Laboratory diagnosis in

hematology

Dr. Enikő Papp

Department Of Laboratory Medicine
Semmelweis University
 ….what sample we measure ?

The sample is anticoagulant whole blood.

Vacutainer tubes with purple or lavender top -

contains EDTA (the potassium salt or K2EDTA).
This is a strong anticoagulant and these tubes are usually
used for complete blood counts (CBC) and blood films.

Tubes with black top (contains citrate)- used for ESR also known
as Erythrocyte Sedimentation Rate
 When we measure?
• Complete blood test:
– Routine medical examination
– Inflammatory diseases, infections
– Endocrine and malignant diseases
– Bleeding (acute or chronic)
– Hematological diseases
– Hemostatic disorders
– Autoimmune and allergic diseases
– Monitoring of salt and water homeostasis
Complete blood test (CBC and WBC diff)
in healthy adult men
• WBC 4-10 G/l • PLT 130-400G/l
• RBC 4-5,5 T/l • MPV 7-11 fl
• HGB 130-160 g/l • PDW 25-65%
• HCT 0,4-0,52 l/l • PCT 0,12-0,36%
• MCV 80-95 fl • FRAGM
• MCH 28-32 pg • %MICRO
• MCHC 330-370 g/l • %MACRO
• (CHCM) 330-370 g/l • %HYPO
• RDW 11,5-14,5% • %HYPER
• HDW 2,2-3,2 g/dL
 Calculating parameters

• MCV (mean corpuscular volume) = Htc/RBC

Ref: 80-95 fl

• MCH (mean corpuscular hemoglobin) = Hb/RBC

Ref: 28-32 pg

• MCHC (mean corp. hemoglobin contrentation) =

Hb/Htc
Ref: 330-370 g/l
 WBC differential
NeNewborn Children Adult

Lymphocyte 20-70% 25-50% 20-40%

Monocyte 1-10% 1-6% 3-8%

Neutrophil 15-60% 25-60% 40-70%

granulocyte
Band form 1-8% 3-6% 1-2%

Eosinophil 1-5% 1-5% 1-5%

granulocyte
Bazophil 0-1% 0-1% 0,5-1%
granulocyte
 Other important laboratory tests in
hematology diagnostics:
• ESR • Se Iron, sTfR, ,
• CRP • transferrin,ferritin
• PCT • Se bilirubin
• LDH
• Haptoglobin • Electrophoresis
• Folic acid, • Immunofixation
B12, Calcium, • Immunoglobulins
ALP, • Light chains
Phosphate
 Interfering factors
• Hemolysis: RBC ↓, Hct ↓
– repeat a new sample
• Transfusion, iron and B12 therapy: RDW ↑, bimodal RBC
histogram
• Lysis resistant red blood cells: WBC↑, lymphocyta ↑
– Dilution, longest lytic time
• Fragmentocytes: RBC↓ PLT ↑
– Blood smear, fragmentocyte measure
• Nucleated red blood cells NRBC :WBC↑,lymphocyte ↑
– NRBC program, blood smear
 Interfering factors
• Extreme high WBC : RBC ↑, incorrect Hgb and calculated
parameters
- dilution the sample
• Cryoglobulins: WBC ↑
- 37⁰ incubation, repeat the measure
• Cold agglutinins: RBC ↓,MCV ↑ ,MCHC ↑
– 37⁰ incubation, repeat the measure
• Lipemic blood sample: Hgb ↑, MCHC ↑, MCH↑
– Repeat a new sample
• PLT aggregates (EDTA induced): WBC ↑, PLT ↓
– Blood smear, repeat the measure by a sample administering
citrate as anticoagulant
• Giant trombocytes: WBC ↑, MCV ↑, PLT ↓
– Blood smear
 Measuring ESR
ESR Westergren method

automated analyser

Ref. 2-10mm/h (man), 2-20 mm/h (woman)

 Erythrocyte sedimentation rate (ESR)
„sed rate” increased:

• Increased levels of plasma proteins (except albumin)

- Infection
- Inflammation
- Malignant conditions
• Anemia – decreased numbers of RBCs
• Autoagglutination – similar to rouleaux formation, clumps
fall faster
• Macrocytes – increased surface area (heavier), RBCS fall
faster
• Hemolysis – destruction of the RBCS (decreased number)
How do analysers work?
 Principles of measurement

