Plasma Hemoglobin And Urine Hemoglobin Estimation 543m6h

PLASMA Hb AND URINE Hb Presented by: PRATEEMA Moderater: Dr. ANSHU PALTA

Normally red cell undergo lysis at the end of their life span of about 120 days. In haemolytic anaemia the red cell life span is shortened. The causes can be divided into three groupsI. 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 & drug associated antibody damage. III. Damage by outside factors such as mechanical trauma , microangiopathic conditions, thrombotic thrombocytopenia purpura, and chemical toxins.

At the end of a normal life span, the red cell are destroyed 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 to lead to Hb being excreted in urine (haemoglobinuria). However , there is often a combination of both mechanisms.

INVESTIGATION OF HAEMOLYTIC ANAEMIA---• The clinical & laboratory phenomena of increased haemolysis reflect the nature of the haemolytic mechanism , where the haemolysis is taking placeand the response of bone marrow to anaemia, resulting from the haemolysis , namely erythroid hyperplasia and reticulocytosis.

Evidence of increased haemolysis Hb estimation : decreased Reticulocyte count : increase due to erythroid hyperplasia. Plasma Hb : increased S. haptoglobin levels : decreased Stained PBF : fragmented RBC’s , spherocytes , macrocytes , polychromatophils. Test for increased unconjugated serum bilirubin by Urinary urobilinogen excretion by erhlichs method.

Test performed to detect plasma/urine Hb:a) Peroxidase method b) Direct measurement of Hb by spectrophotometry -----------------------------------------------------------------------PEROXIDASE METHOD Principle : in the peroxidase method , the catalytic action of haem containing proteins brings about the oxidation of 3,3,5,5 tetramethylbenzidine by hydrogen by hydrogen peroxide to give a green colour. The intensity of reaction is compared in a spectrophotometer with that produced by solutions of known Hb concentration.

Sample collection : every effort must be made to prevent haemolysis during the collection and manipulation of blood. A clean venipuncture is essential, a plastic syringe and relatively wide bore needle sideshould be used. Haemolysis can be reduced to a minimum if the blood is collected through 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 upto 100 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 coloured bottle. c) Standard : it is convinient to use HiCN standard solution as the source of Hb . It is made to a concentration of 60mg/l

Method : take three clean dry test tubes and label as t, s ,c Test standard control Bezidine 1 ml 1 ml 1 ml Sample 0.02 ml standard 0.02 ml d/w0.02ml 1% hydroden 1 ml 1ml Peroxide 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.

Colour of the reaction .

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 nil Interpretation : increased plasma/urine Hb is seen in intravascular haemolysis seen in PNH , PCH , cold haemagglutin syndrome, black water fever, march haemoglobinuria, and mechanical haemolytic anaemai. Mild to moderate rise in plasma Hb is seen in AIHA and sickle cell anaemia. Physiological conditions like voilent exercise and ingestion of certain food and food derivatives.

Haemosiderinuria: significance- haemosiderinuria is a sequal to the presence of Hb in the glomerular filterate. It is a valuable sign in intravascular haemolysis because the urine will be be found to contain iron containing granules even if there is no haemoglobinuria at that time. However, haemosiderinuria is not found in the urine at the onset of haemolytic attack even if this is accompanied by haemoglobinemia and haemoglobinuria because Hb first has to be absorbed by the cells of the renal tubules. The intracellular breakdown of Hb liberates iron which is then re excreted. Haemosiderinuria may persist for several weeks after haemolytic episode.

Method for demonstration of haemosiderinuria: pearls prussian blue reaction Collection of sample: done in iron free tube and collect 5ml of urine sample in iron free comntainer. Principle : heamosidrin reacts with potassium ferrocyanide to form a blue coloured compound ferriferrocyanide : the reaction is the basis of positive prussian blue test. Reagents : a) 2g% pot. Ferrocyanide b) .2 M HCL For use mix equal volume of a & b Counterstain with .1% neutral red

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.