KIMIA KLINIK DAN

BIOANALISIS
Edy Meiyanto
Fakultas Farmasi UGM
http://edymei.blog.ugm.ac.id
1
I. KIMIA KLINIK
1. PENDAHULUAN: Pengertian, batasan,
ketepatan diagnostik, dan sample klinik.
2. PENANGANAN SAMPEL DARAH DAN URIN
3. ANALISIS SEL LIMFOSIT DENGAN FC
4. ANALISIS HEMATOLOGI
5. ANALISIS KIMIA DARAH
6. ANALISIS FUNGSI GINJAL
7. ANALISIS FUNGSI HATI
2
II. BIOANALISIS
1. TEKNIK DASAR KULTUR SEL
2. UJI GENOTOKSIK-1
3. UJI GENOTOKSIK-2
4. UJI MENGGUNAKAN SEL PRIMER
5. UJI MENGGUNAKAN SEL LINES
6. UJI MENGGUNAKAN MIKROBA
7. OVARIEKTOMI
3
Tema Praktikum dan Dosen
Pembimbing
No Tema Keterangan Dosen Pembimbing
1 Analisis kerusakan
genetik
Parameter MNPCE darah
perifer & bone marrow
Dr. Ag.Yuswanto, SU., Apt.
2 Analisis fungsi
hepar A
Kolesterol total dan
Trigliserida
Prof. Dr. Ediati S.E., Apt.

3 Analisis fungsi
hepar B
Parameter SGPT, SGOT, Prof. Dr. Sudibyo M., MS., Apt.

4 Analisis fungsi
ginjal
Kreatinin, Ureum, He Dr. Riris Istighfari Jenie, M.Si., Apt.

5 Analisis fungsi
jantung
SGPT, SGOT, NQ Prof. Dr. Edy Meiyanto, M.Si., Apt.
PENDAHULUAN
TUJUAN KIMIA KLINIK
Mengukur level senyawa yang secara normal
terdapat dalam darah yang memiliki fungsi biologi.
Misal. Glukosa, kolesterol
Mengukur level senyawa metabolit non-fungsional di
dalam darah. Misalnya ureum, kreatinin
Mendeteksi indikator adanya kerusakan sel. Misalnya
SGPT untuk sel hati
Mendeteksi adanya senyawa/obat dalam cairan
biologis
KONSEP DIAGNOSTIK
SISTEM
BIOLOGI
ORGAN TUBUH SEL
GANGGUAN
MACROMOLE
CULS
DNA/RNA
PROTEIN
SISTEM
DETEKSI
TARGETED-
SPECIFIC
TEKNOLOGI
MOLECULAR
DIAGNOSTICS
6
MAKNA DIAGNOSTIK
ILMU DASAR TUBUH
STATUS
KESEHATAN
KIMIA KLINIK
INPUT
TEORITIK
DASAR
ANALISIS
BEAYA KESEHATAN
TERAPI
PENYAKIT
SISTEM
DETEKSI
PREVENSI
PENYAKIT
INTERPRETASI
KLINIK
PENEGAKAN
DIAGNOSIS
7
8
Keberhasilan dalam kedokteran modern
bergantung pada kecermatan dalam diagnosis
spesifik molekular pada:
Viruses
Bacteria
Fungi
Parasites
Proteins
In water, plants, soil and humans.
TESTING PRE-NATAL: apakah janin sehat?


PREDISPOSISI PENYAKIT:
punya risiko penyakit apa?

DETEKSI PENYAKIT: Sakit apa?

SELEKSI OBAT: obat apa yang tepat?


