Basic Technique in

Haematology

Dr Eow Geok Im

MD(UKM), MPath(Haemtology)(UM)

• Aim– Brief background on basic laboratory

techniques in clinical hematology, with emphasis on common manual and automated hematological laboratory tests

Introduction

• Why we need laboratory tests?– Diagnosis, understanding pathophysiology,

monitoring of treatment

• Haematological tests– Is the marrow producing sufficient no of

mature cells? – Are they qualitatively normal?

• What are the common tests?– Full Blood Count

• Red cells – RBC,PCV, Hb, red cell indices, reticulocytes

• White cells – WBC, leukocyte differential• Platelets

– Morphological examination• Blood film preparation• stain

• Collection – Anticoagulant

• Ethylenediamine tetra-acetic acid (EDTA), dipotassium salt 1.5±0.25mg/ml

• Excess may cause– Decrease PCV, increased MCHC– Increase MCV– Plts swell and disintegrate

– Venous or capillary• Capillary – infants <1 year; free flowing, arteriolar• PCV, RBC, Hb, WBC(neutrophils, monocytes) higher in capillary• plts are higher in venous

– Pre-analytical variables• Correct amount• Effects of storage – morphology, quantitative

– Red cell- crenation– White cell- nuclei stain homogenous with ragged cytoplamic margin

• Sample homogeneity• Inherent factors eg. Age, sex, genetic background…

• Reference ranges or normal values?– ‘Normal value’ can be ‘abnormal’– Statistical procedures

• Normal distribution• Mean ± 2 SD

• Manual– Low cost– Labor intensive– Lower precision– as standard

• Automation – High capital– Rapid performance– Less labor– Precise– Need calibration and

maintenance

Red cells

• Quantitative measurement– Hb– RBC– HCT/PCV– RBCs indices (sizes and haemoglobin content

of RBC)– Reticulocytes

Haemoglobin

• Erythrocytes content– Mixture of haemoglobin, oxyhaemoglobin, carboxy

haemoglobin, methaemoglobin and other– Properties: Colour, combination with O2/CO, iron

content

• Principle of measurement (Cyanmethaemoglobin method)– RC are lysed– Hb variants (except sulphaemoglobin) are converted

to the stable compound cyanmethaemoglobin – Quantitation by absorption at 540nm (spectro)

• Hb(Fe2+) K3Fe(CN)6 Hi(Fe3+) KCN HiCN

Haemoglobin

• Clinical implication– The iron-containing protein attached to RBCs that transports

oxygen from the lungs to the rest of the body – Diagnosis of anaemia

• Other method of measurement– Oxyhaemoglobin method, direct reading haemoglobinometry

• ICSH recommendation– Cyanmethaemoglobin method– Very broad absorption peak (535-545nm)– Allow direct comparison with HiCN std

• Quality control– Commercial controls, proficiency testing, control to be run with

each batch of specimens

Haemoglobin

• Problems and error– Cyanide – potential hazard, alternative: sodium azide,

sodium lauryl sulphate (no stable std); HiCN reagent is sensitive to light

– Technical error – pipetting, cuvette, deteriorated reagent

– Turbidity in mixture will cause falsely elevated value (hyperleucocytosis, lipemia, abnormal plasma protein)

– RC which are relative resistance to lysis (HbS, HbC)

• Assignment / Practical– Reagent used, procedure, standard curve

preparation, calculation of Hb concentration, Hb reference ranges

Haemoglobin

• Automation– Direct measurement– Modification of manual HiCN method

• Concentration of reagent, temperature and pH of reaction

• Addition of non-ionic detergent to ensure rapid cell lysis, reduce turbidity

• Measurement at set interval before the reaction is completed

• Utilization of non cyanide reagent

Packed cell volume or Haematocrit

• The ratio of volume occupied by the packed red blood cells to the volume of whole blood

• Use for– Screening for anaemia – Estimation of red cell indices– Calibration of automated blood counter

