Diagnosis Laboratorium Anemia dan Permasalahannya

Agus Susanto Kosasih

Laboratorium Patologi Klinik

RS Kanker Dharmais1

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Definition of anaemia Anaemia is defined as a reduction in the

haemoglobin concentration of the blood This results in a decreased oxygen

carrying capacity

PATHOLOGY, SYMPTOMS, AND SIGNS OF ANEMIA

Normal values for peripheral blood

Female Male Erythrocytes (per µl) 4.8±0.6x106 5.4±0.8x106

Hemoglobin (g/dl) 14 ± 2 16 ±2Hematocrit (%) 42 ±5 47 ±5Reticulocytes (%) 1 1___________________________________________Mean corpuscular volume (MCV; µm3) 82-92Mean corpuscular hemoglobin (MCH; pg) 27-32Mean corpuscular hemoglobin concentration (MCHC; %) 32-

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Etiologic classification of anemias (1)

I. Impaired red cell productionA. Disturbance of proliferation and differentiation of stem cells ( aplastic anemia, pure red cell aplasia)B. Disurbance of proliferation and maturation of

erythrocytes:1.Defective DNA synthesis (megaloblastic

anemias)2.Defective Hb synthesis:

a/. Deficient heme synthesis (iron deficiency)

b/. Deficient globin synthesis (thalassemia)

3. Unknown or multiple mechanisms (anemia of chronic disease, anemia of marrow replacement)

Etiologic classification of anemias (2)

II. Increased rate of destruction (hemolytic anemias)A. Intrinsic abnormalities Hereditary

1. Red cell membrane defects (hereditary spherocytosis, hereditary eliptocytosis)

2. Red cell enzyme deficiencies a/. Glycolytic enzymes: pyruvate kinase,

hexokinase b/. Enzymes of hexose monophosphate shunt: G-6PD, glutathione synthetase3. Disorders of globin synthesis a/. Deficient globin synthesis (thalassemia) b/. Structurally abnormal globin synthesis (sickle cell anemia, unstable hemoglobins)

Acquired1. Membrane defect: paroxysmal nocturnal

hemoglobinuria

Etiologic classification of anemias (3)

B. Extrinsic abnormalities1. Antibody mediated a/. Autoantibodies (idiopathic, drug-

associated, SLE, malignancies) b/. Isohemagglutinins (transfusion reactions,

erythroblastosis fetalis)

2. Mechanical trauma of RBC a/. Microangiopathic hemolytic anemias

(thrombotic thrombocytopenic purpura, DIC) b/. Cardiac traumatic hemolytic anemia3. Chemicals and microorganisms4. Sequestration in mononuclear phagocytic

system - hypersplenism

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Classification of Anemia

Classification of Anemia

Morphologic classification of anemias

Type MCV MCHC Common cause________________________________________________________Macrocytic anemia increased normal Vitamin B12 deficiency

Folic acid deficiency Microcytic anemia

- hypochromic decreased decreased Iron deficiency Thalassemia

- normochromic decreased normal Spherocytosisor normal

Normocytic anemia normal normal Aplastic anemia- normochromic Chronic renal failure

Some hemolytic anemia

Classification of Anaemia: Microcytic Hypochromic MCV <80fl MCH <27pg Iron deficiency

Microcytic anaemiaFerritin >25ug/L Thalassaemia Sideroblastic anaemia (some cases) Anaemia of chronic disease (some

cases) Lead poisoning

Classification of Anaemia: Normocytic Normochromic MCV 80-100fl MCH >26pg Often incidental finding in systemic disorders May be first manifestation of a systemic

disorder Many haemolytic anaemias Anaemia of chronic disease (some cases) After acute blood loss Bone marrow failure, e.g. Post-chemotherapy,

infiltration by carcinoma etc

Classification of Anaemia: Macrocytic MCV >100fl Megaloblastic: vitamin B12 or folate

deficiency Non-megaloblastic: alcohol, liver

disease, myelodysplasia, aplastic anaemia

Iron deficiency anaemiaAssess for Dietary Iron deficiency Malabsorption- coeliac Chronic blood loss Gastrointestinal Menorrhagia

Iron deficiency anaemia

Thalassemia-: -globin chain synthesis or ()

