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

FULL BLOOD COUNT

OTHER INVESTIGATIONS

• Erythrocyte sedimentation rate

• Platelet count

• Reticulocyte count.

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.

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.

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.

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.

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

HEMOGLOBINOMETRYHEMOGLOBINOMETRY

• Colorimetric methods.

• Gasometric methods / Van Slyke method.

• Specific Gravity method.

• Chemical methods. (obsolete).

1 gm of Hb holds 1.34 ml oxygen.

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

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.

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.

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.

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.

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.

TOTAL WBC COUNTTOTAL WBC COUNT

• N : 4000 – 11000 / cmm.

• Increased – Leucocytosis.

• Decreased – Leukopenia.

• WBC pipette uses : CSF, Body fluids, Sperm counts.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.

MCHC = average Hb. conc. per unit volume of RBC.

• MCHC = MCH / MCV x 100 or Hb / PCV x 100.

• Unit = %.

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.

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.

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.

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.

• Absolute Retic count :

Retic count % x RBC count / 100.

• Retic – fine deep violet filaments & granules arranged in a network. RBC’s – pale blue / green.

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.

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.

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.

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.

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.

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.

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.

• 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.

• 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.

• 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.

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%

• 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.

• 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.

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.

• 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.

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.