Principle & Technology-AP July05

3-Part Diff Technology

pocH-100iAutomated Haematology Analyser

2pocH-100i / Product Training July 2005

Specifications Measurement principles Histogram interpretation Flagging

pocH-100i: 3-Part Diff Technology


pocH-100i - Specifications


Parameters

Whole blood mode (WB): 19 parameters WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLTLYM%, MXD%, NEUT%, LYM#, MXD#, NEUT#, RDW-SD, RDW-CV, PDW, MPV, P-LCR+ WBC, RBC & PLT histograms

Prediluted Mode (PD): 8 parametres

Throughput 148 seconds/sample

Sample Volume

•Whole blood mode: 15 µL aspirated 1 mL sample required, 500 µL in case of micro tubes•Pre-diluted mode: 20 µL (auto-dispense is available) 20 µL cap. blood with 1:26 dilution

Measurement Principles

Direct current (DC) detection method: WBCHydrodynamic focusing DC detection method: RBC/PLTNon-cyanide HGB analysis: HGB



Sample ID 15 digits

Printer Built-in thermal printer

Interface Host communication by TCP/IP (LAN) or serial port

Options Bar code reader

Multilanguage English, French, German, Italian, Spanish, Japanese

Dimensions(W x D x H (mm)Weight (kg)

Main Unit: 185 x 460 x 350,approx. 14

Data Storage 100 sample results with histograms6 QC files. Each files can store up to 21 parameters by 60 points



WBC: DC detection method

RBC/PLT: Sheath flow DC detection method

HGB: Non-cyanide HGB detection method

HCT: Cumulative pulse height detection method

Measurement principles



WBC: white blood cell count RBC: red blood cell count HGB: haemoglobin HCT: hematocrit (%RBC vol. to whole

blood vol.) PLT: platelet count MCV: mean corpuscular volume

(HCT/RBC) MCH: mean corpuscular hemoglobin

(HGB/RBC) MCHC: mean corpuscular hemoglobin

concentration(HGB/HCT)

Analytical Parameters


RDW-SD: RBC distribution width – standard deviation

Anisocytosis (RBC) Monitoring of blood transfusion

RDW-CV: RBC distribution width – coefficient of

variation Anemia of spherule shaped

PDW: platelet distribution width PLT agglutination RBC overlap



MPV: mean platelet volume Hematopoiesis function of PLT Movement of PLT inside the body

P-LCR: platelet large cell ratio PLT agglutination RBC overlap Hematopoiesis function of PLT



SCR%: lymphocyte ratio (LYM%) MCR%: mixed leucocyte ratio (MXD

%)(monocyte, eosinophil, basophil)

LCR%: neutrophil ratio (NEUT%) SCC#: lymphocyte count (LYM#) MCC#: mixed leucocytes count (MXD#) LCC#: neutrophil count (NEUT#)



Reproducibility (WB)

Reproducibility study for all parameters by 10 consecutive analyses in WB mode

Low SD Low CV (%) For exact data please refer to scientific

documents (product file)


Reproducibility (PD)

Reproducibility study for all parameters by 10 consecutive analyses in PD mode

Low SD Low CV (%) For exact data please refer to scientific

documents (product file)


Correlation with KX-21

WB and PD: very good correlation with other instruments (KX-21 or SE-9500)

