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Estimation of Sr.TP and AG RATIO 24-25.pptx

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Estimation of serum Total proteins by Biuret method , Albumin by BCG binding method and Determination of A/G ratio. DR. Sharda D. Sarwade (Deshmukh )
Most of the proteins are synthesise by the liver ,these proteins are present In our blood .They are important for health. Two of the key proteins are : Albumin : Exert osmotic pressure , transport medicine and hormones through out our body . Helps in tissue growth and healing. Globulins : This is a group of proteins (alpha1,alpha2,beta,and gama gobulins) some of them synthesize in liver except gama globulins, Which are synhesize by immune system. Gobulins helps in transport nutrients and fight against infection.
The serum protein estimation measures all the proteins as well as Amount of albumin and A/G RATIO Why we need to measure ? To diagnose a variety of diseases that affect protein level such as •Liver ,kidney and blood disease . •Check nutritional status •Risk for an infection . •Find the cause of symptoms like Edema or swelling due to excess tissue Fluid . •Difficulty urinating •Nausea or vomiting •Unexpected weight loss •Loss of appetite •Fatigue
1. Estimation of serum total proteins by Biuret method Principle- When serum is treated with biuret reagent, the peptide bonds of protein react with cupric ions in an alkaline medium to form a violet coloured complex. The absorbance of this complex is measured calorimetrically at 530nm. A standard protein solution is also treated similarly and the colour intensities are compared. Vacutainer used - Red
Procedure- I. Dilution of serum • 0.2ml serum + 3.8ml normal saline, Dilution factor 0.05 II. Colour Development • Label 3 test tubes as “Blank”, “Standard” and “Unknown” and make following additions • Mix and keep at room temperature for 10min and read at 530nm green filter. Blank(B) Standard(S) Unknown(U) Normal saline 1.0ml - --------- Protein Standard (5mg/ml) - 1.0ml - Diluted Serum - - 1.0ml Biuret reagent 5.0ml 5.0ml 5.0ml
 Observations  Calculations: Serum total proteins(Gm/dl)  O .D . of U - O.D OF B 100 1  --------------------------- X conc .of std./ml X vol.of std .X ---------------- X --------  O.D of S - O.D of B vol .of serum 1000 O.D Blank Standard Unknown
Estimation of serum albumin by Bromocresol green binding method  Principle  Sr.albumin is binds with BCG at PH 4.1 to form green colour complex,compared with standard treated similarly and OD is read on colorimeter using red filter (620nm)  Procedure : sr.dilution  0.2ml serum +1.8ml normal saline and mix.  Dilution factor is 0.02 ml
Colour development BLANK STANDARD UNKNOWN Normal saline 0.2ml Albumin std.5mg/ml 0.2ml Diluted serum 0.2ml BCG dye 5ml 5ml 5ml Mix and keep at room temperature for 10minute and read O.D at 62onm(red filter)
Observation O.D BLANK STANDARD UNKNOWN
Calculations: Serum albumin (Gm/dl ) = O.D of U- O.D of B 100 1 ------------------------- x conc.of std/mlxvol.of std X ------------------ X --------- O.D of S -O.D of S vol.of serum 1000 U/S X 5 Total protein = Albumin +Globulin Sr . Globulin =S r. Total Protein – Sr. Albumin A/G RATIO =ALBUMIN/GLOBULIN
Normal range Sr.Total protein : 6.3 – 7.9 Gm/dl Sr.Albumin : 3.7 - 5.3Gm/dl Sr.Globulin : 1.8- 3.6Gm/dl A/G RATIO : 1.5 -2.5
Increase sr.Total protein (hyperproteinemia ) :Dehydration (Hemoconcentration ) Inflammation from infections ,such as HIV or viral hepatitis Cancer such as Multiple myeloma Tuberculosis and Kala aazar Decrease Sr.Total protein usually due to decrease in Sr.Albumin . •Water intoxication(Hemodilution ), Glomerulonephritis or neprotic syndrome(loss of Albumin in in urine), •Liver cirrhosis (Impaired protein synthesis),Severe burns(loss of protein from skin), • Kwashiorkar (Dietary deficiency of protein), •Malabsorption , •Prgnancy induced hypertension
Albumin-fatty acid complex cannot cross blood brain barrier Bilirubin- aspirin interaction Drug interactions Kernicterus mental retardation Biological availability of drugs Protein bound calcium Hypoalbuminemia Decreased calcium in blood Clinical significance of albumin Hypoalbuminemia--- decreased serum albumin ----malnutrition, nephrotic syndrome and cirrhosis of liver. Albumin is excreted into urine (albuminuria) --- nephrotic syndrome and in certain inflammatory conditions of urinary tract. Microalbuminuria (30-300 mg/day) is clinically important for predicting the future risk of renal diseases . Albumin is therapeutically useful for the treatment of burns and hemorrhage.
