Routine Hematology

Download Routine Hematology

Post on 17-Jan-2016




4 download

Embed Size (px)


Hemoglobin MeasurementHematocrit MeasurementBlood Cell Counts (RBC, WBC)Preparation and Staining for Blood SmearsErythrocyte Sedimentation RateReticulocyte CountEosinophil CountSickle Cell TestsHEMOGLOBIN MEASUREMENT Procedures


<p>Introduction</p> <p>ROUTINE HEMATOLOGY PROCEDURES</p> <p>Hemoglobin MeasurementHematocrit MeasurementBlood Cell Counts (RBC, WBC)Preparation and Staining for Blood SmearsErythrocyte Sedimentation RateReticulocyte CountEosinophil CountSickle Cell Tests</p> <p>HEMOGLOBIN MEASUREMENT</p> <p>HEMOGLOBIN MEASUREMENTHEMOGLOBIN: main component of the RBC that serves as the vehicle for the transportation of oxygen and carbon dioxide.- It imparts red color to the blood.- It buffers blood pH.</p> <p>Purpose of estimating hemoglobinTo detect the oxygen carrying capacity of blood.The result assists in detecting diseases, which causes a deficiency or excess of hemoglobin.Studying changes in hemoglobin concentration before or after operations and blood transfusions.To detect anemia and its severity and to monitor an anemic patients response to treatment.To check hemoglobin level of blood prior to donating blood.To calculate red cell indices.</p> <p>Normal Values</p> <p> The normal value depends on the age and sex of the individuals:</p> <p>MALE: 13-18 g/dlFEMALE: 12-16 g/dlAT BIRTH: 15-20 g/dl</p> <p>Methods of Estimation of Hemoglobin</p> <p>Colour based (Colorimetric): Based on the colour of hemoglobin or a derivative of hemoglobin.Physical method (Gravimetric): Based on specific gravity.</p> <p>Chemical method: Based on iron content of hemoglobin.</p> <p>Gasometric method: Based on oxygen combining capacity of hemoglobin.Spectrophotometric method: Based on measurements using spectrophotometric devices.</p> <p>COLORIMETRIC METHOD:1. Sahlis Method or Acid Hematin method</p> <p> Principle</p> <p>Hemoglobin is converted to acid hematin by N/10 HCl, the resulting brown colour is compared with standard brown glass reference blocks. The intensity of the brown colour depends on the amount of acid haematin produced, which in turn depends on the amount of hemoglobin in the blood sample.</p> <p>Reagents:</p> <p>N/10 Hydrochloric acid (HCl)</p> <p>Distilled water for dilution.</p> <p>Blood anticoagulated with EDTA</p> <p>N/10</p> <p>Procedure</p> <p>Place N/10 HCl in the tube up to the lowest mark.</p> <p> Draw blood up to 20 mm mark in the pipette and transfer it to the acid in the tube.</p> <p> Rinse the pipette well by drawing up the acid and re expressing it. Mix the acid and blood by shaking the tube well.</p> <p>Allow it to stand for at least 10 minutes - to allow brown colour to develop due to the formation of acid hematin.</p> <p> Now dilute the solution with distilled water drop by drop with continuous mixing, using the glass rod provided.</p> <p>Procedure (contd....)</p> <p> Match the color with that of the glass plates in the comparator.</p> <p> Reading is taken when the color of the solution in the tube exactly matches the comparator. Matching should be done at eye level against natural light.</p> <p> The level of the fluid at its lower meniscus is noted and the reading on the scale corresponding to this level is read as gram/dl.</p> <p>Sources of Errors</p> <p>Technical errors Improper mixing of blood, Errors in pipetting, Tissue fluid contaminating capillary blood.</p> <p>Visual errors Taking the reading is very subjective, as it is a comparison of colours. It can vary from person to person. So the results may not be accurate.</p> <p>Quality of the color comparators can affect the reading If the glass blocks are old or faded it can cause wrong results.</p> <p> Insufficient time allowed for the conversion of Hb to acid haematin. A minimum of 10 minutes is required for the reaction to be almost complete, otherwise biological false negative result is obtained.