ANAEMIAS

CLINICAL PATHOLOGY DEPARTMENT

Objectives:
By the end of this lesson, the students will be able to:
Differentiate between different types of anaemia. Choose the proper laboratory tests to diagnose different types of anaemia. Interpret laboratory test results of a case of anaemia.

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How to approach a case of anaemia?

A case of anaemia is diagnosed first by complete blood count with special stress on the indices RBC morphology Reticulocyte count Leukemia must be excluded.

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Hypochromic microcytic anaemia

1. 2. 3. 4.

MCV < 75 fl, MCH < 26 pg Iron deficiency anaemia Anaemia of chronic disease Sideroblastic anaemia Thalassaemia (, )

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Lab diagnosis of hypochromic microcytic anaemia

Iron def. Chronic dis. Thalassemia Sideroblastic Serum iron TIBC Reduced Increased Normal/ Reduced Reduced Normal/Increased Normal Increased Normal

Serum Ferritin BM Iron stores BM ringed sideroblasts Hb electroph.

Reduced Absent Absent Normal

Increased Increased Absent Normal

Increased Increased Absent

Increased Increased Ringed sideroblasts

Raised Hb A2, Hb F Normal in thalassaemia

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Iron deficiency anaemia

Thalassemia

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Sideroblastic Anaemia

Ringed Sideroblasts
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PB in a case of Sideroblastic anaemia

Macrocytic Anaemia
A-Macrocytic Anaemia with reticulocytosis 1-Acute bleeding 2-Haemolytic anaemias B-Macrocytic Anaemia without reticulocytosis 1. Megaloblastic anaemia (B12 or folate def.) 2. Myelodysplasia 3. Aplastic anaemia 4. Liver disease 5. Thyroid disease
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Megaloblastic Anaemia
A group of anaemias in which the erythroblasts in the bone marrow show asynchronous maturation of nucleus & cytoplasm i.e. the nucleus maturation is delayed due to DNA synthesis . It is due to vit. B12 or folate deficiency (commonest folate )

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Lab tests for B12 & folate deficiency

1.

Complete blood picture:

Macrocytic anaemia MCV>100 fl Leucopenia, neutrophil hypersegmentation Thrombocytopenia

2. 3.

B.M. examination:
Hypercellular, with megaloblasts, giant metamyelocytes.

Other tests:
Serum B12 Serum folate, Red cell folate Anti-parietal Ab & anti-intrinsic factor Ab.

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Megaloblastic features in the BM:

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Lab Approach to Haemolytic Anaemia

Screening tests
1. Increased reticulocyte count 2. Increased urine urobilinogen 3. Increased indirect bilirubin

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Lab Approach to Haemolytic Anaemia

Specific tests: 1. Hemoglobin Electrophoresis to detect abnormal hemoglobins. 2. Osmotic fragility test for heriditary spherocytosis 3. Coombs test for immune hemolytic anaemias 4. G-6-PD assay for favism 5. Red cell morphology 6. B.M. examination.

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Two Types of Haemolysis

1-Extravascular (in RES e.g spleen) 2-Intravascular (inside vessels) Main cause : ??????
Specific tests include : Hemoglobinaemia Methemoglobiaemia Hemoglobinuria Decreased haptoglobin

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Normochromic Normocytic anaemia

Normal MCV , MCH & MCHC Haemolytic anaemias other than thalassemia Ex :sickle cell , hereditary spherocytosis, G6PD deficiency. Acute bleeding Acute systemic disease

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Normochromic, normocytic anaemia in a case of anaemia of chronic disease

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Hereditary Elliptocytosis

Sickle cell Anaemia

G6PD deficiency

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Sickle cell Anaemia

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Hereditary Spherocytosis
Red cell membrane defect Diagnostic criteria: 1-Normochromic Normocytic anaemia with increased MCHC 2-Blood film show micro-spherocytes 3-Reticulocytosis
4-Increased Saline Osmotic Fragility

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Hereditary Spherocytosis

Spherocytes in blood film

Control

Test Saline Osmotic Fragility Test

Spherocytes in blood film CLINICAL PATHOLOGY DEPARTMENT 19

Immune haemolytic anaemia

Autoimmune Alloimmune

Coombs test positive No history of blood transfusion

Coombs test positive -Positive history of blood transfusion -Haemolytic disease of the newborn.

