Collection of blood and Anticoagulants

Purposes of Blood Collection Haematological study includes TLC DLC Hb % ESR Blood viscosity PCV Coagulation profile study Study for haemolytic disorder

Biochemical Analysis Bacteriological culture eg. Culture for detection of infecting bacteria in septicemia and detection of protozoa. Immunological study includes eg. Detection of antigen and antibodies in blood. Blood transfusion services eg. Detection of blood group. Cross matching of blood. Immunological study for detection of infecting viruses and organisms.

Collection of Blood Blood is collected from Veins- From antecubital vein(most commonly), veins of arm and dorsum of hands. Long saphenous vein is used in situations of nonavailability of the former veins. In infants, femoral vein is the commonest site. In newborn child, umbilical vein and scalp vein are commonly used.

Capillaries are used where small quantity of blood is required eg. in micromethods and in preparation of blood film on the slides. This is collected from the finger tips or ball of finger or lobule of ear. In infants, the heal and thumb is used for collection of capillary blood.

PROCEDURES FOR COLLECTION OF BLOOD Blood should be drawn with all aseptic precautions with sterile dry syringe and needle. The needle should not be too fine or too thick. 21-23 G, 11.5" needle is used foe adults. A short needle with shaft of about 15 mm long is most suitable for infants and children. The technicians drawing the blood should use gloves and gown during the blood collection and should be careful to avoid needle prick.

A blood pressure calf is applied in the arm and pressure is raised up to 40 mm of Hg. In situation of non-availability of blood pressure calf, a tourniquet is sufficient to make the vein prominent.

Commonly used anticoagulant Disodium or di potassium ethelene diamine tetra acetic acid Ammonium and potassium oxalate (3:2) Sodium citrate in appropriate conc. Heparin Acid citrate dextrose Citrate phosphate dextrose sol. with amino acid (Adenine) Sodium fluoride in appropriate conc. Potassium oxalate in appropriate conc.

Anticoagulant act as a preservativeACD- is a mixture of citric acid , trisodium citrate and dextrose. Advantage of CPDAIProvide nutrition to the RBCs through the aminoacid adenine.It contains Trisodium citrate, citric acid, Disodium hydrogen phosphate, dextrose and adenine.

Anticoagulants used for coagulation profile Trisodium citrate sol( 3.8% conc.) Heparin- used as a dry anticoagulant. Siliconised vial or tube Disadvantage of Sodium citrateSodium citrate is always used in soln which will alter the conc. of blood and thus actual PCV, Hb%, TLC. DLC will not be obtained .

Advantage of mixture in Ammonium oxalate & Potassium oxalateAmmonium salt causes swelling of RBCs and Potassim salt causes shrinkage of RBCs. Thus to prevent swelling and there by bursting of RBCs and also shrinkage of RBCs double oxalate is used.

Stains used for blood film: Romannowskys group of stains are most commonly used which containmethelene blue and its oxidation product in combination with eosine and its derivative. Components are Azure B( trimethyle thionine), Azure C, Azure A Methyle violet

Others dye Stains included are Jenners stain, simplest Giemsas method most complex but cost effective Leishmans stain ( in between) Staining result is best with combined Jenner Giemsa method Wrights stain is comparable to Leishmans stain.

Colours of the granules of polymorph Eosinophilic granules are bright red as they contain spermine derivative. The granules gives red colour by Staining strongly with acidic component of the dye eg Eosin Y. Basophils granules are deep violet.The granules contain heparin which has the affinity for basophilic dye, Azure B. Neutrophil granules are pink as they stained weakly with azure complex.

Procedure of Leishmans stain The film is better stained after drying. A longer gap of more than an hour induces artifacts. It is desiarable to fix the film with acetinone free methanol if the blood slide is to be stained later. The film does not require any fixative if the staining is done with RS group of stain.

The blood slide, after drying in air is flooded with Leishmans stain so as to cover the smear only. After 1-2 m of fixation, buffer soln or fresh distilled water is added on the film slowly so that the stain is not lost from the slide by over flooding and is now the whole slide is now covered with the stain. It is the left for 5-7 minutes.

