HEMATOLOGY AUTOANALYZER

Prof. Dr. Adi Koesoema Aman SpPK(KH).

Departement Patology Klinik FK USU /
RSUP H.A.Malik , Medan .
Komponen darah

Darah
Plasma Substansi terlarut
- Protein mayor (Alb, Glob, Fib)
- Non protein nitrogen (ur, Kr)

Air (92%) - Produk metabolisme (glu, aa,as.lemak)

- Regulator (enzim)
- Elektrolit (Na, K, Ca, Cl

Slide 2
Karakteristik
Pria 4,5-5,5 x 106 /L

RBC Wanita 4-5 x 106 /L

280 x 106 mol Hb
Usia 120 hari
140-450 x103 /L

PLT Life span 8-10 hari

Hemostasis primer dan
sekunder
5-10 x103 /L

WBC Life span 7-14 hari

Granular dan non granular WBC

Slide 3
 Gambaran darah tepi

-Rerata volume sirkulasi

orang dewasa sehat
sekitar 5 L (5.28 quarts)

- Darah sekali bersirkulasi dalam

tubuh dari jantung kiri ke seluruh
tubuh dan kembali ke jantung
kanan hanya memerlukan waktu
sekitar 90 detik

Slide 4
Stadium pematangan RBC

Bain B. A beginners guide to blood cells.2004

Slide 5
Stadium pematangan WBC

Slide 6
Stadium pematangan PLT

Slide 7
 The 10 Most Important Blood Tests

LE Magazine May 2006-2011

1. CHEMISTRY PANEL AND COMPLETE BLOOD COUNT

2. FIBRINOGEN
The complete blood count (CBC) is one of
3. HEMOGLOBIN A1C
the most commonly ordered blood tests.
The complete blood count is the calculation 4. DHEA

of the cellular (formed elements) of blood. 5. PROSTATE-SPECIFIC ANTIGEN (PSA) (MEN ONLY)
These calculations are generally
6. HOMOCYSTEINE
determined by special machines that
7. C-REACTIVE PROTEIN
analyze the different components of blood
in less than a minute. 8. THYROID STIMULATING HORMONE (TSH)

9. TESTOSTERONE (FREE)

10. ESTRADIOL

Slide 8
Dulu vs Saat ini
1.0
0.9
0.8
0.7
0.6 Hematocrit
0.5 value
00.4
0..3
0.0
2

Slide 10 PT Sysmex Indonesia

Masa Kini
Manual method
Cover slip
Hemocytometer
Thoma pipette
Methods in performing CBC
Manual method - utilizes a hemacytometer
1. 2.
W - WBC
R - RBC
P - Platelets
3.
W W 4. 5.
R R

W W R
R

R
P
Slide 12
 Preparation of blood smears for examination
(manual methods)
A well prepared smear should
Wedge Type have the following characteristics:

cover at least half the length of

the slide
should be narrower than the slide
so that the side edges maybe
examined
homogenous spread with a
gradual transition from thick to thin
areas.
Should have a straight or a very
slightly
curved feathered end
the smear should be thin enough
to allow proper fixation during
staining.

Slide 13
 Diff Count CLSI 1992

Whole venous blood in K3EDTA is the required specimen

Macroscopically visible clots rejection for analysis
Microscopically visible platelet clumps consisting only of
platelets are acceptable
Prepare 3 blood films from each specimen on clean, dry and
dustfree : 2 will be used for the procedure and 1 kept as a spare
Prepare blood films within 4 h of the blood collection in K3EDTA
On each slide, 200 leukocytes should be counted

Slide 14
 Staining the Blood Smear
Romanowski stains - are the most
common stains that are used in the
hematology laboratory.
composed of methylene blue and eosin
dyes
Wrights stain is the most common
modification of Romanowki stain.
Cells have acidic and basic elements
which interact with
the basic and acidic dyes in the stain.