Warm type : at 37 C Cold type : at 4 C -Primary -Secondary to lymphoma, SLE, drugsetc

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Cold Agglutinins

RBCs fragments in haemolytic anaemia

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Aplastic anaemia & BM failure

:Definition
.Pancytopenia resulting from aplasia of the bone marrow

:Classification
Primary: Congenital (Fanconi, non Fanconi types) and acquired (idiopathic) Secondary: Ionizing radiation, drugs, chemicals & infections.
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:Laboratory findings of aplastic anemia

1. 2. 3. 4. 5.

Normochromic normocytic anaemia with low reticulocyte count. Leucopenia Thrombocytopenia No abnormal cells are seen in blood films. Hypocellular bone marrow with replacement of haemopoietic tissue by fat spaces.

Aplastic anaemia
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Haemostasis

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Bleeding disorders
Bleeding disorders may be due to abnormalities of the vascular system, platelets, coagulation system or fibrinolytic system.

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Lab tests for bleeding disorders

SCREENING TESTS: 1. BT 2. CT 3. PT /INR to standardize PT results among different labs 4. PTT 5. TT
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Lab diagnosis of vascular causes

1. BT: prolonged 2. CT : normal 3. Hess test: > 5 purpuric spots 4. Platelet count : normal 5. Platelet function: normal

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PROLONGED BLEEDING TIME

1. CBC to diagnose thrombocytopenia 2. PT normal & PTT prolonged..?

VWD?? 3. BM for thrombocytopenia 4. Platelet function tests (aggregometer) to diagnose qualitative platelet disorders.eg. Glanzman disease
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Clotting cascade

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Prolonged PTT
1. WITH NORMAL PT: DEFECT IN INTRINSIC

SYSTEM
1. FACTOR VIII &IX ASSAY

(factor VIII deficiency=hemophilia A ) (factor IX deficiency=hemophilia B)

1. PROLONGED PT &PTT

Defect in the common pathway: factor X,II,I

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PROLONGED PT
1. With normal PTT 1. Hereditary:DEFECT IN extrinsic pathway: factor VII deficiency 2. Acquired:
1. Hemorrhagic disease of the newborn due to vit K

deficiency 2. Liver disease (synthetic function of the liver)

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Prolonged thrombin time

In cases of fibrinogen defects : dysfibrinogenemia, afibrinogenemia, DIC

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Monitoring of patients on anticoagulant therapy

1. Heparin therapy by PTT 2. Oral anticoagulant therapy by PT 3. LMWH (CLEXAN) by anti factor X

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LAB INVESTIGATIONS OF DIC

1. All screening tests are prolonged: BT,CT 2. 3. 4.

PT PTT THROMBOCYTOPENIA D-DIMER /FDPS positive Factor assay : decreased activity

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Interpretation Of Lab Tests

1.

2. 3. 4.

Prolonged PT Defeciency in any factor of the extrinsic or common pathway:7,10,2,1 Vit K defeciency Liver disease Oral anticoagulant therapy
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Prolonged PTT 1. defeciency of any factor in the intrinsic or common pathways: most common VIII, VW, IX 2. Heparin therapy 3. Presence of inhibitors
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ACUTE& CHRONIC LEUKAEMIAS

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HAEMATOLOGIC MALIGNANCY
ACUTE LEUKAEMIAS CHRONIC MYELOPROLIFERATIVE DISORDERS CHRONIC LYMPHOPROLIFERATIVE DISORDERS MULTIPLE MYELOMA and PLASMA CELL DYSCRASIAS
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?What are Leukemias

Leukemias are malignant counterparts of normal hematopoietic cells at different stages of maturation. Leukemias are primary disorders of bone marrow. Malignant cells replace bone marrow, may infiltrate spleen, liver, lymph nodes and circulate in blood stream

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Classification of leukemias
According to the course of leukemia
ACUTE LEUKAEMIA: -Proliferation of immature cells -If untreated is rapidly fatal. CHRONIC LEUKAEMIA: -Proliferation of mature cells -chronic course.

According to the cell of origin

LYMPHOID (T, B or NK) ALL and CLPD MYELOID AML and CMPD

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ACUTE LEUKAEMIAS

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ACUTE LEUKAEMIA
EPIDEMIOLOGY
AML is predominantly a disease of adults. ALL is predominantly a disease of childhood Acute leukaemia comprises 1/3 cases of childhood cancer.

