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ROUTINE HEMATOLOGICAL ROUTINE HEMATOLOGICAL INVESTIGATIONSINVESTIGATIONS
CBC includesCBC includes•Hemoglobin estimationHemoglobin estimation•RBC countRBC count•Packed cell volumePacked cell volume•IndicesIndices•Total WBC countTotal WBC count•Differential WBC countDifferential WBC count•Platelet adequacyPlatelet adequacy
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FULL BLOOD COUNT
OTHER INVESTIGATIONS
• Erythrocyte sedimentation rate
• Platelet count
• Reticulocyte count.
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HEMATOLOGYHEMATOLOGY
• HAEM - blood LOGOS – study
• Study of formed elements of blood eg. BBC, WBC, Platelets.
• Serum : blood allowed to clot.
Plasma : centrifuge.
Plasma has FIBRINOGEN but serum does not.
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COLLECTION OF BLOODCOLLECTION OF BLOOD
• Skin puncture / Capillary blood
Eg. ear, heel etc.• Venous blood.
ANTICOAGULANTS - prevent blood from clotting
EDTA - Ethylene diamine tetra acetic acid.• Best for platelets.
Oxalates - Ammonium & Potassium Oxalate 3:2.
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Tri Sodium Citrate : ESR 1:4, BB 1:9, liquid.
Sodium Fluoride : Sugar estimation .Inhibits glycolysis.
Heparin : Liquid, in- vivo also, expensive. Bluish background, rouleaux. Osmotic Fragility test.
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BLOOD COLLECTION APPARATUSBLOOD COLLECTION APPARATUS
VACUTAINERS (Becton Dickinson) B.D.
• Needle, needle holder, glass / plastic vacuum.
• Vacuum tube instead of syringe barrel.
• Single use, quicker, cleaner, safer.
• Microtainers – infants for skin puncture.
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COLOUR CODE OF STOPPER OF COLOUR CODE OF STOPPER OF VACUTAINER.VACUTAINER.
Oxalate – blue
EDTA – lavender
Citrate – blue/ yellow
Heparin – green
Fluoride – grey
No anticoagulant – red
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HEMOGLOBINOMETRYHEMOGLOBINOMETRY
• Colorimetric methods.
• Gasometric methods / Van Slyke method.
• Specific Gravity method.
• Chemical methods. (obsolete).
1 gm of Hb holds 1.34 ml oxygen.
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COLORIMETRIC METHODSCOLORIMETRIC METHODS
Principle : Hb is converted into acid hematin, alkali hematin, cyanmethhb, oxyhb, carboxyhb etc. Colour of the compound produced compared with glass standards / other standards in a photocolorimeter. eg. Sahlis method
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SAHLIS METHOD : Based on the conversion of Hb to acid hematin which has a brown color & compared with the standard plates.
CYANMETHHEMOGLOBIN METHOD :Principle : Whole blood (5ml) is taken & mixed with
Drabkin’s soln. (20ul) - Potassium cyanide & Potassium Ferricyanide. ---- chemical product formed of stable colour - Cyanmethhb.
Method approved By International Committee for Standardization in Hematology.
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CYANMETHHEMOGLOBIN METHOD CYANMETHHEMOGLOBIN METHOD (CONTD)(CONTD)
• Intensity of colour is proportional to Hb. conc. & obeys Beer’s law.
• Absorbance of the soln. is measured in a photoelectric colorimeter at 540 nm.
• NB : Drabkins soln. to be made once a month & stored in dark colored bottles.
• Highly poisonous & never be mouth pipetted.
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Advantages of CyanmethHb methodAdvantages of CyanmethHb method
• Readings need not be taken immediately after dilution as the colour is stable.
• Almost all forms of HB. (except sulfHb) are converted to cyanmethHb.
• Method is accurately standardizable.• Soln. of cyanmeth is very stable.• Drabkin’s soln. can be preserved for many
months.
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Disadv.of Sahlis Method.Disadv.of Sahlis Method.
• Complete conversion to acid hematin takes 1 hour - impractical. Results taken after 10 mins.
• Color development is not stable & starts fading immediately after the peak.
• Glass used for comparison is not satisfactory.
