Human Pancreatic Polypeptide (PP)

Human pancreatic polypeptide (PP) is a polypeptide hormone secreted by the pancreatic polypeptide (PP) cells of the islets of Langerhans in the endocrine portion of the pancreas. It consists of a chain of 36 amino acids.

The function of PP is to self regulate the pancreas secretion activities (endocrine and exocrine) and it also has effects on hepatic glycogen levels and gastrointestinal secretions. It is triggered in humans by protein-rich meals, fasting, exercise, and acute hypoglycemia and is inhibited by somatostatin and intravenous glucose.

The purpose of this test is for the non-radioactive quantitation of human pancreatic polypeptide in human serum and plasma. The principle of this test is by sandwich enzyme-linked immunosorbant assay (ELISA) based sequentially on:

1. Capture of human PP molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti-PP antibody.

2. Wash away of unbound materials from samples.

3. Binding of second biotinylated anti-PP polyclonal Ab to the captured molecules.

4. Wash away of unbound materials from samples.

5. Conjugation of horseradish peroxidase to the immobilized biotinylated Ab.

6. Wash away of free enzyme conjugates.

7. Quantification of immobilized Ab-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of substrate 3, 3’, 5, 5’-tetramethylbenzidine.

The enzyme activity is measured spectrophotometrically at 450nm with 630nm background correction. Increase in absorbency is directly proportional to the amount of captured human PP in the unknown sample.

A standard curve is generated by plotting the absorbance versus the respective reference standards of known concentrations of human PP. The concentration of human PP in the samples is then determined directly from this standard curve.

Range of this assay: 12.3pg/mL to 3000pg/mL human PP (50uL sample size)

Natasha.
0703883I

Immunofixation Electrophoresis

Immunofixation Electrophoresis ( IFE) is a method to detect monoclonal band (M-band). The M-band consist of monoclonal proteins (M-proteins), which are abnormal proteins produce by continous proliferation of plasma cells. The purpose of implementing IFE is to investigate the M-band, that is to know which particular immunoglobulin heavy chains, gamma(IgG), alpha(IgA) and mu(IgM) and light chains (kappa or lambda) that make up the M-proteins. In the laboratory that I am attached to, usually the test that require IFE is myeloma test. In addition, IFE also provides urine protein electrophoresis, which is useful in the study of renal excretion of proteins.

Steps involve:

1. Proteins in the samples (urine and serum) are separated by electrophoresis on agarose gel.

2. Immunofixation of electrophoresed proteins includes, the monospecific antisera diffuse into
agarose gel and bind to the antigens (proteins), if present. Thus, resulting in the precipitation
of the antigens.

3. The unprecipitated antigens are removed from the gel by blotting and washing.

4. The precipitated antigens are stained and can be evaluated visually. M-band is seen when one
of the fractions is dark in colour.

IFE only provides qualitative determination of results.

Siti Shahimah Bte Samat
0702717J

blood collection

In this post, I am going to write about a tutorial given by one of our colleague. All of us have to attend this tutorials unless we are busy at our work stations. These tutorials are given at least once every 2 weeks.

Topic for today: blood collection

The Clinical and Laboratory Standards Institudes (previously known as NCCLS) sets guidelines of procedure to ensure that quality specimens are collected for laboratory testing. Upon collection, all plastic tubes must be gently inverted 5-10 times to provide thorough mixing of the additives. Also, it is important to note that shaking of blood tubes may cause hemolysis of the blood.
Order of draw of blood collection tubes procedure:

1st) Blood culture bottles
Culture bottles should always be drawn first. this is to reduce contamination of the blood specimen which might cause the analyzer to detect a positive bottle. thus, leading to a wrong treatment for the patient.

2nd) Citrate (light blue) tube
blood to be taken as soon as possible before formation of microclots. also, this tubes must be drawn before the collection of serum tubes (red and yellow) to prevent comtamination with clot activator which are present in them.

3rd) Serum (red/yellow) tube


4th) Heparin tube

5th) EDTA (purple) tube
they contain potassium hence, it is not advisable to collect blood into this tube before the serum tubes (chemistry tests). this is because serum tubes might be contaminated with potassium causing inaccurate results. for eg, K+ level will be falsely high and Ca+/Mg+ levels will be falsely low.

6th) Fluoride/Oxalate (grey) tube
oxalate pesent in this tube can be obstructive to cell membrane. thus, it is not advisable to collect blood into this tube before both serum tubes (chemistry tests) and EDTA tubes (haematology tests). if both tubes are contaminated with oxalate, it will also lead to inaccurate results.

wendy/0701158h

answers (there's some prob i couldnt post a comment)
1. nyzah,
heparin tube is the green top tube. most chemistry tests can be done using this tube. for eg, renal panel, lipid panel etc.

2. shemeema,
the citrated tube is used for running the coagulation panel (PTINR/aPTT). hence, the presence of clots will affect the result.

thanks,
wendy.