Thalassemia-: -globin chain synthesis or ()

THALASSEMIAS: Defects of Hb synthesis

heme

heme

Hemoglobin molecule

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Globin chain synthesis: developmental phases

Gen & sejenis Kromosom 16

Gen & sejenis Kromosom 11

2 2 1

G A

Hemoglobins

MasaPerkembangan

Hb Gower 1 (2 2) Hb Gower 2 (22) Hb Portland (22)

Embrio

HbF(22)

Janin

HbA2 HbA(2 2) (2 2)

Dewasa

GEN PENYANDI SINTESIS RANTAI GLOBIN DAN

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embryonic fetal adult

Hb Gower 1 (22)Hb Gower 2 (22)Hb Portland 1 (22)Hb Portland 2 (22)

Human Hemoglobins

Hb F (22) Hb A (22): 95-97% Hb A2 (22): 4%Hb F (22): <1%

Hb switches: 2 major•2 months gestation: embryo to fetus

•birth: fetal to adult

Normal Hb F levels

newborn 70-90%

6 months old 2-13%

1 year 1-5%

2 years <2%

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information on age of patient is important:interpretation of Hb levelspremature: delay in Hb switch (fetal to adult)

How is Thalassemia Classified?

Thalassemia

thalasemia hemoglobinopathies

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Genotypes to phenotypes of thalassemia

Molecular defects (mutations)

-globin gene(-thalassemia)

-globin gene(-thalassemia)

-globin chains /- -globin chains /-

Excess of-globin chains (fetus)-globin chains (adult)

Excess of -globin chains

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Pathophysiology of thalassemia syndromes

globin chain imbalance (excess or chains)

RBC damage

•ineffective erythropoiesis•peripheral hemolysis

anemia

excess chains precipitate in RBC

Clinical aspects: thalassemia syndromes

syndrome clinical age of presentation

need forBT

trait(normal

Hb)

asymptomatic any age none

thal-intermedia

(7-10 gm/dl)

moderatelysevere

after age 2 or later

none, occsome

thal-major

(<7 gm/dl)

(0/0) severe

(0/0)) fatal

1-2

in-utero / birth

regular

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Beta-thal syndromes

trait : + or 0

thalassemia-intermedia

+ / +

+ / 0

thalassemia-major: 0 / 0

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Identification of classical beta-thalassemia trait in an adult

hallmark for classical beta-thalassaemia trait:

raised Hb A2

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information on age of patient is important: full-term or premature

Hb subtypes in normal newborn

F(22): 70-90%

A(22): 10-30%

*A2(22): 0% (not measurable by HPLC)

Hb subtypes in the newborn

• Asimptomatik• Anemia ringan s/d

berat• Hb 8-15 g/dL (rata-rata

12)• MCV < 80 fl• HbA2 > 3.5%• HbF ≥ 1%

HETEROZIGOT HOMOZIGOT βo

THALASSEMIA-β

• Anemia berat sejak usia < 1 tahun

• Perlu transfusi rutin• Hati dan limpa membesar• MCV < 70 fl• Jenis Hb hanya HbA2 and

HbF• HbF > 90%

HOMOZIGOT β+ dan β+

• Anemia ringan sampai berat• Hati dan limpa normal atau membesar• Masih mempunyai HbA• HbF dari 10 s/d > 90%

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functional globin genes

trait mild (+)

severe (0)-thal 2 (-/)-thal 1(--/)

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Hb H diseasemoderate

more severe0 / + (--/-)0 / T (--/ T)

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Hb Bart’s hydrops fetalis

0 / 0 (--/--) 0

Clinical severity of -thal syndrome: related to number of functional -globin genes

0 or -thal 1 trait

H inclusion test positive: 1 RBC cell per 5000 or 10,000 RBC

may be negativecannot be done in newborn

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`golf ball inclusions’

Presumptive identification of -thalassemia in newborns

anemia Hb

MCV<105 fl

MCH<27pg

Hb Bart’s >5%

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Levels of Hb Bart’s (4) in -thalassemia in newborns

syndrome genotype Hb Bart’s (%)

normal / 0

trait -/ 0.5-3

--/ 4-8

-/- 9-13

Hb H disease --/- 19-27%

Hb Barts hydrops fetalis

--/-- 97-100

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Pootrakul et al 1975

BioRad Variant HPLC: -thal short program

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Normal/

FENOTIP THALASSEMIA - (CARRIER)

