A COMPARATIVE STUDY OF RENAL FUNCTION IN BETWEEN HEALTHY AND

THALASSAMIC POPULATION

PRESENTED BY:Name :

Aniruddha GaziStream : BiotechnologyRoll No: 21008210013Reg. No: 10102410002Super vised by :Dr. Sonali Paul

Thalassemia is an inherited disorder.

It leads to the decreased production and increased destruction of red blood cells.

  Hemoglobin in the red blood cells carry oxygen for all organs in the body.

The loss of red blood cells results in low hemoglobin. This leads to anemia. The decreased oxygen will impair the ability to maintain normal functions.

What Is Thalassemia

Types Of ThalassemiaThe various types of thalassemia have specific names related to the severity of the disorder. 

Alpha ThalassemiasAlpha thalassemia silent carrierAlpha thalassemia minor, also called alpha thalassemia trait.Hemoglobin H diseaseAlpha thalassemia major, also called hydrops fetalis.

Beta ThalassemiasBeta thalassemia minor, also called beta thalassemia traitBeta thalassemia intermediaBeta thalassemia major, also called Cooley's anemia or beta-zero (ß0) thalassemiaBeta-plus (ß+) thalassemiaMediterranean anemia

Of Thalassemia

Alpha Thalassemia:-Alpha globins are coded on chromosome 16(Four genes in each diploid cell).3 or fewer deletions have only mild anemia.4 deletions incompatible with lifeUsually no treatment indicated

Types Of Thalassemia

Alpha Thalassemia:-Alpha globins are coded on chromosome 16(Four genes in each diploid cell).3 or fewer deletions have only mild anemia.4 deletions incompatible with lifeUsually no treatment indicated

Beta Thalassemia:-Point mutations result in Beta-

ThalassemiasDisease results in an overproduction of a-globin chains, Erythropoiesis increases, sometimes becomes extra medullary.b-Thal- Types:-

b-Thalassemia Major-Cooley’s Anemia

b-Thalassemia Intermediab-Thalassemia Minor

Epidemiology of Thalassemia There are an estimated 60-80 million people in the world

who carry the beta thalassemia trait alone. This is a very rough estimate and the actual number of thalassemia Major patients is unknown due to the prevalence of thalassemia in less developed countries in the Middle East and Asia. Countries such as India, Pakistan and Iran are seeing a large increase of thalassemia patients due to lack of genetic counseling and screening.

In West Bengal:

Beta- thalassaemia is more common in West Bengal of India.

9.16% showed symptomatic patients of which commonest was E beta- thalassaemia followed by beta- thalassaemia.

Asymptomatic patients with abnormal haemoglobin was found in 15.99% of which beta- thalassaemia minor was the commonest. 

Thalassemia In India

Thalassemia is a serious inherited blood disorder. An estimated 40 million Indians are Thalassemia carriers and over 10,000 Thalassemia major patients are born every year.

Survival of a Thalassemia major depends upon repeated life long blood transfusions and iron chelation.

The disease can be prevented by creating awareness, genetic counseling and screening at pre-conceptual stage.

Thalassemia In West Bengal

Beta- thalassaemia is more common in West Bengal of India

9.16% showed symptomatic patients of which commonest was E beta- thalassaemia followed by beta- thalassaemia.

Asymptomatic patients with abnormal haemoglobin was found In 15.99% of which beta- thalassaemia minor was the commonest. 

Symptoms Of Thalassemia

Symptoms most often begin within 3-6 months of birth. Symptoms may include:• Anemia, which may be mild, moderate, or severe.• Jaundice.• Enlarged spleen, Tiredness, Reduced appetite.• Thickening and roughening of facial bones.• Bones that break easily, Teeth don't line up properly.•Increased susceptibility to infection•Growth problems•Hormone problems like Diabetes, Thyroid problems.

