User:Mohan khatiwada/sandbox

Glucose (sugar) test Introduction: Glucose is a monosaccharide which is used as major source in human body.Estimation of blood glucose is generally done for diagnosis and monitoring of dibetes mellitus. Principle: Glucose oxidase convert glucose into gluconic acid and hydrogen peroxide. peroxidase catalyses. Conversion of H2O2 into water and oxygen.Oxygen then binds with colorless dye to form colored derivative (pink red color). The intensity of color derivative is directly proportional to concentration of glucose in serum and is measured at 510-520 nm. Various dyes are used eq:4-aminoantipyrene,4-aminophenazone,Quinone mine dyes. Glucose+O2+H2O  GOD      gluconic acid + H2O2 H2O2 + 4 amino antipyrene + phenol   POD    pink red complex Procedure: 	Three test tube were labelled as blank (B), standard (S) and test(T). Blank(B)	Standard(S)	Test(T) Glucose reagent	1ml	1ml	1ml Glucose standard	-	10ml	- Serum	-	-	10ml 	Mixed well and incubate at 37°C for 10-12 min. 	After 15min, reading was taken in colorimeter at 510-520 nm. Normal range: 	Fasting= (60 to 110)mg\dl 	Post prandial=(60-140)mg\dl 	Random=(60 to 150)mg\d Calculation: 	Concentration of glucose=O.D. of test\O.D> of STD*concentration of STD Clinical signification: Increased condition:

Bilirubin tests Introduction: Bilirubin is formed by degradation of haeme predominantly present in haemoglobin in RBC.it is transported to liver by binding with albumin.In liver it under goes conjugation with glucorunide molecules to form conjugation bilirubin which is water soluble and can be excreted easily. Two form of bilirubin are hence present; Conjugation bilirubin = Also known as direct bilirubin, since it can react in Van-De Bergh reaction without the use of alcohol. Unconjugation bilirubin= Also known as indirct bilirubin, since it is water insoluble and requires alcohol for reaction in Van-De Bergh reaction. Principle: Bilirubin reacts with diazotized sulphanilic acid to form azobilirubin which is violet colored complex in prence of DMSO (Dimethyl sulphoxide) Total bilirubin: Bilirubin + sulphanic acid  DMSO  Azobilirubin+Nitric acid Drirect bilirubin: Bilirubin + sulphanic acid                Azobilirubin+Nitric acid Working solution: R1=sulpanilic acid + hydrochloric acid R2=sodium nitrate R3=caffine +sodium benzoate Procedure: 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were added as follows; Blank	Direct 	test R1	100µl	100µl	100µl R2	-	1 drop	1 drop R3	-	-	1000µl Normal saline	1000µl	1000µl	- Sample	100µl	100µl	100µl 	All reagent were mixed well and incubated at room temperature (5 minutes) 	Absorbance was measured at 550nm Calculation: 	Total bilirubin= (O.D of total – O.D of blank)*17.5 	Durect bilirubin=(O.D of test – O.D of blank)*17.5 Normal range: 	Bilirubin total=(up to 1.1)mg\dl 	Bilirubin direct=(up to 0.4)mg\dl Clinical signification: Increased condition: 	Excessive hemolysis eg : hemolytic anemia 	Liver diseases eg: hepatitis, cirrhosis 	Obstruction to bile duct

Uric acid test Introduction: Uric acid is end product of purine nucleotide metabolism.It is excreted by kidney via urine. It is also one of the test used in assessing renal function, generally elevated level of uric acid is associated with gout, besides renal disease. Principle: Uricase convert uric acid into allation and H2O2 then react with 4-amino antipyrene in presence of phenolic compound to form colored compound (red pink)-Quinoniemine dye. Uric acid + H2O    uricase         Allation + H2O H2O2 + 4-amino antipyrene  peroxidase   Quinoniemine dye + Phenolic compound (red pink color) Working reagent preparation: 	Buffer Reagent (R1)= 1 part 	Enzyme Reagent= 2 part Procedure: 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were pipetted as follows; Blank(B)	standard(T)	Test(T) Uricase reagent	1ml	1ml	1ml Standard	-	25 µl	- Sample	-	-	25 µl •	Mix well and incubated at 37°C for 5 min. •	Take reading at 520 nm Calculation: Concentration of uric acid in sample =O.D. of test\O.D> of STD*concentration of STD (6mg\dl) Normal range: 	Male =(3.5-7.2)mg\dl 	Female=(2.6-6.0)mg\dl Clinical signification: Increased condition: 	Gout 	Renal disease 	Increased cell turn over(eg:leukemia,cytotoxic drugs etc) Decreased condition: 	Fanconi’s syndrome 	Xanthine oxidase deficiency