Acidic elements: + Basic dyes Color

nucleoproteins methylene blue blue

Basic elements: + Acidic dyes Color

hemoglobin eosin orange-red
Slide 15
 Principles
The Coulter Principle
Electrical Conductivity or Radiofrequency
Optical Scatter
Light Absorption
Fluorescence
VCS Technology (Volume, Conductivity, and Scatter)
Hydrodynamic Focusing: Both optical and impedance
methods of cell counting employ hydrodynamic
focusing (focused flow)
 The Coulter Principle
Using this technology, cells are sized and counted by
detecting and measuring changes in electrical
resistance when a particle passes through a small
aperture. This is called the electrical impedance
principle of counting cells.
A blood sample is diluted in saline, a good conductor
of electrical current, and the cells are pulled through
an aperture by creating a vacuum. Two electrodes
establish an electrical current. The external electrode
is located in the blood cell suspension. The second
electrode is the internal electrode and is located in the
glass hollow tube, which contains the aperture.
 Low-frequency electrical current is applied to the
external electrode and the internal electrode. DC
current is applied between the two electrodes.
Electrical resistance or impedance occurs as the cells
pass through the aperture causing a change in voltage.
This change in voltage generates a pulse (Fig. ). The
number of pulses is proportional to the number of
cells counted .The size of the voltage pulse is also
directly proportional to the volume or size of the cell.
 This was the principal parameter used in
earlier analyzers for characterizing all cell
types, but it is now used primarily for
counting and sizing red blood cells and
platelets.
 Performance
Whole blood is aspirated, diluted, and then divided into
two samples. One sample is used to analyze the red
blood cells and platelets while the second sample is used
to analyze the white blood cells and hemoglobin .
Electrical impedance is used to count the white blood
cells, red blood cells, and platelets as they pass through
an aperture. As each cell is drawn through the aperture, a
change in electrical resistance occurs generating a
voltage pulse. The number of pulses during a cycle
corresponds to the number of cells counted.
The amplitude of each pulse is directly proportional to
the cell volume.
 In the RBC chamber, both the RBCs and the platelets
are counted and discriminated by electrical
impedance Particles between 2 and 20 fL are counted
as platelets, and those greater than 36 fL are counted
as RBCs.
Lyse reagent is added to the diluted sample and used
to count the white blood cells. The lysing reagent also
cause WBC's membrane collapse around the nucleus,
so the counter actually measuring the nuclear size.
After the white blood cells have been counted and
sized, the remainder of the lysed dilution is
transferred to the Hgb Flow Cell to measure
Hemoglobin concentration.
 Hemoglobin Measurement
Using cyanide free Hb chemistry methods, rapid
RBCs lysis followed by the formation of an
imidazole-hemoglobin complex with an absorption
peak at 540 nm.
The Cell-Dyn uses electronic sizing to determine a
three part automated differential. The percentage
and absolute counts are determined for
lymphocytes, neutrophil, and mid-size population
of monocytes, basophils, eosinophils, blasts, and
other immature cells.
Results will be used to monitor patients cell counts
and absolute neutrophil count and to determine if
further chemotherapy should be administered.
 Specimen Requirements
Whole blood collected in an EDTA tube.
Minimum sample volume is 0.5 mL using the
Open Sample Mode. The instrument aspirates
30 L of patient sample.
Samples are stable at room temperature for
eight hours.
 Overview of Analysis Modes
Whole blood mode
This is the mode of analyzing collected blood sample
in the whole blood status. The tube cap is opened and
the sample is aspirated through the sample probe one
after another.

