MED5539 Clinical and Research Laboratory Skills

Introduction

The analysis of serum albumin for many years has been used across the world in the diagnosis of disease. It is particularly important in the consideration of liver and kidney diseases for example; nephropathy and cirrhosis. Levels of albumin have also been crucial in monitoring diseases for example kidney dialysis and transplants, and to contribute to the prognosis of mortality and morbidity after a stroke or heart surgery.

In addition, serum albumin has been used as an indicator of malnutrition although it is not considered reliable and history and clinical presentation of the patient is more important in making a diagnosis. Debate on which dye is the best when analysing the human blood serum and specifically the albumin component continue to thrive and as much as many clinical medics still insist on using the BCG, other Biologists have adopted BCP.  BCP results though highly specific may underestimate albumin in patients who are suffering from renal diseases and those with serum albumin complications. BCG is however consistent and give more accurate results. Apart from this fact BCG is also easy to learn how to use.

Principle of the Experiment

The Bromcresol green (BCG) dye-binding assay for the determination of serum albumin was developed in 1965 by Rodkey. It was again revised (Bartholomew & Delaney, 1965; Doumas, Watson & Biggs, 1971) to give out a more reliable, sensitive and inexpensive assay that was used widely in clinical biochemistry laboratories for many years. Increasing concern that the technique was limited in its specificity and provided an overestimation of values determined by immunoassay led to the introduction of an a different dye-binding assay founded on Bromcresol purple  ( abbreviated as BCP) (Louderback, Mealy & Taylor, 1968; Carter, 1970; Pinnell & Northam, 1978). However, the bromocresol purple assay, though more specific, is widely variable than analysis by BCG and also gives a lower serum albumin reading in patients undergoing hemodialysis. Despite the shortcomings of the assays both are still routinely used, the particular technique adopted depending on the preference of each individual laboratory.

Planning

The task before us was to conduct an investigation to find out and compare the usage of BCG and BCP in the analysis of albumin concentrations in the serum of human beings. Standard curves were performed in the first session thereafter the planned experiment took place in the session that followed.

The major points that were considered during the experiment were: standard curves, incubation times, patient samples and QC. In our individual log books we designed an experiment comparing the two methods.

The following Materials were provided:

1. Stock standard solution of BCP.
2. Stock standard solution of BCG.
3. Samples from 10 patients.

The experiment was carried out with a lot of precaution because human blood is considered to be potentially infectious. The samples were handled carefully then disposed off appropriately.

Outline Protocol for Bromocresol Green Assay

• Set the spectrophotometer to 628nm wavelength and zero using BCG reagent.
• In a test tube add 24 μl of standard or 10μl of sample or QC to 2mls of BCG reagent and mix well.
• Incubate at room temperature.
• Measure the absorbance for each and use the data to determine the concentration of albumin.

The Procedure for Bromocresol Green Assay Preparation

The spectrophotometer was set to give a reading of 628nm wavelength and then zeroed using a BCP reagent. 10μl of sample was then added to the test tube containing 2ml of BCG reagent. The mixture was then slightly shaken to mix it well. It was then incubated at room temperature. The absorbance was then measured for each sample and resulting data used to calculate the concentration of albumin.

The Procedure for Bromocresol Purple Assay

The spectrophotometer was set to give a reading of 600nm wavelength and then zeroed using a BCP reagent.  10μl of sample was then added to the test tube containing 2ml of BCG reagent. The mixture was then slightly shaken to mix it well. It was then incubated at room temperature. The absorbance was then measured for each sample and resulting data used to calculate the concentration of albumin.

Part 2: The information in the table below was used to construct a set of standard curves. The table below shows the volumes of stock serum and saline needed to make up and appropriate set of standards.