-/-

- /

N/anemia ringan< 25 pgnormal or ↓↓ atau (-)

Normal25-27pgnormal or ↓↓ atau (-)

NormalMCH : 27-31 pgHbA2 : 2.5-3.5%HbF : <1%

• Kematian janin (28-32 minggu)• Hydrops fetalis

• HbH disease• mild to severe anemia

--/--

4=HbBart

- -/-

4=HbH

MCH : < 26-32 pgHbA2 : 2.5-3.5%HbF : <1%

FENOTIP THALASSEMIA - (PENDERITA)

Normal/

Adult Fetus

HbBart

HbH

AFFECTED

Normal fetus

HbA

HbF

Hb variant analysisHbBart’ hydrops fetalis

32 weeks gestational age

Diagnosis Thalassemia

Anamnesis/pedigree

Pemeriksaan Fisik

Pemeriksaan laboratorium

Anamnesis/Pedigree

Pucat lama (kronik)

Riwayat keluarga dengan penyakit serupa

Ras

Anorexia

Gangguan Pertumbuhan

Probability Thalassemia

Orang Tua Thalassemia Trait Orang Tua

Thalassemia Trait

Normal

ThalassemiaMayor

ThalassemiaTrait

Orang Tua Thalassemia

Trait

Orang Tua Thalassemia

Mayor

ThalassemiaMayor

ThalassemiaMayor

ThalassemiaTrait

ThalassemiaTrait

Probability Thalassemia

Probability Thalassemia

Orang Tua Thalassemia

Trait

Orang Tua Normal

ThalassemiaTraitNormal

Pemeriksaan fisik► Sangat bervariasi (ringanberat)► Pucat ► Gizi kurang► Pertumbuhan kurang/lambat► Facies mongoloid/ Cooley► Hepar dan limpa membesar► Fraktura patologis

Thalassemia Mayor

Hairy SkullCooley’s face

Pemeriksaan laboratorium (1)Darah perifer:

- Hb rendah / normal- MCV <80fl dan MCH <27pg,- Retikulosit agak meningkat- Jumlah leukosit normal- Hitung jenis normal

Pemeriksaan Hematologi MCV : ukuran eritrosit dibandingkan dengan inti

limfosit kecil (Normal) MCH : warna eritrosit atau content dari

hemoglobin RDW : perbedaan ukuran eritrosit.

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semakin besar variasi eritrosit semakin besar RDW

Pemeriksaan Hematologi RDW-SD: mengukur lebar kurva. Bila kurva makin lebar

makan RDW SD semakin lebar Nilai Normal :

perempuan: 36.4 – 46.3fL Laki-laki: 35.1 – 43.9fL

RDW-CV: dihitung dengan formula:

RDW-CV = 1SD/MCV x 100 % 1SD merupakan variasi eritrosit sekitar mean ukuran eritrosit Nilai Normal :

perempuan: 11.7 – 14.4 % Laki-laki: 11.6 – 14.4 %

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Iron Deficiency Anemia

Anemia

Messwerte

RBCHGBHCTMCVMCHMCHCRDW-CV

4.48 x1012/L8.8g/dl29.3%65.4fl

19.6pg30.0g/dl

18.2%

–––

+

MesswertePLTPDWMPVP-LCR

235 x109/L11.7fl

9.4fl21.7%

(x 1000)

Suspected Thalassemia

Messwerte

RBCHGBHCTMCVMCHMCHCRDW-CV

5.97 x1012/L12.7g/dl

41.1%68.8fl

21.3pg30.9g/dl

14.7%

+

–––

PLTPDWMPVP-LCR

391 x109/L12.0fl10.3fl

27.3%

(x 1000)

