ANEMİYE LABORATUVAR VE MORFOLOJİK YAKLAŞIM

(1)

ANEMİYE

LABORATUVAR VE MORFOLOJİK

YAKLAŞIM

PROF. DR. MELTEM KURT YÜKSEL AÜTF İÇ HASTALIKLARI

HEMATOLOJİ BİLİM DALI 01.02.2018 DÖNEM III

(2)

ANEMİ NEDİR?

• Kansızlık

(3)

(4)

HEMATOPOEZ

(5)

(6)

Blood cells

Granulocytes

(7)

Periferik Yayma

(8)

Periferik Yayma

(9)

Periferik Yayma Hazırlanması

Kötü

İyi

(10)

(11)

İncelenecek alan

Örnek

uygulama noktası 6

2 1 3

4

7 1

5 1

(12)

Lenfosit Monosit

Bazofil Nötrofil Eozinofil

Atipik Bant

Segment

18

5 65 6

2 4

100 LÖKOSİT FORMÜLÜ

(13)

İstenen yayma kalitesi

Kalın Uygun

Kriyoglobulin Soğuk Aglutininler +

(14)

%

Blastlar 0-5

Promiyelositler 1-8

Miyelositler 5-18

Metamiyelositler 13-32

Nötrofiller 7-30

Eozinofiller 0-4

Basofiller 0-1

Monositler 0-5

Lenfositler 3-17

Plazma hücreleri 0-2

Megakaryositler 0-1

Makrofajlar 0-2

Proeritroblastlar 1-8

Normoblastlar 7-32

Miyelosit:Eritrosit seri oranı 2:1-4:1

Normal Miyelogram

(15)

(16)

RETİKÜLOSİT

• Kemik iliğinin durumunu gösteren hücre

• AZALMASI

• ARTMASI

(17)

(18)

(19)

H E M O LİT İK A N E M İ N E D E N LE R İ

(20)

(21)

(22)

(23)

(24)

(25)

KAN HÜCRELERININ

MORFOLOJISI

(26)

Normal Eritrositler

•

Normal red cells or erythrocytes show

only slight variation in

size and shape. The

blood film should be

examined in the area

where the red cells

are touching but not

often overlapping.

(27)

Red cells

• . In this area many red cells have an area of

central pallor which may be up to a third of the

diameter of the cell. This is consequent on the

shape of a normal red cell, which resembles a disc that is thinner in the centre.

(28)

Artefacts

• Fixation artefact is the term used for the

artefact that occurs when there is water in the methanol used for fixation of the blood film.This leads to

refractile rings in red cells and makes it quite impossible to assess red cell morphology.

(29)

Heat artefact

^• Inadvertent heating of a blood

sample, for example during

transport in a hot car, can lead to a heat artefact.

• Red cells bud off vesicles and

microspherocytes seen.

• White cells disintegrate and

• proteins coagulate, producing weakly basophilic particles,

which are similar in size to platelets.

(30)

Storage artefact

• Prolonged storage of blood before making the blood film, particularly storage at room temperature,leads to storage artefact.

• White cells become fragile and may form smear cells [deep red arrow].

• Neutrophil nuclei round up and form homogeneous round masses or a single

mass [blue arrow].These cells have a resemblance to

NRBC.

• Red cells undergo an echinocytic change or crenation.

(31)

PY değerlendirirken dikkat edilmesi gerekenler (Tüm hücreler)

• Boyut

• Çekirdek şekli

• Kromatin özellikleri

• Sitoplazma özelliği

• Sitoplazma içerisinde granül, inkluzyon olup olmadığı

• Renk

(32)

Eritrosit boyu

• Normal: biconcave disc of 7.5 mm diameter

• Has central pallor (1/3 of diameter) on smear

• No inclusions

(33)

(34)

Good Area to Evaluate Morphology

(35)

Anisocytosis: Variation in Size

Microcytes Macrocytes

(36)

Poikilocytosis: Abnormal Shape

Target Cells Hypochromic

(37)

Poikilocytosis

Sickle Cell Schistocyte

(38)

Poikilocytosis

Teardrop Cell Spherocyte

(39)

Poikilocytosis

Elliptocyte Stomatocyte

(40)

RBC Inclusions, etc.

Polychromasia Howell-Jolly Bodies

(41)

RBC Inclusions

Cabot Ring Basophilic Stippling

(42)

RBC Inclusions, etc.

Rouleaux Malaria

(43)

Too Thick Too Alkaline

Too Thin Too Acidic

(44)

Unacceptable Slides

(45)

Sıtma teşhisi

(46)

RECORDING RBC MORPHOLOGY

1. Scan area using ×100 (oil immersion).

2. Observe 10 fields.

3. Red cells are observed for size, shape, hemoglobin content, and the presence or absence of inclusions.

4. Abnormal morphology: Red cell morphology is assessed

according to See the following sample grading system. Note that red cell morphology must be scanned in a good counting area.

