g/day
1.53
23-191 nmol/day
Porphyrin-Uroporphyrin
< 30 g/day
1.2
Up to 36 nmol/day
Protein
0.001
Up to 0.150 gm/day
0.5-7 mg/day
5.05
2.5-35.3 mol/day
0.005-0.013 mmol/day
Current units
Factor
SI units
2.5-9.5 min
60
150-570 sec
25-41 sec
25-41 sec
10.8-13.0 sec
10.8-13.0 sec
11.3-18.5 sec
11.3-18.5 sec
Hematocrit - Male
40.7-50.3%
0.01
Hematocrit - Female
36.1-44.3%
Hemoglobin - Male
13.8-17.2 gm/dl
Hemoglobin - Female
12.1-15.1 gm/dl
4.5-5.7 X106/ l
3.9- 5.0X106/ l
Leukocyte count
3.8-9.8X 10/ l
1.2-3.3X 10/ l
0.1-0.7X10/ l
1.8-6.6X 10/ l
8.56-10.7 mmol/L
7.50-9.36 mmol/L
106
4.5-5.7 X 10/L
3.9-5.0 X 10/L
106
3.8-9.8 X 109/L
106
190-405 X 109/L
26.7-33.7 pg/cell
0.062
1.66-2.09 fmol/cell
32.7-35.5 gm/dl
0.62
20.3-22.0 mmol/L
80.0-97.6 cu
Platelet count
80.0-97.6 fl
11.8-14.6%
Up to 30 mm/hr
Up to 30 mm/hr
Erythrocyte Sedimentation
rate
0.2-2.0%
Reticulocyte count
Practical Sheet #( )
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