Creatinine test

Blank(B)	standard(T)	Test(T) Supernatant	-	-	3.0ml Working creatinine	-	50ml	- Distilled water	3.0ml	-	- Picric acid	1ml	1ml	1ml Sodium hydroxide	0.5ml	0.5ml	0.5ml Mix well incubates at 37°C for exact 15 min or 30 min at room temperature. Read the OD of test & STD solution using Blank ‘O’. Urea test (dam method) Introduction: Urea is end product of protein metabolism. It is synthesized in liver and excerted by kidney via orine. Urea is one of the tests used for assessing kidney function (test of renal function \ RFT). Principle At high temperature, diacetyle monoxime degrades to produce diacetyle and hydroxyl amine. Urea then condenses with diacetyl to form pink purple color complex in presence of thiosemi carbozide The intensity of color developed is directly proportional to concentration of urea in solution and is measured at 520 nm. Procedure 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were added as follows; Blank(B)	standard(T)	Test(T) Mixed acid reagent	2ml	2ml	2ml Mixed color reagent	2ml	2ml	2ml Distill water	2ml	2ml	10 ml 	Mix well, incubated at 37°C for 3 min. Sample 	-	-	20µl Standard 	-	20µl	- 	Mix well, incubated at 37°C for 3 min. 	Take reading at 578nm Normal range

Clinical signification 	Increased condition 	Renal cause \ kidney diseased •	Glomerulonephritis, pyelonephritis 	Pre renal caused •	Dehydration, fever, high protein diet, shock 	Decreased condition 	Liver disease (hepatitis, cirrhosis) 	Haemodilution (eg:pregnancy) Total protein Introduction: Principle Protein in an alkaline medium, bind with the cupric ions present in Biuret reagent to form a blue violent complex. The intensity of color formed is directly proportional to amount of protein present in sample. Procedure 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were added as follows; Blank(B)	standard(T)	Test(T) Biuret reagent	1ml	1ml	1ml Standard	-	20 µl	- Sample	-	-	20 µl 	Mix well, incubated at 37°C for 10 min. 	Take reading at 550nm Calculation: 	Concentration of protein in sample=O.D. of test\O.D> of STD*concentration of STD Clinical signification 	Increased level mainly found in dehydration.

	Decreased level id found mainly in malnutrition, impaired synthesis, protein, protein losses as in Haemorrhage or excessive catabolism. Albumin Introduction: Albumin consists of approximately 60% of the total protein in the body, the other major part being globulin. It is synthesized in the liver and maintains the osmotic pressure in blood .Albumin also helps to transport druges, hormones and enzymes. A variety of test that detect the presence of albumin protein in the urine. which could indicate renal or functional disorders. Principle Albumin binds with the dye Bromoceresol green in buffered medium to formed green color complex. The intensity of color formed is directly proportional to the amount albumin present in the sample.

Procedure 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were added as follows; Blank(B)	standard(T)	Test(T) BCG reagent	1ml	1ml	1ml Standard	-	10 µl	- Sample	-	-	10 µl 	Mix well, incubated at 37°C for 5 min. 	Take a reading at 630nm.

