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ORIGINAL ARTICLE
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Choice of assay affects serum albumin measurements in HIV patients: A comparison of bromocresol green and bromocresol purple dye binding methods


 Department of Biochemistry, Armed Forces Medical College, Pune, Maharashtra, India

Date of Submission25-Feb-2021
Date of Decision20-Jul-2021
Date of Acceptance20-Jul-2021

Correspondence Address:
Pratibha Misra,
Department of Biochemistry, Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_157_21

  Abstract 


Background: Dye-binding assays – bromocresol green (BCG) and bromocresol purple (BCP) –are widely used for serum albumin measurement. Differences between the results reported by these assays can have important clinical implications. There are limited studies elucidating the differences between the results of two assays in HIV patients. We aimed to determine whether the choice of assay affects serum albumin measurements in HIV patients. Materials and Methods: Hundred newly diagnosed HIV patients were included in the study. The subjects were followed up for 6–12 months after initiating ART. Hundred HIV-negative healthy controls matched for age and body mass index were taken as controls. Serum albumin and three acute phase proteins (APPs) (Alpha-1 acid glycoprotein, C-reactive protein, ceruloplasmin) were measured. Serum albumin measurement was done by both BCG and BCP dye binding methods. Paired t-test was used to compare the groups. Results: The concentration of APPs reduces with ART indicating reduced inflammation, and is lowest in the control group. The difference in serum albumin concentrations measured by the two dye binding methods reduces with reduction in APPs. The study further indicates that the BCG method gives higher values of serum albumin as compared to BCP method in the presence of other proteins in the sample. Conclusion: The choice of albumin assay affects the serum albumin assessment of HIV patients. We recommend that the BCP assay be used to measure serum albumin in HIV patients, particularly in patients not on ART.

Keywords: Acute phase proteins, albumin estimation, anti-retroviral therapy



How to cite this URL:
Parmar A, Agarwal D, Saha TK, Somani BL, Misra P. Choice of assay affects serum albumin measurements in HIV patients: A comparison of bromocresol green and bromocresol purple dye binding methods. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 7]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=337799




  Introduction Top


Albumin, a classic nutritional index marker is the most abundant serum protein.[1] Albumin concentration is commonly measured in clinical laboratories using two dye-binding techniques, the bromocresol green (BCG) and bromocresol purple (BCP).[2],[3],[4] Differences between laboratory assays can have important clinical implications. For creatinine assays, this became apparent soon after introduction of the Modification of Diet in Renal Disease formula and resulted in international efforts towards standardization. Although the BCP method is more specific than the BCG method, the latter is still used worldwide due to its lower cost. According to a survey by the College of American Pathologists in 2011, the BCG method was used in 48% of the participating institutions.[5] Since differences between these assays have received limited attention in immunocompromised patients, we compared BCG and BCP assays using blood samples from patients with HIV infection. Thus, the purpose of this study was to elucidate whether the choice of albumin assay affects serum albumin measurements in HIV-infected patients.


  Materials and Methods Top


The study was carried out in the Department of Biochemistry of a tertiary care center in western India between December 2011 and Jul 2013. The therapy was started by following the WHO 2010 criteria.[6] Patients were treated with a combination of zidovudine, lamivudine, and nevirapine (2 NRTIs + 1 NNRTI).

Study subjects

The study was carried out in two groups as follows:

  1. HIV group - One hundred newly diagnosed HIV-infected patients in the age group of 18–55 years were enrolled into the study from the ART center of the institution, after complete history taking and detailed clinical examination
  2. Control group - Hundred HIV-negative healthy controls matched for age and body mass index (BMI) with the above groups were taken as controls.


Exclusion criteria

Patients with BMI >30 kg/m2, diabetes mellitus, preexisting renal or liver disease, and any active opportunistic infection.

Written informed consent was obtained from all the study participants.

The study was approved by the Institutional Ethical Committee (IEC No is 2248/MRU/IEC/2011NA).

Sample collection

Baseline blood samples were collected through a clean venepuncture after 12 h overnight fast. Albumin, alpha-1 acid glycoprotein, C-reactive protein (CRP), ceruloplasmin, and CD4 cell counts were estimated in each group. Repeat measurements of the above parameters were carried out in HIV group after 6, and 12 months of institution of ART.

Biochemical investigations

All the analytes were measured by fully automatic analyzer ERBA XL-600, except CD4+ cell counts which were done by BD FACS counter.

Albumin

Albumin was measured by both BCG and BCP dye binding methods.

Bromocresol green dye binding method

The principle of the method involves binding of albumin with BCG at pH 4.2 causing the change in yellow color of dye to blue – green, intensity of color so formed being measured at 630 nm (Reagent composition-BCG 0.08 mmol/L, succinate buffer (pH ± 0.1 at 25°C) 50 mmol/L, and sodium azide 1 g/L).

