Hematology revision notes pathology

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Hematology
Hematopoesis sites

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TONY SCARIA 2010 kmc

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Fetal to adult HB
switch over @ 30
weeks
• I. Although beta chains can be detected
in amniotic fluid by 16-20 wks of
gestational age, the rate ogf beta chain
synthesis occurs at a slow rate till 30-32
weeks of gestational age.
• II. At 30-32 weeks sudden rise in rate of
beta chain synthesis occurs. Since
alpha chains are already abundant,
sudden rise in beta chain synthesis
leads to sudden increase in adult
hemoglobin synthesis, and this is called
fetal to adult hemoglobin switch over.

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• Earliest evidence of haematopesis @ 3 weeks
• First cel formed is RBC

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• Haematopetic stem cells  CD 34+
• Hb first appears in early normoblast
• Detected by geimsa staining in intermediate normoblast

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Erythropoesis
• Whole stage takes 7 days
• Initial 3 days
• Pronormoblast to reticulocyte
• Last 4 days
• Reticulocyte to mature RBC
• Out of this 4 day reticulocyte spends last day in peripheral circulation

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Reticulocyte can be stained with
• Supravital staining
• New Methylene blue is best
• Crystal violet

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Reticulocyte index
• Poor mans bone marrow aspirate
• Reflects bone marrow activity

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Site of bone marrow biopsy
• In infants anteromedial tibia
• In adults & children
• Posterior superior iliac crest

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In obese person
• Anterior superior iliac spine
Bone marrow biopsy needle
• Jamshidi

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• Largest number of myeloid cells are promyelocytes
• In erythroid compartment
• Orthochromatophilic & polychromatophilic are maximum
Inclusion bodies RBC

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polychromasia
• Polychromasia (young red cells –
reticulocytes present)
• a. Hemolysis
• b. Acute hemorrhage
• c. Increased Red cell turnover

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Pappenheimer bodies
• In sideroblastic
anemia
• Iron deposits bluish in
colour in periphery of
RBC
• Stained with Prussian
blue

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• Purple-blue inclusions visible
only after supravital stain
• Composed of denatures
hemoglobin (usually oxidized
hemoglobin)

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Bite cells
• Bite of RBC membrane is missing
• In G6PD deficiency

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Bite cells
Howell jolly bodies

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Howell jolly bodies
• Nuclear remnants
• Smooth round dark blue-purple cytoplasmic
inclusion
• May be centrally located or sometimes in the
periphery
• Composed of fragments of DNA
• Iron stain negative
Hemoglobin H disease
• Multiple small blue-purple granules distributed within an RBC and
visualized with special stain
• Composed of precipitated chains of beta-hemoglobin (tetramer of
beta chain) seen in some alpha thalassemia

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Cabots ring

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• Basophilic stippling is ribosomal inclusions in RBCs, while
Pappenheimer bodies are iron deposits, Heinz bodies are precipitated
hemoglobin seen with special stain (He for Heinz and He for
Hemoglobin), and Howell Jolly bodies are DNA remnants
Target cell RBC
• Central area of haemoglobinisation
• bulls eye appearance
• Thalassemia
• Post splenectomy
• Hb C ds
• Thalassemia

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Tear drop cells/ dacrocytes
• Myelofibrosis
• Bone marrow infiltration
by hematologic or non-
hematologic malignancy
• Artifact of slide
preparation (in which all
the tails usually point in
the same direction
Tear drop cells are also seen in
• Myelofibrosis
• Myelodysplasia
• BM ifiltration
• Pernicious anemia
• IDA
• Thalassemia

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Schistocyte / fragmented RBC
• Usually associated with an intravascular hemolytic
process (as opposed to spherocytes which are usually
associated with an extravascular hemolytic process)
• Usually smaller than a normal RBC
• Different RBC fragment variants (helmet cell,
irregular fragments, etc.)
• Usually lacks a central pallor
• Microangiopathic Hemolytic Anemia (e.g. as seen in DIC,
TTP, or HUS)
• Severe burns
• Mechanical valve-induced
• Uremia
• Malignant hypertensio
Schistocyte