KX-21N vs pocH-100i WBC

0 20 40 60 80

100 120 140

0 20 40 60 80 100 120 140 KX-21N WBC X 10^9/L

pocH

-100

i WBC

X 1

0^9/

L

KX-21N vs pocH-100i RBC

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8 KX-21N RBC X 10^12/L

pocH

-100

i RB

C X

10^

12/L

KX-21N vs pocH-100i HGB

0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 20 KX-21N HGB g/dL

pocH

-100

i HG

B g

/d/L

KX-21N vs pocH-100i HCT

10 15 20 25 30 35 40 45 50 55 60 65

10 15 20 25 30 35 40 45 50 55 60 65 KX-21N HCT %

pocH

-100

i HC

T %

KX-21N vs pocH-100i MCV

40 50 60 70 80 90 100 110 120 130 140

40 50 60 70 80 90 100 110 120 130 140 KX-21N MCV fL

pocH

-100

i MC

V fL

KX-21N vs pocH-100i MCH

10 15 20 25 30 35 40 45 50

10 15 20 25 30 35 40 45 50 KX-21N MCH pg

pocH

-100

i MCH

pg

KX-21N vs pocH-100i MCHC

20 25 30 35 40 45

20 25 30 35 40 45 KX-21N MCHC g/dL

pocH

-100

i MCH

C g

/dL

KX-21N vs pocH-100i PLT

0 200 400 600 800

1000 1200

0 200 400 600 800 1000 1200 KX-21N PLT X 10^9/L

pocH

-100

i PLT

X 10

^/9/

L

KX-21N vs pocH-100i W-SCC

0 25 50 75

100 125

0 25 50 75 100 125 KX-21N W-SCC

pocH

-100

i W-S

CC

r2 = 0,9968 r2 = 0,9943 r2 = 0,9945

r2 = 0,9293 r2 = 0,9923 r2 = 0,9856

r2 = 0,8737 r2 = 0,9928 r2 = 0,9973


Carryover

Carryover study for CBC 5 parameters with control blood (WBC, RBC, HGB, HCT, PLT)

Procedure: Sample is measured 3 times consecutively WB and PD Carryover ratio = (B1-B3) / (S3-B3) x 100 (%)

B1: 1st measurement of background countB3: 3rd measurement of background countS3: 3rd measurement of sample

Results: no carryover For exact data please refer to scientific documents

(product file)


Haemoglobin Detection with pocH-100i


Haemoglobin

Haemoglobin is the red blood dye Chemically, haemoglobin is an iron-

containing protein It is produced in the erythroblasts of the bone

marrow Haemoglobin is present in the erythrocytes The haemoglobin concentration is directly

linked to the HCT and the RBC concentration Haemoglobin is responsible for transport of

oxygen and CO2


New borns 14 - 26 g/dl

Children 10 - 26 g/dl Women 12 - 16 g/dl Men 14 - 18

g/dl

0

5

10

15

20

25

New borns Babies Children Women Men

Haemoglobin – Reference Ranges

Conversion factors: g/dl mmol/L x 0.6206mmol/L g/dL x 1.611


Erythrocytes are lysed in a dilution of 1:500 1. Reaction step:

HBG with the bound Fe2+ is oxidised by the Potassium hexocyanoferrate (K3(Fe(CN)6) to become Methemoglobin (with Fe3+).

2. Reaction step:Methemoglobin reacts with Potassium cyanide (KCN), building the red and stable Cyan hemoglobin-complex with a maximum in absorption at 546 nm.

The extinction at the maximum of absorption is directly proportional to the Hemoglobin content of the whole blood

Haemoglobin Reference Method (DIN 58931)


Haemoglobin Detection History

Identical measurement principle as the reference method.

Measurement in the photometer under the same conditions.

Also the reaction conditions are identical. Cyanide containing reagent for haemolysis.

Semi automated systems = QuicklyserFully automated systems= Stromatolyser-C

Disposal of cyanide containing waste needs a permission.


pocH-pack L (II) is used for the haemoglobin and the leukocyte analysis

pocH-pack L(II) contains quaternary ammonium salts (MTAC and LTAC)

Very good correlations between the Sysmex method and the reference method

Haemoglobin – Cyanide-free Method


1. Lysis of RBC by quaternary ammonium salts

2. Change of conformity

3. Oxidation of Fe2+ at the haeme group (Fe2+ Fe3+)

Haemoglobin – Cyanide-free Method


Haemoglobin molecule

RBC

Ammonium salts

Fe2+Fe2+

RBC

Fe2+ Fe2+

1. Lysis of RBC

Fe2+ Fe2+ Fe2+ Fe2+



RBC

Fe2+Fe2+

RBC

Fe2+ Fe2+

2. Change of Conformity

Fe2+ Fe2+ Fe2+ Fe2+



Methemoglobin-complex

Fe3+ Fe3+

Fe2+Fe2+

O2

3. Oxidation

Fe2+ Fe2+ Fe2+ Fe2+


Wavelength (nm)

540 630

Abso

rptio

n

Spectrum of Haemoglobin (pocH-pack L (II)

max = 555 nm

Hemoglobin – Spectral Analysis


photometrical analysis at 555 nm

HGB Photometer

lens

Sample stream Cellpack

Flow cell photosensorLED


Start

HGB-chamber is rinsed with diluent

HGB-blank is determined and stored

Measurement of the samplein a dilution of 1:500

Sample - Blank = HGB-result

Printing of the result

End

HGB Measurement – Flow Chart


HGB Measurement

Mixing Chamber

Transducer Chamber

HGB Flow Cell


Haematocrit is the ratio (%) of total volume of RBCs to whole blood volume

Determination by centrifugation or calculation

Besides haemoglobin, hematocrit is a measure for anaemia

Haematocrit (HCT)


