hema chakraborty

8/4/2019 hema chakraborty

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Tests used in the diagnosis of

various disease states

Dr. Hema Chakraborty

Consultant PathologistAMRI Hospitals, Kolkata


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Tests used in the diagnosis ofvarious disease states

Diagnosis of a disease needs a proper workupstarting from the history, proper clinicalexamination by a physician or surgeon andfinally a set of various investigations includingradiological and pathological.

Some times the diagnosis is done on history andclinical examination, but confirmation of thediagnosis is done by pathological tests orradiological examination. Pathological tests arethe basis of final diagnosis because they are thedirect evidences of the disease., eg.


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Demonstration of malaria parasite in the blood, LDbodies in the bone marrow.

Demonstration of Acid fast bacilli in the sputum

Culture of micro organisms from the specimens likesputum, urine, blood, pus, throat swabs, body fluids etc.

Diagnosis of leukaemias by peripheral blood or bonemarrow examination

Demonstration of parasites in the stool sample. Diagnosis of cancer by FNAC or tissue biopsy etc.


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Haematological tests done for the diagnosis of disease

states

Hb, TC, DC, ESR are the commonest routine blood tests

done for each and every patient and gives lot of

information

If the clinicians suspect that the patient is anaemic

following tests are added

Peripheral blood smear comment

Red cell indices (MCV, MCH, MCHC)

Platelet count

Reticulocyte count


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All these tests will give us an idea whether the

anaemia is

Normocytic normochromic

Microcytic hypochromic

Macrocytic

Haemolytic

Aplastic

Only thrombocytopenia Leukaemia

Malaria prasite filaria are diagnosed on peripheral

smear


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Above tests are supplemented with

Serum Fe, TIBC, ferretin

Serum B12, folic acid

Haemoglobin electrophoresis

Coombs test

Bone marrow examination If the patient has bleeding disorder PT, APTT,

fibrinogen, FDP, D-dimer, factor VIII etc.


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Causes of anaemia not falling in above categoriesare anaemia of chromic disorders like

Autoimmune diseases

Renal failure

Chronic infectious

Malignancies (non haematological)

In all these disease other tests related to the diseases are

done for confirmation of the diagnosis


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Haemoglobin

Normal Values

Adult Men 15 20 g%

Adult Women 12 15 g%

Low haemoglobin anaemia

High haemoglobin is associated with increase in number

of red cells and PCV Polycythemia

Polycythemia is suspected in men with Hb > 19.5 gm%

in women > 18.0 gm%


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Causes of Polycythemia

Relative Dehydration fluid loss or diminished intake

Polycythemia of stress

True polycythemia

Primary or idiopathic polycythemia vera Secondary

Secondary to hypoxia

High altitude

Congenital heart disease

Chronic pulmonary disease Secondary to non neoplastic kidney diseases cysts, hydronephrosis,

ischaemia

Secondary to tumours of liver, kidney, pheochromocytoma, adrenal

adenoma, uterine fibroid, virilizing ovarian tumours.


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RBC morphology and red cell indices

These are the basis of diagnosis of the cause of anaemias.

Red cell indices (MCV, MCH, MCHC) tell us whether the

anaemia is microcytic hypochromic, normocytic normochromic

or macrocytic.

Detailed study of red cell morphology is done by examining

the blood film.

Red cell morphology in well spread, dried and stained film

normal red cells have round smooth contours diameter rangesfrom 6.0 to 8.5 mm. As a rough guide normal red cell size

appears to be same as that of the nucleus of a small

lymphocyte.


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Abnormal Erythropoiesis

Amisocytosis and poikilocytosis

These are non specific features of almost any blood disorder

Anisocytosis means presence of both macrocytes and microcytes

poikilocytosis is found in megaloblastic anaemia, Fe deficiency

anaemia, haemolytic anaemia, congenital dyserythropoietic anemia,

myelodysplastic syndromes, myelofibrosis.