Pre-diluted mode
This mode is used in analyzing a minute amount of
childs blood, for instance, collected from the earlobe
or fingertip. In this mode, blood sample diluted into
1:26 before analysis is used. The sample aspiration
procedure is the same as in the whole blood mode.
 Note:
In the pre-diluted mode, particle distribution
curve and particle distribution analysis data
are not output, and the output is confined to
only the CBC 4 parameter (dependent
parameter on MCV) but the remainder
parameter multiply by dilution factor.
 Sources Of Errors
In cell count include:
Cold agglutinins - low red cell counts and high MCVs can
be caused by a increased number of large red cells or red cell
agglutinates.
If agglutinated red cells are present, the automated
hematocrits and MCHCs are also incorrect. Cold agglutinins
cause agglutination of the red cells as the blood cools.
Cold agglutinins can be present in a number of disease states,
including infectious mononucleosis and mycoplasma
pneumonia infections.
If red cell agglutinates are seen on the peripheral smear,
warm the sample in a 37C heating block and mix and test
the sample while it is warm. Strong cold agglutinins may not
disperse and need to be redrawn in a pre-warmed tube and
kept at body temperature .
 Fragmented or very microcytic red cells
These may cause red cell counts to be
decreased and may flag the platelet count as
the red cells become closer in size to the
platelets and cause an abnormal platelet
histogram. The population is visible at the left
side of the red cell histogram and the right end
of the platelet histogram .
 Platelet clumps and platelet satellitosis:
These cause falsely decreased platelet counts.
Platelet clumps can be seen on the right side of
the platelet histogram. Decreased platelet
counts are confirmed by reviewing the
peripheral smear. Always scan the edge of the
smear when checking low platelet counts.
 Giant platelets:
These are platelets that approach or exceed the
size of the red cells. They cause the right hand
tail of the histogram to remain elevated and
may be seen at the left of the red cell
histogram .
 Nucleated red blood cells:
These interfere with the WBC on some
instruments by being counted as white
cells/lymphocytes.
In Measuring Hemoglobin Include
Anything that will cause turbidity and interfere
with a Spectrophotometry method.
Examples are a very high WBC or platelet
count, lipemia and hemoglobin's that are
resistant to lysis, such as hemoglobin's S
and C .
 Basic automated hematology analyzers
provide an electronic measured
Red cell count (RBC),
White cell count (WBC),
Platelet count (plt),
Mean platelet volume (MPV),
Hemoglobin concentration (hb),
And the mean red cell volume (MCV).
From these measured quantities, the hematocrit (Hct),
mean cell hemoglobin (MCH), mean cell hemoglobin
concentration (MCHC), and the red cell distribution
width (RDW) are calculated.
 Red Cell Indices
Hematocrit calculation
Hematocrit (Hct) or (PCV) is the volume of the red
cells as compared to the volume of the whole blood
sample. Hematocrits on the automated systems are
calculated .
The volume of each red cell is measured as it is
counted and a mean cell volume is derived. The
calculations are not precisely the same. But, they can
be summarized as mean corpuscular red cell volume
(MCV) multiplied by the red cell count (RBC(.
Hematocrits are reported in L/L or the traditional .%
 Sources Of Errors In Hematocrit:
Hematocrits calculated by automated
instruments depend on correct red cell
counts and red cell volumes to arrive at an
accurate hematocrit .
Hence, anything affecting the red cell count
or volume measurement will affect the
hematocrit .
This method is not as sensitive to the ratio
of blood to EDTA as the centrifuged
hematocrit
 Correlating Hemoglobin and Hematocrit Values
The hemoglobin times three roughly equals the
hematocrit in most patients.
Example: 14.8 x 3 = 44 (patient's hematocrit result
is 45 L/L)
11.0 x 3 = 33 (patient's hematocrit result is 32 L/L)
The exception to this rule is in patients with
hypochromic red cells. These patients will have
hematocrits that are more than three times the
hemoglobin
 MCV The counter provides us with MCV which is
derived from the histogram (sum of pulse height /
sum of pulse). Not: 1 L= 109 fL
MCH is Mean Corpuscular Hemoglobin weight in
picograms. This is the average weight of the
hemoglobin in picograms in a red cell. It is a
calculated value.
Not: 1g = 1012pg, 1L = 10 dL
MCH =hemoglobin in pg/L / red cell count in
pilions/L
MCHC is Mean Corpuscular Hemoglobin Content.
This indicates the average weight of hemoglobin as
compared to the cell size. It is traditionally a
calculated
MCHC = (Hemoglobin in g/dL / HCT) x 100
 RDW: The RDW (red cell distribution width) is a
measurement of the width of the bases of the RBC
histogram the red cell size distribution and is
expressed as the coefficient of variation
percentage.
The RDW is increased in treated iron deficiency,
vitamin B12 deficiency, folic acid deficiency, post-
transfusion.
MPV: The MPV is a measure of the average
volume of platelets in a sample and is analogous to
the erythrocytic MCV.
Pct: (plateletcrit) analogues to HCT for RBCs
 How Data Are Reported
In most automated systems, the complete
blood count is numerically reported..
The differential is numerically recorded and
then graphically displayed
 RBC and Platelet Histograms

The black line represents normal cell distribution. The red

line on the RBC histogram graphically represents a
Microcytic red cell population.
 Red Cells Histogram
histogram sel normal merah menampilkan bentuk sel (36-
360) fl
(24- 36 fl ) mungkin karena:
1- Fragmen RBCs
2- Fragmen WBC
3- Giant plts
4- Microcyte
Shift to right :
- Leukemia
- Macrocytic anemia
- Megaloblastic anemia
Shift to left :
- Microcytic anemia (IDA)
Bimodal
- Cold agglutinin
- IDA, Megaloblastic anemia dengan transfusi.
-Sideroblastic anemia.
Trimodal
- Anemia dengan transfusi
-Disbtribusi RBC abnormal akan ditandai
-Review mungkin diperlukan berdasarkan
MCV dan RDW
Tindakan: Pemeriksaan Apusan untuk
morfologi RBC
Penyebab RBC dismorfik atau heterogenik:
- Post-traqnsfusi, Post Terapi defisiensi, atau
dua defisiensi sedang berlangsung.
 Histogram Trombosit
Normal platelet histogram (2-20 fl).