MONITORING PENYAKIT: bagaimana penyakitnya akan kambuh?
1
2
3
4
9
5
Molecular
diagnostics is
>$3 billion
market WW and
growing at >20%
annually
CONTOH APLIKASI DIAGNOSIS TERKINI
Prenatal
Diagnostics
Oncology
Infectious
Disease
Transplantation
Medicine
Genetic Testing High throughput testing for genetic
disorders including SNPs, insertions,
deletions
Examples: Factor II, Factor V, CFTR
Non-invasive detection of fetal diseases
Examples: Down syndrome, RhD, cystic
fibrosis
Early diagnosis of cancer
Example: circulating tumor DNA
Non-invasive, early detection of organ
rejection
Example: urine testing for kidney rejection
Pathogen identification and early detection
Examples: identification of multi drug
resistant mycobacteria, early detection of
drug-resistant viral strains, e.g. HIV, HBV,
HCV
Progress is being made
in all of these areas

Each of these areas are
commercially attractive

In some cases, the
MassARRAY platform is
uniquely qualified for
specific tests

More tests will be added
to the platform as these
tests are rolled out
11
Proporsi pasien yang mendapat diagnosis
sehingga memberikan informasi
yangbenar sesuai dengan tujuan
diagnosis.

Untuk menguji ketepatan alat uji
diagnostik,digunakan alat uji pembanding
yang standar yang dinamakan Gold
Standar
12
13
A good detection system should have 3 qualities:
Sensitivity
Specificity
Simplicity
Sensitivity means that the test must be able to
detect very small amounts of target even in the
presence of other molecules.
Specificity: the test yields a positive result for the
target molecule only.
Simplicity: the test must be able to run efficiently
and inexpensively on a routine basis.
1. Ketepatan : jumlah hasil uji yang benar
dibagidengan jumlah pasien yang diuji.
2. Sensitivitas : jumlah hasil uji positif benar
(truepositive)dibagi dengan jumlah orang yang
teruji sakit.
3. Spesifitas : jumlah hasil uji negatif benar
(truenegative) dibagi dengan jumlah orang teruji
tidak sakit.
4. Nilai prediksi positif: jumlah hasil uji positif
benar dibagi dengan jumlah hasil semua uji
positif.
5. Nilai prediksi negatif: jumlah hasil uji negatif
dibagi dengan jumlah hasil semua uji negatif
14
Contoh kasus: skrining bayi lahir yang membawa penyakit
genetik di Amerika
15
PPV= 250/2249 = 11 %
NPV= 997750/997751 = 100 %
Maknanya??? Kalau + perlu revalidasi
Hasil uji

Status sakit total
ada tidak
Hasil uji positif
Hasil uji negatif
Total
TP:250
FN:1
TP +FN:250
FP:1.999
TN:997.750
FP+TN:
999.75
TP+FP:2.249
FN+TN:999.751
Semua bayi yg
diuji:1.000.000

Contoh kasus: Uji penyakit genetik
16
PPV= 299/300 = 99,5 %
NPV= 699/700 = 99,99 %
Maknanya?? Kedua hasil sudah dapat dipercaya
Hasil uji

Status sakit total
ada tidak
Hasil uji positif
Hasil uji negatif
Total
TP:299
FN:1
TP +FN:300
FP:1
TN:699
FP+TN:
700
TP+FP:300
FN+TN:700
Semua bayi yg
diuji:1.000

