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International Journal of Current Research inMedical Sciences

ISSN: 2454-5716www.ijcrims.com

Volume 2, Issue 4, April -2016

Original Research Article http://s-o-i.org/1.15/ijcrms-2016-2-4-5

Assessment of the level of haemoglobin and erythropoietin inpersons living with HIV in Umuahia

*Obeagu, E.I1., Ochei, K.C.2, Okeke, E.I.3 and Anode, A.C.3

1.Diagnostic Laboratory Unit, Health Services Department, Michael Okpara University of Agriculture,Umudike, Abia State, Nigeria.

2. Department of HSS & Laboratory, FHI 360 country office, Garki -Abuja Nigeria.3.Haematology Department, Federal Medical Centre, Umuahia, Abia State,Nigeria.

*Corresponding author

Abstract

The study was done to assess the level of and haemoglobin and erythropoietin in persons living with HIV inUmuahia. A total number of one hundred and ten (110) subjects were recruited for the study. The subjects weredivided into two groups; the first group consists of HIV positive subjects (test group) and the second group consists ofHIV negative subjects (control group).Serum sample was used for the estimation of erythropoietin. Sandwich ELISAmethod was used to estimate erythropoietin e estimation of haemoglobin. while EDTA anticoagulated blood was usedfor t. The results were analysed using t-test with level of significance set at P<0.05.The results showed significantdecrease (p<0.05) in both erythropoietin and haemoglobin level of the HIV patients (6.21±3.46 Mu/Ml,10.42±1.85g/dl) compared to HIV negative subjects (13.63±5.19 Mu/Ml,14.31±2.36 g/dl) respectively. The study showed thatthere was derangement in the kidney of the HIV patients which decreased erythropoietin synthesis and release andinduced anaemia in the patients.

Keywords: Erythropoietin, Haemoglobin, HIV, Umuahia.

Introduction

Erythropoietin is one of the primary regulators oferythropoiesis (Bunn,2013;Mastromario et al.,2011). In the bone marrow, erythropoietinpromotes the proliferation of erythroid progenitorcells and increase the production of erythrocytes.According to Kertesz et al.(2004),erythropoietinis a principal haematopoietic growth factorresponsible for the proliferation, survival anddifferentiation of erythroid progenitor cells.Erythropoietin binds through specific binding toerythropoietin receptor (EpoR).Erythropoietintriggers a chain of intracellular signalling

events, including activation of the receptor-associated tyrosine kinase JAK2 andphosphorylation and nuclear translocation ofSTAT5,leading to progenitor cell proliferation,inhibition of apoptosis and differentiation(DʹAndrea et al.,1989;Watowich et al.,1996).

Anaemia is one of the most common blooddisorders seen in people living with HIV. Theprevalence of anaemia ranges from 10% in peoplewho have no symptoms to 92% in individuals

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with advanced AIDS according to Levine andLeitz (2003).

The pathogenesis of anaemia during HIVinfection is often multifactorial, and contributingfactors include iron deficiency,the anaemia ofchronic disease associated with HIV,malaria andopportunistic infections (Semba,2001);however,itis responsible to iron and epoietin alpha therapy.

The pathogenesis of HIV infection affects manyorgans of the body including kidney. This maylikely affect the level of erythropoietin which willaffect erythropoiesis in the patients(Martinez andGattell,1999).Erythropoietin level in blood arequite low in the absence of anaemia around10Mu/Ml. However, in hypoxic stress,erythropoietin synthesis may increase 1000-fold,reaching 10,000Mu/Ml of blood.

Aim

To determine the level of erythropoietin inpersons living with HIV in Umuahia..

Materials and Methods

Study Area: The study was done inUmuahia.HIV patients were recruited from thoseconfirmed of HIV infection in Health ServicesDepartment of Michael Okpara University ofAgriculture, Umudike, Abia State, Nigeria andDaughters of Mary Mother of Mercy HospitalAhieke, Ihe Ndume, Umuahia.

Ethical consideration: Informed consents wereobtained from the subjects prior to samplecollection and confidentiality assured to them.

Study population and enrolment: A totalnumber of one hundred and ten(110) subjectswere recruited for the study. The subjects weredivided into two groups, consisting of fifty (50)HIV positive subjects(test group) and sixty (60)HIV negative subjects (control group).They wererecruited from the HIV patients and theapparently healthy subjects attending HealthServices Department, Michael Okpara Universityof Agriculture, Umudike, Abia State, Nigeria andDaughters of Mary Mother of Mercy Hospital,Ahieke, Ihe Ndume, Umuahia.

Selection criteria: The test subjects wererecruited after been established of having HIVinfection using national serial algorithm and notreactive to any other viral infection and withoutany AIDS indicator conditions. The control groupon the other hand was recruited after beenestablished of not having HIV or any other viralinfection and were apparently healthy.

Exclusion criteria: The subjects showing anyunderlying chronic illness other than HIVinfection (test group) and reactive to any otherviral infections were excluded from the study andalso exluded were pregnant women.

Sample collection: Using 5ml syringe,5ml offresh venous blood was drawn from each studyparticipant by a clean vein puncture from theantecubital vein and delivered into plain tubes andallowed to clot and retract.Serum was separatedand transferred into clean test tubes for estimationof serum erythropoietin.

Laboratory methods and procedures

The reagents were commercially purchased froma reputable supplier and manufacturers’ standardoperating procedures were duelly followed.Erythropoietin was estimated using ab119522Erythropoietin Human ELISA kit(AbcamPlc,Science Park,Cambridge,UK.).

HIV Screening: Two different HIV screeningkits were used (determine and Unigold test kits)for the detection of HIV-seropositivity followingserial algorithm.