New borns 0,51 - 0,65

Children 0,35 - 0,43 Women 0,38 - 0,48

Men 0,42 -

0,52

0

0,1

0,2

0,3

0,4

0,5

New borns Babies Children Women Men

Hematocrit – Reference Ranges


Volume determination of the corpuscular particles in the blood

Normally, equivalent to the pure erythrocyte mass Blood is aspirated into special capillaries, which

contain dried heparin The capillaries are sealed and put into a dedicated

centrifuge In the high-turning centrifuge the non-coagulating

blood is separated into its solid and fluid parts using the different specific weights of plasma and blood particles

Hematocrit Centrifugation



Total volume VT

Centrifuge

VT

V

HCT (%) = (V / VT) x100


00,10,20,30,40,50,60,70,80,91,0

Leukocytes(Buffy Coat)

Erythrocytes

The reading is done on a scale, which is basedon the principle of similar triangles.

The guide, which cuts through the border between erythrocyte-and leukocyte-layer givesthe Hematocrit-value.



Transducer

Start-Sensor

Stop-SensorDefined volume VT

Hematocrit – Cumulative Pulse Height Detection Method


Ph = k x VEry

Ph pulse heightk constantVery volume of one RBC

VEry = 1 / k x Ph

Ph



VT

V

HCT (%) = (V / VT) x 100

V = VEry

= (1 / k) Ph

HCT (%) = (1 / VT k) Ph x 100

Ph Pulse heightk Constant VEry Volume of one RBC

Ph = k x VEry

VEry = (1 / k) x Ph


VT Total volumeV Volume of all RBCs


Transducerchamber

Ph

HCT (%) = V / VT x 100

HCT (%) = (1 / VTk) Ph x 100

V

VT


Start-Sensor

Stop-SensorDefined volume VT


Mean Cell Volume (MCV)HCT (%)

RBC (x 106/µl)MCV (fl) =

Mean Cellular Hemoglobin (MCH)HBG (g/dl)

RBC (x 106/µl)MCH (pg) =

Mean Cellular Hemoglobin Concentration (MCHC) HBG (g/dl)

HCT (%)MCHC (g/dl) =

RBC Quotients: RBC, HGB, HCT


100Deviation from median in %

-40

-20

0

20

40

60

80n = 411 , confidence range 2,5 % to 97,5 %taken from : R. Haeckel, Rationalisierung des med.Laboratoriums, 2. Auflage GIT Verlag, Darmstadt

WBC

103/µl

5,0 190

PLT

103/µl

14

HGB

g/dl

4,5

RBC

x 106/µl

42

HCT

%

90

MCV

fl

34

MCHC

g/dl

31

MCH

pg

Biol. Variation of CBC Parameters


Sample Processing - WB

Blood aspiration: 15µL Dilution ratio:

RBC/PLT: approx.1365 times WBC/HGB: approx. 500 times

1st dilution: 15µL blood + 1.935mL diluent (130 times)

2nd dilution: RBC/PLT: 200µL 1st sample + 1.9mL diluent

(10.5 times; 130 x 10.5 = 1365) WBC/HGB: 461µL 1st sample + 720µLdiluent + 590µL lyse

(3.85 times; 130 x 3.85 = 500)


Sample Processing - PD

Blood collection: 20µL Dilution ratio:

RBC/PLT: approx. 2730 times WBC/HGB: approx. 1000 times

Pre-dilution: 20µL blood + 500µL diluent (26 times)

1st dilution: 195µL PD sample + 1.755mL diluent (260 times)

2nd dilution: RBC/PLT: 200µL 1st sample + 1.9mL diluent (10.45

times; 260 x 10.45 = 2730) WBC/HGB: 461µL 1st sample + 720µL diluent + 590µL

lyse (3.85 times; 260 x 3.85 = 1000)


DC Detection Method

From pulse to histogram


vacuum

blood cells

resistance

direct current(approx. 100 V)

internal electrodeexternal electrode

aperture

sample beaker

sample

Principle DC Detection Method


external electrode

internal electrode

aperture

vacuumU = R x I

DC Detection Method


Samples are passing through the centre of the aperture with sheath flow solution

Accurate RBC/PLT count Axial cell flow Recirculation and coincidence are prevented Sheath reagent: pocH-pack D (II)

