Normal range of urine protein is less than 150mg/24 hour sample (varying from 2 to 10mg/dL)
What type of sample is preferred for urine protein examination
Early morning sample is preferred
What are the grades of proteinuria
Mild proteinuria – less than 500mg/dL
Moderate proteinuria – upto 4g /day
heavy proteinuria – More than 4g/day
What are the different types of proteins found in urine
Albumin
Globulin
Mucin
Blood proteins (hemoglobin)
Bence-Jones proteins
What are the tests for detection of protein in urine
Qualitative tests
Heat coagulation test
Hellers nitric acid test
Sulphosalicylic acid test
Quantitative test
Esbach’s albuminometre test
Other tests
Protein reagent strip test
Biuret test
Urine protein electrophoresis
Explain the Heat coagulation test
Reagent – 3%% of Acetic acid
Priciple – proteins are coagulated on heating
Procedure –
Fill 3/4th of the test tube with urine
Heat upper part of tube with urine
Coagulum can be formed due to proteins, phosphates or urates
Add 3 to 4 drops of 3% Acetic acid. If the coagulum persists, then it is due to proteins and if it disappears it is due to phosphates ( Excess of Acetic acid may dissolve traces of proteins giving false negative reaction)
Add 2 drops of nitric acid to detect the presence of mucin. If the coagulum is due to mucin it disappears on adding nitric acid.
What are the advantages and disadvantages of the heat coagulation test
Advantage – cheap test which does not require technical expertise
Disadvantage – specific type of proteinuria cannot be judged
Why should the test tube be heated in upper part?
Urine in lower part act as control and is used to compare the haziness developed in the upper part with rest of urine
How do we interpret the heat coagulation test
No cloudiness – absence of proteins
Haziness – traces of proteins present (upto 10mg/dL)
Thick cloudy precipitate (4+) – more than 500ng/dL
What is the procedure of Heller’s nitric acid test for detection of proteins
Reagent required – nitric acid
Principle – coagulation of proteins by chemicals
Procedure –
Take 3ml of concentrated nitric acid in a test tube.
Then add 2 ml of urine slowly along the sides of test tube.
White colour ring is formed at the junction of two fluids which indicates presence of proteins in urine
What are the advantages and disadvantages of nitric acid method
Advantages –
Simple and cost effective method
Small amount of urine is required for test
Disadvantage
Handling nitric acid is dangerous as it is corrosive
Describe Sulphosalicylic acid test for detection of proteins
Reagents – Sulphosalicylic acid (20%)
Principle – Precipitation of proteins by chemicals
Procedure –
Assess the pH of urine by litmus paper
If the urine is alkaline then it should be acidified
2ml of acidic urine is taken in test tube.
To it 2 ml of Sulphosalicylic acid reagent is added
If the proteins are present, cloudiness appears.
Interpretation of test is similar to the heat coagulation test
What are the disadvantages of Sulphosalicylic acid method
Some of the metabolic products present in urine may give false positive reaction. They are
Tobutamide (oral hypoglycemic drug)
Para-aminosalicylic acid
Penicillin
High concentration of urates
Sulphonamides
Drugs used for IV pyelogram
How do we estimate protein by Esbach’s Albuminometer
Reagents –
Esbachs reagent
Picric acid – 10gm
Citric acid – 20gm
Distilled water – 1000ml
24 hour urine sample – Urine should be filtered and acidified with 10% acetic acid
Procedure
Albuminometer is filled with urine upto the mark ‘U’
Esbach reagent is added upto the mark ‘R’
Mix well and keep the tube vertically in the stand and close it with cork
Leave it for 24hrs
Interpretation of result
Precipitate in the tube is measured against the graduations in tube
Reading is given in gms/L (Divide by 10 to get gm%)
If the specific gravity of urine is more then it should be diluted in 1:2 or 1:4. Then final reading should be multiplied by dilution factor
What are the advantages and disadvantages of Esbachs albuminometer
Advantages –
Easy and cheap method
Quantitative estimation of albumin is possible
Disadvantages
Picric acid is hygroscopic and hence cannot be used repeatedly once the pack is opened
Measurement of picric acid should be done quickly
Describe the protein reagent strip method for detection of proteins