<RBC-Histogram>Measurement Data

<PLT-Histogram>

Measurement Data

<RBC-Histogram> Measurement Data

<PLT-Histogram> Measurement Data

Case 1 Case 2

Anisocytosis

RBC-Histogramm Messwerte

RBCHGBHCTMCVMCHMCHCRDW

4.15 x1012/L

14.0g/dl40.8%98.3fl

33.7pg34.3g/dl

22.7%

+

Messwerte

PLTPDWMPVP-LCR

328 x109/L12.4fl10.2fl

26.5%

(x 1000)

RBC-Histogramm Messwerte

RBCHGBHCTMCVMCHMCHCRDW

2.95 x1012/L9.9g/dl28.7%97.3fl

33.6pg34.5g/dl

26.4%

+

PLTPDWMPVP-LCR

PL*DWPLPL

98 x109/L---.-fl---.-fl

-.---%

(x 1000)

<PLT-Histogram>

<PLT-Histogram> Measurement Data

Measurement Data

Measurement DataMeasurement Data<RBC-Histogram> <RBC-Histogram>

Pemeriksaan laboratorium (2)Sedian hapus darah tepi :

Khas, gambaran hemolitik kronik - Mikrositik Hipokrom- Anisositosis- Poikilositosis- Sel target- Fragmentosit- Eritrosit berinti (berat)

Gambaran Sediaan Hapus Thalassemia

Mikrositik Hipokrom Anisositosis Poikilositosis

Sel Target Fragmentosit Eritrosit berinti

Mikrositik Hipokrom Bukan Thalassemia

Anemia def besi Elliptocytosis

SpheroscytosisStomatocytosis

Pemeriksaan laboratorium (3)

Elektroforesis Hb

Analisa Hb (HPLC)

DCIP

Analisis DNA

Elektroforesis Hb

+

-

+

-

HBart’sPortland

AF

S, D, G,LeporeA2, E, C

Constant springOrigin

C

OriginHS

A, A2, D, E, G, OLepore

F, Bart’s, Portland

pH alkali (8,5) pH asam (6.0)

Diagram migrasi fraksi Hb hasil elektroforesa pada pH alkali & asam

(Pemeriksaan Laboratorium Klinik Thalassemia & Hemoglobin Varian; Riady Wirawan, Dr, SpPK FKUI, 1997)

Analisis Hb HPLCDigunakan alat HPLC Variant

Tes-tes Hb yang dapat dilakukan di alat Variant : Beta Thal Short Sickle Cell Short Alpha Thal Short Globin Chains HbA1c

A

FS

A2

C

300 points with 15 zones Curve

Normal Hb Type

Beta thal heterozygote

Hb E heterozygoye

E

A2

Hb Constant Spring homozygote

Hb Bart’s

(Baby’s blood)

Hb F

Hb A

Hb Bart’s

Hb Bart

Alpha thalassemia with Hb H

Hb A

Hb A2Hb H

Alk.

N

A

Anh. Car.

H

A2

Hb H

Badan Inklusi HbHHbH adalah hemoglobin yang tidak stabil akan mengalami denaturasi oksidatif dan presipitasi jika eritrosit terpapar dengan zat warna new methylene blue atau brilliant cresyl blue dan membentuk gambaran seperti “bola golf”

Badan inklusi HbH dijumpai pada eritrosit penderita :

- HbH - Thalassemia a-1 trait, (1/100–1/10.000 eritrosit),

HbH disease HbH inclusion bodies

Limitations in new born

adult with thalassemia normal newborn

Blood counts Hb: N or MCV<80 fl, MCH<27pg

High Hb (14-20 g/dl)MCV fl>105 fl, MCH>27pg

Blood films HypochromiaThalassemia picture

Effects of relative hyposplenism

Hb subtypes Hb A2:4%-thal trait

Hb F(95-100%):-thal major

Hb F: 90-100%Hb A: 5-20%Hb A2: not measurable

H-inclusion test -thalassemia: + cannot be used

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Thalassemia merupakan penyakit yang dapat menurunkan kualitas hidup

Memerlukan biaya yang cukup besar untuk terapi thalassemia (terutama pasien thalassemia yang membutuhkan transfusi sepanjang hidupnya

Insiden thalassemia dapat diminimalkan melalui pemeriksaan skrining yang optimal (Total Solution)

Meningkatkan kesadaran masyarakat mengenai penyakit thalassemia dan akibatnya

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terima kasih