DMLT (1st Year) : Blood Hemoglobin, Blood Glucose estimation and Urine routine examination - Some basic concepts (U. P. State Medical Faculty syllabus) in ENGLISH & HINGLISH

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SOME BASIC CONCEPTS OF BIOCHEMISTRY FOR DMLT FIRST YEAR STUDENTS (U. P.
State Medical Faculty syllabus) in English & Hinglish
[Blood Hemoglobin (Hb or Hgb) Estimation, Blood Glucose Estimation and Urine
routine examination]
IN ENGLISH
BLOOD HEMOGLOBIN (Hb or Hgb) ESTIMATION
Hemoglobin (Mr 64,500) is a tetrameric protein (four polypeptide chains) present in RBCs and it
consists of two α (each made of 141 amino acids) and two β polypeptide chains (each made of
146 amino acids) and to each polypeptide chain (globin) is attached a 'Heme' prosthetic group
(a complex of iron in ferrous form and porphyrin).
Forms of Hemoglobin - The normally occurring forms of hemoglobins are:
● Hemoglobin A (adult hemoglobin) - α2β2 - found normally in adults and is 95% of total
hemoglobin.
● Hemoglobin A2 - α2δ2 - 1.5–3.5% in adults. It may be increased in people with beta
thalassemia or in people heterozygous for beta thalassemia gene.
● Hemoglobin F (fetal hemoglobin) α2γ2 - found in fetuses and newborn babies in
proportions up to 90%, falls gradually within 2 years to 1% of total hemoglobin and less
than 1% throughout adult life.
Function of Hemoglobin -
1. Hemoglobin binds oxygen reversibly in the lungs where oxygen concentration is high (to
form oxyhemoglobin) and then delivers oxygen to the body tissues where oxygen
concentration is low (to form deoxyhemoglobin).
2. It also facilitates the exchange of carbon dioxide between the lungs and tissues.

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Methods of Hemoglobin estimation :
There are several methods for hemoglobin estimation :
● Sahli’s or acid hematin method
● Cyanmethemoglobin method
● Sodium Lauryl Sulphate method (cyanide free method)
● Vanzetti Azide-methemoglobin method
● Oxyhemoglobin method
● Alkaline-hematin method
● Measurement of oxygen-combining capacity
● Measurement of iron content
The most commonly used methods for hemoglobin estimation are Sahli's and
Cyanmethemoglobin methods.
Sahli’s or acid hematin Method -
Principle: Blood is mixed with N/10 HCl (0.1 N HCl) resulting in the conversion of Hb to
acid hematin which is brown in color. The solution is diluted till it’s color matches with the brown
colored glass of the comparator box of hemoglobinometer. The concentration of Hb is read
directly as g%. It is an inexpensive, quick and easy to perform method but suffers from
disadvantages such as less accuracy, individual variations in colour matching, lack of true
standard and not all hemoglobins (oxyhemoglobin, sulfhemoglobin) are converted to acid
hematin.
Cyanmethemoglobin method - is the internationally recommended method for determining
hemoglobin. The basic principle of this method is as follows : When blood is diluted in a solution
(Drabkin's reagent) containing potassium ferricyanide, potassium cyanide and potassium
dihydrogen phosphate, the potassium ferricyanide oxidizes the iron in heme of hemoglobin to
the ferric state to form methemoglobin, which is converted to hemiglobincyanide or
Cyanmethemoglobin (HiCN) by potassium cyanide. HiCN has a maximum absorbance at 540
nm and strictly obeys Beer-Lambert’s law. The absorbance of the diluted sample at 540 nm is
compared with absorbance of a standard HiCN solution whose equivalent hemoglobin
concentration is known.
K3[Fe(CN6)]
Hemoglobin (Fe++) ------------------> Methemoglobin (Fe+++)
KCN
Methemoglobin (Fe+++)-----------> Cyanmethemoglobin (HiCN)

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Reagent:
Drabkin's reagent preparation :
In a 1 liter volumetric flask add -
Potassium ferricyanide K3[Fe(CN)6] - 200 mg
Potassium cyanide (KCN) - 50 mg
Dihydrogen potassium phosphate (KH2PO4) - 140 mg
Non-ionic detergent (e.g. Triton X-100) - 1 ml
Add distilled water to dissolve and dilute to 1 liter with distilled water. Check the pH which
should be 7.0 - 7.4. Store in a dark coloured polythene bottle or brown coloured glass (actinic)
bottle at 4 - 20 °C (do not freeze). Discard the solution if found to be turbid or the pH is outside
range.
Cyanmethemoglobin (HiCN) standard -
Commercial HiCN standard (normally 60 mg/dl) calibrated with international standard solution
(primary calibrant) as per specifications defined by the International Council for Standardization
in Hematology (ICSH) is available.
Procedure -
● Bring Drabkin's solution and standard solution to RT
● Take 5 ml Drabkin's solution in a clean test tube (mouth pipetting is prohibited)
● Mix the blood sample (EDTA tube) by gentle inversions,
● Draw 20 µl blood in a micropipette, wipe the outer surface of the tip (very carefully
without touching the orifice) to remove the excess blood,
● Deliver the blood into the Drabkin's solution in the test tube by dipping the tip in the
solution and rinse the tip several times as shown below :
● Mix well (vortex mixer may be used) and leave it for at least 5 minutes.
● Set the colorimeter or spectrophotometer absorbance at 0 using blank solution
(Drabkin's solution) at yellow green filter or 540 nm

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● Now read the absorbance of standard solution; alternatively, a standard curve can also
be drawn by using different concentrations of standard made by diluting the standard
solution with Drabkin's solution.
● Then read the test solution absorbance
Calculation -
Absorbance of unknown × concentration of standard(mg/dl) × 251
Blood Hemoglobin(g/dl) = -----------------------------------------------------------------------------------------
Absorbance of standard × 1000
(251 is the dilution factor because 20 µl (0.02 ml) blood sample was added to 5 ml Drabkin's
solution, so the final volume was 0.02 ml + 5.0 = 5.02 ml. Therefore, the blood is diluted with a
dilution factor 5.02 ÷ 0.02 = 251. Standard was not diluted)
Advantages of this method
● Accurate International standard available worldwide ;
● Easily adapted to automated hematology analyzers;
● Well established and thoroughly investigated – ICSH recommended method ;
● Inexpensive reagent.
Disadvantages
● Manual method requires accurate pipetting and spectrophotometer;
● Reagent (cyanide) is hazardous;
● The above limitations, further limit its use outside the laboratory;
● Subject to interference from raised plasma lipids, proteins concentration and leukocyte
numbers causing turbidity (Centrifuging the diluted blood can, however, help overcome
the turbidity);
● Does not distinguish those hemoglobin derivatives which have no oxygen-carrying
capacity (MetHb, COHb, SHb). Thus may overestimate the oxygen-carrying capacity of
blood if these are present in abnormal (more than trace) amounts.
Sodium Lauryl Sulphate method (cyanide free method) - Sodium Lauryl Sulphate (SLS) is a
surfactant which lyses erythrocytes and then hemoglobin released from RBCs rapidly forms a
complex with SLS. The product SLS-MetHb is stable for a few hours and has a characteristic
spectrum with maximum absorbance at 539 nm. The results of hemoglobin concentration by the
SLS-Hb method correlate very closely with the reference HiCN method. This method has been
adapted for automated hematology analyzers also.
Advantage - non-toxic reagent and less affected by lipemia and leukocytes interference.
Disadvantage - SLS-MetHb is less stable than Cyanmethemoglobin.
Vanzetti Azide-methemoglobin method - It is similar to HiCN reference method except that
sodium azide (less toxic) is used in place of more toxic potassium cyanide. Just like the HiCN
method, hemoglobin is converted to methemoglobin by potassium ferricyanide and the azide
then forms a complex with methemoglobin (azide methemoglobin) . This is an acceptable
alternative manual method. Some Point-of-care (POC) hemoglobin measurement devices are
based on this method.