Hydrodynamic focusing system: RBC/PLT

Hydrodynamic Focusing


Sample can be more concentrated with HDF: Dilution

KX-21N: RBC/PLT: 25.000 times (WB und PD) pocH-100i: RBC/PLT: 1365 times (WB); 2730

times (PD)

Hydrodynamic focusing system: RBC/PLT

Hydrodynamic Focusing


DC Detection Method

Absolute counting

Defined volume of the diluted blood sample is brought into the RBC/PLT detector line for measurement

Valves separate this defined volume

Electric stepping motor drives the syringe, which pumps the defined volume into the detector unit


DC Detection Method

Syringe Unit No.21(diluent)

Syringe Unit No. 22(sample and sheath)


aperturesample

defined volume

vacuum

startstop

counter

off

display: S pulse / volume

on

pulse management digital

discriminator

signal-pulses

noise-pulses

signal-pulses

Not to be changed by the user:sample volume;dilution ratio;analysed volume;diameter of the orifice

The instrument automatically checks: counting time, condition of the orifice during the analysis (noise-control)

Advantages:no calibration of the counts

DC Detection Method

Absolute counting


Counting of particles in a defined volume

Counting of particles per time unit

Absolute Counting Relative Counting

Cell concentration is determined by a known sample volume and a known dilution

The counting rate is determined indirectly by a reference sample. A similar behaviour of sample and calibrator - resulting in comparable results - is assumed.

No calibration of the counting results needed

Calibration of the counting results is mandatory.

DC Detection Methods - Comparison


1 2 3 4 5 6 7 8 9 10 11 12 13 14tim

e

pulse height

From pulse to histogram: pulse diagram

DC Detection Method


10

20

301 2 3 4 5 6 7 8 9 1011121314

1 2 3 4 5 6 7 8 9 1011121314

tim

e

pulse height

Cumulative Distribution Curve

Pulse Diagram(30 cells)

4 1 0 0 0 1 2 3 4 5 4 3 2 1

cells

DC Detection Method


1 2 3 4 5 6 7 8 9 1011121314

Histogram

10

20

301 2 3 4 5 6 7 8 9 1011121314


4 1 0 0 0 1 2 3 4 5 3 2 14

cells

DC Detection Method



Histogram

0

500

1000

1500

2000

2500

3000

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102108114120

Maximum

From pulse to histogram

DC Detection Method - Histogram


25-75 fl 200-250 fl

Erythrocyte (RBC) Histogram

RBC size: 80-100 fL RBC detection: between 25 and 250 fL Distribution curves are separated by flexible

discriminators

RL RU

RBC

PLT


2-6 fl 12-30 flfixed at 12 fl

Platelet (PLT) HistogramPL PU

PLT

RBC

100%

20%

PLT size: 8-12 fL PLT detection: between 2 and 30 fL Fixed discriminator at 12 fL


Measurement Flow Chart

Detector

WBC Channel

HGB Channel

RBC/PLT Channel

Whole Blood Sample

Information

WBC Scattergram

RBC Scattergram

PLT Scattergram

Method

DC Detection Method

Non-cyanide HGB Method

HDF DC Detection Method

Parameters WBC LYM# LYM% MXD# MXD% NEUT# NEUT%

HGB MCV MCH MCHC

RBC HCT RDW-SD RDW-CV

PLT PDW MPV P-LCR


Summary: Principles and Technologies pocH-100i

RBC / PLT / WBC: DC Detection Method HGB: cyanide-free photometric analysis at 555 nm HCT: cumulative pulse height detection method

3 Histograms: RBC, PLT, WBC Histogram flagging: suspect flags

RBC parameters: RBC, HGB, HCT, MCV, MCH, MCHC, RDW-SD, RDW-CV

PLT parameters: PLT, PDW, MPV, P-LCR WBC parameters: WBC, W-SCR, W-MCR, W-LCR,

W-SCC, W-MCC, W-LCC


Principles & TechnologiesEnd of Part I

Time for questions…


Histogram Interpretation

CBC - parameter

Leukocyte histogram

Lymphocytes in % and absoluteEo, Mono, Baso in % and absoluteNeutrophiles in % and absolute

Erythrocyte histogramErythrocyte distribution width

Thrombocyte histogramThrombocyte distribution widthMean Platelet VolumePlatelets - Large Cell Ratio

