diagnostic logy

8/6/2019 Diagnostic logy

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CLINICAL PATHOLOGYCLINICAL PATHOLOGYDEPARTMENTDEPARTMENT 22

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.




How to approach a case of anaemia?

A case of anaemia is diagnosed first bycomplete blood count with special stress on the

indices

RBC morphologyReticulocyte count

Leukemia must be excluded.




Hypochromic microcytic anaemiaHypochromic microcytic anaemia

MCV < 75 fl, MCH < 26 pgMCV < 75 fl, MCH < 26 pg

1.1. IIron deficiency anaemia2. Anaemia of chronic diseasehronic disease

3.3. Sideroblastic anaemiaSideroblastic anaemia

4.4. Thalassaemia (Thalassaemia (αα,, ββ))




Lab d agnos s o hypochrom c

microcytic anaemia

Iron def. Chronic dis. Thalassemia Sideroblastic

Serum iron Reduced Normal/ Reduced Normal/Increased Increased

TIBC Increased Reduced Normal Normal

Serum Ferritin Reduced Increased Increased Increased

BM Iron stores Absent Increased Increased Increased

BM ringedsideroblasts

Absent Absent Absent Ringed sideroblasts

Hb electroph. Normal Normal Raised Hb A2, Hb Fin β thalassaemia

Normal




Iron deficiency anaemia Thalassemia




Sideroblastic Anaemia

Ringed Sideroblasts PB in a case of

Sideroblastic anaemia




Macrocytic AnaemiaMacrocytic 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




Megaloblastic AnaemiaMegaloblastic 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 )




Lab tests for B12 & folate deficiency

1. Complete blood picture:

Macrocytic anaemia MCV>100 flMacrocytic anaemia MCV>100 flLeucopenia, neutrophil hypersegmentationLeucopenia, neutrophil hypersegmentation

ThrombocytopeniaThrombocytopenia

2. B.M. examination:

Hypercellular, with megaloblasts, giant metamyelocytes.Hypercellular, with megaloblasts, giant metamyelocytes.3. Other tests:

Serum B12Serum B12

Serum folate, Red cell folateSerum folate, Red cell folate

Anti-parietal Ab & anti-intrinsic factor Ab.Anti-parietal Ab & anti-intrinsic factor Ab.




Megaloblastic features in the BM:



Lab Approach to HaemolyticLab Approach to Haemolytic

AnaemiaAnaemiaScreening testsScreening tests

1.1. Increased reticulocyte countIncreased reticulocyte count

2.2. Increased urine urobilinogenIncreased urine urobilinogen

3.3. Increased indirect bilirubinIncreased indirect bilirubin


L b A h H l i



Lab Approach to HaemolyticLab Approach to Haemolytic

AnaemiaAnaemia

Specific tests:Specific tests:1.1. Hemoglobin Electrophoresis to detect abnormalHemoglobin Electrophoresis to detect abnormal

hemoglobins.hemoglobins.

2.2. Osmotic fragility test for heriditary spherocytosisOsmotic fragility test for heriditary spherocytosis

3.3. Coombs’ test for immune hemolytic anaemiasCoombs’ test for immune hemolytic anaemias

4.4. G-6-PD assay for favismG-6-PD assay for favism

5.5. Red cell morphologyRed cell morphology

6.6. B.M. examination.B.M. examination.

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Two Types of HaemolysisTwo Types of Haemolysis

1-Extravascular (in RES e.g spleen)1-Extravascular (in RES e.g spleen)

2-Intravascular (inside vessels)2-Intravascular (inside vessels)

Main cause : ??????Main cause : ??????Specific tests include :Specific tests include :

HemoglobinaemiaHemoglobinaemia

MethemoglobiaemiaMethemoglobiaemia

HemoglobinuriaHemoglobinuria

Decreased haptoglobinDecreased haptoglobin





Normochromic Normocytic anaemia

Normal MCV , MCH & MCHC

Haemolytic anaemias other than thalassemia

Ex :sickle cell , hereditary spherocytosis, G6PD deficiency.

Acute bleeding

Acute systemic disease




Normochromic, normocytic anaemia in a

case of anaemia of chronic disease




Hereditary Elliptocytosis

G6PD deficiencySickle cell Anaemia

Sickle cell Anaemia




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




Immune haemolytic anaemia

Autoimmune Alloimmune

Coombs’ test positiveCoombs’ test positive Coombs’ test positiveCoombs’ test positive

No history of blood transfusionNo history of blood transfusion -Positive history of blood transfusion-Positive history of blood transfusion-Haemolytic disease of the newborn.-Haemolytic disease of the newborn.