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Point-of-care (POC) hemoglobin measurement devices -
● Portable hemoglobinometers - are used for bedside hemoglobin determination. In these
equipments disposable microcuvettes coated with dried reagents necessary for both
release of Hb from erythrocytes and conversion of Hb to a stable colored product are
used. A small amount of sample (typically 10 µl) of capillary, venous or arterial blood is
introduced to the microcuvette which is then inserted into the instrument and it gives the
results in less than a minute. The instrument is factory pre-calibrated using HiCN
standard.
● CO-oximetry – The measurement of ctHb (the total hemoglobin concentration is typically
defined as the sum of oxygenated hemoglobin, deoxygenated hemoglobin,
carboxyhemoglobin and methemoglobin) by CO-oximetry is based on the fact that
hemoglobin and all its derivatives are colored proteins which absorb light at specific
wavelengths and thus have a characteristic absorbance spectrum. In the CO-oximeter
absorbance measurements of a hemolyzed blood sample at multiple wavelengths across
the range that hemoglobin species absorb light (520-620 nm) are used by the installed
software to calculate the concentration of each of the hemoglobin derivatives (HHb,
O2Hb, MetHb and COHb). ctHb is the calculated sum of these derivatives. This method
is utilized in POCT blood gas analyzers. CO-oximetry provides an acceptable means of
urgent estimation of ctHb in a critical care setting.
● WHO Hemoglobin color scale (HCS)- The HCS test is based on the simple principle that
the color of blood is a function of concentration of hemoglobin (ctHb). A drop of blood is
absorbed onto a paper and its color compared with a chart of six shades of red, each
shade representing an equivalent ctHb: the lightest 4 g/dl and the darkest 14 g/dl. It is
generally used in economically deprived countries of the developing world, where
anemia is most prevalent. It is an acceptable clinical tool to screen for anemia in the
absence of more sophisticated technology and is significantly more sensitive and more
specific than clinical examination.

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BLOOD GLUCOSE ESTIMATION
Choice of Blood Specimen -
Capillary Blood from finger
Whole Blood (venous)
Plasma /serum
Plasma or serum more accurately reflect the glucose content of extracellular fluid. The glucose
concentration in the water of cells and plasma is the same but plasma's water content (93%) is
higher than the water content of cells (73%), therefore, plasma glucose is about 12-13% higher
than that of whole blood.
Plasma glucose (mg/dl) = (whole blood glucose × 1.15) + 6.0
Plasma glucose (mmol/l) = (whole blood glucose × 1.15) + 0.33
[This correction is reasonably accurate if the hematocrit is normal and for each change in
hematocrit of 10 units there is a change in the opposite direction of blood glucose of 0.20 mmol/l
(3.6 mg/dl). However, it should be noted that unlike the whole blood glucose concentration, the
plasma glucose concentration is unaffected by hematocrit].
Different methods of glucose estimation -
● Alkaline copper reduction method - Folin and Wu method
● Ferricyanide methods
● o- Toluidine method
● Hexokinase method (reference method)
● Glucose Oxidase - Peroxidase method
Hexokinase method -
This is proposed as "reference method" for glucose estimation.
Hexokinase
Glucose + ATP -------------------> Glucose-6-phosphate + ADP
G-6-P dehydrogenase
Glucose-6-phosphate + NAD -----------------------------> 6-phosphogluconate + NADH + H+
NADH is quantitated spectrophotometrically at 340 nm which is directly related to glucose
concentration.
Why it is a reference method - because:
● it is unaffected by hemolysis, lipaemia, urate, ascorbic acid, bilirubin or drugs affecting
the oxidase method.
Glucose oxidase-peroxidase (GOD-POD) method -
Principle - In this method glucose is oxidized by glucose-oxidase (GOD) in the presence of
atmospheric oxygen to gluconic acid and hydrogen peroxide. The hydrogen peroxide is then
oxidized by peroxidase (POD) in the presence of 4-aminophenazone (4-aminoantipyrine) and
phenol to form the red coloured quinoneimine dye which is quantitated spectrophotometrically
(absorbance/OD) at 505-510 nm and is directly proportional to glucose concentration.
GOD
Glucose + H2O + O2 --------> gluconic acid + H2O2
POD
H2O2 + 4-aminophenazone + phenol ---------> quinoneimine complex(red colored) + H2O

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Reagents -
● Glucose colour reagent; it contains GOD, POD, 4-aminoantipyrine, phenol & phosphate
buffer (pH 7.5)
● Glucose standard - 100 mg/dl or calibrator traceable to the Standard Reference Material
(SRM) of the National Institute of Standards and Technology (NIST).
Procedure -
● Bring the reagent and the standard solution to RT
● Label the test tubes as B (Blank), S (Standard) and T (Test)
● Take 1ml (1000μl) glucose reagent in each tube
● Add 10 μl distilled water to B tube
● Add 10 μl standard to S tube
● Add 10 μl plasma/serum
● Mix well the contents of all the tubes
● Keep the tubes in a water bath at 37 °C for 15 minutes or 30 minutes at RT
● Take reading (absorbance/OD) of S and T tubes against the B tube on a colorimeter with
a green filter or at 505-510 nm on a spectrophotometer.
Calculation -
OD of T
Glucose concentration (mg/dl) = ------------ × concentration of standard (100 mg/dl)
OD of S
Conversion of results from mg/dl to mmol/l -
result in mg/dl × 0.0555 = result in mmol/l
Some important points -
● The reagent, standard solution and plasma/serum sample should be at RT before
running the test.
● Pipetting of the reagent, standard and the samples should be very accurate.
● Since the linearity of GOD-POD test is normally up to 500 mg/dl glucose concentration,
the samples with glucose concentration above 500 mg/dl should be diluted ½ or,⅓ with
normal saline and 10 μl of diluted sample should be tested and the result should be
multiplied by 2 or 3 (as per dilution) to get actual glucose concentration.
● The temperature of the water bath should be 37 °C.
● The reagent gradually develops colour due to exposure, it should not be used if its OD
against distilled water is above 0.300.
● Serum/plasma should be separated within 30 minutes as the glucose level in
serum/plasma decreases gradually at the rate of about 7 mg/dl per hour at RT.
Therefore, plasma separated from fluoride tube blood is preferred due to antiglycolytic
action of fluoride.
● Internal quality control (IQC) sample (s) should be run just like the unknown samples
with every batch of glucose test for quality check purposes.
Glucose meter (Glucometer) - The principle of a glucose meter is based on electrochemical
technology. The important parts of a glucometer are enzymatic reaction and a detector. The
enzyme portion of the Glucose meter is normally packaged in dehydrated form in a disposable