• Two general principles

 Electronic resistance ( impedance)
 Light scattering
 Principles of measurement
• The RF/DC detection method simultaneously
detects RF (radio frequency) impedance and
DC (direct current ) impedance changes, when
blood cells pass through the aperture. This
occurs, while both direct and radiofrequency
currents being supplied to the detector.
• The RF impedance change reflects
intracellular information, the DC impedance
change is proportional to cell size (volume)
Electrical impedance (DC) method:

Disrupts electrical current – proportional to the size of

the cell
•Hydrodynamic focusing
•Laminar flow
Three-part WBC differential
Issues:
•Aperture size
•Coincidence passage loss (coincidence correction)
•Orientation of cell in aperture
•Low hemoglobin (RBC parameter)
Radiofrequency impedance (RF) method:
Impedance - size cells
Conductivity (RF) – proportional to cell
interior density (granules and nucleus)
Five-part WBC differential
•Scatterplot (RF X DC)
•Computer cluster analysis provides
absolute counts

( impedance)
 Optical detection:

Flow cytometry (measurement in a ‘flow’)

Deflects light beam (‘mirrors ‘ bouncing light)
•Cells in solution run through quartz flow cell
•Hydrodynamic focusing (uses sheath fluid) using principle of
laminar flow
•Focused laser beam (coherent, monochromatic)
•Computer cluster analysis of cytograms provide quantitative
and qualitative information
Forward angle light scatter correlates to cell volume/size
Side angle (orthogonal) light scatter correlates to degree of
internal complexity (granules and nucleus)
Automated hematology analysers

Advia 2120 Siemens Sysmex XE

 ADVIA 2120

• Hematology Analyzer
•Complete Blood Count (CBC)
•White Blood Cell Differential (Diff)
•Reticulocyte Analysis (Retic)

• Analysis on one aspiration of whole blood

• 120 CBC/Diff samples per hour
• 3 Modes of Aspiration
• Random Access Test Selectivity
 Unified Fluids Circuit (UFC)

The UFC assembly uses Unifluidics technology.

The UFC block is made up of eight acrylic

plates. Machined within these plates are the
pathways for the fluids and air flow, valves, and
four reaction chambers. An additional chamber
is mounted on the outside surface of the UFC
block.

The reagent pump assembly, mounted to the

bottom of the UFC block, is also acrylic.
Unified Fluids Circuit (UFC)
 The HGB Method
- Red blood cells are lysed to release hemoglobin.
- The heme iron in the hemoglobin is oxidized from the ferrous to the ferric state,
and then it is combined with cyanide in the ADVIA 120 HGB reagent to form
the reaction product.

CYANISATION

HEMOGLOBIN + HGB reagent METHEMOGLOBIN CYANIDE HGB

Fe++ Fe+++ Fe+++.CN

SLS hemoglobin detection method uses cyanide-free sodium lauryl sulphate (SLS).
The reagent lyses red blood cells and white blood cells in the sample.
The chemical reaction begins by altering the globin and then oxidising the haeme group.
Now the SLS’ hydrophilic groups can bind to the haeme group and form a stable,
coloured complex (SLS-HGB), which is analysed using a photometric method.
 The HGB Method

HGB reaction chamber

Lamp
assembly
UFC Filter + Photodiode

Optical readings are obtained colorimetrically at 546 nm.

 The HGB Method

Calculating reported parameters

• HGB Hemoglobin (directly measured)

• MCH (HGB ÷ RBC) x 10

• (Mean Corpuscular Hemoglobin)
•
• MCHC (HGB ÷ [RBC x MCV]) x 1000
• (Mean Corpuscular Hemoglobin Concentration)
 FLOWCELL TECHNOLOGY

Shuttle chamber Sheath stream

Sample stream

ADVIA 120 SHEATH encases the sample stream

as the two fluids pass through the flowcell. Light
detects the cells as they pass through the light path.
 The RBC method

No matter
what your shape
or size ....
We can make you
a SPHERE

Izovolumetrically sphering of RBCs

 The RBC Method
Low angle scatter 2o - 3o (Volume)

High angle scatter 5o - 15o (HGB concentration)

The RBC Method
Laserdiode Sample stream Beamsplitter Dark stop Mirror

Referentie signaal

Absorption Low-angle High-angle

detector scatter scatter
detector detector
Front view of the dark stop
The RBC Method

The RBC Scatter cytogram is the graphical

representation of two light-scatter measurements:
the high-angle light scatter (5° to 15°) is plotted along
the x axis, and the low-angle light scatter (2° to 3°) is
plotted along the y axis.

