renal_function_test_2018.ppt

PRESENTED BY H GROUP
Mohini, monika R, Ankita, Kinjal, Kinal,
monika

 Kidneys are the organ that filter waste
products from the blood.
 The kidneys serve three essential function:
1. They function as filter, removing metabolic
product and toxins from the blood and
excreting them through the urine.
2. They regulate the body’s fluid status,
electrolyte balance and acid-base balance.
3. The kidney produce or activate hormones
that are involved erythrogenesis, Ca²˖
metabolism and the regulation of blood
pressure and blood flow.

 Renal function may be assessed by measuring
blood urea and serum creatinine. Renal
function decreases with age , which must be
taken into account when interpreting test
values.
 These tests primarly evaluate glomerular
function by assessing the glomerular
filtration
 In many renal diseases, urea and creatinine
accumulate in the blood because they are not
excreted properly
 These tests also aid in determining drug
dosage for drugs excreted through the
kidneys

 Renal function tests are use to detect the
presence of renal diseases and assess their
progress.

1. Urea
2. Ammonia
3. Para Thyroid Hormone
4. Calcium
5. Uric acid
6. Potassium
7. Creatinine clearance
8. Glomerular filtration rate

 In the muscles creatine is converted to creatine
phosphate which becomes the source of a high
energy phosphate bond for the immediate
reformation of ATP.
 Creatinine is the byproduct of muscle energy
metabolism and is produce at a constant rate
according to the muscle mass of the individual.
It is the substance that is easily excreted by the
kidney.
 By this method we can also estimate serum
creatinine and urine creatinine.

 Jaffe method
Principle:
In this colorimetric method creatinine reacts
with picrate ion formed in alkaline medium
to develop a red-orange colour. The colour
produced from the sample is then compared
in a colorimeter at wavelength of 520nm
with that produced by known amount of
creatinine under the same condition.
 Creainine + picric acid →creatinine picrate
(orange)

This divides into two types
1. Manual method and
2. Automated kinetic method
The automated kinetic assay of creatinine is a
method with the Jafee reaction but by using
special type of spectrometer or autoanalyser.

 To get clear supernatant, take 1ml of distilled
water, serum, sodium tungstat and sulfuric
acid.
 Mix it well and centrifuge it for 5 min.
 Label three tubes as blank, standard and test
respectively.
Blank Standard Test
Sample - - 2ml
d/w 2ml 2ml 2ml
NaOH 1ml 1ml 1ml
Picric acid 1ml 1ml 1ml

 Mix it well and incubate at room temperature
for 10 min.
 Take the OD against the blank at 520 nm.
Calculation formula:
Creatinine(mg/dL)= abs. of test/ abs. of
std.*std. conc.

 A measure of the amount of creatinine
eliminated from the blood by the kidney.
 The value is given in unit of millions per
minute, representing the volume of blood
cleared by the kidney per minute.
 Calculation:
C cr=U x V/P
Where, U = Urine creatinine concentration in
mg/dl, P = Serume creatinine in mg/dl and V =
Volume of urine in ml/mt.

 Male: 0.7-1.3 mg/dL
 Female: 0.4-1.1 mg/dL

High creatinine level causes:
 Acute and Chronic kidney disease
 Ureter obstruction
 Dehydration
 Glomerulonephritis

PTH is a hormone secreted by the
parathyroid gland
There are four parathyroid glands
located behind the thyroid

 To regulate calcium levels (in blood)
 The parathyroid glands major function – regulate
the calcium level in the body within a very narrow
range (8.5 – 10.2 mg/dl)so that the nervous and
muscular system can function properly.
 Activation of vitamin D is very essential for calcium
absorption from GI tract.
 Vitamin D has to be converted into 1,25-
dihydroxycholeciferol in the liver and kidney in the
presence of PTH.

Vitamin D 1,25- dihydroxycholeciferol
 PTH is also increased the formation of 1,25-
dihydroxycholeciferol from 25-
hydroxycholecalciferol.

 PRINCIPLE OF THE TEST
The DRG Intact PTH Immunoassay is a two-site
ELISA [Enzyme-Linked ImmunoSorbent Assay] for
the measurement of the biologically intact 84
amino acid chain of PTH.
One antibody is
prepared to bind only
the N- terminal PTH
1-34. and this
antibody is labeled
with horseradish
peroxides(HRP) for
detection.
And the another end
c-terminal bind with
biotinlaylded anti-
PTH.
Make a sandwich
complex necessary for
detection.
-incubation
-washed unbound
components
-add substrate (TMB)
-stopping solution(
1N sulfuric acid)
Yellow color which is
praposnal to PTH
concentration.