Reagents
Strip with test area impregnated with indicator tetrabromophenol blue or tetra bromophenolphthalein
Acetic acid
pH paper
Procedure
pH of the urine is recorded
If it is alkaline, it should be acidified by adding few drops of Acetic acid
Then the strip is dipped in the urine
If the protein is present in urine, colour of the strip will be changed depending upon the concentration of proteins
Interpretation of result
Spectrum of colour chnages will be from light yellow to green-blue
Light blue – mild proteinuria (1+)
Green – moderate proteinuria (2+)
Green-blue – Severe proteinuria (3+)
What are the advantages and disadvantages of Strip test
Advantages
This method is sensitive and can detect evn 10mg% of albumin also
Easy to perform and does not require expertise
Can be performed as bed side technique in hospitals
Turbidity of urine does not affect the test
Disadvantages
Usage of strip beyond the expiry date may give false results
False positive may occcur in the usage of following
Chloroquine
Quinidine
Trimethoprim
IV infusion with plasma expanders
Cetrimide
Strongly alkaline urine should be acidfied which may give false result due to semiquantitation
What are the causes of proteinuria
Physiological causes
Excercise induced
pregnancy
Orthostatic (when standing for long time in upright position)
Pathological
Pre-renal causes
Vomitting
Severe diarrhoea
Intestinal obstruction
Diabetic coma
Addison’s disease
Fever
Ascites with intraabdominal tumor
Severe anemia
Renal causes
causes of glomerulonephritis
Glomerular and tubular diseases
Nephrotic syndrome
Post renal causes
Lesions of prostate
Lesions of the urethra
Severe urinary tract infection
Lesions of reanl pelvis and urinary bladder
What is the selective proteinuria
Selective excretion of low molecular weight proteins like albumin (MW 66000) or transferrin (MW 76000) through the kidney is called Selective proteinuria
Excretion of high molecular weight proteins along with albumin is called – Poorly selective proteinuria
Enumerate the causes of selective proteinuria
Minimal change glomerulonephritis
Focal segmental glomerulosclerosis
Membranous glomerulonephritis
Causes of nephrotic syndrome in initial stage
What are Bence-Jones proteins
Bence-Jones proteins are low molecular weight globulins containing either Kappa or Lambda light chains of immunoglobulin
What are the causes of Bence-Jones proteinuria
Multiple myeloma
Waldenstrom macroglobinemia
plasmacytoma
Chronic myeloid leukemia
Malignant lymphoma
Osteomalacia
Osteosarcoma
Generalized carcinomatosis
How are the Bence -Jones proteins detected in urine
Heat coagulation method
On heating urine at 40° – 60° C white coagulum is formed, which dissolves on further heating and boiling
On cooling, the precipitate reappears again at 40° -60° C
Protein electrophoresis
A homogenous band of Bence-Jones protein is formed on electrophoresis
For the identification of light chain immune elctrophoresis should be performed
What is the test for detecting Globulin in urine
Urine should be alkaline. If it is acidic, then add few drops of ammonium hydroxide.
Equal volume of urine and Ammonium sulphate solution are mixed
Precipitate appears if globulin is present
What is microalbuminuria?
Persistent elevation of albumuin excretion in the urine to 20 – 200 mg/litre is called micralbuminuria
What is the clinical significance of microalbuminuria
Persistent microalbuminuria indicates probability of damage to the glomerular filtration capacity of the kidney and is of great diagnostic relevance
In diabetes, for early diagnosis of diabetic nephropathy
In patients with hypertension, as an indicator of end-organ damage associated with a lowered life expectancy
In pregnancy, as a possible predictor of developing preeclampsia
What are the methods to assay microalbuminuria
Immunometric assay
Nephelometric assay
What is the test for chyle in urine
Chyle in urine gives turbid appearance to urine and is also positive for albumin
The following are methods to detect chyle
5 to 10 ml of urine is mixed with equal volume of ether or chloroform. On shaking the mixture urine becomes partially or completely clear
10ml of urine is centrifuged and supernatant shows fat globules
Microscopic examination of Chylous urine shows fat globules in cover slip preparartion