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strip or a reaction cuvette. Glucose oxidase, glucose dehydrogenase or hexokinase is used for
enzymatic reaction. Blood glucose reacts with enzyme in presence of the moisture of the
patient's blood sample and thus this reaction gives the product which can be detected by the
glucometer. Some glucose meters produce hydrogen peroxide or an intermediary which react
with a dye which changes the colour and this change in colour is directly proportional to the
glucose concentration. Other glucose meters incorporate the enzyme in a biosensor which
generates electrons resulting in a small electric current which is detected by the meter and
displayed as glucose concentration. The value measured by a glucose meter may be about
±15-20% different from the value measured in a laboratory.
Blood glucose level - Plasma glucose under fasting condition (at least 8 hrs fasting) is normally
less than 100 mg/dl and 2 hours after meals it is less than 140 mg/dl.
Oral glucose tolerance test (OGTT) - This is done for the diagnosis of diabetes, gestational
diabetes or diseases related to carbohydrate metabolism. The test indicates whether our body
properly metabolises the sugar taken in excess amounts or not. Glucose tolerance test
procedure - It is necessary that the patient should be empty stomach (10-12 hrs fasting) for this
test (water can be taken). This is why this test is done in the morning. First the fasting blood
sample is taken, then the patient is given 75 g glucose dissolved in 250-300 ml of water orally (it
should be drunk slowly in five minutes to avoid vomiting). Then another blood sample is taken 2
hrs after drinking glucose. Sometimes the blood samples are taken at 1 and 2 hrs after glucose.
Then plasma glucose is estimated in all these samples. It is important that nothing should be
eaten (not even paan masala), drunk or smoked during these 2 hours and all the blood samples
should be taken in the same posture (sitting or lying).
World Health Organization criteria for an oral glucose tolerance test for the diagnosis of
diabetes, impaired glucose tolerance and gestational diabetes :
● Normal levels : Fasting plasma glucose - < 100 mg/dl, post glucose (75 g) - <140 mg/dl
● Diabetes: Fasting plasma glucose (FPG) ≥126 mg/dl or Oral glucose tolerance test
(OGTT) 2-hour post glucose (PG) plasma glucose ≥200 mg/dL
● Impaired fasting glucose (IFG): FPG 110 mg/dl to 125 mg/dl and OGTT 2-hour PG <
140 mg/dl
● Impaired glucose tolerance (IGT): FPG < 126 mg/dl and OGTT 2-hour PG 140 mg/dl to
200 mg/dl
● Gestational diabetes : Fasting - ≥ 92 mg/dl, 1 hour post glucose - ≥180, 2 hour post
glucose - ≥ 155 mg/dl
Besides, the diagnosis of diabetes is also done on the basis of HbA1C and random blood
glucose levels -
HbA1C ≥ 6.5 % or random blood glucose ≥ 200 md/dl in presence of signs and symptoms of
diabetes.
On the basis of HbA1C level eAG (estimated average glucose) can also be calculated using this
formula :
eAG (mg/dl) = (28.7 × A1C) – 46.7

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URINE ROUTINE EXAMINATION (Urine routine analysis)
Urinalysis is generally done for the diagnosis of disease, monitoring of the disease and for the
assessment of the effect of drugs.
Urine collection :
● Early morning urine is considered as the best specimen for urinalysis because it is
concentrated and acidic.
● The patient should be told to clean external urethral orifice using towelettes (sanitizing
wipes) before the collection of urine.
● The patient should be told to collect the middle portion of the urine flow because the
initial fraction may be contaminated with cells and bacterial cells present around external
urethral orifice. So do not collect the first or the last part of urine that comes out.
● Urine sample should be collected in a clean, dry and capped container preferably a
sterile container.
● The sample collected should be analyzed as soon as possible because delay may
change its chemical composition and the bacterial growth may also occur. If any delay in
urinalysis is expected then the sample should be kept in refrigerator.
Urine routine examination includes its physical and chemical examination :
Physical examination - includes :
● Urine clear or turbid (normal urine clear/transparent होती है)
● Urine colour (normal urine is straw coloured)
● Specific gravity of urine by an urinometer or an urine strip
Chemical examination - A number biochemical tests are done in urine using paper strips or
test tube tests.
By plastic or paper strips : These strips may be monofunctional (having one indication zone for
one urine component) or polyfunctional (having several indication zones for different urine
components). These plastic or paper strips have pads impregnated with chemicals which react
with compounds present in urine to give specific colours. Polyfunctional strips are used for the
test of the following compounds in urine -
pH, glucose, proteins, bilirubin, urobilinogen, ketone, hemoglobin (blood), nitrite, ascorbic acid,
leucocytes. The semi-quantitative values of these tests are reported as trace, 1+, 2+, 3+ and 4+
or in mg/dl, g/dl.
Procedure -
First urine is mixed well and then the test strip is dipped in it in such a way that all the zone pads
of the strip are soaked in the urine. After soaking a few seconds the strip is removed by running
the edge of the strip against the rim of the container to remove the excess of urine on the strip.
Now keep this strip horizontally on a tissue paper for 1-2 minutes so that all the reactions are
complete and the reagents of different reaction zones do not intermix. Then the colours of all the

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zone pads are matched with the colour scale given by the strip manufacturer to find the
presence and the amount of different components in the urine sample.
By Test tube tests: The different analytes of the urine are tested by individual methods for their
detection -
Urine Analyte Test method Method principle
Sugar Benedict's test reduction of Cu++ to Cu+ in alkaline
medium by reducing sugar
Proteins Heat test denaturation of proteins by heat
Sulphosalicylic acid test precipitation of proteins by sulfosalicylic
acid resulting in turbidity
Ketone bodies Rothera's test Acetoacetic acid and acetone react with
alkaline solution of sodium nitroprusside to
form a purple colored complex.
Bile pigments Fouchet's test Bilirubin is converted to the green
colored biliverdin by the oxidative action of
ferric chloride in the presence of
trichloroacetic acid
Urobilinogen Ehrlich's test dimethylaminobenzaldehyde reacts with
urobilinogen in acid medium to produce a
pink color
Hemoglobin Benzidine test The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing
nascent oxygen which oxidizes
benzidine to give blue color
Some individual biochemical test tube tests in urine
Urine sugar
Urine sugar is tested by Benedict‘s test.
Principle - The reducing sugar upon heating in an alkaline medium reduces cupric ions (Cu++)
to cuprous ions (Cu+) and these cuprous ions form brick red precipitate of cuprous oxide
(Cu2O).
Procedure - Take 2 ml Benedict's reagent (a solution containing copper sulfate, sodium citrate
and sodium carbonate) in a test tube and add 0.2 ml (4 drops) urine. Mix well and heat on a
spirit lamp or a burner carefully and boil for 2 minutes. The change in the colour of the reagent
indicates the presence of sugar in the urine sample :-

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Colour of reagent sugar amount in urine
Clear blue : nil (sugar absent)
Green with no ppt : trace (<0.5%)
Green with ppt. : + (0.5%-1%)
Yellow ppt. : ++ (1%-1.5%)
Orange red ppt. : +++ (1.5%-2%)
Brick red : ++++ (>2%)
Urine proteins
Sulfosalicylic acid test
Principle - In this test the proteins are precipitated by sulfosalicylic acid resulting in turbidity.
Procedure -
2 ml clear urine + 0.2 ml (4 drops) 20% sulfosalicylic acid solution → mix well
Or
1 ml urine + 1 ml 3% sulfosalicylic acid solution → mix well
[If the urine sample is already turbid then centrifuge it before running the test
and use the clear urine obtained after centrifugation]
Interpretation of turbidity -
Clear Urine : nil (protein absent)
Slight turbidity : +
Mild turbidity : ++
Moderate turbidity : +++
(With flocculation)
Visible white precipitate : ++++
Heat test
Principle - proteins get coagulated when they are boiled in an acidic medium.
Procedure -
Take 5 ml urine in a test tube and heat the upper portion of the urine on a spirit lamp or a burner
till boil. Now compare this heated portion of urine with the lower part of urine (which was not
heated). If the upper part has cloudiness or turbidity then this may be due to the presence of
proteins or phosphate /carbonates in the urine sample. Now add 2-4 drops 10% of glacial acetic
acid to it and boil again. If the turbidity persists then it confirms the presence of proteins in the

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urine sample. But if the turbidity disappears after addition of acetic acid then it indicates that the
initial turbidity was due to the presence of phosphate or carbonate in the urine sample and not
due to proteins.
Interpretation of Turbidity (cloudiness) :-
No cloudiness : negative (no protein)
Barely visible cloudiness : Trace amount
Definite cloud without granular flocculation : +
Heavy and granular cloud without granular flocculation : ++
Densed cloud with marked flocculation : +++
Thick curdy precipitation and coagulation : ++++
Urine ketone bodies
Ketone bodies are produced by the liver and used peripherally as an energy source when
glucose is not readily available. These are acetoacetic acid, acetone and
β-hydroxybutyric acid. The ketone bodies in urine are tested by modified Rothera's test.
Modified Rothera's test
Principle - Acetoacetic acid and acetone react with alkaline solution of sodium nitroprusside to
Procedure I -
Reagent - Mix 1 g sodium nitroprusside + 20 g ammonium sulfate + 20 g anhydrous sodium
carbonate
Put a little amount of the above reagent powder (about 5 mm in diameter) on a glass slide or a
piece of white paper. Now add 1-2 drops of urine on this powder reagent. If the color of the
powder changes to violet color then it indicates the presence of ketone bodies (acetone and
acetoacetic acid) in the urine sample.
Procedure II -
5 ml urine is saturated with solid ammonium sulfate (till ammonium sulfate does not dissolve
further). Then add a small amount of sodium nitroprusside (0.5 ml 2% solution or a little amount