The slide is now washed in a stream of buffer and distilled water and then tap water. The back of the slide and the area free from the film is cleaned and dried in air. The blood film may show indistinct nucleus with scattered granules if chlorinated tap water is used instead of buffer or fresh distilled water.

The granules of eosinophils take bluish violet colour instead of bright stain if old distilled water whose pH become acidic due to dissolved CO2 or due to use of buffer whose pH is less than 6.8. A pH of 6.8 is recommended for staining of granules of leucocutes.

A pH of 7.2 is best for staining the Schuffners dot. An alkaline pH accectuate the azure component at the expense of eosin and an acidic pH accentuate the eosin Y component at the expense of azure B. An acidic pH disturbs the colouration of the granules of eosinophil.

Estimation of Haemoglobin

Methods are: Cynameth haemoglobin( HiCN) method Acid haematin method Oxyhaemoglobin Method Alkali haematin method Acid alkali method Automated or semi-automated method

Most suitable method is Cynamethhaemoglobin method becauseIt is most stable Almost all Hb except sulphaemoglobin is converted to cynamethhaemoglobin. A stable reference standard is available. Large number of samples can be tested at a time.

Shortcomings of Acid haematin method Acid haematin is unstable. The colour fades away immediately after reaching the peak conc. The colour intensity has to be matched with the brown colour of comparator box visually. There is no International standard for this brown colour comparator. There is more chance of personal error as it is a visual method. Not all type of Hb is converted to acid haematin eg. sulphhaemoglobin, methaemoglobin.

ESTIMATION OF Hb BY ACID HAEMATIN METHOD Blood Collection: Blood may be collected from finger prick or from vein. Blood collected in EDTA or double oxalate in appropriate proportion may also be conveniently used for estimation of Hb.

Principals Hydrochloric acid converts Hb into soluble unstable acid haematin. The color intensity of this acid haematin is measured comparing with a coloured glass comparator.

Instruments required Haemoglobinometer tube with comparator box, stirrer, dropper Haemoglobin pipette Reagents required are N/10 HCL and distilled water.

Procedure: N/1O HCL is taken into haemoglobinometer upto mark 20. Then 20 microliter blood is drawn with Hb pipette, poured in the acid and mixed well with stirrer and kept for 5-10 minutes for complete conversion of Hb to acid haematin. Distilled water is slowly added and mixed gently and the colour is simultaneously matched with the vertical comparator glass or tube. Hb is expressed in gms/dl and also in percentage.

ESTIMATION OF HB BY CYNAMETHAEMOGLOBIN METHOD Principals: When blood is mixed with a sol. containing potassium ferricyanide and potassium cyanide. All types of Hb except sulphaemoglobin is converted to cynamethaemoglobin which is measured at 540 nm in spectrophotometer or green filter in a photoelectric colorimeter against a standard.

Instruments required Hb pipette Small test tubes, stirrer, dropper etc. Spectrophotometer or photoelectric colorimeter. Drabkins solution and Standard solution.

Procedure: 20 microliters of blood is added to 4 ml (1: 200) or 5 ml (1: 250) of Drabkins solution and mixed well. Then measured within 6 hours against blank and standard solution ( 12 gm/dl for dilution of 1: 200; 15 gm/dl for dilution of 1: 250) in colorimeter with green filter or spectrophotometer at 540 nm.

Sources of errors: Errors of sampling including presence of microclot in EDTA/ oxalated blood due to inadequate mixing. There may be excess of tissue fluid diluting the capillary blood due to squeezing in finger prick method. While diluting the blood In estimation of colour intensity Improperly lysed red cells, nucleated red cells. paraproteins, lipids may give erroneously high results.

