Kimia Klinik Dan Bioanalisis-1

7/22/2019 Kimia Klinik Dan Bioanalisis-1

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KIMIA KLINIK DAN

BIOANALISIS

Edy Meiyanto

Fakultas Farmasi UGM

http://edymei.blog.ugm.ac.id

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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 HATI2


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II. BIOANALISIS

1. TEKNIK DASAR KULTUR SEL

2. UJI GENOTOKSIK-13. UJI GENOTOKSIK-2

4. UJI MENGGUNAKAN SEL PRIMER

5. UJI MENGGUNAKAN SEL LINES6. UJI MENGGUNAKAN MIKROBA

7. OVARIEKTOMI

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Tema Praktikum dan Dosen

PembimbingNo 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.


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PENDAHULUANTUJUAN 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


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KONSEP DIAGNOSTIK

SISTEMBIOLOGI

ORGAN TUBUHSEL

GANGGUANMACROMOLE

CULS

DNA/RNAPROTEIN

SISTEM

DETEKSI

TARGETED-

SPECIFIC

TEKNOLOGI

MOLECULAR

DIAGNOSTICS

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Keberhasilan dalam kedokteran modernbergantung pada kecermatan dalam diagnosisspesifik molekular pada:

Viruses

Bacteria

Fungi

Parasites

Proteins

In water, plants, soil and humans.


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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?

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2

3

4

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Moleculardiagnostics is

>$3 billionmarket WW andgrowing at >20%

annually


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CONTOH APLIKASI DIAGNOSIS TERKINI

PrenatalDiagnostics

Oncology

InfectiousDisease

TransplantationMedicine

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 organrejection

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 madein all of these areas

Each of these areas arecommercially attractive

In some cases, theMassARRAY platform isuniquely qualified forspecific tests

More tests will be addedto the platform as thesetests are rolled out


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Proporsi pasien yang mendapat diagnosis

sehingga memberikan informasi

yangbenar sesuai dengan tujuandiagnosis.

Untuk menguji ketepatan alat uji

diagnostik,digunakan alat uji pembandingyang standar yang dinamakan Gold

Standar

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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 positifbenar dibagi dengan jumlah hasil semua uji

positif.

5. Nilai prediksi negatif:jumlah hasil uji negatif

dibagi dengan jumlah hasil semua uji negatif 14


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Contoh kasus: skrining bayi lahir yang membawa penyakit

genetik di Amerika

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PPV= 250/2249 = 11 %

NPV= 997750/997751 = 100 %

Maknanya??? Kalau + perlu revalidasi

Hasil uji Status sakit total

ada tidak

Hasil uji positif

Hasil uji negatifTotal

TP:250

FN:1TP +FN:250

FP:1.999

TN:997.750FP+TN:999.75

TP+FP:2.249

FN+TN:999.751Semua bayi yg

diuji:1.000.000


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1. DARAH UTUH

2. PLASMA3. SERUM

4. URIN

5. CAIRAN SEREBROSPINAL6. BIOPSI

7. JARINGAN

8. DLL17


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KOLEKSI SAMPLE

IDENTIFIKASI PASIEN

PENGGUNAAN TABUNG/TUBE/CONTAINER

PEMBERITAHUAN KEPADA PASIEN TENTANGTATACARA PENGAMBILAN SAMPEL

PASIEN DALAM POSISI YANG NYAMAN

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KESALAHAN UMUM DALAM KOLEKSI SAMPEL

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PELABELAN SALAH ATAU TIDAK LENGKAP

JUMLAH TIDAK MENCUKUPI

KELIRU DALAM MEMILIH TABUNG

INSTRUKSI PADA PASIEN TIDAK TEPAT ATAU

KURANG

LENGKAP

TUTUP TIDAK RAPAT SEHINGGA MENYEBABKANTUMPAH ATAU

TERKONTAMINASI


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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

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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

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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. Alcoholused 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

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LIPEMIA

Normal serum or plasma is a clear and lightyellow to straw in color. Turbid serum or

plasma appears cloudy or milky.

Serum or plasma may be cloudy due tobacterial 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

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FLOWCYTOMETRY

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What Is Flow Cytometry?

Flow ~ cells in motion

Cyto ~ cell Metry ~ measure

Measuring properties of cells while in a

fluid stream


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Cytometry vs Flow Cytometry


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Cytometry vs. Flow CytometryCytometry

Localization of antigenis possible

Poor enumeration of

cell subtypes

Limiting number of

simultaneous

measurements

Flow Cytometry.

Cannot tell you whereantigen is.

Can analyze many

cells in a short time

frame.

Can look at numerous

parameters at once.


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What is Flow Cytometry?

Flow refers to a fluid stream

Cyto refers to a cell

metry refers to measurement.