(0-2)
Gelembung Udara
Debu
Gangguan Elektrik
Over 20 fL
Sel Mikrosit
Schistosit
FragmenWBC
Platelet Besar
Gumpalan Platelet
 Histogram Analisis Histogram
Histogram adalah representasi dari ukuran
leukosit. Diferensiasinya adalah sebagai
berikut :
Jumlah
Relatif
Sel
 Daftar Tabel Berikut Tanda (R) dan Kelainan yang
Mungkin Dimiliki:
Abnormality Region R Flag
Prekursor Erythrocyte (NRBCs) Jauh ke Kiri(<35fL) R1
Nonlysed erythrocytes
Giant and/or clumped platelets
Heinz body
Malaria
Blasts Antara lymphs dan R2
Basophilia monos
Eosinophilia
Plasma cells
Abnormal/variant limfosit
Populasi abnormal Antara monosit and R3
Eosinophilia granulocytes
Immature granulocytes
Peningkatan granulocytes absolut Jauh ke R4
Kanan(>450fL)
 Nilai Normal
Reporting Results
Rentang Normal Parameter
4.8-10.8 x 103/L 1. WBC
Pria 4.7-6.1 x 106/L 1. RBC
Wanita 4.2-5.4 x 106/L
Pria14-18 g/dl 1. Hemoglobin
Wanita 12-16 g/dl
Pria 42-52% 1. Hematokrit
Wanita 37-47%
Pria 80-94 fl 1. MCV
Wanita 81-99 fl
27-31 pg 1. MCH
32-36 g/dl or % 1. MCHC
11.5-14.5% 1. RDW
150,000 - 450,000/L 1. Platelets
7.4-10.4 fl 1. MPV
 Nilai Kritis
Parameter Critical Value

WBC (K/mm3) 1.0 or 30.0

HGB (g/dL) 6.5 or 19.0

HCT (%) 20.0 or 60.0

PLT (K/mm3) 30.0 or 1000

 Linearitas
Parameter Rentang Linear dari
Produsen
1. WBC (K/L) 1.0 99.9
1. RBC (M/L) 1.0 7.00
1. HGB (g/dL) 2.5 24.0
1. MCV (fL) 50 200
1. PLT (K/L) 10 999
1. MPV (fL) 5.0 20.0
Interferensi yang Mungkin Menyebabkan Hasil yang Salah
WBC
1. Kelainan RBC yang
menyebabkan gagal lysis
2. Nucleated RBCs
3. Fragmented WBCs
4. Partikel tidak lisis (Ukuran
>35 fL)
5. Platelet sangat besar
6. Spesimen yang mengandung
fibrin, fragmen sel atau
debris lain (Spesimen
pediatric/oncologi)
RBC
1. WBC sangat tinggi(>99.9)
2. Konsentrasi tinggi trombosit
yang sangat besar
3. RBC terAgglutinasi, rouleaux
akan hancur saat Istoton
ditambahkan
4. RBC <36 fL
5. Spesimen mengandung fibrin,
fragmen sel atau kotoran
lainnya (spesimen anak /
onkologi)
Interferensi yang Mungkin Menyebabkan Hasil yang Salah
Hgb MCV
1. Jumlah WBC yang sangat 1. Jumlah WBC yang
tinggi sangat tinggi
2. Lipemia berat 2. Konsentrasi tinggi
3. Heparin trombosit besar
3. Sel darah merah
4. Beberapa abnormalitas RBC
yang teraglutinasi
tidak biasa yang sulit lisis
4. fragmen RBC yang
5. Apa pun yang meningkatkan
di bawah ambang
kekeruhan sampel seperti batas 36 fL
tingkat trigliserida
5. RBC kaku
6. Bilirubin tinggi
Interferensi yang Mungkin Menyebabkan Hasil yang Salah

Plt RDW
1. Sel merah sangat 1. WBC sangat tinggi
kecil di dekat 2. Konsentrasi tinggi
trombosit sangat besar
ambang atas atau mengelompok
2. Fragmen sel 3. Sel darah merah di
3. Clumped platelets bawah ambang batas
36 fL
4. Debris seluler di 4. Dua populasi sel darah
dekat ambang merah yang berbeda
trombosit yang 5. RBC aglutinasi
lebih rendah 6. RBC kaku
Interferensi yang Mungkin Menyebabkan Hasil yang Salah
MPV
1. Faktor yang diketahui mengganggu jumlah trombosit dan
bentuk histogram
2. Efek yang diketahui dari EDTA
Hct
Faktor yang diketahui mengganggu parameter perhitungan, RBC dan
MCV
MCH
Faktor yang diketahui mengganggu parameter yang digunakan untuk
perhitungan, Hgb dan RBC
MCHC
Faktor yang diketahui mengganggu parameter yang digunakan untuk
perhitungan, Hgb, RBC dan MCV
 Handling Abnormal Results
Plts < 40,000
1. Check the integrity of the specimen (look for clots, short draw, etc.)
2. Confirm count with smear review for clumps, RBC fragments, giant
platelets, very small RBCs