1. Low-angle light scatter (2° to 3°)

2. High-angle light scatter (5° to 15°)

3. Mie map containing RBCs

4. Platelets detected in RBC method

The RBC Method

28 41
The RBC Method

The Volume/Hemoglobin Concentration

(V/HC) cytogram is a linear version of the RBC
map.
On the V/HC cytogram, hemoglobin concentration
is plotted along the x axis and cell volume is
plotted along the y axis. .

1. 60 fL volume marker

2. 120 fL volume marker

3. 28 g/dL HC marker

4. 41 g/dL HC marker
 The RBC Method

Macrocytic

Hyperchromic
Volume - MCV

Hypochrmic
Normocytic
Normochromic

Microcytic

28 41
HGB Concentration - CHCM
 The RBC Method

Volume - MCV

HGB Concentration - CHCM

The RBC Method
The RBC Volume histogram represents the
distribution of red blood cells by cell volume.
The histogram has a range from 0 fL to 200 fL.

Normal samples have a bell-curve shaped

distribution with a mode channel between
60 fL and 120 fL.

The mean corpuscular volume (MCV) and

the red cell distribution width (RDW) are
determined from this histogram.

• MCV is the mean of the of RBC Volume

histogram.

• RDW is the coefficient of variation of the

population.
 The RBC Method
The RBC hemoglobin concentration (RBC HC)
histogram represents the distribution of red blood
cells by cellular hemoglobin concentration.
The histogram has a range from 0 g/dL to 50 g/dL.

Normal samples have a bell-curve shaped

Hgb concentration distribution with a mean
channel between 28 g/dL and 41 g/dL.

The cell hemoglobin concentration mean (CHCM)

and the hemoglobin distribution width (HDW) are
obtained from this histogram.

• CHCM is the mean of the RBC HC histogram.

• HDW is the standard deviation of the RBC HC

histogram.
The RBC Method

The RBC CH (cellular hemoglobin) histogram

represents the distribution of red blood cells by
the amount of hemoglobin present in each cell
independent of cell volume.

The histogram has a range from 0 picograms to

100 picograms.

• Cellular Hemoglobin Content (CH) is the mean of

the RBC CH histogram.

• Cell hemoglobin distribution width (CHDW)

is the standard deviation of the RBC CH histogram.
 The Platelet Method
Principle of measurement is the same as RBCs.

PLATELET CYTOGRAM
2
1 Platelets
5 2 Large platelets
3 Red blood cells
4 RBC fragments
3
5 RBC ghosts
1
4
 The Retic Method

Reticulocytes – after the isovolumetrically sphering – are stained by

Oxazine 750 according to their RNA content.
The Retic Method
The RETIC Scatter ABS cytogram is the graphical
representation of the absorption and light-scatter
measurements:
• absorption (cell maturation) is plotted along the
x axis
• light scatter (cell size) is plotted along the y axis.

1 RTC Platelet threshold

2 RTC Coincidence threshold
3 RTC threshold
4 Low/Medium RTC threshold
5 Medium/High RTC threshold
A Mature RBCs
B Low absorption retics
C Medium absorption retics
D High absorption retics
E Platelets
F Coincidence events
 The Retic Method

The RETIC Volume histogram represents the

overlaid
distributions of mature RBCs and reticulocytes
by cell size only.

The histogram has a range from 0 fL to 200

fL..
The RETIC hemoglobin concentration (RETIC HC)
histogram represents the overlaid distributions of
mature
RBCs and reticulocytes by cellular hemoglobin
concentration only.

The histogram has a range from 0 g/dL to 50 g/dL.

Mature RBC population (red)

Reticulocyte population (blue)
The Retic Method

The RETIC cellular hemoglobin (RETIC CH)

histogram represents the overlaid distributions of
mature RBCs and reticulocytes by the actual
weight or mass of hemoglobin present in each
cell.

The histogram has a range from 0 pg to 100 pg.