Macrocytes

Classically found in megaloblastic anaemia (macro-ovalocytes)

Myelodysplastic syndrome (dimorphic)

Excess alcohol intake

Chronic liver disease

Congenital dyserythropoietic anaemia.

Haemolytic anaemias because of presence of reticulocytes


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Microcytes

Iron deficiency anaemia

Thalassaemia Anaemias of chronic disease

Basophilic stippling numerous basophilic granules

Thalassaemia

Megaloblastic anaemia Infections

Liver disease

Lead poisoning

Hypochromasia due o lowered haemoglobin synthesis Iron deficiency

Thalassaemia

Chronic infections


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-thalassaemia, specially trait isdifferentiated from Fe def. anaemia by

The presence of target cells

Normal Fe, TIBC & Ferretin

Beta-thalassaemia major differs

More anisopoikilocytosis

Nucleated red blood cells


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Spherocytosis

These cells are more spheroidal than normal

RBCs but maintain regular outline. Their

diameter less and thickness greater than normal

Hereditary spherocytosis

ABO haemolytic disease of new born

Auto immune haemolytic anaemia

Action of bacterial toxins eg. Clostridium perfringens


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Schistocytes of erythrocyte fragments

They are smaller than normal and of varying shopes,have sharp angles or spines or microspherocytes. They

occur

In genetic disorders like thalassaemias

Severe burns

Haemolytic uraemic syndrome

Target cells

Thalassaemia (more often splenectomy)

Haemoglobin C disease and other haemoglobinopathies

Sickle cells

Sickle cell anaemia


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Polychromasia

Red cells stain shades of bluish grey. They

are reticulocytes. They are seen when there

is excessive haemolysis and bone marrow

regeneration in cases of Fe, B12 deficiency

anemia, when respective deficiency is

corrected.


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Erythroblastaemia

These are immature forms of red blood cells or

they are nucleated RBCs. They are found in

Haemolytic anaemias

Myelofibrosis

Haemolytic disease of new born

Leukaemias

Carcinomatosis

After splenectomy


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Leucocytes

Normal TL count

Adults 400 11000/cumm or 4 11 x 109/litre

Infants (at birth) 10000 25000/cumm

Infants (1 year) 6000 18000/cumm

Childhood (4 12 yrs) 5000 15000/cumm

Differential leukocyte count

Neutrophils 40 75%

Lymphocytes 20 50%

Monocytes 2 10%

Eosinophils 1 6%

Basophils < 1%


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Causes of leucocytosis

Neutrophil leucocytosis (size 13 h) Pyogenic infectious like abscess, pneumonia, meningitis, UTI, Pyelonephritis,

septicaemia.

Non pyogenic infections

Rheumatic fever, Scarlet fever, Diphtheria, Cholera, Acute poliomyelitis

Hamorrhage Trauma, surgical operations, fractures, crush injuries, burns

Malignant diseases

Myocardial infarction

Drugs & chemical poisoning

Metabolic disturbances, renal failure, diabetic coma, acute gout, eclampsia

Myeloid leukaemia, polycythemia, myelosclerosis

Collagen diseases

Acute intravascular haemolysis


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Toxic changes seen in neutrophils ininfections

Toxic granules

Cytoplasmic vacuoles

Shift to left (presence of immature cells)

Bacteria rarely in septicaemia bacteria may be found in

the cytoplasm


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Hypersegmented neutrophils seen in

megaloblastic anaemia

Eosinophils 12 17 in diameter two

nuclear lobes, cytoplasm packed with

distinct red granules. Normal absolute

count 40 400/cumm


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Eosinophilia

Allergic conditions Asthma, hay fever, drug allergy, urticaria, show

moderate eosinophilia

Parasitic infestations moderate to severe eosinophilia occurs in

parasites which cause tissue invasion than with those causing

intestinal infestation. Malaria sometimes causes moderate

eosinophilia. Drug allergy

Skin diseases eczema, pemphigus, psoriasis, exfoliative

dermatitis, dermatitis herpetiformis

Tropical pulmonary eosinophilia may be due to filarial infection.