WBC ++++
Dilute 1:2 with Isoton or further until count is within linearity (for final result, multiply
diluted result by dilution factor); subtract final WBC from RBC; perform spun hct,
calculate MCV from correct RBC & Hct (MCV = Hct/RBC x 10), do not report
HGB, MCH, MCHC. Plt counts are not affected by high WBC. Add comment,
Unable to report Hgb, MCH, MCHC due to high WBC.
 Handling Abnormal Results
Plt ++++
Check smear for RBC fragments or microcytes.
If present, perform plt estimate. If they do not agree,
perform manual plt count.
If not present, dilute specimen 1:2 with Isoton or further
until count is within linearity, multiply diluted result by
dilution factor.
RBC > 7.0
Dilute 1:2 with Isoton or further until count is within linearity,
multiply dilution result by dilution factor; perform spun
hct, review Hgb, recalculate MCH, MCHC
 Instruments
The newer analyzers include white cell
differential counts, relative or percent and
absolute number, and reticulocyte analysis.
The differential may be a three-part differential
that includes granulocytes, lymphocytes, and MID
or a five-part differential that includes neutrophils,
lymphocytes, monocytes, eosinophil's, and
basophils. The new generation of analyzers now
offers a sixth parameter, which is the enumeration
of nucleated RBCs (nRBCs).
 Instruments

Automated full
blood counters with
a five-part or more
differential counting
capacity[*]
 Automated Akurat, Presisi dan Aman

Safe & Precise result

Hydrodynamic Focusing DC
SLS HB (Non Cyanide HB)

Slide 65
 Metoda Otomatik
Electrical Impedance
Sel lewat melalui apertura sehingga ketika terjadi perbedaan resistensi
melalui apertura itu, maka tertangkap sebagai sinyal listrik. Besarnya sinyal
yang ditangkap tersebut menentukan jumlah dan ukuran sel yang lewat

Slide 66
Slide 67
RBC and PLT

Slide 68
Slide 69
Slide 70
WBC

Slide 71
 Diff-Channel Abnormal Distribution

Atypical Promyelo
Lymph
Myelo

Metamyelo
Blast

Band

PT Sysmex
Company Indonesia Copyright 2011 PT Sysmex Indonesia
Confidential Slide 24
 5 DIFF Channel - Normal Scattergram

Mono
Lymph

Neut

Baso
Eo

Ghost
Slide 73
5 DIFF Channel- Abnormal Scattergram

Cell Stage Cell diameter

(m)

Myeloblast 14 - 20

Promyelocyte 15 -21

Myelocyte 16 - 24

Metamyelocyte 12 -18

Band 10 -15

Neutrophil 10 -15

Slide 74
Kadar

6.000.000

5.000.000

4.000.000

3.000.000

2.000.000

1.000.000

0
Erythrocytes Leukocytes Thrombocytes
Concentration/l 5.000.000 6.000 250.000

Slide 75
Hemoglobin
Komponen molekul hemoglobin:
Globin - protein composed of 2 sets of dimers: a and b.
4Heme molecules each of which consist of:
protoporphyrin IX
Iron atoms in the ferrous state (Fe)2+

Fe+2 Fe+2

Fe+2 Fe+2 Globin chains
Heme molecule
Slide 76
Metoda Cyanmethemoglobin
Sampel darah dicampur dengan larutan
potassium ferricyanide (K3FeCN6) untuk
mengkonversi Hgb dalam ferro menjadi ferri
Ferri atau methemoglobin berikatan dengan
potassium cyanide (KCN) membentuk
cyanmethemoglobin.
(K3FeCN6) (KCN)

Hb(Fe+2) Hi(Fe+3) HiCN

Serapan larutan HiCN pada panjang gelombang

540nm berbanding lurus dengan kadar Hb yang
ada
Slide 77
Penetapan kadar Hemoglobin dengan metoda spektrofotometer
Spectrophotometry - mengukur cahaya monokromatik
yang ditransmisikan melalui larutan yang berkorelasi
linear dengan kadar zat sesuai serapannya
Komponen dasar suatu spectrophotometer:

Entrance slit Exit slit

Light source Monochromator Cuvette Detector Readout device

Slide 78
Penetapan kadar Hemoglobin
Memperkirakan kemampuan darah membawa oksigen
Rekomendasi ICSH recommended menggunakan
metoda Cyanmethemoglobin
Metoda ini digunakan karena memiliki spektrum
serapan yang lebar (535 - 545 nm)
Absorbance

Wavelength
 Metoda pengukuran Hb otomatik

SLS Methods

Slide 80
Slide 81
Hematokrit
Merupakan ratio volume RBC terhadap volume
darah
Digambarakan dalam satuan prosentase atau
fraksi desimal
Dapat diukur menggunakan metoda mikro
ataupun makro
Pengukuran secara tidak langsung dari hitung
MCV x RBC

Slide 82
Hematokrit (micromethod)

Centrifuge

VT
Capillary tube
VE
VE
Hct %= V x 100
T
Buffy coat
V-Evolume of packed erythrocytes
V-Ttotal volume of blood
Slide 83
Microhematocrit
1.0
0.9
The reading could be done on a
0.8 scale, which is based on the
0.7 principle of similar triangles.
0.6
0.5 Hematocrit
0.4 value
00.3.