Mature RBC population (red)

Reticulocyte population (blue)
 The Retic Method
Calculating new retic parameters
•

CHr (Cellular Hemoglobin content reticulocytes) : Mean of the RETIC CH

histogram for the reticulocyte population

CHr : sensitive indicator for amount of utilizable iron. It shows the functional iron
level of the previous 3-4 days ( iron deficiency: CHr < 28 pg)
Diagnostical: iron deficiency anemia, evaluation of the response of iron therapy,
monitoring of EPO treatment

IRF-M+H (Immature Reticulocytes Fraction Medium + High) :

100 x ([#HRetic + #MRetic] ÷ RETIC Count)

IRF: indicator of changing in erythropoietic activity (mainly in chronic renal

disease, monitoring after bone marrow , stem cell and renal transplantation,
in different type of anemies)
 The Retic Method
Erythropoietin Treatment

Beginning After 4 days

Beginning After 4 days

After 2 weeks After 1 month

After 2 weeks After 1 months

 WBC methods
• Differential of WBCs goes on two paralell
channel: the peroxidase and basophil channel

- The PEROX reagent serves as a substrate that enables the

hydrogen peroxide to form a dark precipitate at sites of peroxidase
activity in the granules of white blood cells.
 The Perox Method

If you have the granules - we have the stain

I’m
melting

PEROX
STAIN

But you
still look
pale
Boy,
your granules
look great !
 The Perox Method
Number of neutrophil granules

Bone marrow Blood

Promyelocytes
Myelocytes
# granules

Metamyelocytes
Band cells
Mature PMN

Blasts

Cell maturation
 The Perox Method
Cytochemical classification according to peroxidase activity

Cel type Peroxidase

Myeloblasts -, sometimes ½+ (especially micromyeloblasts)

Promyelocytes 3+
Myelocytes 3+
Metamyelocytes 3+
Band cells 2-3+
Neutrophils 2+
Eosinophils 4+
Basophils ½-1+ (stay unstained in the ADVIA 120)
Lymphoblasts -
Prolymphocytes -
Lymphocytes -
Atypical lymphocytes -
Monoblasts -
Promonocytes ½-1+
Monocytes 1+
Plasma cells -
Nucleated red blood cells -
 The Perox Method
The PEROX cytogram

When the light scatter and absorption data are

plotted, distinct populations or clusters are formed.
Light scatter = Cell Size

Cluster analysis identifies each population based on

its position, area, and density, and then the number
of cells in each population is processed.
1 Noise
2 Nucleated Red Blood Cells
3 Platelet Clumps
4 Lymphocytes and Basophils
5 Large Unstained Cells
6 Monocytes
7 Neutrophils
Absorbed light = Peroxidase Activity 8 Eosinophils
The Perox Method
 The Baso Method

The ADVIA 120 BASO reagent contains phthalic acid and a surfactant which lyses
the red cells, platelets, and the cytoplasm of all white cell types except basophils.
 The Baso Method
The high-angle light scatter (nuclear configuration) is
plotted on the x axis, and the low-angle light scatter
(cell size) is plotted on the y axis, Cluster analysis
identifies each population based on its position, area,
and density, and then counts the number of
cells/nuclei in each population.
The BASO cytograms is representative of a
patient specimen.

1 Noise
2 Blast cell nuclei
3 Mononuclear WBCs (Monocyte and
Lymphocyte nuclei)
4 Basophils
Nuclear Configuration 5 Baso Suspect
6 Saturation
7 Polymorphonuclear WBCs (Neutrophil and
Eosinophil nuclei)
The Baso Method
 WBC morphological „flags”
• LUC (Large unstained cells): perox negativ blasts, atypical
lymphocytes, hairy cells, plasmacytes, myeloperoxidase
deficiency
• BLASTS The presence of blasts is suspected.
• IG (Immature Granulocytes) : The presence of immature
granulocytes (myelocytes, metamyelocytes etc.) is suspected.
• NRBC (Nucleated Red Blood Cells) : The presence of
nucleated red blood cells is suspected.
• LS (Left Shift) The presence of nonsegmented
neutrophils (bands) is suspected.
Pathological scattergrams

All scattergram
AML M5b scattergram
Peripherial smear
Morphological anomalies of RBCs
Automated slide preparation unit and digital
cell morphology system

Sysmex SP 1000i
Cellavision DM1200

The system provides digital

May Grünwald-Giemsa stain image analysis technology to
locate and examine cells in blood
and other body fluids.
Morphological anomalies in white
blood cells

Toxic granulation Auer rods AML M2