Bromocresol purple dye binding method

This method is based on binding of albumin with BCP dye at pH 5.2–6.8 changing the yellow color of the dye to blue – green, intensity of color so formed being measured at 630 nm (Reagent composition-BCP 40 mmol/L of absolute ethanol, sodium acetate trihydrate [AR grade], Brij-35 solution [poly-oxyethylene lauryl ether], glacial acetic acid).

C reactive protein and alpha-1 acid glycoprotein

Were analyzed by Immuno-turbidimetry method.

Ceruloplasmin-was analysed by ferroxidase kinetic method

CD4+ cell counts-were estimated in whole blood by BD FACS counter which works on the principle of flow cytometry.

Statistical analysis

Data were entered into Microsoft excel and analyzed using SPSS Statistics for Windows, version 17.0 (SPSS Inc., Chicago, Ill., USA). Descriptive statistics were expressed as mean ± standard deviation (SD). Paired' t-test was used to find the statistical significance between various groups.


  Results Top


Out of a total of 100 subjects enrolled in the study, 52 were male and 48 were female. Mean age (±SD in years) was 33.3 (±8.4) and mean BMI (±SD in kg/m2) was 20.46 (±4.432). The mean values of albumin by BCG method were found to be 3.96 ± 0.52, which is much higher than those obtained by BCP method 2.64 ± 0.99, the difference in the mean of the two methods being statistically significant (P < 0.05) in all the study groups [Table 1].
Table 1: Comparison of bromocresol green and bromocresol purple dye binding methods in HIV patients and control groups

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The difference in the mean levels of albumin by BCG and BCP method was found to decrease with therapy in cases. The difference in the mean levels of albumin by BCG and BCP method was lowest in the healthy control group. The difference was found to be 1.32 g/dL before starting ART; reducing to 0.94 g/dL in 06 months after instituting ART; further decreasing to 0.71 g/dL after 12 months of ART; and the lowest 0.47 g/dL in the healthy control group [Figure 1].
Figure 1: Comparison of albumin levels by BCG and BCP methods in various study groups

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  Discussion Top


The mean levels of serum albumin reported by BCG method were significantly higher (P < 0.05) than those reported by BCP method in all the study groups. This finding is in accordance with the studies of Gustafsson,[7] Slater et al.[8] and Webster[9] which explained the higher values of serum albumin reported by BCG method as a result of interference by the other acute phase proteins (APPs) present in the sample. No such interference was reported in the BCP method, indicating BCP method to be more specific for measuring serum albumin in patients with HIV infection.

The difference in the mean levels of serum albumin between the two methods was seen to reduce with therapy, being the lowest in control group. This can be explained by the simultaneous decrease in the mean values of positive APPs with therapy, being lowest in the control group. This is in accordance with a recent study by Ueno et al.,[10] which showed the difference of serum albumin values between BCG and BCP methods to be decreasing with decreasing concentrations of CRP.


  Conclusion Top


The choice of assay affected the values of serum albumin in patients with HIV infection, especially those not on ART. We recommend that the BCP assay be preferred over BCG assay for measuring serum albumin in HIV patients, particularly in patients not on ART.

Acknowledgments

We would like to acknowledge the contribution of the health workers of ART Centre of our institution for their help at each and every step during the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Peters T Jr. Serum albumin. Adv Clin Chem 1970;13:37-111.  Back to cited text no. 1
    
2.
Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chim Acta 1971;31:87-96.  Back to cited text no. 2
    
3.
Pinnell AE, Northam BE. New automated dye-binding method for serum albumin determination with bromcresol purple. Clin Chem 1978;24:80-6.  Back to cited text no. 3
    
4.
Frederichsen P, Kierulf P. A more accurate dye-binding method for the routine determination of serum albumin. Clin Chem 1979;25:1180.  Back to cited text no. 4
    
5.
College of American Pathologists. Survey Participant Summary, Chemistry/Therapeutic Drug Monitoring C-C2011. Northfield, IL: College of American Pathologists; 2011.  Back to cited text no. 5
    
6.
World Health Organisation. Antiretroviral Therapy for HIV Infection in Adults and Adolescents: Recommendations for a Public Health Approach-2010 Revision.  Back to cited text no. 6
    
7.
Gustafsson JE. Improved specificity of serum albumin determination and estimation of “acute phase reactants” by use of the bromcresol green reaction. Clin Chem 1976;22:616-22.  Back to cited text no. 7
    
8.
Slater L, Carter PM, Hobbs JR, Scientific and Technical Committee, Association of Clinical Biochemists. Technical Bulletin No. 34. Measurement of albumin in the sera of patients. Ann Clin Biochem 1975;12:33-40.  Back to cited text no. 8
    
9.
Webster D. The immediate reaction between bromcresol green and serum as a measure of albumin content. Clin Chem 1977;23:663-5.  Back to cited text no. 9
    
10.
Ueno T, Hirayama S, Ito M, Nishioka E, Fukushima Y, Satoh T, et al. Albumin concentration determined by the modified bromocresol purple method is superior to that by the bromocresol green method for assessing nutritional status in malnourished patients with inflammation. Ann Clin Biochem 2013;50:576-84.  Back to cited text no. 10
    


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