Protein Free Filtrate Preparation by Folin Wu Method

PFF Stands for protein free filtrate. It is protein free blood. When we remove all the proteins from blood for some blood test, then the remaining specimen is called protein free filtrate or simply PFF.

Removing proteins from blood can be done via some techniques. The one method which is widely used is precipitation by large anions. Below you would find procedure of this method in detail. But first lets learn more about PFF.

Why proteins are removed from bood:

It is one of the most asked question that why we prepare protein free filtrate? The reason is that, proteins interfere with some blood tests and may alter the results. Therefore, it is necessary to remove proteins or make a PFF before performing these tests.

In Which tests we make PFF:

This is yet another question that in which tests we should removed all the proteins from blood. So, below is the list of all the major tests in which you must prepare a protein less filtrate:

Estimation of Blood Urea.
Estimation of Blood Creatinine
Estimation of Blood Uric Acid etc

Preparation of protein free filtrate:

PFF is prepared by the precipitation of proteins. And as discussed above, there are two methods for precipitating proteins. These two methods are:

Precipitation By Heavy Metals:

Protein acts as an anion when its pH shifts towards alkalinity. Therefore they can be precipitated using heavy metals like Copper, Silver etc. Bio medically this principle is used in AgNO3 cautery and for metallic poisoning etc.

Precipitation By Large Anions:

Proteins can also be precipitated using large anions such as tungstic acid, tri chloro acetic acid, phospho tungstic acid, tennic acid etc.

This method is the basis of folin wu method and other similar methods for PFF preparation.

Folin wu Method:

As mentioned above, folin wu method is one of the most widely method used for the preparation of protein free filtrate. This method is done before doing laboratory estimation of creatinine, urine and uric acid in blood. This is method is based on precipitation of proteins in blood by large anions.

Principle of Folin wu Method:

As mentioned above, all the proteins in the blood are precipitated by anions. The anions that are used in this test is tungstic acid.

Apparatus:

Here are the apparatus that is used for this method:

Volumetric flask
Test Tube
Funnel
WhatMan Filter Paper
Beaker
Pipette

Reagents of Folin wu method:

There are a few reagents used for this. They are the chemicals that are mentioned in principle of this test and some other supporting chemicals. These are the following:

Blood
10 percent sodium tungstate
2/3 Normal H2SO4.
Potassium Oxalate crystals.
Distilled Water.

Procedure of Preparation of protein free filtrate:

Procedure of folin wu method for protein free filtrate preparation is quite easy and much like other tests. There are are few steps in this test that are given below:

First of all take 2ml of blood.
Then shift this blood to a container containing potassium oxalate.
Then dilute the blood with distilled water. Add 14ml of Dist water.
Then add 2ml of 10 percent sodium tungstate.
Then add 2/3 normal H2SO4.
Color of solution will change from dark red to dark brown.
Filter this solution using Whatman filter paper.
After filtration, you will get colorless protein free filtrate.

Here the dilution factor is 10.

Why we Add Sulfuric Acid?

There are two reasons for adding H2SO4. First is to convert sodium tungstate to tungstic acid which helps in protein precipitation (as sodium tungstate cannot precipitate proteins).

The second reason for adding sulfuric acid is to rupture the RBC’s so that the color of solution changes from dark red to dark brown.

Precautions:

Here are some of the precautions of this test:

Add only potassium oxalate as anti coagulant.
Add sulfuric acid carefully. It may cause injury.
Measure the exact 2ml of blood and Na-Tungstate and 14ml of water.
Check for change in color of solution.
Filter the solution accurately.

Results:

After you do all the steps correctly, you would get colorless protein free filtrate. You can then check if proteins are present in filtrate or not by doing a general test for proteins.

Final Words:

If you want to perform laboratory test for blood creatinine, urine or uric acid then you must use PFF instead of serum or blood. And to make PFF you should follow the above step by step guide.