Hb conc in normal individual Adult male-15.0 2.0 gm/dl Adult female 13.5 1.5 gm/dl Newborn child 15.0 1.5 gm/dl

Causes of physiological variations Strenuous physical exercise Diurnal variation- highest in morning and lowest in the evening High altitude

Hb is falsely raised inBurns, severe dehydration due to haemoconcentration Immediately after acute hemorrhage If the blood is taken during the intravenous infusion of iron containing drugs.

Conditions where PCV and Hb is raised: Chronic obstructive airway disease Congenital cyanotic heart disease Cushings sundrome Renal cell carcinoma Pheochromocytoma Smokers polycythemia due to carboxyhaemoglobinaemia.

Conditions of disproportionate anaemia Pregnancy due to increase in plasma volume leading to a fall of 1-2 gm/dl. Hypervolaemia- there is a fall of Hb level due to disproportionate increase in plasma volume and RBC volume. Plasma volume varies widely in renal failure leading to hydraemia or dehydration. This may give the false report of anaemia or polycythaemia.

ERYTHROCYTE SEDIMENTATION RATE & BLOOD VISCOSITY Erythrocyte sedimentation rate may be defined as the rate of sedimentation of red cells in unit time . Normal level Westergrens original upper limit of normal ESR is 10 mm /hour for men and 20 mm/hour for female. But it may be as high as 20 mm/hour for men and 30 mm/hour for women above the age of 50 years, as the ESR increases with age.

Stages of sedimentation areStages of rouleaux formation, takes only a few minutes (approx 10 min). Sinking of rouleaux at a constant speed for next 40 minutes normally. Slowing of the sedimentation rate as the rouleaux packs at the bottom of the tube for final 10 minutes.

Factors responsible for rouleaux formation Concentration of fibrinogen. Concentration of alpha-2 and gammaglobulin in plasma. Concentration of glycoprotein in plasma.

Factors influence the ESR The difference of specific gravity between red cells and plasma. Rouleaux formation of the red cells , ie. RBCs forming rouleaux rapidly and longer rouleaux will fall rapidly and hence higher is the sedimentation rate. Ratio of red cells to plasma influences ESR, ie. Increase in the ratio causes decrease in ESR and the decrease will cause an increase.

Plasma viscosity is mainly controlled by the concentration of fibrinogen and to a lesser extent by 2 globulin and globulin in the plasma. Increased conc. of glycoprotein causes increase in the rouleaux formation. Verticality of the tube. Base of the tube, ie. perfect horizontal position is required for a correct ESR. Dilution of blood with either oedema fluid or iatrogenic glucose saline causes decrease of the ESR.

Methods of estimation of ESR Wintrobes method Westwegrens method-measured in venous blood diluted accurately with 31.3 g/l trisodium citrate in the proportion of 1:4.

Merits and demerits: Wintrobes method is more sensitive when ESR is low but higher results are deceptive.Westergrens method, on the otherhand is more reliable when ESR is high. This is because of the fact that sedimentation occurs in three stages with a longer second phase, ie. sinking of RBCs occur better in longer tube and a longer second phase gives accurate result in case with high ESR. So this is better evident in Westergrens tube. In Wintrobes tube sinking occurs rapidly and packing is fast as the length is shorter.

Different methods areZeta sedimentation rate- requires only 100 microlitres of blood, takes only 3 minutes, not affected by anaemia making it easier to interpret. Micro ESR method- useful in child patient.

ESR IS MARKEDLY RAISED IN Infective- TB, Kalazar and other chronic infection. Inflammatory-Rheumatoid arthritis, Rheumatic fever, and other connective tissue disorder. Neoplastic- Multiple myeloma, lymphoma, and paraproteinamias. Miscellaneous-Aplastic anaemia, autoimmune disorders, mixed connective tissue disorders. ESR IS LOW IN Polycythaemia Sickle cell anaemia

Determination of ESR BY Westergrens method: Instruments required Westergrens tube- 30 cm in length and 2.55 mm in diameter. The bore must be uniform to 0.05 mm throughout. A scale graduated in mm extends over the lower 20 cm. Specially made racks with adjustable leveling screws for holding the sedimentation tubes firmly in an exactly vertical position. A mechanical device for drawing the blood in Westergren tube.