Serum Erythropoietin Assay (SandwichELISA Method)

Principle: Erythropoietin specific antibodies havebeen precoated out in the well plates. Standardsand test samples were added to the wells alongwith a biotinylated erythropoietin detectionantibody and the microplate is then incubated atroom temperature. Following washing with washbuffer a streptavidin-HRP conjugate is added toeach well, incubated at room temperature andunbound conjugates are then washed away usingwash buffer.TMB is then added and catalysed byHRP to produce a blue colour product that

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changes to yellow after addition of an acidic stopsolution. The density of yellow colouration isdirectly proportional to the amount oferythropoietin captured in plate.

Procedure

Microplate was washed twice with 400µl washbuffer per well with thorough aspiration ofmicroplate contents between washes.Wash bufferwas added and allowed remain in the wells for10seconds before aspiration. After the last washstep, wells were emptied and microplate tappedon absorbent pad to remove excess washbuffer.100µl of prepared standard was added tothe appropriate wells.50µl of sample diluent wasadded to appropriate wells. 50µl of Biotinconjugated antibody was added to all wells. Thewells were covered with adhesive film andincubated at room temperature for 1 hour. Afterincubation, the adhesive film was removed andthe wells were emptied. The microplate stripswere washed 6 times with 400µl wash buffer perwell with thorough aspiration of microplatecontents between washes.100µl of sreptavidin-HRP was added immediately to all wells,including the blanks wells. Microplate wascovered with an adhesiv film and incubated atroom temperature for 15 minutes. Afterincubation, microplate was removed and wellswere emptied. Microplate was washed 6 timeswith 400µl wash buffer per well with thoroughaspiration of microplate contents betweenwashes.100µl of TMB substrate solution wasadded to all wells immediately after washing.

Then the microplate was incubated at roomtemperature for 10 minutes. Enzyme reaction wasstopped by adding 100 µl of stop solution intoeach well. Absorbance of each each microplatewas read on a spectrophotometer using 450nmwavelength. The plate reader was blanked withblank wells. The absorbance of both the samplesand the standard were determined.

Haemoglobin Estimation (Cynmeth Method)

Principle: The conversion of heamoglobin ironfrom the ferrous to the ferric state by theferricynanide component of Drabkin’s solution toform methaemoglobin combines with thepotassium cynanide in the solution to produce thestable cynamethaemoglobin. The resultingcoloured solution which is produced is thenmeasured in a spectrophotometer at a wavelengthof 540nm.

Procedure:1 in 201 dilution of blood was madeby adding 20µl of blood to 4ml of diluent(Dbrabkin’s solution) into a test tube. The testtubes were stoppered and inverted several timesand were then poured into a cuvette and theabsorbance were read in a spectrophotometer at540nm against the reagent blank. Thehaemoglobin level was calculated.

Statistical Analysis: The analysis was done usingt-test with the statistical package for socialscience(SPSS) version 17 and the values wereexpressed as mean ± standard deviation (SD) andstatistical significance set at P<0.05.

Results

Parameters HIV Positive HIV Negative p-levelErythropoietin (mu/ml) 6.2±3.46 13.63±5.19 p<0.05Haemoglobin (g/dl) 10.42±1.85 14.31±2.36 p<0.05

Discussion

The study showed that there was significantdecrease (P<0.05) in serum erythropoietin level ofHIV positive subjects when compared with HIVnegative subjects. The result agreed with thestudy done by Adebola et al. (2003), whichreported reduced EPO response among HIVanaemic subjects and also agreed with the work of

Kanyeki et al. (2013), which showed decreasedserum erythropoietin level in HIV positivesubjects. These decrease in serum erythropoitinlevel in HIV patients could be due to derangementof the kidney caused by the oxidative effect ofHIV with release of free radicals on the organs inthe body (Martiuez and Gattel, 1999, Salman andBerrula, 2012).

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This study is in agreement with the work ofChristiani et al. (2009) who showed that at a cutoff of 12.0g/dl, 82.40% of the test subjects wereanaemic with 4.10% of the control group alsobeen anaemic. Also Rapaport (1987) reported theerythropoiesis fail to increase in chronic disease.

It was reported that HIV status could result todecrease in erythropoiesis, which can have effecton other related parameters (haemoglobin andhaematocrit), independent of the effect on serumiron (Walsh et al.,2010). Haemoglobin is low inHIV-infected women compared to HIV-uninfected women according to Walsh etal.(2012).

The pathogenisis and pathophysiology of anaemicin HIV infection is multifactorial as this rangesfrom direct infection of the erythroid progenitors(Krenzer and Rockstroh, 1997). There are threemajor mechanisms involved in the pathogenesisof anaemia in HIV infection, these are : decreasedred blood cell production which is consequent toinfiltration of the bone marrow by neoplastic cells(Sipas et al., 1999), or infection(Hambleton,1996), decreased production ofendogenous erythropoietin, a blunted response toerythropoietin (Spivak et al.,1989) andhypogonadism. The second mechanism isincreased red blood cell destruction (Levine,1999). Thirdly, ineffective deficiencies mostcommonly in iron, folic acid, vitamin B12(Levine, 1999).

Conclusion

The study showed a significant decrease in bothserum erythropoietin level and Haemoglobin levelin the persons living with HIV in Umuahia. Thisshows that the kidney of these patients arederanged and should be taken care of because thatwill hinder survival of some of the patients due toanaemia that was seen in them. Proper care andattention should be given to them both by thesociety and the health workers who are involvedin the management of the patients. The patientsshould eat balanced diets to build up theirhaemoglobin level.

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How to cite this article: Obeagu, E.I., Ochei, K.C., Okeke, E.I. and Anode, A.C. (2016). Assessment ofthe level of haemoglobin and erythropoietin in persons living with HIV in Umuahia. Int. J. Curr. Res.Med. Sci. 2(4): 29-33.

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