Warm type : at 37º CWarm type : at 37º C

Cold type : at 4º CCold type : at 4º C

-Primary-Primary

-Secondary to lymphoma, SLE,-Secondary to lymphoma, SLE,

drugs…etcdrugs…etc




RBCs fragments in haemolyticanaemiaCold Agglutinins




Aplastic anaemia & BM failureAplastic anaemia & BM failure

DefinitionDefinition::

Pancytopenia resulting from aplasia of the bone marrow.

ClassificationClassification::

Primary:

Congenital (Fanconi, non Fanconi types) and acquired

(idiopathic)Secondary:

Ionizing radiation, drugs, chemicals & infections.




Laboratory findings of aplastic anemiaLaboratory findings of aplastic anemia::

1. Normochromic normocytic anaemia withlow reticulocyte count.

2. Leucopenia

3. Thrombocytopenia

4. No abnormal cells are seen in blood films.

5. Hypocellular bone marrow withreplacement of haemopoietic tissue by fatspaces.

Aplastic anaemia




Haemostasis



2525 CLINICAL PATHOLOGYCLINICAL PATHOLOGYDEPARTMENTDEPARTMENT

Bleeding disordersBleeding disorders

Bleeding disorders may be due toBleeding disorders may be due to

abnormalities of the vascular system,abnormalities of the vascular system,

platelets, coagulation system or fibrinolyticplatelets, coagulation system or fibrinolyticsystem.system.



Lab tests for bleeding disordersLab tests for bleeding disorders

SCREENING TESTS:SCREENING TESTS:

1.1. BTBT

2.2. CTCT3.3. PT /INR to standardize PT resultsPT /INR to standardize PT results

among different labsamong different labs

4.4. PTTPTT5.5. TTTT





Lab diagnosis of vascular Lab diagnosis of vascular

causescauses1.1. BT: prolongedBT: prolonged

2.2. CT : normalCT : normal

3.3. Hess test: > 5 purpuric spotsHess test: > 5 purpuric spots

4.4. Platelet count : normalPlatelet count : normal

5.5. Platelet function: normalPlatelet function: normal



PROLONGED BLEEDINGPROLONGED BLEEDING

TIMETIME

1.1. CBC to diagnose thrombocytopeniaCBC to diagnose thrombocytopenia

2.2. PT normal & PTT prolonged…..?PT normal & PTT prolonged…..?

VWD??VWD??

3.3. BM for thrombocytopeniaBM for thrombocytopenia

4.4. Platelet function tests (aggregometer) toPlatelet function tests (aggregometer) to

diagnose qualitative plateletdiagnose qualitative plateletdisorders.eg. Glanzman diseasedisorders.eg. Glanzman disease

2828 CLINICAL PATHOLOGYCLINICAL PATHOLOGY

DEPARTMENTDEPARTMENT



2929 CLINICAL PATHOLOGYCLINICAL PATHOLOGY


Clotting cascade



PROLONGED PTPROLONGED PT

1.1. With normal PTTWith normal PTT

1.1. Hereditary:DEFECT IN extrinsic pathway:Hereditary:DEFECT IN extrinsic pathway:

factor VII deficiencyfactor VII deficiency

2.2. Acquired:Acquired:

1.1. Hemorrhagic disease of the newborn due to vit KHemorrhagic disease of the newborn due to vit K

deficiencydeficiency

2.2. Liver disease (synthetic function of the liver)Liver disease (synthetic function of the liver)

3131

CLINICAL PATHOLOGYCLINICAL PATHOLOGY




Prolonged thrombin timeProlonged thrombin time

In cases of fibrinogen defects :In cases of fibrinogen defects :