Hyper eosinophilic syndrome

Lymphomas, leukaemias, Hodgkins disease

Malignant disease


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Basophilia

Chronic myeloid leukaemia

Myelosclerosis

Polycythemia vera Hypersensitivity states

Myxoedema

Fe def. anaemia Haemolytic anaemia


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Lymphocytosis 7 8 N 1.5 4 x 109/litre

Normally children have higher lymphocyte count and low

neutrophil count

Causes

Infections in infants and young children, infections which in

adults cause neutrophil leucocytosis not uncommonly result in

lymphocytosis, moderate lymphocytosis occurs in typhoid,

influenza, brucellosis, tuberculosis, secondary syphilis, pertusis

Viral infectious Rubella, Mumps, Measles, Chicken pox.

Infectious mononucliosis

Infectious hepatitis

Lymphocytic leukaemia, lymphoma


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Monocytosis 15 - 18 Normal adults 0.2 0.8 x 109/litre

Bacterial infections Tuberculosis

Bacterial endocarditis

Typhoid fever

Brucellosis

Protozoal and Rickettsial infection Malaria

Typhus

Kala azar

Infectious mononucleosis

Hodgkins disease, NHL Monocytic leukaemia

Rheumatoid arthritis

Ulcerative colitis, regional enteritis

Carcinoma


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Leucopenia

Reduction in the number of leucocytesbelow 4 x 109/litre. In most of the casesleucopenia is mainly due to reduction in

neutrophils. In marked leucopenia there isreduction in both neutrophils andlymphocytes.

Infections


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Infections

Bacterial typhoid fever, paratyphoid

Viral influenza, measles, rubella, infective hepatitis, atypical viralpneumonia

Rickettsial typhus Protozoal malaria, kala azar

Over whelming infections milliary TB, severe infections in elderly ordebilitated

Acute and subacute leukaemia

Drug induced neutropenia Aplastic anaemia

Hypersplenism

Bone marrow infiltration or sclerosis

Metastatic carcinoma

Malignant lymphoma Myelosclerosis

Multiple myeloma

Megaloblastic anaemia

SLE



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Platelet Morphology

Normal platelets are 1 3 in diameter. They are irregular in outline with fine red granules.Large platelets are seen when platelet production is increased.

Thrombocytopenia Primary

Idiopathic thrombocytopenic purpura (ITP)

Secondary

Commoner causes (mod. to severe thrombocytopenia)

Drugs and chemicals

Leukemia (acute)

Aplastic anaemia

Bone marrow infiltration by carcinoma, multiple myeloma, lymphoma, myclosclerosis

Hypersplenism

SLE

Less common causes

Infection


Liver disease

Alcoholism

Massive blood transfusion

DIC

Neonatal & Congenital


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Thrombocytosis

Haemorrhage (acute)

Surgery and trauma particularly fracture


Splenectomy

Polycythemia vera, myelosclerosis

Chronic myeloid leukaemia

Idiopathic haemorrhagic thrombocythemia

Infection occasionally

MalignancyHodgkins disease, carcinoma

Abnormally large platelets and extreme platelet anisocytosis is found in

myeloproliferative disorders


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Bone marrow examination

This is an indispensable adjunct in the study of diseases

of blood specially anaemias with leucopenia,

leucocytosis, thrombocytopenia and presence of

immature cells in the blood, eg.

Leukemias (acute) can present as pancytopenia i.e. low Hb, TLC

& Platelets cells in the peripheral blood. We call this condition

aleukaemic leukaemia. This can not be differentiated from

aplasic anaemia unless we do bone marrow. In acute

leukaemias bone marrow in hypercellular with presence ofimmature cells (blasts) of either myeloid or lymphoid series.