</p> <p>Carboxyhemoglobin, methemoglobin and sulfhemoglobin are not converted to acid haematin.</p> <p> Non-hemoglobin substances such as protein, liquid and cell stroma interfere with the colour of blood diluted with acid and hence give false results.</p> <p> Time delay - The brown colour of acid haematin is not stable, so undue delay in reading the test result is not allowed.</p> <p> 2. Alkaline Hematin method</p> <p>In this method the Hb is converted to alkali hematin by the addition of N/10 NaOH.The alkali hematin gives a brown colour that can be read against comparator standards or in a colorimeter.</p> <p>Apparatus:Photo electric meter with green filter.N/10 NAOH0.05 ml pipetteStandard (Gibsons and Harrisons): This is a mixture of chromium potassium sulphate, cobaltous sulphate and potassium dichromate in aqueous solution. The solution is equal in colour to 1 in 100 dilution of blood containing 16.0 Hb per dl.</p> <p>Technique:Add 0.05 ml of blood to 4.95 ml of N/10 NAOH.</p> <p>Mix well and boil for 4 minutes, along with 5 ml standard solution.</p> <p>Cool quickly in cold water,and match the test against standard using colorimeter using green filter.If the test give too high value add 5.0 ml of water and read again.</p> <p>Advantage 1) Unlike Sahlis method, carboxyhemoglobin, sulfhemoglobin are converted to alkali hematin.2) Fetal haemoglobin is resistant to denaturation by alkali and this method is used to determine the level of fetal haemoglobin in blood.Disadvantage The solution of Hb in alkali has to be heated to ensure complete denaturation.Note:Matching should be done within 30 minutes after boiling</p> <p>20</p> <p>Spectrophotometric method</p> <p> The esimation is based on Beers and Lamberts law ( OD= HGB CONC)1) Cyanmethemoglobin method 2) Oxyhemoglobin method</p> <p>Cyanmethaemoglobin method This is the preferred and the most accurate method for determining the hemoglobin concentration in laboratory. </p> <p> Principle: ***Blood is diluted in a solution of potassium ferri cyanide and potassium cyanide. ***The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540 nm or in a colorimeter using a yellow-green filter.</p> <p>Reagents</p> <p>Hemoglobincyanide standardDetergent modified Drabkins solution Potassium Ferricyanide Potassium Cyanide Potassium Dihydrogen Phosphate(ORIGINALLY, NaHCO3) Non-ionic detergent/Sterox SE/Triton X-100/Saponin Distilled water</p> <p> ProcedureTake 5ml of Drabkins solution in a large sized test tube.</p> <p>Add 20 micro litres of well mixed anticoagulated venous blood.</p> <p>Rinse the pipette and mix well.</p> <p>Allow it to stand at room temperature for 3 - 10 minutes.</p> <p>Absorbance is measured against reagent blank at 540 nm either in a spectrophotometer or in colorimeter.</p> <p>Advantages</p> <p>All forms of Hb except SHb are readily converted to HiCN. Direct comparison with HiCN standard possible. Stability of the diluted sample. Easy to perform the test. Reagents are readily available. The standard is stable.</p> <p> DisadvantagesPotassium cyanide in the solutions is poisonous, though it is present only in a very low concentration hence the reagents should be handled carefully. Explosion can occur if undiluted reagents are poured in the sink. Hydrogen cyanide is released by acidification and the gas if it accumulates can result in explosion. Reagents and samples should be disposed along with the running water in the sink. Increased absorbance not due to hemoglobin may be caused by turbidity due to abnormal plasma proteins, hyperlipemia, high WBC count or fat droplets, and the presence of HbS and Hb C.