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Spherocytosis
• Dense cells no central pallor
• Hereditary spherocytosis
• Clostridial infections
• Auto immune haemolytic anemia
• G6PD deficiency
• Splenomegaly a/w cirrhosis
Target cell RBC
• Central area of haemoglobinisation
• bulls eye appearance
• Hb C ds
• Asplenia
• Post splenectomy
• Liver disease
• Thalassemia

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• two types of spiculated red cells
• echinocytes and
• Echinocyte typically have a serrated outline with small uniform projections more or less
evenly spread over the circumference of the cell
• Acanthocytes
• have a few spicules of varying size that project irregularly from the red cell surface

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Spur cell
• Hepatocellular dysfunction
acanthocyte
• Irregular narrow-base sharp projections of red cell membrane
(looking like cowboy boot spurs)
• Abetalipoproteinemia
• Neuroacanthocytosis
• Liver disease

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RBC
RBC
• If central pallor > 1/3 rd 
hypochromia

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MCHC 33- 37 g/dl
Red blood cell distribution width
• Measure of anisocytosis
• RDW increased in megaloblastic
anemia / microcytic
hypochromic anemia
• RDW normal in thalassemia

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Haematocrit or packed cell volume
• Blood volume proportion occupied by RBC
• Normal
• male45%
• Female42%
• Decrease in size or number will lower haematocrit
• Increased RBC count or size  increase haematocrit
• Thalassemia though RBC number increased size is decreased haematocrit
is also decreased
• Megaloblastic anemia with increase RBC size but decreased haematocrit

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Microcytosis
• MCV<80 fL
• Sideroblastic anemia
• Pyridoxine deficiency
• Lead poisoning
• Iron deficiency
• Thalassemia

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macrocytosis
• MCV of >100 fL
•

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• Integral proteins
• Band 3
• Glycophorin A
• Peripheral proteins
• 4.2
• Ankyrin
• Actin
• Spectrin
• Spectrin is the major skeletal protein of
filamentous nature.
• It consists of two polypeptide chains a
and which are intertwined.
• It is connected laterally via two
additional proteins namely protein 4.1
and actin.
• This two dimensional cable meshwork is
tethered (bounded) to the cell
membrane by another protein called
ANKYRIN which forms a bridge between
spectrin and cell membrane protein 3.

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• Glycophorin
• Most abundant integral protein in RBC membrane
• Not involved in hereditary spherocytosis
Anemia

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• Anemia may be defined as a state in which the blood hemoglobin
level is below the normal range for the patient’s age and sex
• Males < 13gm/dL;
• females < 12 gm/dL.
• Pregnancy <11 gm%
• Hemoglobin at birth is about 20 gm/dl and it gets reduced to 10
gm/dl at 3 months of age.
• Extra Edge: Symptoms and Signs
• i. Fatigue,
• ii. breathlessness on exertion,
• iii. palpitation,
• iv. angina,
• v. tachycardia,
• vi. cardiac dilatation,
• vii. systolic flow murmurs,
• viii. edema.

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• Microcytosis (Reduced average cell size, MCV < 76 fl)
• a. Iron deficiency
• b. Sideroblastic anaemia
• c. Thalassaemia
• d. Lead toxicity
• e. AOCD(+/-)

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Normocytic anemia
• Recent Blood loss
• Aplastic anemia
• Myeloplastic anemia
• Normocytic with high reticulocyte count
• i. Blood Loss ii. DIC iii. Hemolytic anemia iv. Sickle cell anemia v. Hemangioma
• Normocytic with low reticulocyte count
• i. RBC aplasia (Diamond Black fan syndrome, Parvo virus)ii. Malignancy iii.
Fanconi Anemia iv. AOCD v. CRF

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Macrocytic anemia
• Hypothyroidism
• Alcoholism
• Liver disease
• Vitamin B12 FA deficiency
• Drugs  methotrexate / Zedovudine
Impaired RBC production
• Megaloblastic anemia

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Iron deficiency anemia
• Mc cause of anemia in india
• Iron absorbed in SI mainly duodenum & upper jejunum
• Absorbed mainly in Fe2+ form
• fe3+  Fe2+ by cytochrome B
• By apical divalent metal transporter (DMT1)
• Also facilitates uptake of Mn2+/Cu2+/Co/zn2+/Pb2+/Cd

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Diphyllobothridium latum
• Causes B12 deficiency