• Carboxy, meth, sulphb cannot be converted to acid hematin (not all hb.).
Adv of Sahlis methodCheap, simple to perform, quick, reagents easily available.
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TOTAL WBC COUNTTOTAL WBC COUNT
• Blood diluted with acid to remove RBC by hemolysis. Nuclei of WBC is accentuated by acid.
• Count on Neubauers chamber, 10x.• Diluting Fluid : Glacial acetic acid, methylene blue.• Calculation : No. of WBC x dilution /Area counted x
depth of fluid.• Dilution = 20. • Area counted = 4x1 sqmm=4sq mm.• Depth of fluid = 0.1mm (constant).• FACTOR = 20/4 x 0.1 = 50.• Unit = cu mm.
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TOTAL WBC COUNTTOTAL WBC COUNT
• N : 4000 – 11000 / cmm.
• Increased – Leucocytosis.
• Decreased – Leukopenia.
• WBC pipette uses : CSF, Body fluids, Sperm counts.
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HEMATOCRIT / P.C.VHEMATOCRIT / P.C.V..
• Defn : amount of RBC’s following centrifugation and is expressed as % of the total volume of blood.
• Reasonable index of RBC population & good to detect anemias.
• Method – Microhematocrit, Macrohematocrit --- Wintrobes & Westergren
methods.
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WINTROBE’S METHODWINTROBE’S METHOD
• Anticoagulant : oxalate.• Take blood up to mark. Centrifuge.• Read on the graduation mark on the tube.• 110 x 3 mm tube.• 1 ml blood.• 3000 rpm x 30 min.• Layers : RBC – red, WBC – grey, Platelet
– creamy, plasma – straw coloured.
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MICROHEMATOCRIT METHODMICROHEMATOCRIT METHOD
• Anticoagulated blood is centrifuged in sealed capillary tubes.
• Volume of packed cells to % of whole blood determined by hematocrit reader.
• Tube 7mm long. Bore 1mm.• Venepuncture samples- plain capillary tubes.• Skin puncture – Heparinised capillary tubes.• 12,000 rpm x 3min.
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MICROHEMATOCRIT METHOD ( contd.)MICROHEMATOCRIT METHOD ( contd.)
• Suitable for small volume specimens.
• Requires disposable capillary tubes, special centrifuge, reading device cost.
• N : M = 42 - 52; F 36 - 48%.
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ERYTHROCYTE ERYTHROCYTE SEDIMENTATION RATESEDIMENTATION RATE
• DEFN : When whole blood is allowed to settle, sedimentation of the RBC’s will occur and the rate at which the RBC’s fall is the ESR.
• Non specific test which reflects changes in plasma protein which accompany both acute & chronic infections.
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ERYTHROCYTE SEDIMENTATION ERYTHROCYTE SEDIMENTATION RATE (contd)RATE (contd)
• Methods : Westergren’s method. Wintrobe’s method. Zeta sedimentation rate. Micro ESR method.• Anticoagulant : Westergren – Na citrate 1:4 Wintrobe – Oxalate.• N Values : Westergren 5 – 15; 5 - 20. Wintrobe 0 – 10; 0 - 20.• Units mm / hr.
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ESR (contd)ESR (contd)
• Westergren - more accurate .
• Wintrobe - PCV can be done later.
Stages of ESR.
• Stage of rouleaux formation – 10 min.
• Stage of fast settling – 40 min.
• Period of packing – 10 min.
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ESR ( contd)ESR ( contd)
Increased ESR• old age, pregnancy.• anemias.• chronic diseases – TB, RA.• collagen vascular disease.• neoplastic dis. • DD of angina from MI.• multiple myeloma.
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HEMOCYTOMETRYHEMOCYTOMETRYEnnumeration of the formed elements of blood.Ennumeration of the formed elements of blood.
• Manual methods.• Automated Methods of blood cell counting
- Electrometric system, Photometric system.
Automatic:• accurate, reliable, faster, more information in
some parameters than manual methods.• Difficulty in differentiating betn. diff. types of
WBC’s.
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PRINCIPLES OF BLOOD CELL PRINCIPLES OF BLOOD CELL COUNTERCOUNTER
• Blood cells suspended in an electrolyte soln.