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of powder). Mix it and add about 0.5 ml conc. ammonia. If a purple coloured ring forms then it
indicates the presence of ketone bodies in the urine sample.
Urine bile pigment
Fouchet’s Test for bilirubin
Principle - Barium chloride precipitates the sulphate radicals present in urine to form precipitate
of barium sulphate. If bile pigments are present in urine, they adhere to these molecules of
barium sulfate. Ferric chloride present in Fouchet's reagent then oxidises yellow bilirubin, in the
presence of trichloroacetic acid to green biliverdin.
Fouchet's reagent - dissolve 25 g trichloroacetic acid (TCA) in 50 ml distilled water in a 100 ml
volumetric flask, add 10 ml ferric chloride solution (10%) and make up to 100 ml with distilled
water.
Procedure -
10 ml urine + about 5 ml barium chloride solution (10%) → mix well → filter or centrifuge it →
add 1-2 drop of Fouchet's reagent on to the precipitate → appearance of bluish green color
shows the presence of bilirubin in the urine sample.
IN HINGLISH
HEMOGLOBIN (Hb or Hgb) ESTIMATION
Hemoglobin (Mr 64,500) एक tetrameric protein (four polypeptide chains) है जो RBCs में होती है
और इसमें दो α (प्रत्येक chain में 141 amino acids) और दो β polypeptide chains (प्रत्येक chain में 146
amino acids) और प्रत्येक polypeptide chain (globin) से एक 'Heme' prosthetic group (जो ferrous
iron और porphyrin का एक complex होता है) जुड़ा होता है I
Forms of Hemoglobin - The normally occurring hemoglobins are:
● Hemoglobin A (adult hemoglobin) - α2β2 - यह सामान्यतौर पर adults में पाया जाता है और
क
ु ल हीमोग्लोबिन का 95% होता है I

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● Hemoglobin A2 - α2δ2 - यह adults में क
ु ल हीमोग्लोबिन का 1.5–3.5% होता है I यह beta
thalassemia क
े मरीज़ों या beta thalassemia genes क
े heterozygous लोगों में बढ़ा होता है I
● Hemoglobin F (fetal hemoglobin) - α2γ2 - यह fetuses और newborn babies में क
ु ल
हीमोग्लोबिन का लगभग 90% भाग होता है जो दो वर्ष क
े अंदर धीरे - धीरे कम होकर क
ु ल हीमोग्लोबिन का 1%
हो जाता है और व्यस्क क
े पूरे जीवन में 1% से भी कम रहता है I
Functions of Hemoglobin -
1. Hemoglobin lungs में oxygen को उत्क
् रमणीय रूप (reversibly) से bind करता है क्योंकि वहाँ
oxygen concentration high होता है और इस तरह oxyhemoglobin बनता है जो body tissues
(जहाँ ऑक्सीजन का concentration कम होता है) को ऑक्सीजन deliver करता है जिससे
deoxyhemoglobin बनता है I
2. यह carbon dioxide क
े lungs और tissues क
े बीच विनिमय (exchange) को भी सुगम बनाता है I
Methods of Hemoglobin estimation :
There are several methods for hemoglobin estimation :
● Sahli’s or acid hematin method
● Cyanmethemoglobin method
● Sodium Lauryl Sulphate method(cyanide free method)
● Vanzetti Azide-methemoglobin method
● Oxyhemoglobin method
● Alkaline-hematin method
● Measurement of oxygen-combining capacity
● Measurement of iron content
इनमें से Sahli's method और Cyanmethemoglobin method ही सर्वाधिक उपयोग किए जाते हैं I
Sahli’s or acid hematin Method -
जब Blood को N/10 HCl (0.1 N HCl) क
े साथ mix करते हैं तो हीमोग्लोबिन brown color क
े acid hematin में
परिवर्तित हो जाता है I अब इसे N/10 HCl से तब तक dilute करते हैं जब तक इसका color hemoglobinometer क
े
comparator box क
े brown color क
े समान नहीं हो जाता I हीमोग्लोबिन का concentration g% में पढ़ा जाता है I
यह एक बहुत सस्ता, तेज और आसान method है I लेकिन इसमें क
ु छ कमियाँ भी हैं, जैसे - कम accuracy, color
matching करने में व्यक्तिगत विविधता, true standard का अभाव और सभी प्रकार क
े हीमोग्लोबिन (जैसे -
oxyhemoglobin, sulfhemoglobin) का HCl क
े साथ reaction करक
े acid hematin में परिवर्तित न हो पाना आदि I
Cyanmethemoglobin method - is the internationally recommended method for determining
hemoglobin. इस method का basic principle इस प्रकार है : Blood को जब Drabkin's solution (जिसमें
potassium ferricyanide, potassium cyanide और potassium dihydrogen phosphate होते हैं) क
े साथ
mix करते हैं तो hemoglobin methemoglobin (मेथीमोग्लोबिन) में बदल जाता है जो potassium cyanide की
उपस्थिति में Cyanmethemoglobin (साइनमेथीमोग्लोबिन, HiCN) में बदल जाता है I यह
साइनमेथीमोग्लोबिन 540 nm पर maximum absorbance देता है और Beer-Lambert’s का कड़ाई से पालन

15
करता है I Test sample क
े 540 nm पर absorbance को standard HiCN solution क
े absorbance से
तुलना करक
े test sample क
े hemoglobin concentration की गणना कर लेते हैं I
K3[Fe(CN6)]
Hemoglobin(Fe++) ------------------> Methemoglobin (Fe+++)
KCN
Methemoglobin(Fe+++)-----------> Cyanmethemoglobin (HiCN)
Reagent:
Drabkin's reagent -
1 liter volumetric flask में निम्न chemicals डालते हैं :
Potassium ferricyanide K3[Fe(CN)6] - 200 mg
Potassium cyanide (KCN) - 50 mg
Dihydrogen potassium phosphate (KH2PO4) - 140 mg
Non-ionic detergent (e.g. Triton X-100) - 1 ml
अब इसमें distilled water डालकर dissolve करते हैं और distilled water से dilute कर 1 liter solution बनाते
हैं I इस solution का pH 7.0 - 7.4 होना चाहिए I इसे एक dark coloured polythene bottle या brown
coloured glass (actinic) bottle में 4 - 20°C पर रखते हैं (do not freeze) I Solution में ज़रा सी भी
turbidity दिखने या pH क
े निर्धारित range (7.0 - 7.4) से बाहर होने पर इस solution का उपयोग नहीं करते हैं I
Cyanmethemoglobin(HiCN) standard - Commercial HiCN standard (normally 60 mg/dl)
calibrated with international standard solution (primary calibrant) as per specifications defined
by the International Council for Standardization in Hematology (ICSH) is available.
Procedure -
● सर्वप्रथम Drabkin's solution और standard solution को कमरे क
े तापमान (RT) पर आने देते हैं I
● एक साफ़ test tube में 5 ml Drabkin's solution लेते हैं (mouth pipetting नहीं करनी है)
● Blood sample (EDTA tube) को धीरे - धीरे inversion दवारा अच्छी तरह mix करते हैं I
● एक micropipette द्वारा 20 µl blood लेते हैं, tip की बाहरी सतह पर लगे blood को बेहद सावधानी से
(बिना tip क
े छेद को छ
ु ए) tissue paper की मदद से साफ़ करते हैं I
● Pipette की tip को test tube क
े Drabkin's solution में dip करक
े tip को कई बार rinse करते हैं ,
जैसा नीचे चित्र में दिखाया गया है, जिससे tip क
े अंदर का पूरा blood solution में आ जाए :