Chronic myeloid and lymphocytic leukaemias can be diagnosed

by peripheral blood examination


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Bone marrow examination

Pancytopenia cases after bone marrow examination mayshow

Aplastic anaemia

Acute leukaemia (subleukaemic, aleukemic)


Myelodysplastic syndrome

Kala azar (L D bodies)

Infiltration of marrow by secondary carcinoma, malignant

lymphoma, multiple myeloma Tuberculosis

Myelosclerosis

Normal marrow may be found in hypersplenism


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Tests done for acquired haemolyticanaemias

Examination of peripheral smear

Detection of auto antibodies (DAT)

For micro angiopathic haemolytic anaemiaswhich is a generalized disease following tests

are done

Renal function tests Platelet count

Coagulation screen including FDP and D-dimer


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Investigations for Abnormalhaemoglobins and thalassaemias

Haemoglobin molecule

Haemoglobin is formed from two pairs of globin chains

each with a heme group attached.

HbF is the predominant haemoglobin of foetal life (22)

and comprise major proportion of haemoglobin at birth.

HbA is the major haemoglobin found in adults and

children mainly after 6 12 months of age - 22HbA2 is in small proportion in adults, approximately 2

3.3% - 22 HbF in adults is .2 1%


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Thalassaemia

Failure to synthesize one or more of the globin chains thalassaemia

-thalassaemia major 22 (HbF) & minor HbA2

Structural variants of HbA

HbS, HbC, HbD, HbE or combination of these with thalassaemia

eg.

HbS thalassaemia

HbE thalassaemia

HbD thalassaemia

HbSC disease

HbSD disease


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CELLULOSE ACETATE ELECTROPHORESIS

ph 8.5

At alkaline pH, haemoglobin is a negatively chargedprotein and when subjected to electrophoresis will

migrate towards the anode (+) structural variants which

have a change in the charge on their surface will

separate from the HbA


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Sickling Test

Thin wet film of blood sealed with

petroleum jelly shows sickling if HbS ispresent.


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Investigations of haemostasis

There are five different components involved in the haemostasis

which

Maintain blood in a fluid state

Arrest bleeding at the site of injury. Those five different components are

Blood vessels

Platelets

Coagulation factors

Their inhibitors

Fibrinolytic system

Deficiency or exaggeration of any of these may lead to either thrombosis

or haemorrhage.

First line test used in the investigations of acute haemostatic failure


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Commonly used test

Prothrombin time (PT)

(Normal range 11 16 second)

Activated partial thromboplastin time

(APTT)

Thrombin time

Fibrinogen

Platelet count


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Causes of Prolongation of PT

Administration of oral anticoagulant drug (vitamin Kantagonists)

Liver disease, particularly obstructive

Vitamin K deficiency

DIC

Rarely, previously undiagnosed factor VII, X, V orprothrombin deficiency or defect

With prothrombin, factor X or factor V deficiency APTTwill also be prolonged.


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Activated partial thromboplastin time

Normal range 30 40S

Causes of prolonged APTT are

DIC

Liver disease

Massive transfusion with stored blood Administration of heparin or contamination with heparin

A circulating anticoagulant

Deficiency of coagulation factor other than factor VII

Moderately prolonged in patients on oral anticoagulant drugsand in the presence of vitamin K deficiency


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Thrombin Time

Normal range 15 19 seconds

Causes of prolonged TT Hypofibrinoginaemia as in DIC

Raised concentration of FDP as in DIC andliver disease

Extreme prolongation due to presence ofheparin

Dysfibrinogenaemia in liver disease

Hypoalbuminaemia


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Fibrinogen assay

Normal assay 2 4 gm/litre

Low fibrinogen level

Inherited dysfibrinogenaemia

Presence of high levels of FDP in DIC


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If all the first line investigations are normal in a

patient who continues to bleed from the site of

an injury or after surgery there are following

possibilities

Disorder of platelet function, congenital or acquired

Von Willebrands disease in which the factor VIII is

not sufficiently low to cause prolongation of APTT

Mild factor VIII deficiency

Factor XIII deficiency

A vascular disorder of haemostasis

Bleeding from severely damaged vessel


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hema chakraborty

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