Tube 0.9% NaCl (µl) Stock Serum (µl) The Final Concentration (grams/L)

Saline Blank 60 0 0.0

S1 35 25 27.0

S2 30 30 32.4

S3 25 35 37.8

S4 20 40 43.2

S5 15 45 48.6

S6 10 50 54.0

S7 5 55 59.4

S8 0 60 64.8

This experiments helps to understand the considerations necessary when developing biochemical assay for clinical use.

Determination of inter-assay variation

The assay was repeated 5 times using every patients sample for each reagent at a chosen incubation time. The coefficients for each set of assays was then calculated.

Results

Albumin values measured by all the methods had a strong correlation with each other.  The coefficients of correlation of albumin and total protein by the two methods had negligible differences. The colloid osmotic pressure was un-negligibly higher measured by the BCG method or by the long protein method as compared by the albumin measured by the BCP or total protein method. There occurred identifiable differences between the correlation coefficients of albumin as measured by the two methods. The experiment outlined that with a BCG short reaction time, the values of BCG correlated strongly with BCP values (r= 0.964) and was well paralleled over a very wide range of around 5g/L higher and the slope not differing significantly. Thus in a short reaction time of BCG, there is no need to change BCG to BCP in the diagnosis of abnormality since BCG has no clinical advantage over BCP. This is because the range of reference is so narrow to detect any change. The readings also parallel each other and also correlate strongly.

Discussion

There was a big difference in the absorbance values of the serum when different dyes were used. This is caused by the difference in reaction of the dye and the serum. The standard curves of bromocresol purple depict a more linear standard curve (more so when the albumin concentration is higher) as compared to the linear standard curves of bromocresol green method. We found out that bromocresol green method values are more comparable with measurements obtained from other techniques when compared to the values obtained when bromocresol purple is used. This gives BCG an advantage over BCP. In other  experiments such as those involving quality control and assurance, calibrations  methods, BCP will sharply  deviate from the expected values especially when non-human sera was analyzed. Because of accuracy and consistency, methods that are more specific should are recommended.

Research has shown that bromocresol green depicts nonspecific binding to non-albumin protein. However BCP is highly specific as compared to BCG method. The differences between these methods can be reduced but not completely done away with if the rapid-reaction technique is employed where absorbance is values are recorded after short incubation times.

The ideology to adopt a new method in the analysis to BCP is not advisable because it will lead to changed measurements for the similar analysis under investigation. The clinical staff will be forced to learn a new range of reference in the investigation of the abnormality and also to adjust the “limits of action” that have taken them a lot of years to acquire. The change will have to be put into account and BE revised adequately especially when dealing with patients who are under regular care. However, the new methodology can easily be adopted when it can convince medics that will improve the medical benefits of the measurements.

The ideology attached to changing automated dye-binding procedure for the albumin serum from bromocresol green to bromocresol purple is founded wholly on the non-specific reactions of bromocresol green with serum globulins. In the experiments involving long reaction times  the over approximation  of bromocresol green method is particularly marked t very low concentrations of albumin , these measurements show a higher spread  as compared with the reference methods. Analysis with BCP will underestimate the albumin in the blood serum for example in patients of pediatric hemodialysis it would give reading of 13g/L, BCG is however not affected and would give normal readings. Adults suffering from renal insufficiency when their blood serum is analyzed will also affect the BCP. From the experiment we confirmed that albumin can be falsely elevated up to 11g/L when plasma is used with BCP. The resemblance of correlation in turbidity and concentration of albumin in the blood serum is an evidence that the elevated results of the serum is caused by turbidity that increases the absorbance readings and hence leading to larger calculated values of concentrations.  Our data however did not show that the higher readings of results we obtained were due to the elevated color of BCP-albumin complex. It however showed that samples of plasma required blanks that are separate due to turbidity.

We were unable to single out any benefit of the bromocresol purple-albumin over bromocresol green that could convince us that patients will find it useful in the event of change. We found out that laboratories that continue to use BCG in the diagnosis their patients have been criticized for conservatism however, as long as a method that involves a short reaction time is used, we will advocate for conservatism.