dysfibrinogenemia, afibrinogenemia, DICdysfibrinogenemia, afibrinogenemia, DIC

3232





LAB INVESTIGATIONS OF DICLAB INVESTIGATIONS OF DIC

1.1. All screening tests are prolonged: BT,CTAll screening tests are prolonged: BT,CT

PT PTTPT PTT

2.2. THROMBOCYTOPENIATHROMBOCYTOPENIA

3.3. D-DIMER /FDP’S positiveD-DIMER /FDP’S positive

4.4. Factor assay : decreased activityFactor assay : decreased activity

3434






DEPARTMENTDEPARTMENT 3636

ACUTE& CHRONICACUTE& CHRONIC

LEUKAEMIASLEUKAEMIAS





HAEMATOLOGIC MALIGNANCYHAEMATOLOGIC MALIGNANCY

ACUTE LEUKAEMIASACUTE LEUKAEMIAS

CHRONIC MYELOPROLIFERATIVECHRONIC MYELOPROLIFERATIVEDISORDERSDISORDERS

CHRONIC LYMPHOPROLIFERATIVECHRONIC LYMPHOPROLIFERATIVE

DISORDERSDISORDERS

MULTIPLE MYELOMA and PLASMA CELLMULTIPLE MYELOMA and PLASMA CELL

DYSCRASIASDYSCRASIAS





What are LeukemiasWhat are Leukemias??

Leukemias are malignant counterparts of normalLeukemias are malignant counterparts of normalhematopoietic cells at different stages of maturation.hematopoietic cells at different stages of maturation.

Leukemias are primary disorders of bone marrow.Leukemias are primary disorders of bone marrow.

Malignant cells replace bone marrow, may infiltrateMalignant cells replace bone marrow, may infiltratespleen, liver, lymph nodes and circulate in blood streamspleen, liver, lymph nodes and circulate in blood stream





Classification of leukemiasClassification of leukemias

According to the courseAccording to the courseof leukemiaof leukemia

ACUTE LEUKAEMIA:ACUTE LEUKAEMIA:

-Proliferation of immature-Proliferation of immature

cellscells-If untreated is rapidly-If untreated is rapidlyfatal.fatal.

CHRONICCHRONIC LEUKAEMIA:LEUKAEMIA:-Proliferation of mature-Proliferation of mature

cellscells-chronic course.-chronic course.

According to the cellAccording to the cellof originof origin

LYMPHOIDLYMPHOID

((T, B or NK) ALL andT, B or NK) ALL andCLPDCLPD

MYELOIDMYELOID

AML and CMPDAML and CMPD





ACUTE LEUKAEMIASACUTE LEUKAEMIAS





ACUTE LEUKAEMIAACUTE LEUKAEMIA

EPIDEMIOLOGYEPIDEMIOLOGY

AML is predominantly a disease of adults.AML is predominantly a disease of adults.

ALL is predominantly a disease of childhoodALL is predominantly a disease of childhood

Acute leukaemia comprises 1/3 cases of childhood cancerAcute leukaemia comprises 1/3 cases of childhood cancer..





French-American-British (FAB)French-American-British (FAB) ClassificationClassification

Based on morphology assisted by cytochemistry andBased on morphology assisted by cytochemistry and

immunophenotypingimmunophenotyping

WHO Classification (most recent)WHO Classification (most recent)

Based on morphology, cytogenetics, molecularBased on morphology, cytogenetics, moleculargenetics , immunophenotyping & clinical features.genetics , immunophenotyping & clinical features.

Classification of Acute leukemiaClassification of Acute leukemia





FAB Classification of Acute Leukaemia

AMLM0 Minimally differentiated

M1 Myeloblastic

M2 Myeloblastic with differentiation.

M3 Promyelocytic

M4 Myelomonocytic

M5 Monoblastic

M6 Erythroleukemia

M7 Megakaryocytic

ALLL1

L2

L3Sta

ge

of

Diffe

re

ntiati

on

Lineag

eo

f

Blasts





M0 Minimally

differentiated





M1 Myeloblastic





M2 Myeloblastic with differentiation.





M3 Promyelocytic: Hypergranular





M3 Promyelocytic: Microgranular





M4 Myelomonocytic





M5a Monoblastic without maturation





M5 b Monoblastic with maturation





M6 Erythroleukemia





M7 Megakaryocytic

ACUTE LYMPHOBLASTICACUTE LYMPHOBLASTIC





ACUTE LYMPHOBLASTICACUTE LYMPHOBLASTIC

LEUKAEMIALEUKAEMIA

FAB ClassificationFAB Classification ALL-L1ALL-L1

ALL-L2ALL-L2 ALL-L3ALL-L3

Immunologic ClassificationImmunologic Classification

T or B lymphoblastic leukemiaT or B lymphoblastic leukemia





ALL L1





ALL L2





ALL L3





Burkitt’s Lymphoma





LABORATORY DIAGNOSISLABORATORY DIAGNOSIS

4-4- ImmunophenotypingImmunophenotyping

A panel of monoclonal antibodies (CDs) are used toA panel of monoclonal antibodies (CDs) are used to

detect : myeloid markers and lymphoid markers (for B &detect : myeloid markers and lymphoid markers (for B &

T cells).T cells).

