RBC METHODOLOGIES-II

I.ERYTHROCYTE SEDIMENTATION RATE(ESR)

Rate of settling of RBC from the plasma after the addition of anticoagulant.

Importance of ESR1. Good index for the presence of hidden

carcinoma but active diseases.2. It measures the suspension stability of RBC.3. It measures the abnormal concentration of

fibrinogen and serum globulin. Roleaux formation (Packing or piling of RBC)

METHODS

A.Wintrobe and landsberg method Anticoagulant used- Ammonium potassium

oxalate (wintrobe solution/ double oxalate/balanced oxalate/ paul-Heller’s soln.)

Tube – wintrobe tube - Left side – red; 0 on top and 10 cm bottom. - Right side – white; 10 cm top and 0 bottom.

Procedure1. With a long stem pasteur pipet, fill the wintrobe

tube with oxalated blood up to 0 mark.2. Let the wintrobe tube stand perfectly vertical.3. Read result after 1 hour. Reading must be done

on the left red side of the tube.Normal values Male - (0-9) mm/hr Female - ( 0-20 )mm/hr ( bcg less RBC ) Children- (1-13) mm/hr

White 10

Red 0

Red cells

0 at bottom at bottom 10

Layers -Plasma layer-Buffy coat (WBC and platelets)-Packed RBC (hematocrit)

Wintrobe tube

Females have more space in settle down and faster than male and children because they have less RBC.

( 1cm – 10mm/hr)B.Westergren method (200mm) – most

sensitive and most accurate . - Anticoagulant used -3.8% sodium citrate - Tube- westergren tube (through suction method

long tube)

200 mm

0

Procedure 1. Fill the tube with the citrated blood2. Stand the tube vertically and read result at the

end of the 1st hrs. and 2nd hrs.Normal values Male – (3-5) mm/hr 7-15 mm/2hr Female – (4-7) mm/hr 12-17 mm/2hr

Comparision

Wintrobe Wester gren

Bore 3 mm 2.5 mm

Graduation up to 100 mm

up to 200 mm

Anticoagulant Double oxalate

3.8% sodium citrate

Amount of blood

1 ml 2.4 ml

Reading once Twice

Hematocrit

C. Graphic cutler Anticoagulant – 3.8% sodium citrateD. Linzenmeier Anticoagulant – 3.8% sodium citrateE. Roarke- Ernstiene Anitocagulant – HeparinF. Bray’s Anticoagulant- 3.8% sodium citrate

G. Micro methods1) Micro landau Anticoagulant- 5% sodium citrate2) Smith micro Anticoagulant- 5% sodium citrate3) Crista or hellige- vollmerStages of ESR1. Initial rouleaux formation – (first 10 min)2. Period of rapid settling – (next 40 min)3. Period of final settling – (last 10 min) total 60 minutes or 1hr.

Factors in ESR1. Intrinsic Factor - nos of RBC ( less RBC faster settlement) - size of RBC ( Bigger the size is faster the

settlement) - viscosity of Plasma ( less viscous fast

settlement) * nos of RBC- inversely * size of RBC- directly

2. Extrinsic factor Length of tube ( smaller length fast settlement) Diameter of tube (wider diameter fast settlement) Position of tube(vertical or slightly fast settlement Temperature ( high temp. fast settlement) Pipetting ( incorrect pipetting result error) Volume of blood ( less blood faster settle.) Anticoagulant (more anticoagulant slow

settlement)

II Osmotic fragility test Test the stability of RBC in hypotonic solutions. Follows the law of osmosis. Factor affecting OFT - Red cell shape - Red cell volume - Red cell surface Area - State of Red cell membrane *Fragile cells( decrease)- spherocytes *Resistant cells( increase)- sickle cell , target cell,

reticulocytes

METHODS

1. Sanford method Different conc. Of hypotonic solution 12 test-tube is used Initial solution used – 0.5% NaclInterpretation No hemolysis – tubes with compact sediment

and clear solution. Initial hemolysis -1st tube from the left with not

so compact sediment and with dark red solution Complete hemolysis - 1st tube from the left

without sediment and with dark red solution.

Normal values Initial hemolysis- tube 22 Complete hemolysis- tube17Increase OFT Initial hemolysis- tube 24 ( increased-hemolytic

anemia , hereditary spherocyte) Complete – tube 20 { decrease-sickle cell anemia,

thalassemia , jaundice, SIDA(severe ion deficiency Anemia)}.

Decreased OFT Initial hemolysis-tube 19 Complete hemolysis-tube 15

2. Modified Sanford – in terms of ml

3. Griffin and Sanford method

4. Dacies method Hemolysis read is used through

spectrophotometer. (Transparent – fake pink- light pink- red)

III.ERYTHROCYTE INDICES

Important in assessing borderline types of anemia. Computed using 3 determinants Hb, hematocrit and

RBC count.A. Mean corpuscular volume (MCV) Average volume of an individual RBC. volume % Hct x 10 = cubic micra or femtoliter RBC in millions Normal value- 82- 92 cubic micra.

Interpretation 95- 160 cubic micra- macrocyte 72-79 cubic micra – microcyte 50-71 cubic micra – microcyte hypochromic (less Hb)Example Hct = 46 vol % RBC count – 5,000,000/ cumm MCV= 46 x 10 = 92 cubic micra 5

B. Mean Corpuscular Hemoglobin (MCH) Ratio of Hb to red cell count Average weight or amount of Hb in an individual

RBC in millions gm Hb x 10 = uug or picogram RBC in million Normal value – (27-33) uugInterpretation > 33 uug- macrocyte < 27uug – microcyte < 22 uug – microcytic hypochromicExample Hb = 16gm/100ml RBC count = 5,500,000/cumm MCH= 16 x 10 = 29 uug 5.5

C. Mean corpuscular hemoglobin conc.(MCHC) Mean conc. Of Hb in the average RBC. Normal Value = 32-38% Average weight or amount of Hb in an individual

RBC gm Hb x 100 = % vol. % HctExample Hb -16 gm/ 100ml Hct = 46 vol. % MCHC = 16 x 1000 = 34.7% 46

Normal – normochromic < 32 – hypochromic > 38 – hyperchromic