Questions

1. a) Comment on the quality of standard curves.  

Standard curves allow for comparison of known properties of samples which are then used to determine the qualities of unknown samples at interpolation. Comparing the slopes and the coefficients we find that the coefficients are sufficient and therefore we can say that the curves are of good quality.

2. b) What is the usual acceptable CV for an albumin assay to enable its use in clinical diagnosis? 2.9% at mean values of 30.6 and 38.3g/L
   
   
3. c) Under which circumstances can the BCG assay give elevated readings for serum albumin?  Assay of BCG can give slightly higher values in patients who are suffering from hypo-albumin emic and are undergoing hemodialysis.

Or when fibrinogen is involved in the serum, e.g. in heparinized canine plasma due to specific binding to globulins.

1. d) What is the effect of heparin on albumin levels measured by both methods?

Heparin boosts the level of albumin in the samples. Since BCP is more specific it’s readings would not be much affected but BCG readings would be elevated since the concentration of the albumin is increased.

Heparin would only BCG readings since and not BCP because BCP is more specific to globulin

1. e) Which method can be adapted to account for the potential interference by heparin?

Bromocresol purple method.

1. f) Which pathophysiological circumstances lead to albumin being underestimated by the BCP assay?

Renal failure in patients who are undergoing hemodialysis care in hospitals. This because of a uremic toxin that binds to albumin and compete with BCP thus causing temporarily low results.

Reflection

Ligand macromolecule binding is a ubiquitous occurrence of medical importance to numerous disciplines of Science for example; pharmacology, endocrinology, enzymology, clinical chemistry and protein chemistry. Albumin can be investigated in biological fluids using methods such as radioimmunoassay, electro immunoassay, immune cephalometric and radial immunoassay.

These methods when used are very specific and highly sensitive. However, these methods are not recommended for routine use because they are very slow, expensive and very difficult to automate. In clinical chemistry labs, albumin is mostly measured using dye-binding techniques such as BCP and BCG. However, BCG is mostly used because of its strong binding to other serum proteins other than albumin.  BCP only binds with albumin ligand and is highly specific. These experiments acted as an eye opener to us as far as BCG and BCP debate is concerned.  BCP results with albumin gives moderately lower values when compared with BCG. The results however correlate well with specific immunological assays.

When heparin concentration is increased, the concentration of albumin is apparently increased. Consequently, precipitate is formed that may remain in the solution as a suspension if heparin is continuously added. Addition of heparin increases the concentration of albumin in the serum samples. When concentration of serum is increased BCG readings are affected but not BCP readings. This is because BCP readings are more specific to binding with the albumin.  This experiment had to be carried out with a lot of care because human blood is deemed to be very infectious.

References

Bartholomew R.J. & Delaney A., (1964). Spectrophotometric studies and analytical application of the protein error of some pH indicators. Proc Aust Assoc Clin Biochem, 1, 64- 67.

Carter P. (1970). Ultramicroestimation of human serum albumin: binding of the cationic dye 5, 5’dibromo-o-cresolsulfonphthalein. Microchem J, 15, 4, 531-539.

Doumas B.T & Peters T. (1997). Serum and urine albumin: a progress report on their measurement and clinical significance. Clinica Chimica Acta, 258, 3-20

Doumas B.T., Watson W.A. & Biggs H.G. (1971). Albumin standards and the measurement of serum albumin with bromcresol green. Clinica Chimica Acta, 31, 87-96.

Louderback A., & Mealy E.H. & Tayor N.A. (1968). A new dye-binding technique using bromcresol purple for determination of albumin in serum. Clin Chem, 14, 793-794.

Pinnell A.E. & Northam B.E. (1978). New automated dye-binding method for serum albumin determination with bromcresol purple. Clin Chem, 24, 80-86.

Rodkey F.L. (1965). Direct spectrophotometric determination of albumin in human serum. Clin Chem, 11, 478-487

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