02.1
0.0 The guide, which cuts through
the border between packed
RBC and buffy coat gives the
Hematocrit value

Slide 84
Metoda perhitungan hematokrit otomatik

Slide 85
 Complete Blood Count (CBC)
Pemeriksaan yang rutin dikerjakan di
laboratorium hematologi
Pada masa kini, pemeriksaan CBC umumnya
dilakukan menggunakan alat semi-automated
dan automated hematology analyzer
Terdapat 3 langkah dasar dalam teknik ini:
Pengenceran darah
Pengambilan darah sesuai volume yang diukur
Pengukuran sel berdasarkan volume yang diukur

Slide 86
CBC
Darah dengan antikoagulan EDTA diperlukan
Ada 8 parameter dasar dalam tes CBC ,yi:
RBC count
WBC count
PLT count
Hemoglobin concentration
Hematocrit
Mean Cellular Volume
Red cell
Mean Cellular Hemoglobin
indices
Mean Cellular Hemoglobin Concentration

Slide 87
 Indeks RBC:MCV, MCH, & MCHC
Menggunakan teknik perhitungan untuk menentukan ukuran,
isi, dan kadar Hb RBC
Berguna dalam menentukan karakteristik anemia berdasarkan
ukuran RBC
Dihitung berdasarkan jumlah RBC, kadar Hb, dan HCT

Slide 88
 MCV = Hct % x 10
RBC ( x 106/uL)

MCV (Mean Cell Volume) volume eritrosit rerata

(VER) Digambarkan dalam satuan femtoliters (fL)

MCH (Mean Cell Hemoglobin) hemoglobin eritrosit rerata (HER)

Digambarkan dalam satuan picograms (pg)

MCHC (Mean Cell Hemoglobin Concentration) konsentrasi

hemoglobin eritrosit rerata (KHER)
Digambarakan dalam satuan g/dL

Slide 89
 CBC + DIFF
Darah dengan antikoagulan EDTA diperlukan
Parameter hitung CBC+DIFF terdiri dari:
CBC
LYMPH%, LYMPH#
NEUT%, NEUT#
MONO%, MONO#
EO%, EO#
BASO%, BASO#

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 90
Sysmex Indonesia
CBC.
Hitung RBC, WBC, dan PLT dilaporkan dalam
jumlah sel per satuan unit volume darah
International Committee for Standardization in
Hematology (ICSH) merekomendasikan satuan
unit volume darah dalam liter (L)
Contohnya :
RBC: 5.00 x 1012/L or 5.00 x 106/uL
WBC: 7.00 x 109/L or 7.00 x 103/uL
PLT: 300 x 109/L or 300 x 103/L

PT Sysmex Indonesia Slide 91

 Complete assays to suit your needs

Parameters
CBC + PLT +
YES YES YES YES YES YES
WBC Differential (5 part)

MCV, MCH, MCHC,

RDW-CV & SD
YES YES YES YES YES YES

IG % & # (with IG Master NO YES YES YES YES YES

SW)

Retic #, %, Retic Diff & IRF NO NO YES YES YES YES

RET-He (with RET Master NO NO YES YES YES YES

SW)
NRBC % & #,
HPC (with HPC NO NO NO NO YES YES
Master SW), IPF (with
IPF Master SW)

Body Fluid (WBC BF, NO NO NO YES NO YES

PMN (%,#), MNC
Company Confidential Copyright 2011 PT S ysmex Indonesia Slide 44 PT Sysmex Indonesia
(%,#), RBC-BF)
 Analysis methods

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 93
Sysmex Indonesia
New Parameter :
Reticulocyte

Slide 94
 Reticulocyte
Definition
Young red cells, newly released from the bone marrow,
that still contain ribosomal RNA

* Morphology:
- Any non-nucleated red cell containing two or more
particles (dots) of bluestained material corresponding to
ribosomal RNA
- The dots should be at a clear distance from the cell wall
to avoid being mistaken for Heinz bodies
* Cytochemistry:
- Supravital staining, nucleic acid staining
NCCLS, Methods for Reticulocyte Counting ; 2002
Slide 95
Stadium pematangan eritrosit

Bain B. A beginners guide to blood cells.2004

Slide 96
Reticulocyte maturation

Reticulocytes and their significance Sysmex Xtra Online | August 2010

Slide 97
 Morphological Definition of Reticulocyte and the
relative proportions of circulating forms
(Heilmeyer 1932, Sep 1953)
Proerythroblast
Stage 0 Orthochromatic Normoblasts

Polychr.
Stage 1 Dense Coherent Reticulum in non-nucleated Cell
0.1%
Reticulocytes