16
● Tube को अच्छी तरह mix करते हैं (vortex mixer की सहायता ले सकते हैं) और कम से कम 5
minutes क
े लिए RT पर छोड़ देते हैं I
● Colorimeter या spectrophotometer पर blank tube (क
े वल Drabkin’s solution) का
absorbance yellow green filter या 540 nm पर zero पर set करते हैं I
● इसक
े बाद standard solution का absorbance नोट करते हैं ; या फिर standard को कई
concentrations में Drabkin’s solution में dilute करक
े सभी dilutions का absorbance नोट
करक
े एक standard curve plot करते हैं I
● अब इसीप्रकार test solution का absorbance भी नोट करते हैं I
Calculation:
Absorbance of unknown × concentration of standard(mg/dl) × 251
Blood Hemoglobin(g/dl) = -----------------------------------------------------------------------------------------
Absorbance of standard × 1000
(251 is the dilution factor because 20 µl (0.02 ml) blood sample was added to 5 ml Drabkin's
solution, so the final volume was 0.02 ml + 5.0 = 5.02 ml. Therefore, the blood is diluted with a
dilution factor 5.02 ÷ 0.02 = 251. Standard was not diluted)
Advantages of Cyanmethemoglobin method
● पूरे world में accurate International hemoglobin standard उपलब्ध है;
● यह method automated hematology analyzers क
े लिए आसानी से अनुक
ू लित किया जा सकता है;
● यह एक सुस्थापित और गहनता से अनुसन्धानित – ICSH (International Council for Standardization in
Haematology) द्वारा recommended method है;
● Reagent कम खर्चीला होता है I
Disadvantages
● Manual method में accurate pipetting और spectrophotometer की आवश्यकता होती है;
● Reagent (cyanide) ख़तरनाक होता है;
● उपरोक्त कारणो से इस method का उपयोग लैब क
े बाहर सीमित होता है ;
● Plasma में lipids और proteins का बढ़ा हुआ स्तर और leukocyte की बढ़ी हुई संख्या क
े कारण हुई
turbidity test में हस्तक्षेप कर सकती है;
● Hemoglobin क
े वो derivatives जिनमें oxygen-carrying capacity नहीं होती (जैसे - MetHb,
COHb, SHb) उनमें यह method भेद नहीं कर पाता I इसलिए यदि ऐसे derivatives भी blood में
असामान्य मात्रा में उपस्थित होते हैं तो इस method द्वारा blood की oxygen-carrying capacity का
अधिआकलन (overestimation) हो सकता है l
Sodium Lauryl Sulphate method (cyanide free method) - Sodium Lauryl Sulphate (SLS)
एक surfactant है जो erythrocytes का विघटन (lysis) करता है और तब RBCs से निकला hemoglobin SLS
क
े साथ मिलकर एक complex बनाता है l यह complex (SLS-MetHb) क
ु छ घंटों क
े लिए स्थिर रहता है और
इसका maximum absorbance 539 nm पर होता है I इस method द्वारा प्राप्त hemoglobin
concentration क
े परिणाम reference HiCN method से प्राप्त परिणाम से बहुत सहसम्बद्ध (correlate) होते हैं I
यह method automated hematology analyzers क
े लिए भी अनुक
ू लित किया गया है I
Advantage - non-toxic reagent और lipemia और leukocytosis द्वारा कम हस्तक्षेप l
Disadvantage - SLS-MetHb Cyanmethemoglobin की अपेक्षा कम स्थिर होता है I
Vanzetti Azide-methemoglobin method - यह method HiCN reference method क
े समान ही है
किन्तु इसमें ज़्यादा विषैले potassium cyanide क
े स्थान पर कम विषैला sodium azide इस्तेमाल होता है I

17
HiCN method की तरह इस method में भी hemoglobin potassium ferricyanide द्वारा methemoglobin
में परिवर्तित होता है जो फिर sodium azide क
े साथ azide methemoglobin complex बनाता है I यह हीमोग्लोबिन
मापन का एक स्वीक
ृ त वैकल्पिक manual method है I क
ु छ Point-of-care (POC) hemoglobin measurement
devices इस method पर आधारित हैं I
Point-of-care (POC) hemoglobin measurement devices -
● Portable hemoglobinometer - का उपयोग bedside hemoglobin determination हेतु किया जाता
है I इन equipments में disposable microcuvettes होती हैं जो RBCs से Hb को release करने एवं
Hb को एक stable colored product में बदलने हेतु आवश्यक dried reagents से coated होती हैं I
capillary, venous या arterial blood का बहुत ही small amount (आमतौर पर 10 µl) को
microcuvette में डालते हैं और उसे instrument में फिट कर देते हैं I एक मिनट से भी कम समय में
result display हो जाता है I यह instrument factory में HiCN standard द्वारा पहले ही से calibrate
(pre-calibrated) किया हुआ होता है I
● CO-oximetry – द्वारा ctHb (the total hemoglobin concentration is typically defined as the
sum of oxygenated hemoglobin, deoxygenated hemoglobin, carboxyhemoglobin and
methemoglobin) क
े measurement का आधार यह है कि hemoglobin और इसक
े सभी derivatives
colored proteins होते हैं और एक विशेष wavelength पर light absorb करते हैं जिसका एक
characteristic absorbance spectrum होता है I CO-oximeter में एक hemolyzed blood sample
का absorbance विभिन्न wavelengths (520-620 nm) पर अलग - अलग measurement करते हैं
और machine में लगे software की मदद से hemoglobin क
े प्रत्येक derivative का concentration
calculate करते हैं I ctHb इन सभी derivatives क
े concentration का क
ु ल योग होता है I यह method
POCT blood gas analyzers में इस्तेमाल होता है I CO-oximetry critical care setting में urgent
ctHb मापने का एक स्वीकृ त तरीका है I
● WHO Hemoglobin color scale (HCS)- यह HCS test इस साधारण सिद्धांत पर आधारित है कि
blood का colour उसमें hemoglobin क
े concentration (ctHb) पर निर्भर करता है I blood की एक
बूँद test paper पर absorb की जाती है और इसका colour एक chart में दिए red colour क
े 6
shades (प्रत्येक shade ctHb क
े एक concentration को represent करता है) से तुलना करते हैं :
सबसे light colour 4 g/dl और सबसे dark 14 g/dl ctHb को represent करता है I यह method
आमतौर पर ग़रीब देशों में इस्तेमाल होता है, जहाँ खून की कमी (anemia) अधिक पायी जाती है I अन्य
परिष्कृ त तकनीक क
े अभाव होने पर anemia की जाँच (screening) हेतु हीमोग्लोबिन मापने का यह
एक स्वीकृ त तरीका है जो anemia की जाँच हेतु clinical examination से अधिक sensitive और
specific होता है I

18
BLOOD GLUCOSE ESTIMATION
Choice of Blood specimen -
Capillary Blood from finger
Whole Blood (venous)
Plasma /serum
Plasma या serum द्वारा extracellular glucose content अधिक accurately प्रतिबिम्बित होता है I हालांकि,
cells और plasma क
े water में glucose concentration समान होता है किन्तु plasma का water content
(93%) cells क
े water content (73%) से अधिक होता है, इसलिए plasma glucose whole blood glucose
से लगभग 12-13% अधिक होता है I इसीप्रकार, capillary (whole) blood में glucose स्तर venous (whole)
blood क
े स्तर से थोड़ा अधिक होता है I
Plasma glucose (mg/dl) = (whole blood glucose × 1.15) + 6.0
Plasma glucose (mmol/l) = (whole blood glucose × 1.15) + 0.33
[This correction is reasonably accurate if the hematocrit is normal and for each change in
hematocrit of 10 units there is a change in the opposite direction of blood glucose of 0.20 mmol/l
(3.6 mg/dl). However, it should be noted that unlike whole blood glucose concentration, plasma
glucose concentration is unaffected by hematocrit].
Different methods of glucose estimation -
● Alkaline copper reduction method - Folin and Wu method
● Ferricyanide methods
● o- Toluidine method
● Hexokinase method (reference method)
● Glucose Oxidase - Peroxidase method
Hexokinase method -
This is proposed as "reference method" for glucose estimation.
Hexokinase
Glucose + ATP -------------------> Glucose-6-phosphate + ADP
G-6-P dehydrogenase
Glucose-6-phosphate + NAD -----------------------------> 6-phosphogluconate + NADH + H+
NADH is quantitated spectrophotometrically at 340 nm which is directly related to glucose
concentration.
Why it is a reference method - because:
● it is unaffected by hemolysis, lipaemia, urate, ascorbic acid, bilirubin or drugs affecting
the oxidase method.
Glucose oxidase-peroxidase method (GOD-POD) -
Principle - इस method में Glucose oxidase की उपस्थिति में glucose oxidize होकर gluconic acid और
hydrogen peroxide बनाता है I यह hydrogen peroxide, peroxidase (POD) enzyme की उपस्थिति में