1. DARAH UTUH
2. PLASMA
3. SERUM
4. URIN
5. CAIRAN SEREBROSPINAL
6. BIOPSI
7. JARINGAN
8. DLL
17
18
19
KOLEKSI SAMPLE
IDENTIFIKASI PASIEN
PENGGUNAAN TABUNG/TUBE/CONTAINER
PEMBERITAHUAN KEPADA PASIEN TENTANG
TATACARA PENGAMBILAN SAMPEL
PASIEN DALAM POSISI YANG NYAMAN
20
21
22
23
24
25
26
27
28
29
30
KESALAHAN UMUM DALAM KOLEKSI SAMPEL
31
PELABELAN SALAH ATAU TIDAK LENGKAP
JUMLAH TIDAK MENCUKUPI
KELIRU DALAM MEMILIH TABUNG
INSTRUKSI PADA PASIEN TIDAK TEPAT ATAU
KURANG
LENGKAP
TUTUP TIDAK RAPAT SEHINGGA MENYEBABKAN
TUMPAH ATAU
TERKONTAMINASI
KESALAHAN UMUM DALAM KOLEKSI SAMPEL
SERUM
GAGAL DALAM MEMISAHKAN SERUM DARI
SEL DARAH MERAH
DALAM WAKTU 60 MENIT
PENJENDALAN DARAH TIDAK BERHASIL
DENGAN BAIK
TERJADI HEMOLISIS
LIPEMIA: KEKERUHAN PADA SERUM
DISEBABKAN DIET
32
KESALAHAN UMUM DALAM KOLEKSI
SAMPEL PLASMA
KELIRU DALAM MEMILIH ADDITIF
TIDAK DICAMPUR DENGAN CEPAT DENGAN
ADDITIF
TERJADI HEMOLISIS
SAMPEL TIDAK MENCUKUPI
KELUPAAN MELABEL SEBAGAI PLASMA
LUPA MENULIS JENIS ANTIKOAGULAN: EDTA
ATAU SITRAT
33
HEMOLISIS
In general, grossly or even moderately
hemolyzed blood specimens may not be
acceptable for testing.
Hemolysis occurs when the red cells rupture
and hemoglobin and other intracellular
components spill into the serum.
Hemolyzed serum or plasma is pink or red,
rather than the normal clear straw or pale
yellow color
34
MENGHINDARI HEMOLISIS
For routine collections, use a 20- to 22-gauge needle. (On
occasion, however, it may be necessary to use a 23-gauge
needle for patients from elderly and pediatric populations
with small or difficult veins.)
If there is air leakage around the needle or loss of vacuum in
the tube, replace the vacuum tube.
use only clean, dry, sterile needles, syringes, and tubes.
Collect blood in room temperature containers
Do not remove the needle from the vein with the vacuum
tube engaged. This applies to both the last tube collected
during a routine venipuncture and to tubes collected during
a difficult procedure.
Premature removal of the tube causes a rush of air to enter
the tube, which may result in damage to the red cells.

35
MENGHINDARI HEMOLISIS CONT
Be as gentle as possible, drawing the blood evenly.
Too much pressure in drawing blood into a syringe
or forcefully ejecting blood into a collection tube
from a syringe may damage red cells.
Allow collection site to dry after cleaning. Alcohol
used to clean the puncture site may cause
contamination in a tube.
Do not collect a specimen in a hematoma.
Allow specimen to clot completely before
centrifuging.
Do not centrifuge the specimen for a prolonged
period of time.

36
LIPEMIA
Normal serum or plasma is a clear and light
yellow to straw in color. Turbid serum or
plasma appears cloudy or milky.
Serum or plasma may be cloudy due to
bacterial contamination or chronic or
transient high lipid levels in the patient's
blood.
The primary dietary sources of lipids (fatty
substances) are meats, butter, cream, and
cheese.
37
LIPEMIA-2
Patients who consume these foods within the
24-hour period immediately
preceding collection of a blood specimen may
have temporarily elevated
lipid levels, which may be manifested by
cloudy or lipemic serum. Lipemic
serum or plasma may not be a true indicator
of the patient's physiologic state.

38
MENGHINDARI LIPEMIA
Regardless of diet and length of fast, some
patients may produce cloudy specimens.
To avoid dietary-induced high lipid levels prior
to testing, many physicians require patients to
exclude the high-fat foods from their diets or
to fast for 12-14 hours prior to specimen
collection. For morning specimen collection,
the laboratory recommends that the patient
be required to fast from 6 PM on the previous
evening.
39
40
41
42
43
44
45
46
47
FLOWCYTOMETRY
48
What Is Flow Cytometry?
Flow ~ cells in motion
Cyto ~ cell
Metry ~ measure
Measuring properties of cells while in a
fluid stream

Cytometry vs. Flow Cytometry
Cytometry
Localization of antigen
is possible
Poor enumeration of
cell subtypes
Limiting number of
simultaneous
measurements

Flow Cytometry.
Cannot tell you where
antigen is.
Can analyze many
cells in a short time
frame.
Can look at numerous
parameters at once.
Uses of Flow Cytometry
It can be used for
Immunophenotyping
DNA cell cycle/tumor ploidy
Membrane potential
Ion flux
Cell viability
Intracellular protein staining
pH changes
Cell tracking and proliferation
Sorting
Redox state
Chromatin structure
Total protein
Lipids
Surface charge
Membrane fusion/runover
Enzyme activity
Oxidative metabolism
Sulfhydryl groups/glutathione
DNA synthesis
DNA degradation
Gene expression
The use of flow in research has
boomed since the mid-1980s
Medline Publications citing "Flow
Cytometry"
0
1000
2000
3000
4000
5000
1
9
6
0
1
9
6
5
1
9
7
0
1
9
7
5
1
9
8
0
1
9
8
5
1
9
9
0
1
9
9
5
2
0
0
0
Year
P
u
b
l
i
c
a
t
i
o
n
s
Background Info Summary
Flow Cytometry is a quickly expanding
technology
Has continually increased in popularity
since the mid 1980s.
Gives us the ability to analyze many
properties of many cells in very little time