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Tongue manifestation in B12 deficiency

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Neurological manifestation of B12 deficiency
• Vitamin B12 deficiency
causes optic neuropathy
and impaired acuity and
central scotoma

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Blood manifestation
• Neutrophil hypersegmentation
• Pancytopenia
MCHC
Megaloblastic bone marrow

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Neutrophilic hypersegmentation
• Seen in
• B12 /folate deficiency
• CRF
• Myelo proliferative disorder
hypersegmented neutrophil seen in vitamin
B12 deficiency
• multilobed polymorphonuclear leukocytes
• larger than normal neutrophils with >5 segmented nuclear lobes.
• folic acid or vitamin B12 deficiency.
• M>>F
• paresthesias, muscle weakness, or difficulty in walking and
sometimes dementia, psychotic disturbances, or visual impairment
• Oval macrocytes, usually with considerable anisocytosis and
poikilocytosis,
• MCV is usually >100 fL

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Role of b12 in odd chain FA metabolism

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subacute combined degeneration of the spinal cord.
• The combined involvement of the axons
• in the ascending tracts of posterior column
• and the descending pyramidal tract

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Knuckle hyperpigmentation in b12 deficiency
Dorsal column initially dollowed by lateral
column

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Lhermitte sign

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Schilling test to detect b12 absorption
• To detect the cause
• Schilling test (Radioisotope
absorption test)- This test is done to
detect vitamin B12 deficiency as well
as to distinguish and detect lack of
intrinsic factor (IF) and malabsorption.
So schilling test is used for detection
• Good renal function and proper
urinary collection are prerequisite for
schilling test.
• Done in 4 stages
Stage 1 – Oral vitamin B12 + IM vitamin B12
• 1st small dose of a radioactiove form of B12 by mouth [1 mcg] 2nd
larger dose via intramuscularly [1000 mcg] 1 hour later Collection
of urine after 24 hrs
• * Normal individuals secreates more that 10% of the administered radioactive
vitamin b12 in 24hr urine.
• * Patients with pernicious anemia excretes less that 5% of the administered
dose.
• * If stage 1 is abnormal then stage 2 is done 3-7 days later.

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Stage 2 – Vitamin B12 + Intrinsic factor
• Administer oral radioactive B12 along with intrinsic factor  The test
can tells whether low vitamin B12 levels are caused by problems in
the stomach that prevent it from producing intrinsic factor
• If a Stage II test is abnormal, a Stage III test is performed.
Stage 3 – Vitamin B12 + Antibiotics
• If the defect is still not corrected, then the lesion is in the small
intestine likely The abnormal bacterial flora in the small intestine is
corrected by a 7 day antibiotic course with oral tetracycline
• The test then again repeated after 7 days of the antibiotic therapy.

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Stage 4 – Vitamin B12 + Pancreatic Enzymes
• Intake of pancreatic enzymes for 3 days —–> A small dose of vitamin
B12.
• This test determines whether low vitamin B12 levels are caused by problems
with the pancreas

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Pernicious anemia
• Pernicious anemia is associated with increased risk of
• gastric cancer and
• increased chances of atherosclerosis and thrombosis (because of elevated
homocysteine levels)

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Type III Ab against proton pump proteins
affect acid secretion TONY SCARIA 2010 kmc

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• In pernicious anemia  atrophic gastritis  gastric adeno carcinoma
in fundus regions

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Iron deficiency anemia

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Hepcidin is the main regulator of iron
absorption
• Increased hepcidin  causes internalisation& degradation of
ferroportin
• Hence iron is sored with in enterocytes as bound to ferritin and is
shed along with enterocyte

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Hepcidin main regulator of iron absorption
Basolateral transport requires ferroportin &
hephaestin
• Iron from enterocyte is transferred to blood stream via ferroportin
• Hephaestin
• Converts Fe2+ Fe3+
• Helps in loading Fe3+ to transferrin

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Regulation of iron absorption
Increasing iron absorption
• Acidic pH
• HCl
• Citric acid
• Vitamin C
• Aminoacids
• Heme iron
Decreasing iron absorption
• Alkaline pH
• Phytate
• Tannates
• Tetracycline
• EDTA
• Nonheme iron

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Peripheral smear in IDA
• Microcytic and hypochromic red
cells with slight reticulocytosis
• microcytosis (initially)
hypochromia. (MH)
• Poikilocytosis
• Increased red cell distribution
width
• pencil cells
BONE MARROW
• Hypercellular bone marrow (having increased erythroid progenitors)
with depleted bone marrow iron stores.

