hema chakraborty
TRANSCRIPT
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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
Iron deficiency anaemia
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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
Megaloblastic anaemia
Liver disease
Alcoholism
Massive blood transfusion
DIC
Neonatal & Congenital
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Thrombocytosis
Haemorrhage (acute)
Surgery and trauma particularly fracture
Iron deficiency anaemia
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)
Megaloblastic anaemia
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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