red blood.cells

Bone marrow aspirate

Bone Marrow Aspirate

• Needles:

• Salah needle (identified by side screw)

• Klimaneedle (identified by longitudinal screw)

Bone marrow study

Needle used:

• Jamshidi BMB needle (T-shaped)

• Normal bone marrow:

• Consists of bony trabeculae, cells & fat

• Normal cell to fat ratio in an adult - 1:1

• To measure cellularity: (100-age) % cells

Site for bone marrow studies

MC Site: PSIS (Posterior superior iliac spine)

• In obese patients: ASIS (Anterior superior iliac spine)

• In children: Anterior medial surface of upper end of tibia

Periferal smear stains

Peripheral Smear

• Romanowsky family of stains:

• Giemsa stain

• Leishman stain

• Wright Stain

• Jenner Stain

• Field Stain (Stain for Plasmodium falciparum)

Rbc formation

Inside bone marrow, cells with nucleus:

Pronormoblast

(First cell to contain Hemoglobin, seen on electron microscope)

Early normblast/Basophilic normblast (B)

Intermediate normblast/Polychromatophic normblast (P)

(Hemoglobin can be seen on light microscope)

Late normblast/Orthochromic normblast (O)

• Inside peripheral blood, cells lack nucleus:

Reticulocyte

(First cell in peripheral smear)

(First cell without nucleus)

Erythrocyte/RBC

Reticulocyte

Cell that contains a reticulum of RNA

• To stain a Reticulocyte: Supravital stain (In-living stain; no methanol, ethanol fixation)

• Examples:

￫ New methylene blue

￫ Brilliant cresyl blue

• Normal value: 0.5 to 2%

• Corrected Reticulocyte count = Reticulocyte count x (Hbpariem/HbNormalPerson)

• Reticulocyte Production Index = Corrected Reticulocyte count/Maturation factor

00:06:35

FMGE Jan 2024, June, Dec, 202

Normal rbc indices

Mean Corpuscular Volume (MCV)

• Size of RBC

• Normal MCV: 80 - 100 FL (Normocytic)

￫ <80 FL: Microcytic

￫ >100 FL: Macrocytic

• Mean Corpuscular Hemoglobin (MCH)

• Normal value: 27 - 32 pg

• Mean Corpuscular Hemoglobin Concentration (MCHC)

00:10:04

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Red Blood Cells

• Normal value: 33 - 37 g/dl

o Only anemiawhere Hb concentration (MCHC)

will increase: Hereditary Spherocytosis

• Red Cell Distribution Width (RDW)

• Variation of size in RBC: Measure of Anisocytosis

• Normal value: 11.5% - 14.5%

• Packed Cell Volume (PCV)

• Also called Hematocrit (Hct)

• Normal value: & 45%

Wintrobe vs westergren

Wintrobe tube Westergren pipette

• Identification: Openat one end and closed at another • Identification: Open from both ends

• It is used to measure PCV and ESR (erythrocyte • It is usedonly to measure ESR sedimentationrate. (notPCV)

Micro hypo anaemia features

MICROCYTIC HYPOCHROMIC ANEMIA

00:14:53

Features

• S= Sideroblastic Anemia

• I= IDA (Iron Deficiency Anemia)

• T= Thalassemia

• A = AOCD (Anemia of Chronic Disease)

• L= Lead Poisoning (type of Sideroblastic Anemia)

Iron metabolism

Iron Metabolism

• Iron fromFood: Fe

• Iron absorbed from the duodenum can't be inFe" form

• Cytochrome b reductase converts Fe" to Fe?

• Fe" enters the duodenum via Divalent Metal Transporter -1

(DMT-1)

• Fe gets into the blood via Ferroportin

• Fe"is converted back to Fe" by Hephaestin& Ceruloplasmin

• Transportation of iron occurs via Transferrin

• Transferrin Saturation: 33%

• Storage form of iron: Ferritin

• Masterregulator of iron: Hepci

IDA causes

Iron Deficiency Anemia

Causes

• Diet (lack of iron in diet)

• Blood loss:

• Trauma

o PUD

• Colon cancer

• Menorrhagia

Hookworm infestation

Bood findings of ida

Blood Findings

• Low Hemoglobin

• Low MCV (microcytic anemia)

• Low MCH, MCHC (hypochromic anemia)

• High RDW (Anisocytosis)