</p> <p>27</p> <p>Specific gravity method (Physical method)Assesses hemoglobin concentration via blood specific gravity.Mass screeningSG of CuSO4: 1.053 (equivalent to 12.5g/dL)</p> <p>PROCEDURE:Collect blood sampleDrop a blood the copper sulfate solution (distance: 1 cm)Observe the activity of the blood</p> <p>INTERPRETATION: (Within 15 SECONDS describe how the drop of blood behaves in the solution)</p> <p>MAINTAIN: hgb concentration equals to 12.5 g/dL/13.5 g/dLFLOAT: 12.5 g/dL</p> <p> Chemical methods (estimation of the iron content)</p> <p>The principle is based on the fact that each molecule of haemoglobin contains 4 atoms of iron or 3.47 grams of hemoglobin.</p> <p>The iron present is detached from the hemoglobin and measured.</p> <p>The hemoglobin is calculated by using the formula.</p> <p>Hb(gm/dl) = Blood iron content in mg/dl blood 3.47</p> <p>Gasometric methods(Measurement of Oxygen combining capacity)It is done by using van Slyke apparatus.The principle is based on the fact that one molecule of O2 binds to each iron atom.So one molecule of hemoglobin binds 4 molecules of oxygen. Thus oxygen combining capacity thus indirectly measures the amount of Hb.</p> <p>It is estimated that 1 gram of hemoglobin binds about 1.34 ml of oxygen. </p> <p>From this the haemoglobin concentration is calculated by using the following formula.</p> <p>Hb in gm/dl = O2 binding capacity in ml/dl blood 1.34</p> <p>Quality Control</p> <p>The important aspect of quality control is to identify those steps in which the likelihood of error is high and to consider ways to minimize that likelihood. Some of the measures followed are:</p> <p> Duplicating samples.</p> <p> Hemolysate of known value are run with batches of tests.</p> <p> Haemoglobin values are compared with other values. For example PCV = 3 x Hb. This is true unless there is marked microcytosis or macrocytosis. If haemoglobin values are abnormal either too low or high, check peripheral smear to look for other associated abnormalities.</p> <p>RULE OF THREE</p> <p>HEMATOCRIT MEASUREMENT</p> <p>HEMATOCRIT MEASUREMENT (Packed Cell Volume)The percentage of the total volume of WB that is occupied by pRBCs when a known volume of blood is centrifuged at a constant speed for a constant period of time.</p> <p>Relevance:</p> <p> MOST accurate and simplest for detecting presence and degree of anemia or polycythemia.</p> <p>2. Essential in determination of red cell indices that help in detecting and classifying various types of anemia.</p> <p>Principle:GRAVITY/CENTRIFUGAL FORCE:Due to gravity and centrifugal force, the dense particle settles down.</p> <p>1. MacromethodCENTRIFUGE: 2000-2300 for 30mins1st layer: FATTY LAYER: barely visible unless patient is lipemic2nd layer: PLASMA3rd layer: BUFFY COAT (1mm: 10,000 WBCs/mm3)Bottom: PACKED CELLS</p> <p>WINTROBE TUBE:Length: 115 mmLEFTRIGHTBore: 3 cm0-100100-0ESRHCT</p> <p>2. Micromethod *Capillary tube: LENGTH: 70-75 mmBORE: 1 mmHolds app. 0.05 ml of WB/ 50 uL of blood*Centrifuge: 10,000-15,000 for 5 minsTop: PLASMA2nd: PLATELETS3rd: LEUKOCYTES4th: RETICS, nRBCs5th: MATURE RBCsBOTTOM: Clayseal (4-6 mm)</p> <p>RESULTS</p> <p> Height of packed red cells (mm)Hematocrit = ------------------------------------------ 100 Height of packed RBCs and plasma (i.e, height of blood column)</p> <p>HCt. = 45/100 100 = 45 percent</p> <p>This means out of 100 volumes or parts ,45 volumes are red cells and 55 volumes are plasma.</p> <p>3. AUTOMATED METHODSHCT is COMPUTED.HCT= MCV X RBC COUNT</p> <p>Increased PCVPolycythemia-Newborns, High altitude, Hypoxia due to lung and heart diseases.Congestive Heart failure, Burns (loss of plasma), Dehydration, Severe Exercise, Emotional stressDecreased PCVAll types of Anaemia</p> <p>Pregnancy (Hemodilution)</p> <p>Ingestion of large amount of water</p> <p>BLOOD CELL COUNTS</p> <p>BLOOD CELL COUNTS</p> <p>Counting ChambersFUCHS ROSENTHAL: 4 X 4 X 2</p> <p>2. SPEIRS LEVY: 2 X 5 X 4</p> <p>3. IMPROVED NEUBAUER: 3 X 3 X 2</p> <p>Red Cell and White Cell CountsRBC problem: anemia, polycythemiaWBC problem: leukocytosis, leukopeniaRBC