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Classification of Acute leukemia

French-American-British (FAB) Classification
Based on morphology assisted by cytochemistry and immunophenotyping

WHO Classification (most recent)

Based on morphology, cytogenetics, molecular genetics , immunophenotyping & clinical features.
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FAB Classification of Acute Leukaemia

AML
M0 Minimally differentiated
Differentiati on

ALL
L1 L2 L3

Stage of

M1 Myeloblastic M2 Myeloblastic with differentiation. M3 Promyelocytic M4 Myelomonocytic

Lineage of Blasts

M5 Monoblastic M6 Erythroleukemia M7 Megakaryocytic

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FAB CLASSIFICATION
Morphologic classification of AML is based on
Cellular differentiation ( What type of cell?)
(granulocyte, monocyte , erythroid, or megakaryocytic)
Extent of maturation

(Myeloblast, promyelocyte, granulocyte)

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M0 Minimally differentiated

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M1 Myeloblastic
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M2 Myeloblastic with differentiation.

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M3 Promyelocytic: Hypergranular
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M3 Promyelocytic: Microgranular
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M4 Myelomonocytic
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M5a Monoblastic without maturation

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M5 b Monoblastic with maturation

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M6 Erythroleukemia

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M7 Megakaryocytic
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ACUTE LYMPHOBLASTIC LEUKAEMIA

FAB Classification
ALL-L1 ALL-L2 ALL-L3

Immunologic Classification
T or B lymphoblastic leukemia

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ALL L1
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ALL L2
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ALL L3
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Burkitts Lymphoma

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LABORATORY DIAGNOSIS
1- Complete blood picture -Anaemia -White cell count -Platelet count 2- Bone marrow examination -Cellularity -% of blasts 3-Cytochemical stains e.g Myeloperoxidase, Sudan black B

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LABORATORY DIAGNOSIS
4- Immunophenotyping
A panel of monoclonal antibodies (CDs) are used to detect : myeloid markers and lymphoid markers (for B & T cells). CD: cluster of differentiation

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LABORATORY DIAGNOSIS
5- Genetic Analysis (karyotyping, FISH and PCR) Karyotyping
Chromosomal analysis is important for the diagnosis , classification and follow up of haematologic malignancies. Chromosomes are visible only in metaphase ( mitosis). FISH (fluorescence in situ hybridization) Using a labelled probe that hybridizes to DNA Hybridization is visualized
using fluorescent microscope.

PCR ( polymerase chain reaction)

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CML
CML arises from a neoplastic proliferation of a pluripotential stem cell that can differentiate into granulocyte, monocyte, erythroid, megakaryocyte and lymphoid lineages .

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CML
CLINICAL COURSE : The disease is characterized by 3 phases: Chronic phase : 3 % blasts Accelerated phase Blast crisis: PB / BM blasts > 20 %

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Lab diagnosis of CML -CHRONIC PHASE

1-Blood picture a-Normochromic normocytic anaemia b-Leucocytosis - Neutrophil leucocytosis with 2 peaks (myelocytes & neutrophils) - Basophilia - Eosinophilia. - Blasts < 2 % c-Thrombocytosis . 2- Bone marrow examination -Hypercellular Bone marrow -3% of blasts

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Lab diagnosis of CML -CHRONIC PHASE

3-Genetic Analysis (karyotyping, FISH and PCR)
All Cases of CML are Philadelphia chromosome positive : (translocation between chromosomes # 9, & #22 :t (9;22)) This translocation fuses the BCR gene on #22 with regions of the ABL gene from # 9 & results in the formation of a hybrid gene : BCR/ABL fusion gene.

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t (9 ; 22) (q34 ; q11)

BCR-ABL
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BCR-ABL by FISH

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Peripheral Blood

Bone Marrow

CML Chronic Phase

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CML: Blast crisis

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CHRONIC LYMPHOCYTIC LEUKAEMIA

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Chronic lymphocytic leukemia )(CLL

Old age >40y Accidental discovery by routine CBC showing lymphocytosis. Lab diagnosis 1-CBC : Lymphocytosis with characteristic CLL morphology 2-BM xamination : Hypercellular with lymphocytosis 3-Immunophenotyping 4-( trisomy 12)

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B-CLL

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