Sr.no aliqutes Reage
-nt 1
Reage
-nt 2
Cover
with
alumin
um foil
and
place it
on
rotator
set at
170
rpm
for 3
hr at
RT.
Wash 5
times
with
workin
g wash
solutio
n (
reagent
A)
Reagent B
Cove
r it
and
place
on
rotat
or at
170
rpm
for
30
min
at
RT.
Stop
solutio
n
OD
at
450
nm
CALIBRAT
OR A
0.0 50 µL 50 µL 150 µL 100µL
B 0.2 50 µL 50 µL 150 µL 100µL
C 0.3 50 µL 50 µL 150 µL 100µL
D 0.4 50 µL 50 µL 150 µL 100µL
E 0.5 50 µL 50 µL 150 µL 100µL
F 0.6 50 µL 50 µL 150 µL 100µL
CONTROL
0.5 50 µL 50 µL 150 µL 100µL
2 0.5 50 µL 50 µL 150 µL 100µL
sample 0.5 50 µL 50 µL 150 µL 100µL

 Where,
 Reagent 1= biotinylated PTH antibody
 Reagent 2= peroxidase labeled PTH ab
 Reagent A= saline with surfactant
 Stop solution = 1N sulfuric acid
 Referance range := 10-65 pg/ml

Introduction
Uric acid is a naturally occuring waste
product resulting from the breakdown of
purine,crystalline compound found in certain
foods.
Under normal condition, uric acid dissolves
in the blood, passes through the kidney and is
eliminated with the urine.

Sometime the body produces too much uric
acid or doesn’t filter out enough of it and that
time uric acid level increase in blood.
Main two condition are observed,
1. Hyperuricemia
2. Hypouricemia

 Introduction
A uric acid blood test also known as a serum uric
acid measurment, determine how much uric acid
is present in your blood.
The test can help determine how well your body
produce and removes uric acid.

 Intended use
This reagent is for in vitro determination of
uric
acid in serum/plasma.
 Clinical significance
Uric acid is a metabolism of purines, nucleic
acids and nucleo-proteins. Consequently,
abno-
rmal levels may be indicative of a disorder in
the metabolism or in some genetic diseases.

 Causes of increase and decrease of uric acid
level
- High level of uric acidin your blood can
also indicate of a variety of conditions,
inluding:
- diabetes
- gout (acute arthritis)
- chemotherapy & radiation
- leukemia(bone marrow disorders)
- hypoparathyroidism
- kidney disorders(stones)
- multiple myeloma
- metastasized cancer

- low levels of uric acid in the blood may
inluding:
- wilson’s disease
- fanconi syndrome(cystinosis)
- alcoholism
- liver or kidney disease
- a diet low in purines

 Methodology
1. chemical method
phototungstic acid method
2. enzymatic method
The reagent is based on Trinder’s reaction,
enzymatic and colorimetric method.

 Raference range
serum/plasma
for women – 2.5-6.8mg/dl
for men – 3.6- 7.7mg/dl

 Limitations
Limitations of uric acid testing are as follows:
- Methodological interference and in cases
of vitamin C, levodopa, and alphamethyldopa.
- Early purine rich diet
- several exercise increases uric acid
- Rapid degradation of uric acid,which
occurs at room temp.in the plasma of
patients with tumor lysis syndrome.

 Urea is the chief nitrogenous waste of body.
 Urea is the end product of protein
metabolism.
 After filtered by glomeruli. It is partilly
reabsorbed by the renal tubules.
Methodology:
1. Kinetic method
2. Enzymatic method

 Urea in the sample is hydrolized enzymatically
into ammonia(NH4+) and carbon dioxide (co2).
 Ammonia ions formed reacts with α-
ketoglutarate in a reaction catalysed by glutmate
dehydrogenase(GLDH) with simultaneous
oxidation of NADH to NAD+.
Urease
 Urea + 2H₂O 2NH₄⁺+ CO₃²⁻
GLDH
 NH₄ ⁺ +2-Oxoglitarate +NADH L-glutamate +NAD ⁺+H₂O

 The decrease in concentration of NADH, is
proportional to urea concentration in the
sample.
ASSAY PROCEDURE:
Mix well and aspirate standard followed by
samples.
PIPETTE IN TUBES MARKED STANDARD TEST
WORKING REAGENT 1000μl 1000μl
STANDARD 20μl -
SAMPLE - 20μl

Urea (mg/dl)=(∆ Abs of test/∆Abs of
std)*conc. Of standard(mg/dl)
REFERENCE VALUE:
serum/plasma: 13-45mg/dl

 Urea clearance test is less than the GFR and it is
influnced by the protein content of the diet.
 Approximately 40% of filtered urea is normaly
reabsorbed by tibules
 The sensitivity of urea clearance is much less than
the creatinine clearance because plasma
concentration of urea is affected by number of
factors.
 Like, Dietary protien
fluid intake
infaction
surgery, etc.
 Nornaml value of urea clearance: 75% ml/min.
 Urea clearance is defined as the volume(ml) of
plasma that would be completely cleared of urea
per minue.

