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7/30/2019 Plasma Hemoglobin and Urine hemoglobin estimation

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PLASMA Hb ANDURINE Hb

Presented by: PRATEEMAModerater: Dr. ANSHU PALTA


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Normally red cell undergo lysis at the end of their lifespan of about 120 days. In haemolytic anaemia the

red cell life span is shortened. The causes can bedivided into three groups-

I. Defects within the red cell from dysfunction of enzyme controlled metabolism, abnormal

haemoglobins, and thallassaemias.II. Loss of structural integrity of red cell membrane

and cytoskeleton in hereditary spherocytosis,elliptocytosis, PNH, and immune & drugassociated antibody damage.

III. Damage by outside factors such as mechanicaltrauma , microangiopathic conditions, thrombotic

thrombocytopenia purpura, and chemical toxins.


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At the end of a normal life span, the red cell aredestroyed within the RE system in liver, spleen and bone marrow. In some haemolytic anaemias, the

haemolysis may occur predominantly in the RE system(extravascular) and the plasma Hb concentrations is

barely increased.

In other disorders, a major degree of haemolysis takes place within the blood stream (intravascular) , the plasma Hb increases substantially & in some cases

the amount of Hb so liberated may be sufficient tolead to Hb being excreted in urine(haemoglobinuria). However , there is often a

combination of both mechanisms.


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INVESTIGATION OF

HAEMOLYTIC ANAEMIA----

The clinical & laboratory phenomena of increased haemolysis reflect the nature of thehaemolytic mechanism , where thehaemolysis is taking placeand the response of bone marrow to anaemia, resulting from thehaemolysis , namely erythroid hyperplasia andreticulocytosis.


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Evidence of increased haemolysis

Hb estimation : decreasedReticulocyte count : increase due to erythroidhyperplasia.

Plasma Hb : increasedS. haptoglobin levels : decreased

Stained PBF : fragmented RBCs , spherocytes ,

macrocytes , polychromatophils.Test for increased unconjugated serum bilirubin byUrinary urobilinogen excretion by erhlichs method.


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Test performed to detect plasma/urineHb:-

a) Peroxidase methodb) Direct measurement of Hb by spectrophotometry------------------------------------------------------------------------

PEROXIDASE METHODPrinciple : in the peroxidase method , the catalytic action

of haem containing proteins brings about theoxidation of 3,3,5,5 tetramethylbenzidine by hydrogenby hydrogen peroxide to give a green colour.

The intensity of reaction is compared in aspectrophotometer with that produced by solutions of known Hb concentration.


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Sample collection : every effort must be made to prevent haemolysisduring the collection and manipulation of blood. A clean venipuncture isessential, a plastic syringe and relatively wide bore needle sideshould be

used.

Haemolysis can be reduced to a minimum if the blood is collectedthrough a wide bore needle direct into a siliconised centrifuge tube

containing heparin and the plasma is separated without delay.Avoid frothing of blood.

Avoid undue pressure.Method : Reagents----a) Benzidine compound: dissolve 1 gram of 3,3,5,5

tetramethlybenzidine in 90 ml of glacial acetic acid and make upto100 ml with water. The solution will keep for several weeks in dark

coloured bottle at 4 degree.b) Hydrogen peroxide : dilute 1 volume of 3% hydrogen peroxide with

2 volume of water before use and store at 4 degree in dark colouredbottle.

c) Standard : it is convinient to use HiCN standard solution as thesource of Hb . It is made to a concentration of 60mg/l


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Method : take three clean dry test tubesand label as t, s ,c

Test standard controlBezidine 1 ml 1 ml 1 mlSample 0.02 ml standard 0.02 ml d/w0.02ml

1% hydroden 1 ml 1mlPeroxide 1ml------------------------------------------------------------------------

mix well and keep for 20 minutea at room temp.Add 10 ml 10% glacial acetic acid in each tube and

keep for 10 minutes. Take o.d. at 515 nm.


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Colour of the reaction .


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Calculation: o.d. of test-o.d. of blank/o.d.of standard o.d. of blank * concentration of standard

Normal range : plasma Hb 10-40 mg/dl

urine Hb nilInterpretation : increased plasma/urine Hb is seen in

intravascular haemolysis seen in PNH , PCH , cold

haemagglutin syndrome, black water fever, marchhaemoglobinuria, and mechanical haemolyticanaemai.

Mild to moderate rise in plasma Hb is seen in AIHAand sickle cell anaemia. Physiological conditions likevoilent exercise and ingestion of certain food andfood derivatives.


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Method: centrifuge the fresh urine sample for 15-20 minutes at 1200 g in an iron free tube.Transfer the deposit to a iron free slide spread out to occupy an area of 1-2 cm and allow to dry.

Fix by placing the slide in methanol for 15 minites.Stain with pearls prussian blue.

Warm up ferrocyanide mixture upto 50 degree celcius and pour over the slide.

Keep for 30 minutes.Rinse under tap water.Counterstain with neutral red .

Wash under tap water.

Results: haemosidrin if present appears in the form of Isolated or grouped blue staining granules 1-3 micrometer in

size.Note : test should be done for 3 consecutive days before

declaring it as negative for haemosidrin .

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