Two questions should be asked

5.Is the morphology seen in every field?

6.Is the morphology pathologic and not artificially induced?

(47)

Abnormal Erythrocyte Morphology

Is found in pathological states that may be abnormalities in

I. Red cell distribution.

II. Size (anisocytosis).

III. Hemoglobin content – Color Variation . IV. Shape (poikilocytosis).

V. The presence of inclusion bodies in erythrocyte.

(48)

I. Erythrocyte Distribution Abnormalities

Rouleaux formation

Stacking of RBCs due to increased plasma proteins coating RBCs (resembling a stack of coins)

Found in

• Hyperfibrinogenaemia

• Hyperglobulinaemia

(49)

Agglutination

Antibody-mediated Irregular clumping , temperature dependent

Found in

• Cold agglutinins

• Warm autoimmune hemolysis

(50)

(51)

Rouloux Formation Agglutination

(52)

II. Variation In Erythrocyte Size (Anisocytosis)

Anisocytosis

Variations in size (Microcyte and Macrocyte)

Normocytic RBC’s

Normal size of RBC (8 μm) with a range of 7 to 9 μm.

The nucleus of a small

lymphocyte (± 8 µm) is a useful

guide to the size of a red blood

cell).

(53)

Microcytic

RBC cell smaller than the normal RBC ( <7 μm), and is associated with a decrease in hemoglobin synthesis

Found in

• Iron deficiency anemia.

• Thalassaemia.

• Sideroblastic anemia.

• Lead poisoning.

• Anemia of chronic disease.

(54)

Macrocyte

RBC larger than the normal (<9 μm) and is the result of a defect in nuclear maturation or stimulated erythropoiesis.

May be round or oval in shape, the diagnostic significance being different.

Found in

• Folate and B12 deficiencies (oval)

• Ethanol (round)

• Liver disease (round)

• Reticulocytosis (round)

(55)

Example : Film Study

Most erythrocytes presented in the picture are microcytes (compare with the small

lymphocyte). The degree of hemoglobinization is

sufficient. Normal platelets and single ovalocytes are present.

1. Microcyte

2. Normocyte

(56)

III. Variation In Erythrocyte Color

• A normal erythrocyte has a pinkish-red color with a slightly lighter-colored center (central pallor) when stained with a blood stain, such as Wright.

• The color of the erythrocyte is representative of hemoglobin concentration in the cell.

• Under normal conditions, when the color, central pallor, and hemoglobin are proportional, the erythrocyte is

referred to as Normochromic

^.

(57)

Hypochromia

• Increased central pallor and decreased

hemoglobin concentration, the central pallor

occupies more than the normal third of the red cell diameter.

Found in

• Iron deficiency

• Thalassaemia

• any of the conditions leading

to Microcytosis

(58)

Polychromasia

• Red cells stain shades of blue-gray as a consequence of uptake of both eosin (by hemoglobin) and basic dyes (by residual ribosomal RNA). Often slightly larger than normal red cells and round in shape - round macrocytosis.

Found in

Any situation with reticulocytosis – for example bleeding,

hemolysis or response to

heamatinic factor replacement.

(59)

IV. Shape Abnormalities of Erythrocytes

• Poikilocytosis is the general term for mature

erythrocytes that have a shape other than the round, biconcave disk.

• Poikilocytes can be seen in many shapes.(e.g.

Acanthocyte, Spherocytosis,…)

(60)

Shape Abnormalities of Erythrocytes

Terminology Description Condition

Target Cells Central Hemoglobin; target

shaped Liver Disease; Thalassaemia,

Abnormal Hb; Iron Deficiency Echinocyte Short specula's, equally-

spaced Uremia, Hypokalemia, Artifact

Acanthocyte Speculated, Irregular Liver disease (Alcohol), Post- spleenoctomy.

Spherocyte Spherical, no central pallor HS, immune Hemolytic anemia Shistocyte Fragmented RBC, Helmet

cells MAHA, burns

Ovalocyte Oval / Elliptical shaped Hereditary elliptocytosis, Megaloblastic anemia.

Sickle Cell Bipolar speculated shape “

banana” shaped Hb S-containing hemoglobinopathy

Teardrop cell Single elongated extremity Myelophthistic changes Bite cells Irregular gap in membrane G6PD deficiency

(61)

Red cell with a “target” or bull’s-eye appearance. The cell appears with a central bull’s eye that is surrounded by a clear ring and then an outer red ring.

Found in

• Obstructive liver disease

• Severe iron deficiency

• Thalassaemia

• Post splenectomy

• Lipid disorders

• Haemoglobinopathies (S and C)

Target cell

(62)

Red cells are more spherical. Lack the central area of pallor on a stained blood film.

Found in

• Hereditary spherocytosis

• Immune haemolytic anemia

• Zieve's syndrome

• Microangiopathic haemolytic

Spherocytosis

(63)

Red cells with a central linear slit or stoma. Seen as mouth- shaped form in peripheral smear.