What is Flow Cytometry?
Flow refers to a fluid stream

Cyto refers to a cell

metry refers to measurement.

What Is Flow Cytometry?
Simultaneous measurements of
multiple characteristics of a single cell
Measurements are made on a per-cell
basis at routine rates of 500 to 4000
cells per second
Definitions
Flow cytometry: study of cells as they
move in fluid suspension, allowing multiple
measurements to be made per cell.

FACS: fluorescence-activated cell
sorting

What Can a Flow Cytometer
Tell Us About a Cell?
Its relative size (Forward Scatter
FSC)
Its relative granularity or internal
complexity (Side ScatterSSC)
Its relative fluorescence intensity
(FL1, FL2, FL3, FL4, and FL5)
Properties of FSC and SSC

Forward Scatterdiffracted light
Related to cell surface area
Detected along axis of incident light in the forward
direction
Side Scatterreflected and refracted light
Related to cell granularity and complexity
Detected at 90 to the laser beam
Right Angle Light Detector
Cell Complexity
Forward Light Detector
Cell Surface Area
Incident
Light
Source
What measurements can be made?
Forward light scatter (FSC): proportional to
cell size
Side light scatter (SSC): proportional to cell
granularity
Fluorescence:
Binding of fluorescent-labeled antibodies
Ca
++
-sensitive dyes within cells
Fluorescent proteins expressed by cells
Binding of DNA dyes
Scatter profile of lysed whole blood
S
i
d
e

S
c
a
t
t
e
r

Forward Light Scatter
0 200 400 600 800 1000
0

2
0
0

4
0
0

6
0
0

8
0
0

1
0
0
0

Lymphocytes
Monocytes
Granulocytes
largest and most
granular population
smallest and least
granular population
Cytometer fluidics create laminar flow
Sample stream
Sheath stream
Cell
Laser beam
Flow Cell
Typical 2-color cytometer configuration

530/30 nm band
pass filter
488nm band pass filter
488nm laser beam
560nm short pass
dichroic mirror


585/42 nm
band pass filter
FL2
PMT
FL1
PMT
FSC PD
flow cell
1% ND front
surface mirror






488/10 nm
band pass filter
SSC
PMT
What is Fluorescent Light?
The fluorochrome absorbs energy from
the laser
The fluorochrome releases the absorbed
energy by:
Vibration and heat dissipation
Emission of photons of a longer wavelength
= 488 nm
Emitted
Fluorescent Light
Energy
Antibody
Incident
Light Energy
Fluorescein
Molecule
530 nm
HO
CO
2
H
O
C
Absorption and Emission Spectra
of a Fluorochrome
Wavelength (nm)
400 450 500 550 600 650
700
1000
800
600
400
200
0
FITC
A Cytometer Needs
a Combined System of:
Fluidics
To introduce and focus the cells for
interrogation
Optics
To generate and collect the light signals
Electronics
To convert the optical signals to
proportional electronic signals and digitize
them for computer analysis
OpticsFACSCalibur

FL1
Red Diode Laser
~635 nm
SSC
FL3
FL4
670LP
661/16
585/42
488/10
90/10 Beam Splitter
DM 560SP
Fluorescence
Collection Lens
DM 640LP
Half Mirror
488/10
.
488 nm
Blue Laser
FSC Diode
Focusing
Lens
FL2
530/30
Beam Combiner
Flow
Cell
Flow
Cell
Two-Color Direct Staining
Analyze
Wash Incubate
Two-Color Cell Analysis
C
D
1
9