reference rangesMale4.50 - 6.00 x 106/L (x 1012/L)Female4.0 -5.0 x 106/L (x 1012/L)WBC reference rangeBoth4.5 11.0 x 103/L (x 109/L)</p> <p>DIFFERENCES BETWEEN RBC AND WBC PIPETTE</p> <p>RBC pipetteWBC pipette1) It has a red beadIt has a white bead</p> <p>2)It has graduations up to mark 101It has graduations up to mark 113)Size of bulb is largerSize of bulb is smaller</p> <p>4)Size of lumen is smallerSize of lumen is larger</p> <p>RBC PIPETTEWBC PIPETTE</p> <p>Thoma PipetRBCAspirate to 0.5 or 1.0Dilute to 101DF = 200 or 100WBCAspirate to 0.5 or 1.0Dilute to 11DF = 20 or 10</p> <p> IMPROVED NEUBAUER HEMOCYTOMETER</p> <p>Each scale is 3mm wide and 3mm long.Depth of the chamber is 0.1mmThe whole scale is divided into 9 big squares.Each square is 1mm long and 1mm wide.</p> <p>The four corner squares are further divided into sixteen smaller squares and are used for WBC counting.</p> <p>Four corner squares are meant for WBC counting.Total = 64 small squares</p> <p> WWWW</p> <p>Central square is divided into 25 medium sized square and are separated by triple lineThe medium sized square are further divided into 16 small square(tiny)The four corner and central square are used for platelet and RBC count.</p> <p>Counting RuleDo not count cells touchingBottom lineRight line This is to avoid double counting.</p> <p>Principle Dilution of bloodSampling of diluted suspension into measured volumeCounting of cell in that volume</p> <p>FOCUSING4X to see the general formation of slide.10X for WBC counting 40X for RBC/Plt. counting</p> <p>Source of errorFalse high countFalse low countImproper mixingUneven distribution of cellError in pipettingError in calculationBlood taken from area of hemo concentrationYeast, dirt and leucocyte are counted as RBC</p> <p>Blood diluted with tissue fluidUndue delay in counting of cellClumping of cell(AIHA)Uneven distribution of cellFaulty technique of countingImproperly standarized counting chamber</p> <p>*RBC COUNT @ HPORBC diluting fluids are ISOTONIC solutions.DILUTION (RBC pipette): 0.5:100 (blood: diluent) = 1:200RBC count: RBC/mm3= #RBC x Area Correction Factor (ACF) x Depth Correction Factor (DCF) X Dilution Factor (DF) = #RBC X 5 X 10 X 200= RBC X 10,000SI: x 0.001</p> <p>RBC ExampleAspirate blood to 0.5, then dilute to 101DF = 200Counted 500 erythrocytes in 5 small squares</p> <p>RBC count: RBC/mm3= #RBC x Area Correction Factor (ACF) x Depth Correction Factor (DCF) X Dilution Factor (DF)</p> <p>RBC = 500 rbc x 5 x 10 x 200</p> <p> = 5.00 x 106/mm3 (L)</p> <p>*WBC COUNT @ LPOWBC diluting fluids are HYPOTONIC SOLUTIONS to lyse non-nucleated RBCs.Mix for 3 minutes to allow lysis of RBCs.DILUTION: 0.5:10 (Blood:Diluent)= 1:20*Leukocytosis: Use RBC pipette (1:100 or 1:200)WBC count: WBC/mm3= #WBC x AF x DCF X DF = #WBC X 0.25 X 10 X 20= WBC X 50SI: x 0.001</p> <p>WBC ExampleAspirate blood to 0.5, then dilute to 11DF = 20Counted 160 leukocytes in 4 large squares</p> <p>WBC/mm3= #WBC x AF x DCF X DF = #WBC X 0.25 X 10 X 20= 160 x 0.25 x 10 x 20 = 8.0 x 103/mm3 (L)</p> <p>** NUCLEATED RBCs are not lysed by WBC diluents. They are then falsely counted as WBCs.</p> <p>PREPARATION AND STAINING FOR BLOOD SMEARSTypes of blood smears: The cover glass smear.The wedge smear.The spun smear.The are two additional types of blood smear used for specific purposesBuffy coat smear for WBCs &lt; 1.0109/L and LE cell preparation Thick blood smears for blood parasites</p> <p>BLOOD SMEAR PREPARATION</p> <p>Wedge blood smearSpecimen : EDTA blood within 2 to 3 hours &amp; collected to the mark on tube.Note : May change RBCs morphology such as Spiculated (crenated) cells if :Excessive amount of anticoagulant to specimenOld blood - long standing. Warm environment (room temperature) may hasten changes.</p> <p>ProcedurePlace a drop of blood from mixed sample on a clean glass slide.Spreader slide using anoth...</p>