• Mode of measurement– Manual: centrifuged microhaematocrit (PCV)– Automated: Generation of electrical pulses (Hct)

PCV

• Centrifuged Microhaematocrit– A small amount of whole blood is centrifuged

to determine maximum packing of erythrocytes

– Error• Technical: failure to mix, EDTA in excess,

improper sealing, inadequate centrifugation• Physiologic: plasma trapping increased in

hypochromic anaemia, spherocytes…; venous blood 2% higher

pcv

• Automated Hct– Passage of a cell thro the aperture of an

impedance counter or thro the bean of light in a light scattering instrument lead to the generation of an electrical impulse

No of pulses = count

Average pulse height = volume

Summation of height = PCV

RBC count

• Manual counting– Counting red cells microscopically in diluted

sample of blood contained in a counting chamber

– Obsolete, time-consuming, inaccurate

• Automation– Aperture impedance– Light-scattering technology

• Aperture impedance counting– Beckman-Coulter, Sysmex, Abbott, Roche– Rbcs are poor conductors of electricity– Dilute in a buffered electrolyte solution

cells through an aperture, causes a change in electrical resistance, this pulse is detected and amplified by the instrument.

amplitude of the pulse is proportional to cell size

• Light scatter– Diluted cell suspension flow in a single file– A light source in front of the aperture– Scatter light is detected by photomultipier or

photodiode– Convert into electrical impulses

RBC count

• Threshold setting– Lower threshold to discriminate plt but include

microcytic RBCs– Multichannel instrument – threshold are precalibrated

or automatically adjusted

• Errors– Inaccuracy due to coincidence or recirculation

• Sheath flow or hydrodynamic focusing - cells passing in single file

• Sweep flow – directed stream of diluents

– Faulty maintenance – inaccurate aspiration vol– Threshold setting, appropriate diluents– Sample eg.cold agglutinin

Red cell indices• MCH, MCV, MCHC etc.

• Derivative– Mean cell volume (fl)

• PCV(l/l) x 1000 ÷ RBC(1012/l)

– Mean cell haemoglobin (pg)• Hb (g/dl) x 10 ÷ RBC(1012/l)

– Mean cell haemoglobin concentration (g/l)• Hb (g/dl) ÷ PCV (l/l)

RBC indices

• Indices provided by automated system are of considerable clinical importance– Classification of anaemia

• MCV can be a direct measurement using automated system– Factitious elevation in hyperosmolarity, cold

agglutininPulse height average = MCV

Summation of pulse height = PCV No of impulses = RBC

Other RBC indices• Provided by the automated system

– Red cell distribution width (RDW)• Volume distribution histogram

Degree of anisocytosis

Derived from pulse height analysis

CV(%) or SD(fl)

Other RBC indices

– Cellular haemoglobin concentration mean (CHCM)

• Direct measurement using light scattering at different angle in Bayer-Technicon

• To replaced the role of MCHC. Sensitivity to iron deficiency has improved.

• As a internal quality control. If all measurement accurate, MCHC=CHCM

– Haemoglobin distribution width (HDW)• Bayer-Technicon, determine Hb concentration on

individual cell • Degree of variation in red cell haemoglobinization

Reticulocyte count

• Juvenile red cells - remnants of the ribosomal ribonucleic acid

• Used to assess bone marrow erythropoietic activity

reticulocytes

• Procedure: – Stain: NMB, brilliant cresyl blue, purified azure B– Incubate at 37C for 15 min– Make films and examine microscopically– Count using x 100 oil-immersion– Technique

• Subjective with low precision and accuracy– Well spread, well stain, observer, quality of

microscope

• Absolute reticulocyte count

reticulocytes

• Automation:– Stain: auramine O(sysmex), thiazole orange(ABX), CD4K

530(Abbott), Oxaxine 750(bayer-technicon), NMB(Beckman-coulter)