P
E

CD3 FITC
10
0
1
0

0

1
0

1

1
0

2

1
0

3

1
0

4

10
1
10
2
10
3
10
4
Three-Color Direct Staining
Analyze
Wash Incubate
Three-Color Cell Analysis
CD3 APC CD3 APC
C
D
4

F
I
T
C

C
D
8

P
E

CD4 FITC
C
D
8

P
E

10
0
10
1
10
2
10
3
10
4
1
0

0

1
0

1

1
0

2

1
0

3

1
0

4

1
0

0

1
0

1

1
0

2

1
0

3

1
0

4

1
0

0

1
0

1

1
0

2

1
0

3

1
0

4

10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
DNA Histogram
0 200 400 600 800 1000
0
50
100
150
200
250
C
o
u
n
t
s

FL2-Area
G0/G1
S G2/M
Fluorescence data display
P
E

FITC
FITC Fluorescent Intensity
N
u
m
b
e
r

o
f

E
v
e
n
t
s

Negative control histogram
Major components of a flow cytometer
Sample intake port
Sheath and waste reservoirs
Flow cell

Laser(s)
Optical filters

PMTs (photomultiplier tubes) or photodiodes
Signal processor

Cell sorting
Background and autofluorescence
All cells have a certain level of background
fluorescence, due to:
Autofluroescence: from pigments and fluorescent
moieties on cellular proteins
Non-specifically bound antibodies, and free
antibody in the sample stream
The level of autofluorescence varies with the
wavelength of excitation and collection:
Highest in FITC, PE detectors; lowest in far red
(APC, Cy7) detectors
Fluorescence sensitivity
Detection Efficiency (Q): number of
photoelectrons generated per molecule of
fluorophore
Dependent upon fluorophore, filters, PMT
sensitivity, voltage gain setting, etc.
Background (B): non-specific signal intrinsic
to the system
Dependent upon autofluorescence, unbound
fluorophore, stray light, etc.
Common fluorophores for Ab conjugation
FLUOROCHROME Type of molecule Typical excitation laser Approximate emission
peak
Fluorescein
isotyocyanate (FITC)
Small organic 488 nm 518 nm
AlexaFluor 488 Small organic 488 nm 518 nm
Phycoerythrin (PE) Protein 488 or 532 nm 574 nm
PE-Texas Red Protein tandem 488 or 532 nm 615 nm
PE-Cy5 Protein tandem 488 or 532 nm 665 nm
Peridinin chlorophyll
protein (PerCP)
Protein 488 or 532 nm 676 nm
PerCP-Cy5.5 Protein tandem 488 or 532 nm 695 nm
PE-Cy7 Protein tandem 488 or 532 nm 776 nm
Allophycocyanin (APC) Protein 633 nm 659 nm
AlexaFluor 647 Small organic 633 nm 667 nm
AlexaFluor 700 Small organic 633 nm 718 nm
APC-Cy7 Protein tandem 633 nm 784 nm
Pacific Blue Small organic 405 nm 454 nm
AmCyan Protein 405 nm 487 nm
In vivo flow cytometry, which combines confocal microscopy
and flow cytometry, has been developed as a new non-
invasive method of detecting and monitoring circulating cells


The Becton Dickinson Biosciences FACSCalibur
is a four color flow cytometer
Flow Cytometry Clinical
Applications
HIV immunophenotyping (CD4 absolute counts)
Cytokine analysis
Leukemia and lymphoma diagnosis
- minimum residual disease detection
Cell cycle and DNA ploidy analysis
Stem Cell Analysis
HLA B 27 Typing
Residual white blood cell detection
Reticulocyte enumeration
Flow crossmatching (organ transplantation)

Immunophenotyping of leukaemic
cells
The Composition of Whole Blood (4-6L in an adult)
The percentage
ranges for white
blood cells indicate
the normal variation
seen in a count of
100 white blood
cells in a healthy
individual.
Gambaran mikroskopis sel-sel darah
Lymphocyte
Eosinophil
Erythrocyte
Basophil
Neutrophil
polymorph
Monocyte
Sel darah dan fungsinya
Jenis Titer (sel per
uL)
Fungsi Catatan
Sel Darah Merah