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• Serum ferritin and serum iron are decreased
• serum transferrin and TIBC are increased.
• Red cell protoporphyrin levels are increased
• because there is decrease in the availability of heme (due to reduced iron
availability) resulting in elevated free erythrocytic protoporphyrin levels.
• RBC free protoporphyrin is normally 30-50 μg/dl whereas its value reaches >
200 μg/dl in iron defciency anemia.

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TIBC
• TIBC levels at
Top = Iron defciency (In iron
defciency anemia, TIBC is
raised)
Bottom = Chronic disease
(In Anemia of Chronic
Disease TIBC is low).

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Investigations
• Most specific / gold standard  BM biopsy with iron stain
• Most sensitive
• Serum transferrin receptor to log index of ferritin ratio>>>serum transferrin
receptor assay >> serum ferritin

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DMT 1 deficiency
• AR
• anemia – iron overload
syndrome
• Anemia present form birth
• Microcytic hypochromic anemia
• Transferrin saturation is high
• Serum ferritin is mildly elevated
• High serum iron
• Increased free erythrocyte
porphyrin
• Elevated ferritin

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Plummer – Vinson syndrome or Paterson-
Brown-Kelly syndrome.
• in middle-aged female
• Mucosal lesions in mouth & pharynx
• symptomatic hypo pharyngeal websdysphagia
• web in postcricoid region
• May develop postcricoid carcinoma.
• iron deficiency anemia constitute
• Hemoglobin is low
• hypochromic microcytic anemia.
• high iron binding capacity.
• rx
• oral Iron should
Haemoglobinopathies

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Haemoglobinopathies
• Qualitative
• Defect in abnormal structure of Hb with out change in quantity
• Quantitative
• Decreased synthesis & quantity of hb with out change in quality

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40 % HbS rest HbA
Sickling is more in renal hypertonic mileu

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Increased expression of adhesion
molecules on surface of RBC (VCAM)

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CF
• Anemia
• Reticulocytosis
• Splenomegaly is absent
• d/t autosplenectomy
• Increased size of heart  fat deposition
• Crisis
• Vasoocclusive crisis
• Sequestration crisis
• Aplastic
• Hemolytic crisis

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Sequestration crisis
• Most severe & dangerous
crisis
• Massive entrapment of sickle
cells in spleen 
splenomegaly hypovolemia &
shock
• Splenic sequestration 
gamna gandy bodies
Gamna gandy bodies in splenic sequestration
• Nodules formed by organisation of
splenic microhaemorrhages fb
fibrous tissue hemosiderin &
calcium deposition
• Also seen in
• Cirrhosis with Portal HTn
• CML

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Vaso occlusive crisis
• Most common clinical manifestation of sickle cell anemia
• d/t increased expression of adhesion molecules on surface of RBC 
make them sticky  painful ischemic infarction
• Autosplenectomy
• Hand foot syndrome
• a/c chest syndrome
• Fish mouth vertebra
• Crew cut skull

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• Most common cause of
osteomyelitis in sickle cell
anemia is salmonella
Crew cut appearance / hair end appearance
• d/t marrow expansion 
bone resorption & secondary
bone formation
• On Xray skull

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Fish mouth vertebra
• Increased chance of
vaso occlusion in
vertebral A
• Also seen in
Auto splenectomy
• d/t hypoxia & ischemia splenic tissue replaced by fibrous tissue
• Increased chance of infection by capsulated organisms (streptocoocus
pneumoniae & haemophilus influenza)

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Aplastic crisis
• Parvo virus infection
• Cb low reticulocyte count

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Peripheral smear
• Howell jolly bodies (nuclear
remnants)
• Drepanocytes
• Sickle shaped RBC
Howell jolly bodies

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• ESR is raised in all anemia except sickle cell anemia
• ESR is decreased as there is no rouleaux formn
Sickling in post capillary venule

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Sickle cell loose potassium and water but gain calcium