WBC

RBC

PLT


CBC: Reference Ranges

RBC Men 4.6-6.2 x 106/µl x 1012/lWomen 4.2-5.4 x 106/µl x 1012/l

HGB Men 14-18 g/dl 8,5-11,0 mmol/l

Women 12-16 g/dl 7,5-10,0 mmol/l

HCT Men 43-49 % 0,43-0,49 mmol/lWomen 36-46 % 0,36-0,46 mmol/l

MCV 85-95 flMCH 27-33 pg 1,68-2,05 fmolMCHC 32-36 g/dl 19,9-22,4 mmol/l

RDW-SD 37-46 fl (width in 20% peak height)

RDW-CV 11-16 % (calculated from width in 60 % peak height)

Parameter Sex Convent.Units SI-Units


Display of Analysis Results

Analysis data without preceding sign are within preset limits

If analysis error has occurred and a value is not available,one of the following is displayed

Sign Exposition

! Value is out of the linearity limit

+ Result exceeds the upper patient limit

- Result exceeds the lower patient limit

* Result is unreliable

Sign Exposition

“+++.+” Value exceeds display range.

“***.*” Value could not be calculated because of analysis error. At this time, the analysis error flag. {Error] (inverse in coloration display) appears.

“---.-” Value could not be calculated due to data error, or volume distribution analysis parameter is not displayed when the analysis was performed in pre-diluted mode


Histogram error flags

Flag ExpositionRL Error at the lower discriminator for RBC

RU Error at the upper discriminator for RBC

DW Distribution width (20%) can be not calculated

MP There are multiple peaks

PL Error at the lower discriminator for PLT

PU Error at the upper discriminator for PLT

AG The particle count equal to or less than WBC-LD has exceed the range

WL Error at the lower discriminator for WBC

WU Error at the upper discriminator for WBC

T1 Trough discriminator T1 could not be determined

T2 Trough discriminator T2 could not be determined

F1 Small cells are inaccurate

F2 Middle cells are inaccurate

F3 Large cells are inaccurate


Curve does not start at the base line.

flag “ RL “, abnormal height at the lower discriminator

Possible cause:• Large PLT• Fragmented RBC• PLT aggregation

CAUTION:All results flagged with “ RL “ should be checked!!

LDRBC

PLT

RBC Histogram Flagging


Curve does not end at the baseline

UDRBC

flag “ RU “, abnormal height at the upper discriminator

UDRBC

Possible cause:• Cold Agglutinins• Erythroblasts

Normoblasts

CAUTION:The RBC-result and all results flagged with “ RU “should be checked!!




flag “ MP “, Multiple peaks

RBCRBC

Possible cause: Transfusion: patient’s and donor’s RBCs have different

size Unlysed and aged sample


flag “DW “, Distribution width

RBCRBC

It is impossible to determine the distribution width, because the histogram does not cut the 20%-line for a second time.

This curve is only an example and might also show a different course.



flag “DW “, Distribution width

RBCRBC

Various size of cells are present Review by manual method



RDW-SD: 37 - 46 fl

100 %

20 %

[fL]

RDW-CV (%) = 100 x (L1-L2) / (L1+L2) 100 %

L1 L2µ

Turning Points

68,26 % of all values

[fL]

RBC Histogram Distribution Width


PLT 150-350 x 10³/µl x 109/l

PDW 9-14 fl (Width in 20% peak height)MPV 8-13 flP-LCR 15-35 %

Parameter Convent.Units SI-Units

PLT Histogram: Reference Ranges


PLT Histogram

Curve is marked off by two discriminators

The distribution curve should lie between the discriminators and start and end at the base line

The measurement area is between 2 and 30 fl


PLT Histogram Flagging

PLT, Platelet count

Parameter from the thrombocyte-histogramMPV, Mean Platelet Volume between the

upper and the lower discriminatorNormal range: 8 - 12 flUse for control of erythropoiesis

P-LCR, Platelet - Large Cell RatioNormal range: 15 - 35 % Elevation might be hint for:

Thrombocytes aggregates Micro erythrocytes

PDW, Platelet Distribution Width in 20 % of total curve heightNormal range: 9 - 14 flElevation might be hint for:

Thrombocytes aggregates Micro erythrocytes Erythrocyte fragments

Pct (%)PLT (x 103/µl)MPV (fl) =

12 fl

LD UD

PLT P-LCR

100 %

20 %

PDW



Curve does not start at the base line.