19
oxidize होकर 4-aminophenazone (4-aminoantipyrine) और phenol क
े साथ मिलकर red coloured
quinoneimine dye बनाता है जिसका absorbance 505-510 nm पर glucose concentration क
े सीधा
आनुपातिक होता है I
GOD
Glucose + H2O + O2 --------> gluconic acid + H2O2
POD
H2O2 + 4-aminophenazone + phenol ---------> quinoneimine complex (red colored) + H2O
Reagents -
● Glucose colour reagent; it contains GOD, POD, 4- aminoantipyrine, phenol & phosphate
buffer (pH 7.5)
● Glucose standard - 100 mg/dl or calibrator traceable to the Standard Reference Material
(SRM) of the National Institute of Standards and Technology (NIST).
Procedure -
● सर्वप्रथम serum/plasma sample, reagent और standard solution room temperature (RT) पर
आने देते हैं I
● तीन test tubes B (Blank), S (Standard) और T (Test) label करते हैं I
● प्रत्येक tube में 1ml (1000μl) glucose reagent लेते हैं I
● B tube में 10 μl distilled water डालते हैं I
● S tube में10 μl glucose standard डालते हैं I
● T tube में 10 μl plasma/serum डालते हैं I
● सभी tubes क
े contents को अच्छी तरह mix करते हैं I
● तीनों tubes को एक water bath में 37 °C पर 15 minutes या RT पर 30 minutes क
े लिए रखते हैं I
● फिर एक colorimeter पर green filter पर या एक spectrophotometer पर 505-510 nm पर S और
T tubes की B tube क
े विरुद्ध reading (absorbance/OD) नोट करते हैं I
Calculation -
OD of T
Glucose concentration(mg/dl) = ------------ × concentration of standard in mg/dl
OD of S
To convert results from mg/dl to mmol/l -
result in mg/dl × 0.0555 = result in mmol/l
Some important points -
● The reagent, standard solution और plasma/serum sample को test लगाने क
े पूर्व कमरे क
े
तापमान (RT) पर होना चाहिए I
● Reagent, standard और samples की pipetting accurate होनी चाहिए I
● सामान्य तौर पर GOD-POD test की linearity लगभग 500 mg/dl glucose concentration की
होती है, इसलिए यदि किसी sample में glucose concentration 500 mg/dl से अधिक आता है तो
plasma sample को normal saline से ½ या ⅓ dilute करक
े इस diluted plasma sample में पुनः
glucose test करते हैं और प्राप्त result को 2 या 3 (dilution क
े आधार पर) से multiply करक
े actual
glucose concentration निकालते हैं I
● Water bath का तापमान 37 °C होना चाहिए I

20
● Glucose reagent का colour समय क
े साथ क
ु छ बदल जाता है जिसका कारण होता है bottle का बार -
बार खुलना I इसलिए यदि reagent की OD distilled water क
े against 0.300 से अधिक हो जाए तो
उस reagent का उपयोग नहीं करना चाहिए I
● Serum/plasma को 30 minutes क
े अंदर ही पृथक कर लेना चाहिए क्योंकि serum/plasma में
glucose का स्तर धीरे - धीरे 7 mg/dl प्रति घंटे की दर से कम होने लगता है I इसलिए Fluoride tube
क
े blood से पृथक किए गए plasma को प्राथमिकता दी जाती है क्योंकि fluoride क
े कारण glucose का
स्तर आसानी से नहीं घटता (antiglycolytic action of fluoride) I
● Glucose test क
े प्रत्येक batch क
े साथ Internal quality control (IQC) sample (s) को भी
unknown samples की भांति ही अवश्य लगाना चाहिए जिससे glucose test परिणाम की quality
check हो सक
े I
Glucose meter (Glucometer) - Glucose meters electrochemical technology पर आधारित होते हैं और
enzymatic reaction एवं एक detector इसक
े दो आवश्यक भाग होते हैं I Glucose meter का enzyme
portion सामान्यतौर पर dehydrated स्थिति में एक disposable strip या reaction cuvette में packaged
रहता है I Enzymatic reaction हेतु glucose oxidase, glucose dehydrogenase या hexokinase
enzymes का उपयोग किया जाता है I Patient क
े blood sample की moisture क
े कारण glucose enzyme
से react करक
े product बनाता है जिसे glucometer द्वारा detect किया जा सकता है I क
ु छ glucose meters
hydrogen peroxide या एक intermediary (मध्यवर्ती) उत्पन्न करते हैं जो एक dye से react कर colour में
परिवर्तन करते हैं जो कि glucose concentration क
े सीधा आनुपातिक होता है I दूसरे glucose meters
enzyme को एक biosensor में incorporate कर देते हैं जो एक electron generate करता है जिसे एक meter
द्वारा detect किया जाता है I Glucose meter द्वारा मापी गई glucose values लैब में मापी गई values से
लगभग ±15-20% भिन्न हो सकती हैं I
Blood glucose level - यह fasting condition (at least 8 hrs fasting) में सामान्यतः 100 mg/dl से कम
होता है और खाना खाने क
े 2 घंटे बाद 140 mg/dl से कम I
Oral glucose tolerance test (OGTT) - यह diabetes, gestational diabetes या carbohydrate
metabolism से सम्बंधित बीमारिओं की diagnosis हेतु किया जाता है I इस test से यह पता चलता है कि
अधिक मात्रा में sugar लेने पर शरीर उसे उचित रूप से metabolise कर पाता है या नहीं I
Glucose tolerance test procedure - इसक
े लिए व्यक्ति का खाली पेट (10-12 घंटे की fasting) होना
आवश्यक होता है (पानी पी सकते हैं)I इसीलिए यह test सुबह ही किया जाता है I सबसे पहले व्यक्ति का fasting
blood sample लेते हैं, फिर उसे 75 g glucose 250-300 ml पानी में घोल कर पिलाते हैं (धीरे - घीरे 5 मिनट में
पीना चाहिए, वरना उल्टी हो सकती है) और फिर इसक
े 2 घंटे बाद blood sample लेकर उसमें glucose level
मापते हैं I कभी - कभी 1 घंटे और 2 घंटे पर दो blood samples लेते हैं I यह ध्यान रखते हैं इन 2 घंटों क
े बीच
क
ु छ भी खाना, पीना, पान मसाला या धूम्रपान मना होता है और सभी blood samples एक ही posture (बैठे या
लेटे हुए) में ही लिए जाएं I
World Health Organization ने diabetes, impaired glucose tolerance और gestational diabetes की
diagnosis क
े लिए निम्न मानदंड (criteria) तय किये हैं :
● Normal levels : Fasting plasma glucose - < 100 mg/dl, post glucose (75 g) - <140 mg/dl
● Diabetes: Fasting plasma glucose (FPG) ≥126 mg/dl or Oral glucose tolerance test
(OGTT) 2-hour post glucose (PG) plasma glucose ≥200 mg/dL
● Impaired fasting glucose (IFG): FPG 110 mg/dl to 125 mg/dl and OGTT 2-hour PG <
140 mg/dl
● Impaired glucose tolerance (IGT): FPG < 126 mg/dl and OGTT 2-hour PG 140 mg/dl to
200 mg/dl
● Gestational diabetes : Fasting - ≥ 92 mg/dl, 1 hour post glucose - ≥180, 2 hour post
glucose - ≥ 155 mg/dl