Reagents required Trisodium citrate as an anticoagulant. EDTA if EDTA blood is used. The usual practice is that the blood is diluted immediately with the citrate solution. If EDTA blood is used then the trisodium citrate or normal saline is used as diluent immediately before the test is performed. The sedimentation should be set up at room temperature ie. 18-25C. Sedimentation is normally accelerated as the temperature rises.

Procedure Blood sample is collected and mixed thoroughly with trisodium citrate solution 3.8% in a ratio of 1 vol of citrate to 4 vol of blood. The mixture is drawn upto into the Westergrens tube with the help of a mechanical device. The tube is placed exactly vertical and left undisturbed for 60 minutes free from vibration and draughts and not exposed to direct sunlight.

The height of the clear plasma is read above the upper limit of the sedimenting cell column to the nearest mm. This measurement in mm is ESR. A poor delineation of the upper layer of red cells, so called stratified sedimentation is due to presence of many reticulocytes. The test should be carried out within 2 hours of collection of blood, although a delay of about 6 hours is permissible if the blood is kept at 4 C.

Plasma viscosity Viscosity of serum or plasma relative to water is less than 2.0 having the normal range of 1.50-1.70 centipoise at 25 C using plasma separated from blood collected in EDTA. Plasma viscosity can be measured with capillary tube viscometer.

Different types of viscometer areOstwald viscometer Contraves low sheer 30 viscometer Rotational viscometer

Method of determination of plasma viscosity: Instruments required Ostwalds U tube viscometer Capillary pipette Centrifuge machine Reagents required EDTA to prepare EDTA blood Temparature required-25 C or 37 C

Procedure 2 ml of EDTA blood is collected and centrifuged to separate the plasma. The plasma should be collected with minimum stasis ie. as early as possible The centrifuge should be stopped at 3000 g for 5 minutes to obtain the clear plasma. The plasma is then pipetted into the Ostwalds viscometer.

The fluid is then allowed to flow from one arm to the other arm, up to the upper mark, ie. The marking above the bulb. The upper fluid level should touch the upper mark just tangentially. Stop watch is started and time noted for the fluid to reach the lower mark. The procedure is repeated thrice and average is taken.

Significance of blood viscosity The ESR and viscosity increase in parallel. But viscosity is advantageous over ESR because Viscosity is not affected by age and sex whereas the ESR does. Changes in viscosity have been seen to reflect the clinical severity of disease more closely than ESR. Measurement of plasma viscosity can be done on blood several days after collection. But the procedure of ESR measurement involves simple instruments which are easy to clean in contrast to the viscosity instrument.

Packed cell volume Packed cell volume is the amount of packed red cell volume expressed in percentage to that of the volume of whole blood. PCV is accurate simple screening test for anaemia.

Utility of PCV estimation: PCV is used for calculation of absolute values for determination of anaemia eg. MCV & MCHC. MCV is expressed as PCV/total number of RBCs in millions /cm and expressed in cubic microns. MCHC is calculated as Hb in gm/dl of blood /volume of packed cells percent and expressed in percentage.

Normal value In adults- 45-50% or 0.45-0.50 L/L In male-0.45 0.05 L/L In female- 0.41 0.05 L/L Newborn-55-60% or 0.55-0.60 L/L

PCV is increased inPolycythaemia vera Secondery polycythaemia due to chronic cyanotic heart disease and COPD Dehydration and haemoconcentration due to acute gastroenteritis, cholera, severe vomiting, etc. PCV is diminished in Anaemia Haemodilution or overhydration

Methods used for determination of PCV Wintrobes haematocrit method Micromethod Uses of Wintrobes method: Estimation of PCV is the primary use Dtermination of ESR, specially for anaemia correction with the help of correction curve. For demonstration of RE cells by preparing the smear from buffy coat after centrifugation and staining with Leishman stain in diagnosis of SLE

Wintrobes method Most commonly used method because it has the advantage that ESR can also be done before setting up for PCV. Instruments required are Capillary tube with a long capillary to reach the bottom of the Wintrobes method. Cetrifuge machine with a speed of at-least 4000 rpm.