Erythroblast
0
Stage 2 Extended Network of Loose Reticulum
1
Orthochr.
Erythroblast
7.0 %
Stage 3 Scattered Granules with Residual Reticulum
Blood - b2one marrow barrier
32.0 %
Stage 4 Scattered Granules
3
61.0 %
4

Erythrocytes

Slide 98
Reticulocyte Count

Slide 99
Reticulocyte count technic

Manual method
Automatic method

Slide 100
 Manual count

Staining method

- 2 or 3 drops stain into a tube

- Add equal volume blood sample
- Mix and leave in water bath (incubator at 37oC 15-20 min)
- At the end of this time, resuspend by gentle mixing
- Make a thin film
- When dry, examined without counter-staining
- The reticular material stained deep blue and
- The non-reticulated cells shades of pale greenish-blue

http://www.searo.who.int/en/Section10/Section17/Section53/Section480_1733.htm
Slide 101
Manual count

Counting
- Choose an area where the cells are undistorted
- The staining is good
- Using the x100 oil-immersion lens
- Count the number of reticulocytes seen per 100 or
per 1000 red cells Examination area

Body Tail
http://www.searo.who.int/en/Section10/Section17/Section53/Section480_1733.htm
Slide 102
Automated count

Slide 55
CV Reticulocyte count

RET (%) Target of CV

2% 5% 10%

1 247 500 39 600 9 900

2 122 500 19 600 4 900
5 47 500 7 600 1 900
10 22 500 3 600 900
20 10 000 1 600 400
50 2 500 400 100

Slide 61
Comparison of reticulocyte parameters

Reference range
Relative reticulocytes:
- Female: 0.54 2.02 %
- Male : 0.48 1.64 %

Reticulocyte concentration:
- Female: 0.025 0.102 x 106/L
- Male : 0.026 0.078 x 106/L

Slide 62
Reticulocytosis

Slide 63
Reticulocytopenia

Slide 64
 Di MCV N / MCH N

BLOOD FILM

RETICULOCYTE COUNT

Reticulocytes N / Reticulocytes

BONE MARROW MORPHOLOGY

Normal Abnormal

Hypoplastic Infiltration / Dyserythropoietic

fibrosis

SECONDARY ANAEMIA, APLASTIC LEUKAEMIA, MYELODYSPLASIA ACUTE HAEMOLYSIS

eg INFLAMMATION, ANAEMIA MYELOMATOSIS, BLOOD
RENAL DISEASE, LIVER METASTASES, LOSS
DISEASE, ENDOCRINE MYELOFIBROSIS

DEFICIENCY Slide 65
Lewis SM, Bain BJ, Bates I. Dacie and Lewis practical haematology. 9th. London : Churchill Livingstone; 2001.p.583.
 Reticulocyte count

Reticulocyte index (RI)

Retic production Index (RPI)
Immature Reticulocyte Fraction (IRF) (previously called
Reticulocyte maturation index)

NCCLS, Methods for Reticulocyte Counting ; 2002

Slide 66
 Reticulocyte count

Reticulocyte index (RI)

- The relative portion (%, ) of reticulocytes may increase, if the
reticulocytes are in fact increased or the red blood cells
decreased

RI = RET [%] x HCT [%] (patient)

45 [%] (standard HCT)

Slide 67
HCT and maturation time of retics in blood

Slide 68
RETICULOCYTE COUNT

Retic production Index (RPI)

To be useful the reticulocyte count must be adjusted for the
patient's hematocrit, also when the hematocrit is lower
reticulocytes are released earlier from the marrow so one
can adjust for this phenomenon. Thus:
Corrected retic. = Patients retic. x (Patients Hct/45)
RPI = % Retic X Hct/45 X 1/CF (Correction factor )
Hct = 45 CFI = 1.0 Hct = 25 CFI = 2.0
Hct = 35 CFI = 1.5 Hct = 15 CFI = 2.5
Normal RPI = 1 (for non-anemic pts)
RPI < 2 : hypoproliferative
RPI 2 : hyperproliferative
PT Sysmex Indonesia Slide 69
 Interpretation of RPI

RPI < 2 hypoproliferative RPI 2 : hyperproliferative

(inadequate response) (adequate response)

Iron def. anemia Hemolytic disease

B12/folate def Hemoglobinopathy (inc.Thalssemia)
Chronic disease Treated B12/folate def.
Sideroblastic anemia
Aplastic anemia
Myeloproliferative

Slide 70
Reticulocyte count
Immature Reticulocyte Fraction (IRF)
- An early marker for evaluating the regeneration of
erythropoiesis
- A quantitative expression of the maturation state of the entire
reticulocyte population in the peripheral blood

IRF is the sum of MFR and HFR, i.e.