21
इसक
े अतिरिक्त diabetes की diagnosis HbA1C और random blood glucose स्तर क
े आधार पर भी करते
हैं -
HbA1C ≥ 6.5 % या random blood glucose ≥ 200 md/dl in presence of signs and symptoms of
diabetes.
HbA1C क
े आधार पर eAG (estimated average glucose) को calculate करने क
े लिए इस फॉर्मूले का
उपयोग करते हैं :
eAG (mg/dl) = (28.7 × A1C) – 46.7
URINE ROUTINE EXAMINATION (Urine routine analysis)
Urinalysis सामान्यतः बीमारी की diagnosis, बीमारी की monitoring और दवा क
े प्रभाव को देखने हेतु की
जाती है I
Urine sample collection :
● Early morning की पहली urine उत्तम रहती है क्योंकि यह concentrated और acidic होती है I
● मरीज़ को बताना होता है कि urine collect करने से पहले external urethral orifice को towelettes
(sanitizing wipes) से साफ़ करक
े पोछ लेना चाहिए I
● Urine flow का मध्य भाग (middle portion) ही collect करना चाहिए क्योंकि urine flow क
े initial
fraction में external urethral orifice क
े आस-पास क
े cells और bacterial cells का contamination
हो सकता है I इसलिए urine का पहला और आखिरी भाग एकत्र नहीं करना चाहिए I
● Urine sample एक साफ़ एवं सूखे ढक्कनदार container में लेना चाहिए (sterile container बेहतर
होता है) I
● Sample collect करने क
े बाद उसमें जाँच जितनी जल्दी कर ली जाए उतना अच्छा होता है क्योंकि
अधिक देर होने से urine क
े chemical composition में बदलाव हो सकते हैं और bacterial growth भी
हो सकती है I यदि जाँच में अधिक देर हो तो sample को फ्रिज में रखना चाहिए I
Urine routine examination में physical और chemical examination शामिल होते हैं :
Physical examination - इसमें हम urine का appearance देखते हैं -
● Urine clear है या turbid (normal urine clear/transparent होती है)
● Urine का colour (normal urine भूसे क
े रंग (straw colour) की होती है)
● Urine की specific gravity भी urinometer या urine strip द्वारा देखते हैं I
Chemical examination - Urine में कई biochemical tests किये जाते हैं I इन tests क
े लिए paper strips
या test tube tests का उपयोग करते हैं I
By plastic or paper strips : ये strips monofunctional (having one indication zone for one urine
component) या polyfunctional (having several indication zones for different urine components)
होती हैं I इन plastic या paper strips में pads होते हैं जो ऐसे chemical से संसेचित होते हैं जो urine में
उपस्थित compounds से react करक
े विशेष colour देते हैं I urine में present compounds की जाँच क
े लिए
Polyfunctional strips द्वारा निम्लिखित biochemical tests किये जाते हैं -
pH, glucose, proteins, bilirubin, urobilinogen, ketone, hemoglobin (blood), nitrite, ascorbic acid,
leucocytes. इन tests की semi-quantitative values को trace, 1+, 2+, 3+ and 4+ या mg/dl, g/dl में
report करते हैं I
Procedure -
Urine को अच्छी तरह mix करक
े उसमें Test strip को क
ु छ seconds क
े लिए इतना डुबोते हैं कि strip क
े सभी
zone pads urine से भीग जाएं I फिर container की wall से strip को touch करते हुए strip को इस प्रकार
निकालते हैं कि strip पर लगी excess urine निकल जाए I अब इस strip को 1-2 मिनट क
े लिए horizontally
एक tissue paper पर रखते हैं ताकि reactions पूरे हो जाएं और विभिन्न reaction zones क
े reagents आपस

22
में भी न मिलें I फिर सभी zone pads पर आये colours को strip manufacturer द्वारा दिए गए colour scale
से मैच करक
े urine क
े विभिन्न अव्यवों की उपस्थिति एवं मात्रा note कर लेते हैं I
By Test tube tests: इसमें विभिन्न analytes की जाँच अलग - अलग methods द्वारा की जाती है -
Urine Analyte Test method Method principle
Sugar Benedict's test reduction of Cu++ to Cu+ in alkaline
medium by reducing sugar
Proteins Heat test denaturation of proteins by heat
Sulphosalicylic acid test precipitation of proteins by sulfosalicylic
acid resulting in turbidity
Ketone bodies Rothera's test Acetoacetic acid and acetone react with
alkaline solution of sodium nitroprusside to
Bile pigments Fouchet's test Bilirubin is converted to the green
colored biliverdin by the oxidative action of
ferric chloride in the presence of
trichloroacetic acid
Urobilinogen Ehrlich's test dimethylaminobenzaldehyde reacts with
urobilinogen in acid medium to produce a
pink color
Hemoglobin Benzidine test The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing
nascent oxygen which oxidizes
benzidine to give blue color
Some individual biochemical test tube tests in urine
Urine sugar
Urine में sugar की जाँच Benedict‘s test द्वारा करते हैं I
Principle - Alkaline medium में reducing sugar cupric ions (Cu++) को cuprous ions (Cu+) में
reduce करती है और ये cuprous ions cuprous oxide (Cu2O) क
े रूप में brick red precipitate बनाते हैं I
Procedure - एक test tube में 2 ml Benedict's reagent (जो copper sulfate, sodium citrate और
sodium carbonate का एक solution होता है) लेते हैं और इसमें 0.2 ml (4 drops) urine mix करक
े spirit

23
lamp या burner पर 2 मिनट क
े लिए boil करते हैं I तब test tube में reagent क
े colour में हुए परिवर्तन क
े
आधार पर urine में sugar की उपस्थिति का पता चलता है :-
Clear blue : nil (sugar absent)
Green with no ppt : trace (<0.5%)
Green with ppt. : + (0.5%-1%)
Yellow ppt. : ++ (1%-1.5%)
Orange red ppt. : +++ (1.5%-2%)
Brick red : ++++ (>2%)
Urine proteins
Sulfosalicylic acid test
Principle - sulfosalicylic acid urine में उपस्थित proteins को precipitate कर देता है जिससे urine
sample turbid (आविल, गंदला) हो जाता है I
Procedure (विधि) -
2 ml clear urine + 0.2 ml (4 drops) 20% sulfosalicylic acid → mix well
Or
1 ml urine + 1 ml 3% sulfosalicylic acid solution → mix well
Interpretation -
Clear Urine : nil (protein absent)
Slight turbidity : +
Mild turbidity : ++
Moderate turbidity : +++
(With flocculation)
Visible white precipitate : ++++
Heat test
Principle - proteins acidic medium में boil करने पर coagulate (जम जाना) हो जाती हैं I
एक test tube में 5 ml urine लेंगे और urine का upper portion एक spirit lamp या burner पर boil करेंगे I
अब इस गर्म किये गए urine क
े upper part की urine क
े lower part (जिसे गर्म नहीं किया था) से तुलना करेंगे I
यदि upper part में cloudiness या turbidity होगी तो यह proteins या phosphate /carbonates की urine में
उपस्थिति क
े कारण हो सकती है I अब इसमें 2-4 drops 10% glacial acetic acid डाल कर पुनः boil करेंगे I
यदि turbidity बनी रहती है तो यह urine में proteins की उपस्थिति की पुष्टि करता है किन्तु यदि turbidity