Found in

• Alcohol excess

• Alcoholic liver disease

• Hereditary stomatocytosis

Stomatocyte

(64)

Ovalocyte

An elongated oval cell. They are a result of a membrane defect.

Found in

• Thalassaemia major.

• Hereditary ovalocytosis.

• Sickle cell anemia

(65)

Elliptocyte

The red cells are oval or elliptical in shape. Long axis is twice the short axis

.

Found in

• Hereditary elliptocytosis

• Megaloblastic anemia

• Iron deficiency

• Thalassaemia

• Myelofibrosis

(66)

Red cell fragments that are

irregular in shape and size. They are usually half the size of the normal RBC; therefore, they have a deeper red color.

Found in

• DIC

• Micro angiopathic haemolytic anemia

• Mechanical haemolytic anemia

Schistocyte

(67)

Have accentric hallow area. Resemble a women's handbag and may be called pocket-book cell.

Found in

Microangiopathic hemolytic anemia

Blister cell: pre keratocyte

(68)

Part of the cell fuses back leaving two or three horn-like projections. The keratocyte is a fragile cell and remains in circulation for only a few hours.

Found in

• Uraemia

• Severe burns

• EDTA artifact

• Liver disease

Also called helmet cells

Keratocytes (horn cell)

(69)

Degmacyte "bite cell"

• An abnormally shaped red blood cell with one or more semicircular portions removed from the cell margin.

• These "bites" result from the removal of denatured hemoglobin by macrophages in the spleen.

Found In

• G-6-PD deficiency, in which uncontrolled oxidative stress causes hemoglobin to

denature and form Heinz

bodies, is a common disorder

that leads to the formation of

bite cells.

(70)

Sickle shaped red cells.

Found in

Hb-S disease and trait

Sickle Cells

(71)

Red cell with 30 or more, short blunt projections which are regularly distributed on their surface

Found in

Usually artifactual— the result of slow drying under humid conditions.

Sometimes are non - artifactual, indicating uremia or pyruvate kinase deficiency.

• Hemolytic anemia

• Uremia.

• Megaloblastic anemia

Cells retain the central pallor.

Echinocyte “Burr” (crenation ) cell:

(72)

Echinocytes (Burr Cells)

(73)

Red blood cells with irregularly spaced

projections, these projections very in width but usually contain a rounded end

Found in

• Liver disease

• Post splenectomy

• Anorexia nervosa and starvation

Acanthocytosis (Spur Cells):

(74)

Acanthocytes (Spur Cells)

(75)

Resembles a tear and usually smaller than the normal RBC.

Found in

• Bone marrow fibrosis

• Megaloblastic anemia

• Iron deficiency

• Thalassaemia

Dacryocytes (Teardrop)

(76)

Envelope Form Cell

Found in

• Thalassaemia

• Sickle cell anemia

(77)

V. Erythrocyte Inclusions with Wright’s Stain

Inclusion Composition Appearance Condition

Basophilic stippling Precipitated

ribosomes Evenly dispersed fine or coarse granules

- Lead poisoning - Thalassaemia ,

other anemia.

Howell-Jolly bodies DNA in origin Nuclear Fragment

Dense, round blue granule

Post – Splenectomy

Pappenheimer

bodies Iron-containing

granules Small blue granules

in clusters Anemia's Heinz bodies Denatured

Hemoglobin Round blue

precipitates G6PD Cabot Rings Remnants of

Nuclear membrane Reddish-blue thread

like rings Severe anemia, Lead poisoning.

Organism Small blue

inclusion Malaria

Babesiosis

(78)

Small round cytoplasmic red cell inclusion with same staining characteristics as nuclei

Found in

• Post splenectomy

• Megaloblastic anemia

Howell-Jolly Bodies

(79)

These are iron containing granules in red blood cells that are seen because the iron is aggregated with

mitochondria and ribosomes. They appear as faint violet or magenta specks, often in small clusters, due to staining of the associated protein.

They are associated with severe anemias and

thalassemias. Pappenheimer bodies can be increased in hemolytic anemia, infections and post-splenectomy.

Siderotic Granules (Pappenheimer Bodies)

(80)

Considerable numbers of small basophilic inclusions in red cells.

Found in

• Thalassaemia

• Megaloblastic anemia

• Hemolytic anemia

• Liver disease

• Heavy metal poisoning.

Basophilic stippling

(81)

Represent denatured hemoglobin (methemoglobin - Fe+++) within a cell.

With a supravital stain like crystal violet, Heinz bodies appear as round blue precipitates.

Presence of Heinz bodies indicates red cell injury and is usually associated with G6PD-deficiency.

Heinz Bodies

(82)

Heinz Body Preparation. RBC are incubated supravitally in new Methylene blue to identify precipitates of oxidatively denatured hemoglobin.

(83)