IRF = MFR + HFR

Reference range IRF

- Female :1.1 15.9 % , Male: 1.5 13.7 %
In-vitro stability of IRF : 6 hours
Slide 72
Clinical use of IRF

Slide 73
Clinical Utility of Immature Reticulocyte Fraction

NCCLS, Methods for Reticulocyte Counting ; 2002

Slide 74
Ret He

Gives the HGB content of the freshly produced red blood cells
and thus offers real-time information on iron supply to
erythropoiesis
Useful to differentiate between the two most common anaemias
(iron deficiency anaemia and anaemia of chronic disease
(ACD)
RET-He is not affected by the acute phase reaction

Reference range RET-He: 28 35 pg

Slide 75
 Indication

Classification of normochromic and hypochromic anaemias

Differentiation between iron deficiency anemias and functional

iron deficiency

Monitoring therapy of chronic infections or tumours

Monitoring erythropoietin therapy and iron substitution

Slide 77
Clinical utility of Ret-He

Slide 78
Therapeutic implication for
treatment different
phases of iron
deficiency

Slide 79
 Using Ret-He as a guidelines

Identification and Monitoring of Functional Iron Deficiency in ES RD Patients Using an Early Erythroid Population-
Sysmex America, RET-He White Paper
Slide 80
 Using Ret-He as a guidelines

Identification and Monitoring of Functional Iron Deficiency in ES RD Patients Using an Early Erythroid Population-
Sysmex America, RET-He White Paper
Slide 81
 Company Confidential Copyright 2011 PT Sysmex Indonesia Slide 82 PT Sysmex Indonesia
Modified NAAC (National anemia action council, 2008) article.http://www.anemia.org/professionals/feature-articles/content.php
Slide 84
Kasus Anemia Defisiensi Besi

Slide 86
Kasus CML

Slide 87
Kasus ALL 1

Slide 88
 A real life example of the use of
RET-He to monitor r-HuEpo + i.v. iron therapy
Forward Scatter

Forward Scatter
Fluorescence Intensity Fluorescence Intensity
a. A 7-year-old child with severe hypochromic anaemia.

b. 3 days after r-HuEpo plus i.v. iron therapy. RET-He has increased, as well
as the concentration of reticulocytes.
Company Confidential Copyright 2011 PT
PT Sysmex Indonesia Slide 89
Sysmex Indonesia
 Megaloblastic Anaemia

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 90
Sysmex Indonesia
 Megaloblastic Anaemia,
after one week treatment

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 91
Sysmex Indonesia
 Megaloblastic Anaemia, after
two weeks treatment

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 92
Sysmex Indonesia
 Kesimpulan :

Teknologi Hydrodynamic Focusing DC untuk

identifikasi dan per-hitungan sel yang akurat

Konsep
Fully automatic analysis : memastikan standarisasi
pemeriksaan, tanpa adanya preanalitik
merubah paradigma :
kecepatan pemeriksaan meningkat manual ke Automated
Sampel native darah : memastikan bahwa unsur-unsur
Hematology Analyser
darah tidak rusak atau hilang karena adanya proses
seperti sentrifugasi

Informasi morfologi WBC, RBC, PLT, HCT, HgB:

Identifikasi adanya penyakit

Company Confidential Copyright 2011 PT

PT Sysmex Indonesia Slide 93
Sysmex Indonesia
Fitur dan Kelebihan
Fitur Kelebihan

hasil yang accurate dan quantitative.

Hydrodynamic Focusing DC memperbaiki kualitas hasil

Ramah lingkungan, limbah tidak menghasilkan

Non Cyanide HgB Methode
Cyanide sehingga aman untuk user dan lingkungan
tidak membutuhkan preanalitik (sample native)
minimalisasi variasi dalam hal pembacaan
AutomatedAnalysis microscopic
hasil yang objektif

membantu klinisi mendapatkan informasi yang

WBC, RBC, HgB, PLT, HCT
spesifik utk diagnosis

Compact and Light Design Dimensi yang cukup, bisa di tempatkan di mana saja

User Friendly Touch screen panel dg icon yg mudah di mengerti

Slide 94
 Clinical Significance of RET-Channel
Reticulocyte Count
Ret # , Ret %
Ref Range for Sysmex XT*: 0.5 2%
RET-Quantity

RET-He (Hemoglobinization of Reticulocytes)

Reflects the actual iron supply for hemoglobin synthesis in the BM
Early detection of iron depletion in erythropoiesis.
Distinguish Fe def (ID) and functional Fe Def (FID).
RET-Quality
In FID the iron stores are replete, but the iron is not sufficiently
available for Hb systhesis
Ref range** : 24-36 pg

*Wirawan R, Uji ketelitian, ketepatan dan nilai rujukan parameter retikulosit orang Indonesia dewasa di Jakarta menggunakan alat hitung sel darah otomatik
Sysmex XT-2000i. 2006, p 26.**Brugnara, C. et al. Reticulocyte hemoglobin equivalent (Ret-He) and assesment of iron-deficient states.

Slide 95