CD: cluster of differentiationCD: cluster of differentiation





LABORATORY DIAGNOSISLABORATORY DIAGNOSIS

5- Genetic Analysis (karyotyping, FISH and PCR)5- Genetic Analysis (karyotyping, FISH and PCR)

Karyotyping Karyotyping

Chromosomal analysis is important for the diagnosis , classificationChromosomal analysis is important for the diagnosis , classification

and follow up of haematologic malignancies.and follow up of haematologic malignancies.

Chromosomes are visible only in metaphase ( mitosis).Chromosomes are visible only in metaphase ( mitosis).

FISH (fluorescence in situ hybridization)FISH (fluorescence in situ hybridization)

Using a labelled probe that hybridizes to DNAUsing a labelled probe that hybridizes to DNA Hybridization is visualizedHybridization is visualized

using fluorescent microscope.using fluorescent microscope.

PCR PCR ( polymerase chain reaction)( polymerase chain reaction)





CMLCML

CML arises from a neoplastic proliferation of CML arises from a neoplastic proliferation of

a pluripotential stem cell that can differentiatea pluripotential stem cell that can differentiate

into granulocyte, monocyte, erythroid,into granulocyte, monocyte, erythroid,megakaryocyte and lymphoid lineages .megakaryocyte and lymphoid lineages .



Lab diagnosis ofLab diagnosis of




Lab diagnosis of Lab diagnosis of

CML -CHRONIC PHASECML -CHRONIC PHASE

1-Blood picture1-Blood picture

a-Normochromic normocytic anaemiaa-Normochromic normocytic anaemia

b-Leucocytosisb-Leucocytosis

-- Neutrophil leucocytosis with 2 peaksNeutrophil leucocytosis with 2 peaks (myelocytes & neutrophils)(myelocytes & neutrophils)

-- BasophiliaBasophilia

-- Eosinophilia.Eosinophilia.

-- Blasts < 2 %Blasts < 2 %

c-Thrombocytosis .c-Thrombocytosis .

2- Bone marrow examination2- Bone marrow examination

-Hypercellular Bone marrow-Hypercellular Bone marrow-3% of blasts-3% of blasts

Lab diagnosis of Lab diagnosis of





ggCML -CHRONIC PHASECML -CHRONIC PHASE

3-Genetic Analysis (karyotyping, FISH and PCR)3-Genetic Analysis (karyotyping, FISH and PCR)

All Cases of CML are Philadelphia chromosome positive :All Cases of CML are Philadelphia chromosome positive :

(translocation between chromosomes # 9, & #22 :t (9;22))(translocation between chromosomes # 9, & #22 :t (9;22))

This translocation fuses the BCR gene on #22 with regions of This translocation fuses the BCR gene on #22 with regions of

the ABL gene from # 9 & results in the formation of athe ABL gene from # 9 & results in the formation of a

hybrid gene : BCR/ABL fusion gene.hybrid gene : BCR/ABL fusion gene.





t (9 ; 22) (q34 ; q11)

BCR-ABL





BCR-ABL by FISH





Peripheral Blood

CML Chronic Phase

Bone Marrow





CML: Blast crisis





CHRONIC LYMPHOCYTICCHRONIC LYMPHOCYTIC

LEUKAEMIALEUKAEMIA

Chronic lymphocytic leukemiaChronic lymphocytic leukemia





y p yy p y

(CLL(CLL))

Old age >40yOld age >40y

Accidental discovery by routine CBC showing lymphocytosis.Accidental discovery by routine CBC showing lymphocytosis.

Lab diagnosisLab diagnosis

1-CBC : Lymphocytosis with characteristic CLL morphology1-CBC : Lymphocytosis with characteristic CLL morphology

2-BM xamination : Hypercellular with lymphocytosis2-BM xamination : Hypercellular with lymphocytosis3-Immunophenotyping3-Immunophenotyping

4-( trisomy 12)4-( trisomy 12)



B-CLL

diagnostic logy

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