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D fi iti


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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


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What Can a Flow Cytometer

Tell Us About a Cell? Its relative size (Forward Scatter

FSC)

Its relative granularity or internalcomplexity (Side ScatterSSC)

Its relative fluorescence intensity(FL1, FL2, FL3, FL4, and FL5)


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Scatter profile of lysed whole blood


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Scatter profile of lysed whole blood

SideScatte

r

Forward Light Scatter

0 200 400 600 800 1000

0

20

0

400

600

800

1000

Lymphocytes

Monocytes

Granulocytes

largest and mostgranular population

smallest and least

granular population

Cytometer fluidics create laminar flow


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Cytometer fluidics create laminar flow

Sample stream

Sheath stream

Cell

Laser beam

Flow Cell


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Absorption and Emission Spectraof a Fluorochrome

Wavelength (nm)

400 450 500 550 600 650 700

1000

800

600

400

200

0

FITC


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A Cytometer Needsa 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


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OpticsFACSCaliburFL1

Red Diode Laser

~635 nm

SSC

FL3

FL4

670LP

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488/10

90/10 Beam Splitter

DM 560SP

Fluorescence

Collection Lens

DM 640LP

Half Mirror

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.488 nm

Blue Laser FSC Diode

Focusing

Lens

FL2

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Beam Combiner

FlowCell

FlowCell


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Two-Color Direct Staining

Analyze

WashIncubate


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Two-Color Cell Analysis

CD19PE

CD3 FITC

100100

101

102

103

104

101 102 103 104


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Three-Color Direct Staining

Analyze

WashIncubate


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Three-Color Cell Analysis

CD3 APC CD3 APC

CD4F

ITC

CD8PE

CD4 FITC

CD8PE

100 101 102 103 104100

101

10

2

103

104

100

101

10

2

103

104

100

101

10

2

103

104

100 101 102 103 104 100 101 102 103 104


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DNA Histogram

0 200 400 600 800 1000

0

50

100

150

200

250

Counts

FL2-Area

G0/G1

S G2/M

Fluorescence data display


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Fluorescence data display

PE

FITCFITC Fluorescent Intensity

NumberofEvents

Negative control histogram


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Cell sorting


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Cell sorting


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Fluorescence sensitivity


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Fluorescence sensitivity

Detection Efficiency (Q): number ofphotoelectrons 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, unboundfluorophore, stray light, etc.

Common fluorophores for Ab conjugation


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Common fluorophores for Ab conjugation

FLUOROCHROME Type of molecule Typical excitation laser Approximate emission

peakFluorescein

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



APC-Cy7 Protein tandem 633 nm 784 nm

Pacific Blue Small organic 405 nm 454 nm

AmCyan Protein 405 nm 487 nm


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Flow Cytometry Clinical


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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)


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Immunophenotyping of leukaemiccells


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The Composition of Whole Blood (4-6L in an adult)


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The percentageranges for whiteblood cells indicate

the normal variationseen in a count of100 white bloodcells in a healthyindividual.


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Sel darah dan fungsinya


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g y

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 dijaringan, 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


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g y

Jenis Sel Titer (sel per uL) Fungsi Catatan

Monocytes 200-9502-8 %

Masuk ke jaringanberdiferensiasi menjadi

sel macrophage; :

menelan patogen atau

debris yg ada di jaringan,

Bertahan beberapabulan

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 selmegakariotik;

bertahan beberapa

bulan


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Goals

To identify the cell type of the neo-plasticprocess.

Outline the cell lineage and maturation

Used in conjunction as an aide in theclassification of the leukaemia or lymphoma.

Monitor patients following therapy


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M n cl n l ntib d p n l


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Monoclonal antibody panels

Multiparameter immunofluorescence ispreferred

Panels should be designed to resolve normal as

well as malignant cells (normal cells act asinternal reference standards)

Brighter fluorochromes (eg, PE) used in

expected cases of low antigen expression


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Problems with mouse monoclonal antibodies

Mouse monoclonal antibodies are recognised by thehuman body as non-self.

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

Antibody is also rapidly removed from circulation; they

have a short half-life in the circulation.


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Fluorochromes


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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


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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


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panels

CD20/KAPPA/LAMBDA

CD10/CD19/CD5/CD23 CD20/CD103/CD25/CD11c

CD38/CD138/CD45/CD19/CD56

CD3/CD4/CD8

Case 2


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A 16-year old male was referred asa new leukemia. Peripheral blood

had a high WBC with 53%

promyelocytes. Sample was sentfor flow cytometry to role out M3


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Antigen prof i le: Positive for CD45, CD33, CD13,CD9 and cMPO. Negative for HLA-DR, CD117 and

CD34.

Diagnosis:Acute Myeloid Leukemia, FAB(M3)


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Looking at this case display you can tell that there is an abnormal population (green) that is v.bright CD45

brighter than normal lymphocytesand has a high side scatter. There is almost no normal lymphocyte in

this bone marrow specimen.


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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 CD7confirm the T cell lineage of the

abnormal cells.


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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.


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The cytoplasmic staining of TdT and CD3 confirm the immature nature of the blasts.


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Kimia Klinik Dan Bioanalisis-1

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