flag “ PL “, abnormal height of the curve at the lower discriminator

PLTPLT

Possible cause:• Blank is too high• Cell fragments or cell ruins

CAUTION:PLT-result and all results flagged with “ PL “should be checked!Thrombocytes also in the counting chamber,if needed!



flag “ PU “, abnormal height of the curve at the upper discriminator

Possible causes: Coagulation of blood Pseudo-thrombocytosis Fragmented RBC Large platelets Thrombocyte aggregates

CAUTION:PLT-result and results flagged with “ PU “should be checked!Thrombocytes: counting chamber

Curve does not end at the base line



flag “ MP “, Multiple peaks

Possible cause: Transfusions, the patient’s and the donor’s PLTs

havedifferent sizes

Pathological cells: CML (chronic myeloic leukemia)



flag “ DW “, Distribution width

PLT

The distribution curve should lie between the discriminators and start and end at the base line

The measurement area is between 2 and 30 fl



flag “ DW “, Distribution width

PLT

Microcytes or fragmented RBC PLT size dissimilitude and cryoglobulins


WBC grown ups 4-10 x 103/µl x 109/lchildren up to 12 x 103/µl x 109/lbabies up to 15 x 103/µl x 109/l

W-SCR grown ups 25-40 %children, babies up to 70 %

W-MCR grown ups 3-13 %W-LCR grown ups 50-70 %

W-SCC grown ups 1-4 x 103/µl x 109/lchildren up to 5 x 103/µl x 109/lbabies up to 6 x 103/µl x 109/l

W-MCC grown ups 0,2-1 x 103/µl x 109/l

W-LCC grown ups 2-7 x 103/µl x 109/l

Parameter Age range Convent.Units SI-Units

Reference Ranges


Schematic pictureof a leukocyte

Mitochondrion

Nucleus

Nucleolus

Cell membrane

Ribosome

Cytoplasm

Electrolyte-solution

After lysis

WBC Histogram: Influence of the lysing reagent


WBC Histogram: Influence of the lysing reagent

Before lysis

0 2 4 6 8 10 12 14 16 18 20 22

NeutrophilesBasophilesEosinophilesMonocytesLymphocytes

Cell diameter in µm10 - 15 9 - 1411 - 1612 - 20 7 - 12

30 - 80 60 - 120 70 - 130 80 - 140120 - 250

Cell volume in fLLymphocytesMonocytes Basophiles Eosinophiles Neutrophiles

After lysis

0 50 100 150 200 250 300

Lymphocytes

MonocytesBasophiles Eosinophiles

Neutrophiles


• Distribution curve should lie within the discriminators (start and end at base line)

• Lower discriminator (LD) is flexible, but cannot be set below 30 fl

• In the WBC channel WBC and PLT are present (RBC are lysed)

• PLT volume is normally between 8 and 12 fl: LD separates WBC from PLT and PLT are not counted

Curve is marked off by 2 discriminators

0 50 100 150 200 250 300

UD ( fixed)T2T1LD

WBC Histogram


flag “ WL “, curve does not start at the base line

Possible causes: PLT aggregates or large PLT RBC lyse resistance Normoblasts (NRBC) Cold Agglutinins

CAUTION:WBC - results and all results flagged “WL” should be checked

WBC Histogram Flagging



flag “ WU “, curve does not end at the base line

CAUTION:Review by manual method or washing of blood cells

Sample dilution? (high leukocytes value?)

Immature WBC Agglutination of WBC


T1 and T2 are so called trough discriminators:separation of WBC into 3 populations (F1, F2, F3)

flags “T1” and “T2”

0 50 100 150 200 250 300

UD ( fixed)LD T2T1

population 1 = F1 population 2 = F2

population 3 = F3F = fraction

• Discriminators are flexible in certain areas to adapt to the individual sample

• In abnormal cases the troughs are not able to separate between the populations properly




It is not possible in this case to set T1 since there was no valley found: T1 is flagged

flags “T1” and “T2”

CAUTION: • If the sample is

flagged with T1 or T2, the results from the pre-differential should not be reported

• Manual method• The WBC result is

reportable (if there is no other flag). All leukocytes are counted.

Pathological cells (CML) Numerous abnormal blood cells



T1 was found, but no second valley for T2:T2 is flagged

CAUTION: • If the sample is

flagged with T1 or T2, the results from the pre-differential should not be reported.