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• Cardiac enlargement & systolic murmurs
• Pulmonary vas occlusive crisis pulmonary arteria HTn Cor
pulmonale

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Common cause of death
• Infection
• a/c chest syndrome
Diagnosis
• High performance liquid chromatography
• HPLC
• electrophoresis
• 2 bands in sickle cell trait
• HbA (has negatively charged glutamine )
hence move faster towards +vely charged
anode than HbS
• HbS mpves faster towards cathode

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• Prenatal genetic testing can be done using the enzyme MstII
endonuclease.
• Chorionic villus sampling at 10-12 weeks of gestation is used to estimate
fetal DNA abnormality

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• HbS is tested by dithionite test

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HbSC disease
• Milder sickling disorder
• Mild anemia
• Lysine replaces glutamine in 6th position of beta chain
• 50% HbS & 50 % HbC
• Proliferative retinopathy higher incidence in HbSC ds
Hemolytic anemia

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Haptoglobin is an a/c phase protein

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Hereditary spherocytosis
• Autosomal dominant
• extravascular hemolysis
• Intrinsic defect in the red cell membrane that render the red cells spherical,
less deformable, vulnerable to splenic sequestration and destruction
• Mutations in Ankyrin are the most common cause in hereditary
elliptocytosis
• Caused by mutations affecting the proteins of RBC membrane
skeleton
• Ankyrin (most common)
• Band 3
• Spectrin
• Band 4.2 (Palladin)
• Glycophorin A
• Membrane protein defects
• Ankyrin (most common) >> Band 3> Spectrin >Band 4.2 (Palladin)>
Glycophorin A

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Clinical features of Hereditary Spherocytosis
• Anemia, Jaundice, Splenomegaly are classical features
• Gallstones, Leg ulcers
• Aplastic crisis may be triggered by Parvovirus B19 infection
• Hemolytic crisis may be precipitated by Infectious mononucleosis

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Lab features and Diagnosis of Hereditary
Spherocytosis
• Spherocytosis in peripheral smear lacking central pallor
(hyperchromic)
• MCV ↓ MCHC ↑ (the only condition where MCHC is increased)
• Osmotic fragility increased – main diagnostic test (RBCs abnormally
susceptible to lysis in hypotonic media)
• Diagnosis:
• based on red cell morphology and a modified version osmotic fragility test
(pink test)
Spherocytosis
• Dense cells no central pallor
• Hereditary spherocytosis
• Clostridial infections
• Auto immune haemolytic anemia
• Most common cause
• G6PD deficiency
• Splenomegaly a/w cirrhosis

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Treatment of Hereditary Spherocytosis
• Splenectomy is the TOC but not before the age of 4 years
• Whwn health is impaired
• Hemolysis
• Cholelithiasis
• Aplastic crisis in parvo virus infection
• Daily pencillin V 250 mg for atleast 5 yrs
• Pneumococcal vaccination before splenectomy
• Blood transfusion in severe haemolytic crisis
• FA = 5mg/day for the increased erythropoiesis
Hereditary elliptocytosis
• Mc cause is alpha spectrin deficiency

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Thalassemia
Thalassemia
• Autosomal recessive disorders
• Most common haemoglobinopathy

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Cooleys anemia

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Intermedia  Hb is normal >7g/dl

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• Microcytosis
• Poikilocytosis
• Target cells
• Reduced osmotic fragility
• Increased fetal hemoglobin
• Hypochromia
• Poly-chroma sia
• Reticulocytosis
• Iron overload
• Elevated unconjugated serum bilirubin

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NESTROF test
• Naked eye single tube red cell osmotic
fragility test
• Screeening test for beta thalassemia
• Positive in beta thalassemia
• Black lines cant be read
• if negative black lines can be read

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Persistence of fetal Hb on electrophoresis

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• Increased HbA2 in thalassemia minor
• Only diagnostic in thalassemia minor
Mentzer index
• Thalassemia
• Thirteen less semia

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Paroxysmal nocturnal haemoglobinuria