Complications of Thalassemia

Iron Overload : People with Thalassemia can get too much iron in their bodies, either from the disease itself or from frequent blood transfusions. Too much iron can result in damage to your heart, liver and endocrine system, which includes glands that produce hormones that regulate processes throughout your body.Infection : People with Thalassemia have an increased risk of infection. This is especially true if you've had your spleen removed.Bone deformities : Thalassemia can make your bone marrow expand, which causes your bones to widen. This can result in abnormal bone structure, especially in your face and skull.Slowed growth rates : Anemia can cause a child's growth to slow. Puberty also may be delayed in children with Thalassemia.

How Are Thalassemias Treated

Standard Treatments - Blood Transfusions

Iron Chelation TherapyFolic Acid Supplements Blood and Marrow Stem Cell

Transplant

Pathophisiology of Renal Dysfunction In Thalassemic Population

Progressive renal failure is one of the main complications in beta- thalassemia.

Renal dysfunction in thalassemia patients can be attributed to chronic anemia, and iron overload as well as to desferioxamine (DFO) toxicity.

Anemic patients had increased urinary zinc excretion (U(zinc)) and fractional excretion of sodium (FE(Na)) and uric acid (FE(UA)) compared with both controls and patients without anemia.

THE RENAL FUCTIONS MAINLY:Many of the kidney's functions are accomplished by relatively simple mechanisms of filtration, reabsorption, and secretion, which take place in the nephron. 

The kidneys excrete a variety of waste products produced by metabolism. Two organ systems, the kidneys and lungs, maintain acid-base homeostasis, which is the maintenance of pH around a relatively stable value.

Osmolality regulation

Blood pressure regulation

Hormone secretion

Renal Problems In Thalassemic Patient

THE ORIGINAL PROSPECTIVE STUDY SHOWED THAT:

In patient with and without chelation, glomerular Tubular dysfunctions and oxidative stress marker disturbances and were reported higher than controls.

In patients, with chelation glomerular dysfunction was significantly higher while,oxidative stress level was significantly lower than those without chelation.

Hyperuricemia and microscopic hematuria are more common in thalassemia intermedia than thalassemia major. Microscopic hematuria in thalassemia intermedia might be related to either hypercalciuria or hyperuricosuria.

Aims of The Project

To Compare The Renal Function in Between

Healthy and Thalassemic Individuals.

Analysis Performed In Laboratory are :

Urea Level AnalysisUric Acid Level Analysis

Creatinine Level Analysis

Materials Required In Laboratory Test

Study Group : Bloods were collected from 15 Thalassemic patients at IGE patient’s clinic & from 10 healthy controls.

KIT (Reagent) : Ecoline (Urea) Innoline (Uric Acid) Merckotest (Creatinine)

Instruments : Colorimeter , Sprectometer.

Other Requerments : Syringe,Gloves,Ependrops,Fusion Tubes, Tissue Paper Micropipette,Tips,Ependrop stands,Vacutiner, Distilled Water.

Methods for tests performed in lab:

1. Blood sample collection:

200-500 micromililtr venous blood was drawn from thalassemic patients and healthy individuals. Collected blood was left standing at room temperature ,then the sample serum was collected from suspended cells.

2. Then the serum was used for Uric Acid, Urea and Creatinine tests.

Analysis of Urea Level

Principle: Urea+2H2O---------------------2NH4+ + Co3

2-

NH4 + a- ketogluterate +NADH----------------L-Glu +NAD+ + H2O

L- Glu=L-GLUTARMATEGIDH= Gluarmate dehydrogenase

Urease

GLDH

Standard: Urea ------- 50mg/dl

Procedure: Wavelength:340nmTemparature: 370c

ONE –REAGENT PROCEDURE

BLANK STANDARD SAMPLE

Reagent R 1000µL 1000µL 1000µL

Standard ----- 10µL ------

Sample ---- ------ 10µL

Mix after 30sec at 370c ,measure A1.After 90secs. Further measure A2.

CALCULATION

AA

SampleStandard

×n

n= standard conc.

PRINCIPLE: Enzymatic determination of uric acid according to th following reactions:

Uric acid +2H2O + O2----------------------------- Allantoine+CO2+H2O2

2H2O2 + 4-EHSPT --------------------------------------- Quinoneimine +4H2O

EHSPT=N- Ethayl-N-(2-Hydrooxy-3-Sulphopropyl)m- Toluidine 4-AAP=4-Amino antipyrine

Standard: Uric acid: 6mg/dl

URICASE

Peroxidase

Analysis of Uric Acid Level

PROCEDURE:

This reagent can be used on most analysis,semi-analysis and manual method.