Calculation: 	Concentration of albumin in sample=O.D. of test\O.D> of STD*concentration of STD Clinical signification 	Increased values are seen only in rare conditions and are usually associated with dehydration. 	Decreased values are seen in : •	Liver diseased •	Malnutrition •	Kidney disorder •	Increased fluid loss during extensive burn •	Decreased absorption in gastro intestinal

SGPT Introduction: Serum glutamate pyruvate transaminase or SGPT is one of the test enzymes participating in transaminase reaction. It is also known as ALP (Alanine Transaminase).This enzyme is present in various tissues such as skeletal muscle, liver, heart, kidney, brains etc. It is commonly used as one of the test in LFT panel. Principle Alanine and α-ketoglutatarats are interconverted to pyruvated and glutamate.This is a transaminase reaction, calatysed by enzyme SGPT – pyruvate then condenses with 2,4 DNPH to give a hydrazone derivative under alkaline pH.this derivative give brown color and color intensity is directly proportional to SGPT present in serum color intensity is measured at 490-510 nm. Procedure 	 Three tubes were marked as blank (B) and total (T). 	The reagent and sample were added as follows; Blank(B)	Test(T) L1\substrate reagent	500 µl	500 µl 	Allow to warm at 37°C for 2-3 min. Serum	-	100 µl 	Incubate for 30 min at 37°C L2\DNPH reagent	500 µl	500 µl Distill water	100 µl	- 0.4 n NaOH	5ml	5ml Calculation O.D=O.D. of test – O.D of blank The calculated O.D. is checking in calibration curve from which enzyme activity value from a given O.D. can be plotted.

O.D

Enzyme activity (Lu\L) Normal range (Up to 40) Lu|L Clinical signification 	High values are seen in •	Liver diseased Eg;Hepatitis, cirrhosis, obstructive jaundice Alkaline phosphatase Introduction: Alkaline phosphatase is an enzyme present in various tissues and orgen such as liver bone, placenta, intestine etc. It is also present in kidney but alkaline phophatase from kidney does not contribte to serum alkaline phophatase. Alkaline phophatase is presumed to cleave phosphate estrer in alkaline conditions.ALP is physiologically elevated in growing children and pregnant female, the source of which are bone and placenta respectively. Principle ALP cleave phenol phosphatase into phenol and inorganic phosphate.Phenol then combine with anti-amino pyrene to give red colored complex, whose intensity is directly proportional to ALP present in serum and is measured at 540 nm. Procedure 	Three tubes were marked as blank (B), Direct (D) and total (T). 	The reagent and sample were added as follows; Blank(B)	Standard (D)	Test(T) Buffer	2ml	2ml	2ml Standard	-	20 µl	- Serum	-	-	20 µl 	Mix well & allow warming at 37°C for 2-3 min. Color reagent	2ml	2ml	2ml 	Mix well and take reading at 540 nm. Calculation: •	Concentration of test = O.D. of test\O.D> of STD*concentration of STD Normal range •	Male = (80-306)Iu\L •	Femal=(64-306)Iu\L Clinical signification 	High values are seen in; •	Liver disease (hepatitis,cirrhosis) •	Bone disease (Rickets, oesteomalacia) 	Low values are seen in; •	Severe malnutrition •	Scurvey HDL (high density lipoprotein) Introduction: HDL stands for high Density Lipoprotein. It is good cholesterol. Principle In the presence of phosphotungstic acid and magnesium chloride; LDL, VLDL and chylomicrons are pricipited. Centrifugation in HDL fraction can be tested by usual method. Procedure 	1ml cholesterol reagent was kept in tube blank (B). 	500µl HDL cholesterol reagent and 200µl sample was kept in tube test (T) and mixed well 	Tube (T) was centrifuged at 3000 rmp for 10 min 	100µl supernatant was added with 1 ml cholesterol reagent.After mixing the reagent; it was incubated for 5min at 30 degree cent great 	Absorbance was taken. Calculation 	HDL cholesterol=O.D. of test\O.D > of STD*concentration of STD LDL estimation 	LDL cholesterol = total cholesterol – (Tg\5+HDL) VLDL estimation 	VLDL (mg\dl) = Tg\5

TRIGLYCERIDE Introduction:

Triglyceride or Triacyl glycerol is major energy from stored in adipose tissues.IT is formed by esterification of glycerol with 3 molecules of fatty acids High level of Tg is associated with cardio vascular disease. Principle Lipase breaks down Tg into glycerol and three molecules of acids. Glycerol is acted upon by Glycerol kinase to form Glycerol phosphate-Glycerol phosphate is converted into di hydroxy acetone phosphate and H2O2 by the action of glycerol oxidase.H2O2 combine with 4-amino antipyrene to give red colored complex. 	Triglyceride  lipase   glycerol + 3 fatty acid 	Glycerol glycerol kinase    glycerol phosphate ATP              ADP 	Glycerol phosphatase  glycerol oxidase      Dihydroxy acetone phosphatase +H2O2 	H2O2+4-amino antipyrine   peroxidase    Red colored complex (Quinoneimine dye) Procedure 	Three clean test tubes were taken and labeled as blank(B),standard (S),and test(T) 	Reagent and sample were pipetted as follow: Blank(B)	standard(T)	Test(T) Tg reagent	1ml	1ml	1ml Standard	-	10µl	- Sample	-	-	10 µl 	Mixed well and incubated at 37°C for 5 min. 	Absorbance was measured at 540nm Calculation Conc. of test =O.D. of test\O.D> of STD*concentration of STD Normal range 	Up to 150 mg\dl Clinical signification Increased level are seen are 	Familial hypertriglyceridemia 	Hypothyroidism 	Uncontrolled dibetes mellitus 	Nephrotic syndrome Decreased values 	Malnutrition 	Hyperthyroidism CARDIAC FUNCTION TEST Introduction Cardiac function test is the group of test performed to diagnose MI(Mycocardial infraction )in a patient.It consists of Non enzymatic and enzymatic tests. Performed tests 1.	Non enzyme test a)	Myoglobin It is a muscle protein in all type of muscle including cardiac muscle.It is the earliest marker of MI arising at 2 hrs,but not specific. b)	Cardiac troponin •	Cardiac troponin can represent in 3 forms •	Cardiac troponin T(tropomyosin) •	Cardiac troponin I (inhibitory) •	Cardiac troponin C(calcium) Cardiac troponin I is most frequently used for diagnosis of MI ,arising at 3 hrs. 2.	Enzyme test Various enzyme are present in cardiac muscles are released in large quantities and can be detected in blood during MI.