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Triad of PNH
• Hemolytic anemia
• Intravascular hemolysis
• Paroxysmal & nocturnal
• Maximum at acidic pH
• Bone marrow failure Pancytopenia
• Thrombosis
• Hepatic vein thrombosis  Budd Chiari syndrome
• Most common cause of death
PNH
• Aquired mutation in PIGA gene
• PIGA gene is required for synthesis of glycosyl phosphatidyl inositol (GPI
AP)which anchors following complimentary regulatory proteins
• CD 59(membrane inhibitor of reactive lysis)
• Most common
• CD 55(decay accelerating factor)
• C8 binding protein

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Diagnosis of PNH
• Ham’s test
• Best screening test
• Acidic pH will activate compliment pathway
• sucrose lysis test
• Sucrose will reduce pH  activate compliment
• Flow cytometry
• Confirmatory test

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PNH
• Acquired somatic mutation
• Rx
• Splenctomy  not needed
• Prednisolone
• Eculizumab

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Eculizumab is used for Rx of PNH

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• PNH is a/w aplastic anemia
• Will transform in to AML or myelodysplastic syndrome
Immunohemolytic anemia

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Direct coombs test
• Immune mediated
haemolytic anemia
• HDN
• ABO haemolytic
disease
• Mismatch transfusion

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Indirect coombs test
• Detect Ab in serum
• Screen pergannt woman for Ab that may
cause HDN
• Compatibility testing
G6PD deficiency

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• In the absence of G6PD
• Oxidant stress  denaturation of Hb Heinz bodies
• Cannot be stained with romanowsky stained with supra vital staining
• Bite cells

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Sideroblastic anemia
• Caused by enzyme disorder in
synthesis of heme moiety of Hb
 iron is trapped in
mitochondria (in perinuclear
mitochondria)

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• Sideroblastic anemia  ineffective erythropoiesis iron overload
Causes of sideroblastic anemia
congenital
• X linked recessive
• Most common
• Mitochondrial
• Autosomal dominant
Acquired
• AI disorders
• SLE
• RA
• Iron overload & porphyria
• Hematological
• Leukemia
• Myelodysplastic syndrome
• Drugs
• INH
• Chloramphenicol
• Pb
• Alchol
• Pyridoxine deficiency

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• PS
• Dimorphic
• microcytic hypochromic +
macrocytosis
• Basophilic stippling
• Target cells
• Pappenheimer bodies
• BM
• Ringed sideroblasts in Prussian
blue

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Rx of sideroblastic anemia
• Pyridoxine in high doses
• Exogenous erythropoietin
• Transfusion dependant

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Pearson syndrome
• Mitochondrially inherited
• Congenital Sideroblastic anemia
• Pancytopenia with macrocyosis

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Transfusion
• Indication
• Blood loss > 20 %
• PCV falls below 30 %

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Shelf life with anticoagulants
• ACD  21 days
• CPD 28 days
CPDA if used shelf life  35 days
• Citrate as anticoagulant
• Phosphate as buffer
• Dextrose energy for cells
• Adenosine for RBC survival

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SAGM for 42 days
• Sodium chloride
• Adenosine
• Glucose
• Mannitol

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Westegren ESR

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Malaria
Severe PK deficiency

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CRP
• a/c phase reactant
• Synthesised by liver
• Gene is on chromosome 1 q
• Normal value is 10 mg/dl
• Increases after 4- 6 hrs
• Reaches a peak in 24 hrs
• Remains elevated for 48 hrs
• Consistently below 10mg/dl  reliable indicator to r/o sepsis
• Returns to normal earlier than ESR in response to Rx
• Stable half life

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• CRP raised in
• Burns
• Viral & bacterial infn
• Active inflammation
• In late pregnant women
• CRP normal ESR elevated in
• a/c or untreated polymyalgia rhumatica
hs CRP
• Used as a rough Prognosis of heart
ds

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Pb poisoning

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Pb poisoning
• Sideroblastic anemia
• Hemolysis
• Punctate basophilia

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Pure red cell aplasia
• Mostly auto immune
• Affecting red cell precursors
• Diamond Black fan syndrome  congenital red cell erythroplasia
• Thymoma
• AI d/s
• Drugs  NSAIDs/mycophenolic acid
• Large granular lymphocytic leukemia
• Viral  parvo virus B19

22-04-2018
Diamond blackfan syndrome
• Hereditay pure red cell aplasia
• Fanning of upper limb with triphalangeal thumb
Endocrine disorders  anemia

Hematology revision notes pathology

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