– fluorescent cell enumerated using flow cytometry– Better precision (more cells counted, better recognicing of late

reticulocytes)– Error: inclusion of WBC and plt, Howell-Jolly bodies, malarial

parasites– Assessment of reticulocytes maturity: more RNA, fluoresce

stronger• Sign of engraftment, clinical response to treatment (SAA), predicting

optimal time for stem cell harvesting

• Summary– Manual techniques

• Red cells– HB, PCV, RBC, reticulocyte– Principle of measurement, error, clinical application– Derivative: MCV, MCH, MCHC

– Automation• Principle• New parameters (RDW, CHCM, HDW, % of

hypochromic cells)

White Cells and Platelet• Manual cell count

– Acceptable alternative to electronic counter– Simple instrument: microscopes, Neubauer counting

chamber– Error:

Technical Inherent

Technician – mixing, faulty in filling of chamber, careless counting

Uneven distribuition of cells in counting chamber

Instrument – chamber, pippete, microscope

White cell and platelets

• Automation– Impedance or light scattering– WBC count

• Red cells are lysed, residual particles are counted• Threshold are set for WBC to exclude plt• Error for WBC: giant plt, nRBC, white cell agglutination

– Platelet count• Counted in WB suing electrical electro-optical detection• Threshold is set to seperate red cells, debris and electronic

noise• New plts parameter: MPV, PDW…

Differential Counts

• Manual– Visual examination of blood films– Misdistribution of various cells

BODY TAIL

• Counting the differential– From head to tail:

• Error: maldsitribution, misinterpretation (thick film)

– Battlement method

– Assign the leucocytes, express in % (5 part or more) ??nRBC, corrected WBC

– how many cells to count? Precision vs practicality– No best counting method in badly made film

Differential count

Automated Differential

• More precise but sometimes inaccurate• Flow cytometry principle• Diluted WB, RC lysed, WC categorised

into 3 or 5 part diff• Single channel or 2/ more channel• Based on

– Volume– Physical characteristics– Activity of cellular enzyme

Auto diff

• 3 part– Single channel– Based on volume of

various cells– 3 categories

• Granulocytes/LC• Lymphocytes/SC• Monocytes/MNC

– Eosinophil/basophils are included in MNC

• 5 part or more– 2 or more channel– Cell volume and other

characteristics– 5 categories

• Neutrophils• Eosinophils• Basophils• Lymphocytes• Monocytes

– Other may have LIC, AL..

Auto diff

• Instrument principle– Light scattering and absorbance– Impedance measurement with low and high

frequency electromagnetic current or radiofrequency current

– Cytochemical reaction

• Analysis– 2 parameter or more complex– Cells are divided into cluster – Threshold (fixed and variable)

Abbot Cell-Dyne

sysmex

• Automated vs manual– More precise– Accuracy less impressive: unusually cell, aging

sample

• Main function of automation– Performing differential count on normal– Flagging “abnormal” sample

• Graphical display– Histogram of red cell– Histogram of plt sizes– Scatter plot of DC

Peripheral Blood Films

• Sample: from fresh blood or EDTA-anticoagulated sample

• Method: – Manual: clean glass slide, spreader– Automated

• Labelling: name, date• Fixing: immediately with methyl alcohol• Staining:

– automated – Manual: MGG, standardized Romanowsky stain etc

• Mounting– Xylol or cedarwood oil

PBF

• Examination of blood films– Red cells: sizes and shape, haemoglobin,

invlusion and other abnormalities– White cells: quantity, differential count,

abnormal morphology, abnormal granules – Platelets: quantity, morphology

Platelet morphology

Platelet morphology

Neutrophils

Eosinophils, basophils, monocytes

Lymphocytes

Red cell morphology

What are the abnormalities?

• Automated image analyser– Examination of peripheral blood films using

automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96. J Clin Pathol. 2007 Jan;60(1):72-9.

References

• Dacie & lewis Practical haematology

• Blood cells – a practical guide. Barbara bain

• Clinical haematology. Principles, procedures, correlation – Lippincott

• Esssential haematology – AV Hoffbrand, blackwell publishing