• Flow : outer chamber – inner chamber.
• 100 u orifice.
• Electrode in each chamber to sense the current flowing thr’ the orifice.
• As cell passes - imparts resistance to electrical conductivity betn. 2 chambers.
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PRINCIPLES OF BLOOD CELL COUNTER PRINCIPLES OF BLOOD CELL COUNTER (contd.)(contd.)
• Resistance measured as voltage pulse which corresponds to the counting of each cell --- Number.
• Degree of resistance is proportional to the volume of the cell --- size of cell obtained.
• 7 to 18 parameters.
• Measured / calculated.
• HB, PCV, RBC count WBC count, Indices.
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BLOOD CELL INDICESBLOOD CELL INDICES..
MCV = average volume of the BBC’s.• MCV = PCVx10 / RBC count in millions.• Unit = Femtolitres.
MCH = Average Hb content by weight of a RBC.
• MCH = Hb x 10 / RBC count in millions.• Unit = picograms.
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MCHC = average Hb. conc. per unit volume of RBC.
• MCHC = MCH / MCV x 100 or Hb / PCV x 100.
• Unit = %.
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PLATELET COUNTPLATELET COUNT
• Smallest cell in circulation.• N values : 1- 3 lakh / cmm.• EDTA – best for platelet count.• Venous blood better than capillary blood –
clumping.
• Methods :-- Manual – Rees Ecker Method, Estimation of platelet - P.S.
-- Automated.
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PLATELET COUNT (contd.)PLATELET COUNT (contd.)
Rees - Ecker Fluid 1 : 200 dilution.• Trisodium citrate – prevents clotting.• Neutral Formaldehyde – fixes platelets.• Brilliant Cresyl Blue – colours background.
Neubauers chamber can be used Spencer Brightline chamber – white lines on
a dark background.
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PLATELET COUNT(contd)PLATELET COUNT(contd)
Peripheral Smear• No of platelets in an oil immersion field.Check
out for 10 fields.• N 10 –25 adequate. 0 – 5 inadequate.
• Increased : polycythemia vera, CML, splenectomy.
• Decreased : aplastic anemia, acute leukemia, ITP, megaloblastic anemia, hypersplenism, RT, CT.
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RETICULOCYTE COUNTRETICULOCYTE COUNT
• Juvenile RBC that pass into bloodstream from BM.
• Ribosomal & cyto. Remanants – supravital stains.
• No of retics indicates degree of activity of BM.
• Stains; Brilliant Cresyl Blue, New Methylene Blue – stains RBC’s when living.
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• Absolute Retic count :
Retic count % x RBC count / 100.
• Retic – fine deep violet filaments & granules arranged in a network. RBC’s – pale blue / green.
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PERIPHERAL SMEAR PERIPHERAL SMEAR EXAMINATIONEXAMINATION
• Routine, part of complete blood count.• Cellular components can be examined.• Smear – prepared, dried, fixed, stained &
viewed.• PREPARATION – Thick & thin smear.
Thick : Large drop, 0.5 inch square, printed material.
Thin : angle 30-35%. Proper spreader.
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FIXING OF BLOOD FILMSFIXING OF BLOOD FILMS
• Fixed with methanol ( acetone free) x 1-2 min.
• Wright’s & Leishman stain – no fixation is required. Built–in fixative.
• Prevents hemolysis when the cells come in contact with water.
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STAINING OF BLOOD FILMSSTAINING OF BLOOD FILMS
• ROMANOWSKY’S STAIN – Combination of acidic and basic dyes.
• Wright’s, Leishman’s, Jenner’s, May-Grunwald.• Basic stain - methylene blue, toludine blue.• Acidic stain - eosin, azureI, azureII.• Nuclear material – acidic – stains with basic• Cytoplasm – basic – stains with acidic
component.
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STEPS OF STAININGSTEPS OF STAINING
• Cover slide with stain x 2 mins.
• Add buffered water / neutral distilled water (pH 6.8) x 5-7 mins.
• Mix stain & water with a blow pipe (metallic sheen).
• Wash stain off. Colour – Rose pink tinge (Do not tip the stain off – stain deposits).
• Dry slide upright in a staining rack.