Albumin is reduced – decreased in albumin level results in loss of water from blood and its entry in to interstitial fluids causing edema . A) Malnutrition; generalised edema ,synthesis is reduced B) Nephrotic syndrome; facial edema; Loss of albumin in urine. C) Cirrhosis; Ascites; synthesis Albumin is synthesis diminished . 2) Congestive Cardiac Failure Dependent edema, feet, Increased hydrostatic P in vein
Normal Albumin level in blood: 3.5 - 5 g /dl Hypoalbuminemia – sr.albumin level is lower than normal Cirrhosis of liver, albumin synthesis is impaired . Malnutrition--diet deficient in protein (kwashiorkor ). Malabsorption syndrome-- Protein losing enteropathy – loss of albumin from intestinal tract. Nephrotic syndrome– permeability of glomerular membrane is defective .(loss of albumin in urine ) Analbuminemia --- rare condition ,defective gene ,defect in synthesis.
Proteinuria– excretion of protein in urine or presence of protein /albumin in urine* . Glomerular proteinuria Micro albuminuria Minimal albuminuria Pauci (small in quantity ) albuminuria Small quantities of albumin Less than 300 mg per day Diabetes mellitus Hypertension Indicator of future renal failure *Heat coagulation test is used for detection of presence of albumin in urine Reversal of Albumin : Globulin Reduction of albumin Compensatory increase in globulin fractions. cirrhosis of liver and nephrotic syndrome .
REVERSED A/G RATIO :Glomerulo nephritis and Liver cirrhosis A low A/G ratio may be due to an overproduction of globulin, underproduction of albumin or loss of albumin indicates the following An autoimmune disease Cirrhosis. Involving inflammation and scarring of liver Multiple myeloma Nephrotic syndrome kidney disease A high A/G ratio may suggest An underproduction of antibodies. Leukemia or cancer of the bone marrow
Hypergammaglobulinaemias : decrease albumin --- increase globulins 1.Chronic infections (gamma) 2.Multiple myeloma (gamma)--- cancer of plasma cell ,abnormal Abs formed (light chain immunoglobulin ) increase – kidney problem ,thick blood ,mass in bone marrow or soft tissue ,more than one – multiple myeloma – malignant plasmacytoma . 3.Lipoproteinemias (beta) Atherosclerosis 4.Nephrotic syndrome (alpha)2 globulin increase.
Nephroitic syndrome --- characteristics :Proteinuria , Edema,Hyperlipidimia , Lipiduria and Hypercoagubility state. Altered permiability of the glomerular basement membrane leading to proteinuria Both in children and an adults . Analbuminuria– rare congenital disorder of absence of albumin synthesis (homozygus state) Microalbuminura---Excretion of 30 to 300mg of albumin /day.Early markar of Hypertensive and diabetic renal disease.
Cirrhosis of liver---Necrosis of hepatocytes and its replacement by fibrous tissues due to Chronic liver disease like Alcoholism/chronic hepatitis. Multiple myoloma---- Malignant disease of plasma cells , synthesis of abnormal proteins BENCE JONES proteins(Light chain Immunoglobulin),excreated in urine. BJ proteins also link to the cancers of the lymphatic system, lymphoma , macroglobulinemia. The BJ protein is a building block of the antibodies made by cancerous Tumors ,detecting it can help doctors diagnose type of cancer. Detected by urine test :urine protein electrophoresis( UPEP),Urine immunofixation electrophoresis , or immunoassay for free light chains
Case study : A 5 year old child is brought by mother with history of swelling all over the body for past one week . The swelling started on face specially around the eyes on getting up in the morning and slowly it was noticed involving the abdomen ,upper and lower limb . There is no history of fever , rash or sore troat . On examination ,the edema was non pitting type . Following are the results of various investigations : Total protein : 5.6 gm /dl ( normal = 6.3 -7.9 g /dl ) Serum albumin : 2.3 g /dl ( 3 .7 – 5 .3 g / dl ) Total cholesterol : 258 mg /dl (150 – 250 mg /dl ) 24 hrs . Urinary protein : 3.5 g / day ( less than 300 mg /24 hrs .) 1 .What may be the clinical condition this child may be suffering from ? 2.What is the diagnostic criteria of above condition ? 3.What is the reason of low albumin in blood ? Calculate A/G ratio in this case .what other condition reversal of A/G ratio is seen .
Key point clinical case of liver cirrhosis Complaints Fatigue ,abdominal distension ,Jaundice ,leg swelling ,loss of appetite,nausea vomiting,bleeding etc Medical history Heavy alcohol consumption ,hepatitis B or C ,Family history of liver disease Non alcoholic fatty liver Wilsons disease AT deficiency Galactosemia ,obesity ,DM. Physical examination Hepatomegaly or shrunken liver Splenomegaly Ascites Spider like blood vessels on the skin Red ness of palm Lab test LFT,,PT– prolonged Increase AST ,ALP,ALT Low serum albumin ,reversed A:G ratio Elevated sr.bilirubin Electrolyte abnormalities . Electrophoretic pattern
Two brothers one age 3years and other aged 2 years present to OPD For complain of swelling over bthe body with protruded abdomen .They belong to poor family .family is mainly surviving on rice az their staple meal with no access to dal and fruits ,vegetables ,eggs etc . On examination both children were lethargic ,abdomen was noticibly enlarged. Examination revealed mild hepatomegaly with ascites .Lowe limbs showed pitting pedal edema with scally peeling skin at certain places .Hair examination revealed hyp[opigmentation(flagging in hair ) Comment on the above diagnosis 2.Biochemical basis of above symptoms and signs 3.Line of management .
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