 It is calculated by the formula:
Cm= U*V/P
Cm= Maximum Urea clearance.
U = Urea concentration in urine (mg/dl).
V = Urine excreted per minute in ml.
P = Urea concentration in plasma.
 If the output of urine is more than 2ml per
minute.
 This is referred to as maximum urea
clearance.

Standard Urea Clerance:
 the urea clearance drastically changes when
the volume of urine is less than 2ml/min.
 This is known as standard urea clearance(C)
and the and the normal value is around
54ml/min.
Diagnostic importance:
 A Urea clearance value below 75% of the
normal is serious. Since it is an indicator of
renal damage.
 Blood urea level is found to increas only when
the clearance falls below 50% normal.
 Normal level of blood urea:20-40 mg/dl.

Pre-renal condition:
-Dehydration: Severe vomiting, intestinal
obstruction, diarrhea, diabetic coma, severe
burns, fever and severe infections.
Renal diseases:
1. Acute glomerulonephritis
2. Nephrosis
3. Malignant hypertension
4. Chronic pyelonephritis

 Urea concentration in serum may be low in
late pregnancy, in starvation, in diet grossly
deficient in protein and in hepatic failure.
 Azotemia:=
 Increase in the blood level of
NPN(creatinine,urea, uric acid) is referred to
as azotemia and is the hallmark of kidney
failure.

 Calcium plays an important role in:
 nerve impulse transmission,
 muscle contraction,
 pancreatic insulin release,
 as a core factor for some enzyme reactions
and blood coagulation,
 and most important bone and tooth
structural integrity. Normal total calcium
values 8.8‐10.3 mg/dl.

 Hypocalcemia usually implies a deficiency in
either the production or response to
parathyroid hormone(PTH) or vitamin D
 Hypercalcemiais an increased calcium
concentration and it is usually associated with
malignancy or metastatic diseases.

 Younger than 12 months : not established
 Age 1-14 yr : 9.6-10.6 mg/dl
 Age 15-16 yr : 9.5-10.5 mg/dl
 Age 17-18 yr : 9.5-10.4 mg/dl
 Age 19-21 yr : 9.3-10.3 mg/dl
 Age 22 yr and older : 8.9-10.1 mg/dl

 Principle: calcium in an alkaline medium
combines with o – Cresolphthalein
complexone to form a purple coloured
complex. Intensity of the colour formed is
directly proportional to the amount of
calcium present in the sample.
 Calcium + OCPC Purple coloured complex

 Wavelength/filter: 570nm(Hg 578 nm)/yellow
 Temperature : R.T.
 Light path : 1 cm
 Pipette into clean dry test tubes labelled as
blank(B),standard(S), and Test(T)

 Mix well and incubate at R.T.(25 ℃) for 5
min. Measure the absorbance of the
standard(Abs.S), and test sample(Abs.T)
against the blank,within 60 min.
Addition sequence
Buffer Reagent(L1)
ColourReagent(L2)
Distilled water
Calcium Standard
(S)
Sample
0.5 0.5 0.5
0.5 0.5 0.5
0.02 - -
- 0.02 -
- - 0.02
B S T
(ml) (ml) (ml)

 Calcium in mg/dl = Abs.T
______ X 10
Abs.S

 Principle : Calcium combines specifically with
Arsenazo iii at a neutral pH to forma blue
purple coloured complex. Intensity of the
colour formed is directly proportional to the
amount of calcium present in the sample.
 Calcium + Arsenazo iii Blue purple coloured complex

 Wavelength/filter: 650nm(Hg 623nm)/Red
 Temperature : R.T.
 Light path : 1 cm
 Pipette into clean dry test tubes labelled as
blank(B),standard(S), and Test(T)

 Mix well and incubate at R.T.(25 ℃) for 5
min. Measure the absorbance of the standard
(Abs.S), and test sample (Abs.T) against the
blank,within 60 min.
Addition sequence
ColourReagent(L1)
Distilled water
Calcium Standard
(S)
Sample
1.0 1.0 1.0
0.01 - -
- 0.01 -
- - 0.01
B S T
(ml) (ml) (ml)

 Potassium is the most abundant intercellular cation approximately 3500
mEq of potassium is contained in the body of a 70 kg adult. Only10% of
the body potassium is extracellular. Normal values are 3.5‐5.0 mEq/L or
mmol/L
 The role or function of potassium is in the maintenace of proper electrical
conduction in cardiac and skeletal muscles.
 Potassium measurements are used to monitor electrolyte balance in a
diagnosis and treatment of disease condition characterized by low or high
blood potassium levels.