24
गायब हो जाती है तो इसका अर्थ हुआ कि पहले जो turbidity आई थी वो phosphate या carbonate की
उपस्थिति क
े कारण थी I Turbidity (cloudiness) का interpretation इस प्रकार करते हैं :-
No cloudiness : negative (no protein)
Barely visible cloudiness : Trace
Definite cloud without granular flocculation : +
Heavy and granular cloud without granular flocculation : ++
Densed cloud with marked flocculation : +++
Thick curdy precipitation and coagulation : ++++
Urine ketone bodies
Ketone bodies are produced by the liver and used peripherally as an energy source when
glucose is not readily available. These are acetoacetic acid, acetone and
β-hydroxybutyric acid. Urine में ketone bodies की जाँच modified Rothera's test द्वारा करते हैं I
Modified Rothera's test
Principle - Acetoacetic acid और acetone (ketone bodies) sodium nitroprusside क
े alkaline
solution क
े साथ react करक
े purple color का एक complex बनाते हैं I
Procedure I (पहली विधि) -
1 g sodium nitroprusside + 20 g ammonium sulfate + 20 g anhydrous sodium carbonate - तीनों
को mix कर लेते हैं I
एक glass slide या white paper पर थोड़ा सा ऊपर बनाया powder लेते हैं (about 5 mm in diameter) और
उसपर 1-2 drop urine की डालते हैं I यादि तुरंत violet color आता है तो urine sample में acetone और
acetoacetic acid (ketone bodies) present हैं I
Procedure II (दूसरी विधि) -
5 ml urine को solid ammonium sulfate से saturate करते हैं (जब तक कि ammonium sulfate घुलना बंद
न हो जाए) I फिर इसमें थोड़ा सा sodium nitroprusside (0.5 ml 2% solution या थोड़ा सा powder) डालते हैं
I इसे Mix करते हैं और लगभग 0.5 ml conc. ammonia डालते हैं I यदि purple colour की ring बन जाए तो
ketone bodies present हैं I

25
Urine bile pigment
Fouchet’s (फ
ु शेट्स) Test for bilirubin
Principle -
Barium chloride urine में उपस्थित sulphate radicals को precipitate करक
े barium sulphate
precipitate बनाता है I अब यदि urine में bile pigments उपस्थित होंगे तो वे इस precipitate से चिपक जायेंगे
I अब Fouchet's reagent में उपस्थित ferric chloride yellow bilirubin pigments को trichloroacetic acid
की उपस्थिति में green colour क
े biliverdin में बदल देता है I
Fouchet's reagent - dissolve 25 g trichloroacetic acid (TCA) in 50 ml distilled water in a 100 ml
volumetric flask, add 10 ml ferric chloride solution (10%) and make to 100 ml with distilled water]
Procedure (विधि) -
10 ml urine + about 5 ml barium chloride solution (10%) →mix well → filter or centrifuge it →
add 1-2 drop of Fouchet's reagent on to the precipitate → appearance of bluish green color
shows the presence of bilirubin in the urine sample.
Some practice questions
Q. 1. Fill in the blanks:
A. Hemoglobin derivatives which have no oxygen-carrying capacity are , ,
B. Carboxyhemoglobin (COHb) is a stable complex of with hemoglobin.
C. Hemoglobin concentration decreases in and increases in .
D. When sulfur binds to hemoglobin molecule the resulting complex is called .
E. The method recommended by International Committee for
Standardization in Hematology (ICSH) is _______ method.
F. Readings are taken at nm in Cyanmethemoglobin method.
G. Hemoglobin molecule has two polypeptide chains and two
polypeptide chains.
H. The iron in the hemoglobin molecule is in form.
I. The iron in the methemoglobin molecule is in form.
J. The highly poisonous chemical in Drabkin's reagent is .
K. Full form of POCT is .
L. The pH of Drabkin's reagent should be and it should be stored in a

26
bottle at °C.
M. The Drabkin's reagent should be brought to temperature before using it
for hemoglobin estimation.
K3[Fe(CN6)]
N. Hemoglobin (Fe++)------------------ >
KCN
O. Methemoglobin (Fe+++)---------- >
Ans. A-MetHb, COHb, SHb, B-carbon monoxide, C-anemia, polycythemia,
D-sulfhemoglobin, E- Cyanmethemoglobin, F- 540, G- alpha, beta, H- ferrous, I- ferric, J-
potassium cyanide, K- point of care testing, L- 7.0-7.4, dark coloured, 4-20, M- room, N-
methemoglobin, O- Cyanmethemoglobin
Q.2. Fill in the blanks:
GOD
A. Glucose + H2O + O2 --------> ___________ + H2O2
POD
B. _________ + 4-aminophenazone + _______ ---------> quinoneimine complex (red
colored) + H2O
C. plasma's water content is higher than the water content of ______.
D. Plasma glucose is about 12-13% ________ than that of whole blood.
E. The reference method for glucose estimation is ________ method.
F. The full form of GOD is ___________.
G. The full form of POD is __________
H. The reading in GOD/POD is taken at __________nm or ________ filter.
I. The reagent, standard solution and plasma/serum sample should be at ________
temperature before running the test.
J. Every batch of glucose test should include _______ _______ ______ samples for
quality check purposes.
OD of Test
K. Glucose concentration (mg/dl) = ------------------------ × _____________
OD of Standard
L. The blood sample for glucose estimation is taken in _________ tube.
M. Fluoride inhibits enzyme ________ to prevent glucose degradation with time.
N. Glucose in blood can be estimated in serum, _______ and _______ blood.
O. According to WHO diagnostic criteria for diabetes the fasting plasma glucose should be
≥ ______mg/dL.
P. According to WHO diagnostic criteria for diabetes the 2-hour post glucose (75 g) plasma
glucose (PG) should be ≥ ______mg/dL.
Ans.
A- gluconic acid, B- H2O2, phenol, C- cells, D- higher, E- hexokinase, F- glucose oxidase, G-
peroxidase, H- 505-510, green, I- room, J- internal quality control, K- concentration of standard,
L- fluoride, M- enolase, N- plasma, whole, O- 126, P- 200.
Q.3. Match the following :

27
A. Benedict's test द्वारा urine test में
प्राप्त color (ppt) sugar result
(i) green without ppt (a) 0.5 % - 1% (+)
(ii) green with ppt (b) trace amount (<0.5%)
(iii) yellow ppt (c ) > 2% (++++)
(iv) orange red ppt (d) 1.0 - 1.5% (++)
(v) brick red ppt (e) 1.5 - 2% (+++)
Ans. (i) - (b), (ii) - (a), (iii) - (d), (iv) - (e), (v) - (c )
B. Sulfosalicylic acid test द्वारा urine test में
प्राप्त परिणाम Protein result
(i) Clear Urine (a) +
(ii) Slight turbidity (b) nil (protein absent)
(iii) Mild turbidity (c ) +++
(iv) Moderate turbidity (d) ++++
(With flocculation)
(v) Visible white precipitate (e) ++
Ans. (i) - (b), (ii) - (a), (iii) - (e), (iv) - (c ), (v) - (d)
Q.4. Identify the true and false statements :
(a) Heat test में urine को गर्म करने पर यदि turbidity आती है किन्तु glacial acetic acid डालने पर
turbidity गायब हो जाती है तो urine में protein present है -
(b) Urine में ketone bodies की जाँच क
े fलए Fouchet's test करते हैं -
(c) Urine में bile pigments क जाँच क
े fलए Rothera’s test करते हैं -
(d) Rothera's test acetone और acetoacetic की उपस्थिति में positive आता है
Ans. (a) false, (b) false, (c ) false, (d) true
REFERENCES
1. Practical Clinical Biochemistry. Varley H, Gowenlock A H, Bell M. Fifth edition, 1991.
2. Higgins C, Hemoglobin and its measurement. www.acutecaretesting. org (2005)
3. Practical Hematology. Dacie, Sir John V and Lewis S M., Seventh edition, 1991
Disclaimer : The pictures given in the text have been downloaded from Google images and
I am thankful to the persons who have uploaded these pictures.
Dr. P. K. Nigam
Ph. D. (Retired Biochemist)

DMLT (1st Year) : Blood Hemoglobin, Blood Glucose estimation and Urine routine examination - Some basic concepts (U. P. State Medical Faculty syllabus) in ENGLISH & HINGLISH

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