Procedure: 2 ml of venous blood is collected and immediately mixed with double oxalate powder or EDTA powder in the proportion of 1.5 mg/ml of blood and mixed gently. The blood is drawn in a long tipped Pasteur pipette and introduced in Witrobes tube from the bottom displacing the air from down to up to the marks or 10 above. The tube is then centrifuged at 3000 rpm (23G with 22.5 radius) in a centrifuge machine. The packing is done for atleast half an hour.

The packed red cells are measured by markings in ascending order and is expressed as percentage by V/V. When ESR is to be estimated from the same sample, the tube is kept in a stand in vertical position for an hour. Reading noted and measured from descending order of marking, expressed in mm per hour. Then PCV is measured after centrifugation.

Zones separated after centrifugation and their importance: Free plasmatic zone- (topmost layer)- 48-52%. Intermediate zone or buffy coat- (zone of packed WBC )2-3%. Lower most zone of packed red cells- 45-50%. If the buffy coat is increased it indicates severe degree of leucocytosis or Leukaemia etc.If the colour of plasma is red indicates haemolysis ; yellow colour plasma indicates jaundice and milky turbid plasma indicates Lipidaemia.

Sources of error in Wintrobes method The technical factors that lead to inaccuracies areSpecimen handlingEDTA in excess may lead to cell shrinkage. The degree of oxygenation also affects the result as it has been seen that the PCV of venous blood is 2% higher than that of fully oxygenated blood.

PCV should be carried out with as little delay as possible and not longer than 6 hours after collection. Inadequate mixing of the anticoagulant may result in microclot formation and alteration of PCV.

The tube- variation in the bore of the tube may cause error. Trapping of the leucocyte, platelet clamps in the tube may result in defective cell packing. Centrifugation- the speed of packing depends upon the density of red cells and the size of red cells, the viscosity of suspending fluid, ie. on the relative densities of cells and fluid.

Determination of PCV by Micromethod Instruments required Capillary tube of uniform bore of 1 mm and length of 75 mm. coated with heparin, or heparinised. Centrifuge machine of more than 12000 g per minMicrohaematocrit centrifuge Reading device.

Procedure: The blood from the finger is taken in the heparinised capillary tube by capillary action. Blood mixed with anticoagulant may also be taken in non-heparinised capillary tube. The blood should be taken upto 60 mm only leaving the 15 mm unfilled and dry. This dry end is then sealed with plastic seal or paraffin or by heating in fine film. The sealed tube is then centrifuged at 12000 g for 5 minutes. Then PCV is measured by using a reading device.

Swift method for determination of PCV PCV along with other haematological indices can be estimated with the help of multichannel Coulter counter with little amount of blood within 40-50 seconds.

Reticulocytes These are the immediate precursor of mature RBC. They contain basophilic reticulum, which strand as greenish-blue reticulum while stained by the supravital stain. Finally these are reduced to clumps which are scattered in centre of the cell. These cells are bigger than mature RBC. They do not possess any nucleus and show slight basophilia with the romanowsky stains.

USES OF RETICULOCYTE COUNT Reticulocyte count denotes the rate of erythropoisis in the bone marrow. Its number in the peripheral bloods is increased when the erythropoisis is increased. So, its estimation is helpful for the diagnosis and assessment of the treatment of anaemia.

Normal value Normal value varies according to the methods used, it is between 0.5-1.5%. The normal value varies in certain physiological condition ie. Infancy, pregnancy and during spring.

Reticulocytes increased inIn response to anti-anaemic treatment Sickle cell anaemia Spherocytic anaemia Other macrocytic anaemia

Methods of Reticulocyte countNew methylene blue method Rapid method of Schilling Brilliant cresyl method

ESR tube and stand