• The WBC result is reportable (if there is no other flag). All leukocytes are counted.

Numerous abnormal blood cells Aged sample Pathological cells (CML)



All leukocytes are counted: total number of WBC is correct (if no other flags are present)

Valleys were found: T1 and T2 are not flaggedBut valleys are far from the base line (F1, F2, F3)

flags “F1” , “F2” and “F3”

F1 F2 F3



MonocytosisEosinophiliaBasophiliaUnlysedAged sample

flags “F1” , “F2” and “F3”

F1 F2 F3

CAUTION: • Review by manual

method• The WBC result is

reportable (if there is no other flag). All leukocytes are counted.


Histogram Interpretation: Cases (1)Neutrophilia

BandSegLymphMonoEoBaso

8 %77 %

7 %7 %1 %0 %

DifferentialWBCLYM%MXD%NEUT%

Results+ 23.8 x 109/L

8.1%7.9%

84.0%

(x 400)

WBC-Histogram

Clinical diagnosis: NeutrophiliaProminent peak with broad distribution (NEUT%) for large leukocytes.In case of Lymphocytopenia a similar curve is obtained.


Histogram Interpretation: Cases (2)Lymphocytosis

BandSegLymphMonoEoBasoAty-Lym

4 %20 %64 %

4 %5 %0 %3 %

DifferentialWBCLYM%MXD%NEUT%

Results7.9 x 109/L

+ 64.7%15.8%

– 19.5%

(x 1000)

WBC-Histogram

Clinical diagnosis: LymphocytosisHigh, pointed peak in lympho area (LYM%).In case of Neutropenia a similar curve is obtained.


Histogram Interpretation: Cases (3)

WBC-Histogram

PLT-Histogram

Case 1ResultsWBCLYM%MXD%NEUT%

6.0 x109/L27.5%

7.9%64.4%

ResultsPLTPDWMPVP-LCR

+

+

86 x109/L18.6fl12.8fl

43.7%

(x 400)

The smear clearly shows that platelets are aggregating. The WBC histogram shows a peak in the ghost area ( ) , while the PLT histogram shows a wide distribution. Although these large particles usually affect the leukocyte counts, the leukocytes distribution is well separated from the ghost area on the WBC histogram, probably without any effect of small particles in the ghost area. There is no WL Alarm given .


Histogram Interpretation: Cases (4)Case 2

WBC-Histogram ResultsWBCLYM%MXD%NEUT%

WL*WL*WL*WL*

6.4 x109/L41.4%14.0%44.6%

ResultsPLTPDWMPVP-LCR

PUDWDWDW

55 x109/L---.-fl---.-fl

-.---%

PLT-Histogram

(x 400)

This sample contains larger aggregation clusters as shown in the smear. These clusters are considered to affect the leukocyte counts, because the distribution curve on the WBC histogram intersects the discriminator line between the ghost and the Small cell ratio at a high point, and the WL flags are given. The PLT histogram suggests the presence of large particles. Analysis of a fresh blood sample is required to obtain correct platelet values.


Histogram Interpretation: Cases (5)Insufficient Lysing of Erythrocytes

WBCLYM%MXD%NEUT%

WL*WLWLWL

49.4 x109/L-.----.----.---

WBC-Histogram Results

(x 1000)

Case: lyse resistance RBC• Histogram shows a pattern typically seen in insufficient lysing of RBC• On the WBC histogram the distribution curve intersects the WBC lower discrimination line at an abnormally high point. The WL flag is output and asterisk marks are put to the WBC value, warning of low reliability of the data• This is frequently seen with blood samples taken from hepatic disease patients or very early newborns. These problems are solved by diluting the sample or replacing plasma with cellpack (blood cell washing)


Summary: Principles & Technologies

RBC / PLT / WBC: DC Detection Method (HDF) HGB: cyanide-free photometric analysis at 555 nm HCT: cumulative pulse height detection method

3 Histograms: RBC, PLT, WBC Histogram flagging: numerical flags and suspect flags

RBC parameters: RBC, HGB, HCT, MCV, MCH, MCHC, RDW-SD, RDW-CV

PLT parameters: PLT, PDW, MPV, P-LCR WBC parameters: WBC, W-SCR, W-MCR, W-LCR,

W-SCC, W-MCC, W-LCC


Thank you for your kind attention!

Questions ?!

Principle & Technology-AP July05

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