4,4-6,0 juta Mengangkut oksigen Umur sekitar 120
hari, tidak punya
nukleus,
mitokondria
SEL DARAH PUTIH
Neutrofil


1800-7300
50-70 %
Fagosit: menelan patogen
atau debris yg ada di
jaringan, melepas esim
sitotoksik

Bertahan beberapa
hari
Eosinofil


0-700
2-4 %
Fagosit: menelan material
yg terlabel antibodi,
melepas ensim sitotoksik,
mengurangi inflamasi
Bertahan beberapa
hari
Basofil

0-150
<1%
Masuk ke sel yg rusak dan
melepas histamin shg
menyebabkan inflamasi
Mamcu inflamasi
bersama mast cells
Sel darah dan fungsinya
Jenis Sel Titer (sel per uL) Fungsi Catatan
Monocytes


200-950
2-8 %
Masuk ke jaringan
berdiferensiasi menjadi
sel macrophage; :
menelan patogen atau
debris yg ada di jaringan,
Bertahan beberapa
bulan
Lymphocytes


1500-4000
20-30%
Bearada pd sistem
Limfatik, sebagai sel imun,
mengenali patogen
spesifik

Bertahan beberapa
tahun
Platelet


150.000-500.000 Hemostasis; berperan
dalam penjendalan darah
Dihasilkan dari
diferensiasi sel
megakariotik;
bertahan beberapa
bulan
Generation of an immune response
Vaccine
Pathogen
Antigen
CD4
T helper cell
B
cell
CD8
Activated cytotoxic T cell
CD8
Cytotoxic T cell
TCR
Antigen
presenting
cell
CD4
Activated
Th2 cell
CD4
Activated
Th1 cell
Memory
T cell
Infected
target cell
Infected
cell death
Memory
B cell
Antibody
production
Pathogen
neutralization
IMMUNE
MEMORY
Goals

To identify the cell type of the neo-plastic
process.
Outline the cell lineage and maturation
Used in conjunction as an aide in the
classification of the leukaemia or lymphoma.
Monitor patients following therapy

Sample processing
Whole blood lysis is generally the recommended procedure
Remove contaminating red cells, while leaving white cells
intact
Assumes all leucocyte subsets are equally tolerant to the lysis
method
Incubation with monoclonal antibodies can be done before or
after lysis of red blood cells
Density gradient separation eg, ficol. Enrichment for MNC
Tissues: mechanical dissociation is preferred to enzyme digestion
Viability can be performed using trypan blue or PI.
Monoclonal antibody panels
Multiparameter immunofluorescence is
preferred
Panels should be designed to resolve normal as
well as malignant cells (normal cells act as
internal reference standards)
Brighter fluorochromes (eg, PE) used in
expected cases of low antigen expression

Problems with mouse monoclonal antibodies
Mouse monoclonal antibodies are recognised by the
human body as non-self.

Strong immune response is mounted and this is a
problem if prolonged treatment is needed.

Antibody is also rapidly removed from circulation; they
have a short half-life in the circulation.
c19orf10 immunofluorescence staining of fibroblast-like synoviocytes (FLSs). FLSs
were labeled with anti-c19orf10 monoclonal antibody, 1B6, and visualized using red
fluorescent cyanine 3 (Cy3) goat anti-mouse immunoglobulin G (IgG) (heavy and light chain
reactive [H&L]) antibody. The cells were counterstained with green fluorescent Oregon Green
phalloidin to visualize the F-actin.
T-lymphocytes
Yellow-green: cytotoxic
CD8 T cellsRed-orange:
T-helper or CD4

Tubulin antibody
Fluorochromes

Fluorochrome options
Dependant on laser(s) available
Single laser systems 488 nm
Multiple laser systems 488 nm, 635 nm, violet
and UV


Fluorochromes
Small Organic Molecules:
FITC, Texas Red, Cy5, Cy5.5, Cy7
Fluorescent Proteins:
Phycoerythrin (PE), Allophycocyanin (APC), Peridinin
Chlorophyll Protein (PerCP)
Tandems:
PE-Texas Red (PE-TR), PE-Cy5, PE-Cy7, PerCP-Cy5.5,
APC-Cy7