a.	CPK CPK (creatine phosphokinase )is an enzyme present in heart, brain and muscles. Three types of CPK enzyme are found. They are: i.	CPK BB – found in brain ii. CPK MB – found in cardiac muscle iii. CPK MM – found in skeletal muscle Blood CPK primarily constitutes of CPK MM, CPK MB is generally less than 5% of total CPK. During MI, CPK MB may be increased above this level. Total CPK is increased in MI. It is non-specific marker since increased may occur in muscle damage and Cerebal injury too. CPK MB is specific marker for MI. It starts to rise at 4 to 6 hrs. b.	GOT AST\GOT is non-specific marker because increased may occur in liver or muscle diseases. c.	LDH LDH is non-specific marker because increased may occur in liver disease or muscle disease, haemolytic anemia, etc. Calcium Introduction: It is one of the most abundant mineral present in human body. It is present in about 1-1.5 kg in adult human being. Calcium is required for various processes like; development of bone and teeth, muscular contraction, nerve transmission, enzyme activity, hormone action, etc. Principle Calcium reacts with organic compound such as OCPS (O-cresol phthalate complex) to give purple colored complex. The intensity of color complex is directly proportional to calcium present in serum. Procedure 	Three clean test tubes were taken and labeled as blank(B),standard (S),and test(T) 	Reagent and sample were added as follow: Blank(B)	standard(T)	Test(T) OCPS reagent	1ml	1ml	1ml Standard	-	25µl	- Sample	-	-	25 µl 	The pipette reagent were mixed properly and incubated at 37°C for 5 min. 	Absorbance was measured in colorimeter at 520 nm. Calculation: 	Concentration of glucose=O.D. of test\O.D> of STD*concentration of STD Normal range •	(8.4-10.8)mg\dl Clinical significances 	Increased values are seen in; •	Hyper Parathyroidism •	Malignancy •	Excess vitamin D therapy 	Decreased values are seen in; •	Hypoparathyroidism •	Vitamin D deficiency Notes 	Calcium test is carried out in either plastic tube or acid washed glass tube. It is because Ca++ estimation is very susptible to interferences that can result in falsely high values. 	While performing calcium test, blood should be collected without the use of tourniquet which leads to escaping of plasma to extravascular spaces and calcium being bound to albumin (around 50% calcium is bound to albumin) cannot escape, leading to falsely high values. ADENOSINE DEAMINESE ACTIVITY (ADA) Introduction Tuberculosis, though curable, it is infection is on rise. The most specific test is positive bacterial culture of a patients sputum sample X-rays, smears for AFB and Adenosine Deaminese(ADA) is an enzyme widely distributed in mammalian tissues, particularly in T. lymphocyte. Principle Adenosine Deaminase hydrolyses adenosine to ammonia and inosine. The ammonia formed further react with a phenol and hypochlorite in an alkaline medium to form a blue indophenols complex with sodium Nitroprusside acting as a catalyst. Intensity of the blue color complex formed is directly proportional to amount of ADA present in sample. Adenosine + H2O ADA ammonia + Inosine Ammonia + phenol + hypochlorite alkaline blue indophenols complex Reagent preparation Reagent L1, L2 and standard are ready to use. L2 may form crystals at (2-8) °c.Dissolus the reagent by gently warming (37°c - 50°c) for some time before use. Phenol reagent (L3) and Hypochlorite reagent (L4) need to be diluted at 1:5 with distilled water before use. Procedure 	Three clean test tubes were taken and labeled as blank(B),standard (S),standard blank(SB) and test (T) 	Reagent and sample were added as follow: Blank(B)	Standard(S)	Standard blank(SB)	Test(T) Buffer reagent(L1)	200µl	200µl	-	- Adenosine reagent (L2)	-	-	200µl	200µl Standard	-	20µl	-	- Sample	-	-	-	20µl 	Mix well and incubate at 37°c for 60 minutes 	Following are added. Working phenol reagent(L3)	1ml	1ml	1ml	1ml Sample 	-	-	20µl	- Working hypochlorite reagent(L4) 	s1ml	1ml	1ml	1ml Absorbance measured at 580 nm. Calculation Total ADA activity = Abs. T. –Abs. SB\Abs. S – Abs. B * 50 Clinical significances Increased level of ADA are found in variable forms of tuberculosis making it a marker for the same. ADA is also increased in various infection diseased like Typhoid, Viral hepatitis, initial stage of HIV, and in case of malignant tumours.

References values •	Serum, plasma, pleural fluids,pericardial fluids, ascetic fluids. Normal suspect strong suspect Positive	< 30 U\L 30 U\L to 40 U\L >40 U\L to 60 U\L >60 U\L •	Cerebrospinal fluid ( CSF )	Normal positive	<10 U\L >10 U\L

Thyroid function test (TFT) Introduction Thyroid function test is a panel of test performed to assess functioning of thyroid glands. Thyroid verity of physiological aspects and any diseased affecting thyroid gland can seriously affected these aspects. Thyroid function test can be classified into different tests. Performed tests 1.	Thyroid hormones T3 and T4 are the thyroid hormone produced by thyroid. Increased value is seen in hyper thyrodism and decreased value is seen in hypo thyrodism. 2.	Thyroid stimulating hormones (TSH) TSH is secreated by anterior pituitary gland and it regulates activity of thyroid gland. TSH is increased in hypothyroidism and decreased in hyper thyroidism. 3.	Thyroid antibodies Thyroid antibodies are often tested for diagnosising and monitoring diseased affecting thyroid gland. Some thyroid antibodies are : 	Anti TPD (Anti thyroperoxidase) 	Anti Tg (Anti thyroglobulin) 4.	Thyroid scintiscanning Use of radio isotrope is done Thyroid stimulating hormones (TSH) Introduction