Potassium is regulated by:
 Kidneys
 Aldosterone
 Arterial pH
 Insulin
 Potassium intake
 Sodium delivery to distal tubules

 An Ion-Selective Electrode makes use of the
unique properties of certain membrane
materials to develop an electrical potential for
the measurement of ions in solution.
 The potassium electrodes are based on
neutral carriers.

 Serum :
K+ : 3.3 – 5.1 mmol/L
 Plasma :
K+ : 3.3 – 4.5 mmol/L
 Urine :
K+ : 25 – 125 mmol/L (diet dependent)

Interpritation:
 Hypokalemia can occur. The kidneys are
responsible for approximately 90% of daily
potassium loss. Other losses occur mainly
through GI system.
 A low potassium level has many causes but
usually results from vomiting, diarrhea,
adhrenal gland disorders.
 A low potassium level can make muscles feel
weak, cramp, twitch, or even become
paralyzed, and abnormal heart rhthms may
develop.
 Hyperkalemia most common results from
decreased renal elimination, excessive intake
or from cellular break down.

 Ammonia combines with α-ketoglutarate and
NADPH in the presence of glutamate
dehydrogenase (GLDH) to yield glutamate and
NADP+. The corresponding decrease in
absorbance at 340 nm is proportional to the
plasma ammonia concentration.
GLDH
 α-ketoglutarate + NH₃+NADPH
glutamate+ NADP⁺

 1. Assay conditions:
 Wavelength: 340nm
 *Cuvette: 1 cm light path
 Constant temperature:25/30/37ºC
 * Please try not to use flow cell. Exchangeable
cuvettes are suggested to avoid cargover in
manual photometers.
 2. Adjust the instrument to zero with distilled
water.
 3. Pipette into a cuvette:

WR blank standard Sample
Sample - - 0.1ml
Distilled water 0.1ml - -
Standard - 0.1ml -
Reagent 1.0ml 1.0ml 1.0ml
4. Mix, and allow to stand for 5 min. Read initial absorbance of sample and
blank (A1).
5. Then add:
GLDH (R2) = 0.01 mL 0.01 mL 0.01 mL
6. Mix, and incubate for 5 min. Read final absorbance of sample and blank
(A2).
CALCULATIONS:=
Ablank = Blank A1 - Blank A2
Asample = Sample A1 - Sample A2

 Conc. of Asample - Ablank
 Ammonia = ---------------- x Standard conc
 Astandard - Ablank
 REFERENCE VALUES(2)
 Plasma ammonia: 10 - 47 μmol/L
 0.17 - 80 μg/dL
 0.017 - 0,080 mg/dL

Glomerular filtration rate(GFR) is the volume
of fluid filtered from the renal(kidney)
glomerular capillaries in to the Bowman’s
capsule per unit time.
Central of the physiological maintenance of
GFR.

 GFR is equal to the clearance rate when any
solution is freely filtered & is neither
reabsorbed non secreted by the kidneys.
 GFR is typically recorded in units of volume
per tine
 EX: milliliters per minutes(ml/min), compare
to filtration fraction

 The rate at which plasma is filtered by the
kindney glomeruli.
 An important measurement in the evaluation
of kidney fuction
 GFR = 125 ml plasma/min or, 180 L/day
 Plasma volume (70-kg young adult man)=
about 3L, the Kidneys filter the plasma some
60 time in a day.

1. Change in renal blood flow
2. Glomerular capillary hydrostatic pressure
3. Change in capsular hydrostatic pressure
4. Oncotic pressure
5. Glomerular capillary permeability
6. Effective filtration surface area
7. Size, shape & electrical change of the
macromolecules

 Modern imaging techniques
 Measuring renal clearance of various
substances

1. Nephrotic syndrome
2. Nephritic syndrome
3. Single kidney

 Measurement or calculation of the
glomerular filtration reat(GFR)
 Considered the most sensitive chemical test
for assessing function
 Collect a 24 hour or timed urine speciment &
a blood specimen from the patient
 Measure the creatinine in the serum $ urine
soecimen
 Calculate the clearnce

 eGFR=30849.2 X (serum creatinine)-
1.154(age)-0.203
 (If female X (0.742))

renal_function_test_2018.ppt

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renal_function_test_2018.ppt