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EXAMINATION OF SMEAREXAMINATION OF SMEAR
• Screen smear under low power.
• Check background color, distribution of cells, quality of cells etc.
• Oil immersion : plain mirror, lift condensor, open iris diaphragm to light.
• Count at 2/3 & 1/3 junction or where RBC’s just overlap.Count in a serpentine fashion.
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QUALITIES OF A GOOD QUALITIES OF A GOOD SMEARSMEAR
• Gradual transition from head – body- tail
• No holes, waves, water artefacts.
• Not extend to the ends of slide.
• Staining - not overstained, understained or stain deposits.
• WBC’s should not be concentrated in the tail.
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INFORMATION OBTAINED ON INFORMATION OBTAINED ON PERIPHERAL SMEARPERIPHERAL SMEAR
RBC SERIES • Size : normocytes, microcytes & macrocytes. • Shape : Normal biconcave shape with central
pallor 1/3.
Poikilocytes - oval shape, pencil shape, tear drop, sickle cells, crenated shape, schistocytes.
• Hemoglobinization : normochromic / hypochromic.
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• Immature forms : polychromatic RBC’s, stippled RBC’s, nucleated RBC’s.
• Inclusions : Howell - Jolly bodies, malarial parasite (trophozoite, schizont, gametocytes).
• Arrangement : Autoagglutination, excess rouleaux formation.
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• WBC SERIES
• Differential WBC count : N, L, E, M, B. Number : normal, increased or decreased.
• Abnormal / immature cells : band cells, leukemic cells.
• Differential counts of the abnormal cells.
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• PLATELETS• Normal 7-25 platelets / oil immersion field.• Size, abnormal forms, clumping function
assessed.• Low platelet count on chamber / counter to
be confirmed on peripheral smear.• PARASITES : Malaria, filaria (Wucheria
Bancrofti), trypanosomiasis, Leishmaniasis.
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NORMAL VALUES ON P.S.NORMAL VALUES ON P.S.
• Neutrophils : 50 - 70%
• Lymphocytes : 20 - 40%
• Eosinophils : 1 - 4%
• Monocyte : 2 - 10%
• Basophils : 0 - 1%
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• POLYCHROMASIA : Bluish tinge of RBC’s. indicates prematurity of red cells / increased no of retics.
• BASOPHILIC STIPPLING : fine purple granules due to aggregates of ribosomes and mitochondria. Rate of production rbcs high - retics released.
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• HOWELL - JOLLY BODIES : small 1-3 u purple – blue round inclusions, remanants of nuclear material in DNA. Severe pernicious anemia, E.F., Thal major, sickle anemia.
• CABOT’S RINGS : stains purple red, loops & figure of 8 structures representing denatured protein / nuclear membrane. Seen in pernicious anemia, lead poisoning.
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ABNORMAL RBC FORMSABNORMAL RBC FORMS
• MACROCYTES - RBC’s > 9 u, MCV > 96 fl. -megaloblastic anemias, liverdiseases, hypothyroidism & alcoholics.
• MICROCYTES - < 6 u, MCV < 76 fl.- iron deficiency anemia, anemia of chronic diseases, thalassemia.
• TARGET CELLS - Thalassemia, iron def. anemia, obstructive jaundice.
• SCHISTOCYTES - red cell fragments in O. intravascular mechanical trauma, microangiopathic hemolysis.
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• ACANTHOCYTES : irregularly spiculated surface - liver disease, abetalipoproteinemia.
• ECHINOCYTES : regularly spiculated surface - bile acid abnormality, high plasma F.F.A.
• SPHEROCYTES : no central pallor. No biconcave shape. MCHC high – hereditary spherocytosis.
• STOMATOCYTES : Slit like area of pallor of rbc’s - liver disease, hereditary defects in membranes.
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ABNORMALITIES OF WBC’SABNORMALITIES OF WBC’S
• Immature forms like blasts & other leukemic cells. AML, ALL, CML, CLL ETC.
• Toxic granules in WBC’s - severe bacterial infection, Chediak - Higashi syndrome.
• Hypersegmented neutrophils - > 5% with 5 lobes or 1 with 6 lobes - megaloblastic anemia.
• Hyposegmented neutrophils : Pelger - Huet abnormality.