Wavelenghth: 546nm temparature: 370c

BLANK STANDARD SAMPLE

Reagent1 500µL 500µL 500µL

Distilled water

10µL ----- -------

Standard ------- 10µL --------

Sample -------- -------- 10µL

Mix and measure absorbance (A) after 325 sec incubation.

CACULATION

A Sample

A Standard

* n

n= Standard conc.

Analysis of Creatinine Level

Principle: Creatinine forms a yellow-orange compound in alkaline solution with picric acid. At low concentration of picric acid as used in this method. Precepitation of protein does not take place.As a result of rapid reaction between creatinine and picric acid,the secondary Reactions do not cause interference. The method thus distinguishes itself by its high specificity.

REAGENT PREPARATION Sample Start: Mix Reagent1 and Reagent2 in the ratio

1+1(e.g. 1ml of buffer solution and 1ml of picric acid solution.)Leave the monoreagent for t least 10min.at room temp. before using.

Assay Procedure

Wavelenghth (490-510nm) Light path 1cmTemparature

Sample Start

370c

Sample/Standard

Sample/Standard

Monoreagent 100µL 1000µL

Mix & read absorbance A1 after 60 sec read absorbance A2 after further 120sec.

Result Of Urea Level Analysis

2.35 fold higher In healthy than thalassemic Patient

Healthy

Patient0

5

10

15

20

25

30

Avarage

Column1

Patient Healthy0

0.5

1

1.5

2

2.5

3

3.5

Avarage

Series 1

Result Of Uric Acid Level Analysis

1.33 fold higher In patient than healthy

Result Of Creatinine Level Analysis

2.4 fold higher in thalassemic patient than healthy

Patient Healthy0

0.5

1

1.5

2

2.5

3

Avarage

Series 1

DISCUSSIONIn patients with β- thalassaemia major, the most important cause of mortality and morbidity is organ failure due to deposits of iron. In patient with beta- thalassemia major, blood urea level is not high. According to this I found that blood urea level is 2.35 fold higher in heathy population than patient.( By Renzo Galanello, Susanna Barella, Maria P. Turco, Nicolina Giagu, Antonio Cao, Fausto Dore, Nicola L. Liberato)

Serum uric acid was significantly higher in thalassemic patient.& I’ve also found that uric acid level in patient is 1.33 fold higher than healthy.

(Saudi Journal of Kidney Diseases and

Transplantation,)

In the mentioned journl 95.2% had raised serum creatinine levels.Accordingto this I found that there is 2.4 fold higher creatinine level in patient than healthy. (jpma.org.pk/full_article_text .php? article_id=3323)

The present STUDY SHOWED THAT, RENAL PROBLEMS IN THALASSEMIC POPULATION OCCUR DUE TO IRON OVRELOAD.

Conclusion

ACKNOWLEDGEMENTFirst of all I would like to express my sincere thanks to

the Principal of our institution Prof. AMIT CHAKRAVARTY & the vice

principal of our institutionDR. SUDIPA CHACRAVARTY for providing me all necessary

facilities for my project.I’m very much greatfull to my

Teacher Dr.SONALI PAUL for her support & guidance & to complete this seminar.

I would also like to thanks all of my faculty members for their help specially ANIRBAN SIR & PUSHPAL SIR.I also thanks my parents for their unceasing help & support. I also place on record ,my sense of gratitude to one

and all,who ,directly & IIndirectly lent their helping hand for this venture

REFERENCES:www.ncbi.nlm.nih.gov

www.nhlbi.nih.gov › ... › Health Topics

sickle.bwh.harvard.edu/menu_thal.html

www.medicinenet.com › home › alpha thalassemia index

www.ijponline.net/content/36/1/39

www.nephrology.uoi.gr/site/_.../146_Elisaf_Am_J_Hematol_2001.p

www.springerlink.com/index/L455J9JMGEU2MBE4.pdf