Acute Leukaemia; Two Step
Strategy
Screening panel can consist of:
Non lineage; CD45, CD34,HLA-DR
T cell; CD2, CD3, CD7
B cell; CD10, CD19, CD20
Myeloid; CD13, CD33, CD117
Additional Abs to identify maturation, prognostic features or
aberrant phenotypes
Lymphoma / Chronic leukaemia
panels
CD20/KAPPA/LAMBDA
CD10/CD19/CD5/CD23
CD20/CD103/CD25/CD11c
CD38/CD138/CD45/CD19/CD56
CD3/CD4/CD8


Case 2

A 16-year old male was referred as
a new leukemia. Peripheral blood
had a high WBC with 53%
promyelocytes. Sample was sent
for flow cytometry to role out M3
The CD45/SSC display looks different than your typical bone marrow and peripheral blood patterns. You
can see that there are two clusters of cells one is the normal lymphocytes (red) and the other (green) are the
abnormal cells. For the observer the later population may look like neutrophils, but if you look closely you
will see that they are not. Usually the normal PMNs in peripheral blood start from approximately the
channel-600 on the SSC and spread higher (see arrow). In this particular case the distribution of the
abnormal cells start from the area where blasts tend to be present and gradually move higher overlapping
with the area where PMNs tend to be located. So definitely we are not dealing with PMNs but with cells
that are large and very granular.
This display is self explanatory in term of the CD3/CD4/CD8 staining on the normal lymphocytes (red). I would
like you now to pay attention to pattern of staining of the abnormal cells (green) with these monoclonal antibodies.
You can see that there is a considerable increase in the autofluoresence of the blasts (see brackets). This increase in
autofluoresence is characteristic of APL due to the high content of protein in the granules. Care should be taken
when placing the markers so there will be no false results
Here we are looking at more characteristics phenotype of APL. The blasts are homogenous positive for CD33,
heterogeneous positive for CD13, negative for CD34, HLA-DR and CD7. It is the lack of HLA-DR and CD34 that
kindly differentiate APL from other types of AML.
Again we are looking at additional markers , and we find that the blasts are clearly positive for CD9, CD64 and
slightly partially for CD15 and negative for CD117, CD11b and CD2.
Antigen profile: Positive for CD45, CD33, CD13,
CD9 and cMPO. Negative for HLA-DR, CD117 and
CD34.
Diagnosis: Acute Myeloid Leukemia, FAB(M3)
Case 3


Seven (7) year old, male patient, referred with
Clinical history of Mediastinal Mass.
Peripheral Blood Examination showed high
Leukocyte Count with 26% Blasts.

Looking at this case display you can tell that there is an abnormal population (green) that is v.bright CD45
brighter than normal lymphocytes and has a high side scatter. There is almost no normal lymphocyte in
this bone marrow specimen.
This is a very informative slide and probably gave you the diagnosis already. Looking at the CD3/CD4/CD8
patterns we can see that the majority of the gated cells are dual positive for CD4/CD8 and negative for surface CD3.
This pattern is very uncommon in bone marrow and indicate the immature T-cell nature of the blasts. Among the
abnormal cells you can recognize the normal lymphocytes pattern (see red arrow).
Looking at this slide we learn more about the nature of these cells. We find that they are negative for
CD10,CD19,CD34,CD33,CD13 and positive for CD7. The later antibody CD7 confirm the T cell lineage of the
abnormal cells.
The abnormal cells are positive for CD2 and CD11b and negative for HLA-DR. Usually in most cases of the T-cells
they tend to be negative for HLA-DR. The positivity of CD11b is also atypical of this kind of malignancy.
Again the abnormal cells continue to confirm its T-cell lineage; they are positive for CD56, CD5, partially CD1a
and negative for CD117 and CD64. Usually the expression of CD56 in leukemias is associated with worse
prognosis.
The cytoplasmic staining of TdT and CD3 confirm the immature nature of the blasts.
Antigen profile: Positive for CD4, CD8, CD7, CD2,
CD5, CD56, partially CD1a, cCD3 and negative for
HLA-DR.

Molecular: Positive for TCR gamma gene
rearrangement by PCR
Diagnosis: Acute T-cell Leukemia
Terimakasih