• Low Serum Iron

• Low Transferrin Saturation

• Low Ferritin

• High TIBC (total iron binding capacity)

• Most Specific Test: Bone Marrow Studies

• Stain: Prussian Blue Stain

• Peripheral Smear:

• Microcytic hypochromic anemia with anisocytosis (variation in size) & poikilocytosis (variation in shape: penc

Anaemia of chronic disease pathophysiology

Anemia Of Chronic Disease

Pathophysiology

00:24:25

FMGE Jan 2024

Chronic diseases like Rheumatoid Arthritis (RA)

Release of Interleukin (IL6)

Act on the liver and release Hepcidin

Inhibits Absorption of Iron (Inhibits ferroportin) &

Inhibits the release of iron from ferritin

Serum Iron & Transferrin saturation will decrease

TIBC (total iron binding capacity) will decrease, Ferritin will increase

Inv and mx of anaemia of ch disease

Investigations & Management

• Peripheral Smear: Normocytic Normochromic Anemia» Microcytic hypochromic anemia

• Management: Treat the underlying chronic disease

Features Ida Aocd Sid anaemia

Serum Iron Decreased Decreased Increased

Transferrin Saturation Decreased Decreased Increased

Ferritin Decreased Increased Increased

TIBC Increased Decreased decreased

Sideroblastic anaemia

Pathophysiology

Succinyl Coenzyme-A

+ (ALA synthose)

ALA

+ (ALA dehydratase)

Protoporphyrin+ Fe

1 (Ferrochelatase)

Heme

Causes of sideroblastic anaemia

Causes

• Enzyme deficiency

• Vitamin B, (co-factor) deficiency

• Isoniazid consumption

• Lead (Pb) poisoning

• Inhibitsferrochelatase

• Alcohol poisoning

• Inhibitsferrochelatase

Investigation of sideroblastic anae

Nvestigations

• Peripheral Smear

• Pappenheimer Bodies (iron)

• Bone Marrow Studies

• Ringed Sideroblasts on Perl's/Prussian blue stain

￫›= 5 iron granules make a ring around the nucleus, covering

more than 1/3°° of the circumference of the nucleus

Macrocytic anemia causes

MACROCYTIC ANEMIA

Causes

• Lady: Liver Disease

• Harding: Hypothyroidism

• Medical: Megaloblastic Anemia

• Pernicious Anemia

• College: Chemotherapy

Megaloblastic anaemia

Megaloblastic Anemia

• Occursbecause of deficiency of Vitamin Biz and Folic acid (FA)

• Bizis absorbed from the terminal ileum with Intrinsic factor (IF)/Castle factor (parietal cells of the stomach)

• FA absorbed from the je ju

Pernicious anaemia

Pernicious Anemia

• Antibodies form against the parietal cells of the stomach

• Inhibits the formation of intrinsic factors (IF)

• Prevents absorption of Vitamin B,2

Important Information

• Parasite infection that causes Biz deficiency: Diphyllobothrium Latum

• Hookworm infection causes iron deficiency anemia

Pernicious anaemia c/ f

Clinical Features

• Pure vegetarian or vegan patient

• Bi is majorly in eggs, meat, milk in a normal diet

• Hyperpigmentation of phalanges & knuckles

• Neurological problems:

• S- Sub

• A - Acute

o C-Combined (sensory and motor)

• D - Degeneration

Biochemical findings. Megaloblastic anaemia

Biochemical Findings

• Increased Serum Bilirubin and LDH (due to hemolysis)

• Decreased Vitamin Biz

• Decreased Folic Acid

• Increased Homocysteine

• Increased Methylmalonyl CoA

• Anti-IF antibody (Pernicious Anemia)

• FIGLU test (measures folic acid levels in urine)

PBS megaloblastic anaemia

Peripheral Smear Findings

• Macrocytic Macro-ovalocyte

• Howell Jolly Bodies:

• Single

• Composedof DNA remnant Normal

• Seen in:

￫ Megaloblastic Anemia

￫ Post-splenectomy

• Cabot ring:

• Made up of arginine rich mitotic spindle

• Fine Basophilic Stippling

• Seenin:

￫ B, deficiency

￫ Thalassemia

• Coarse Basophilic Stippling is seen in lead (Pb) poisoning

• Hyper-segmented Neutrophil:

• 1 Neutrophil with 6 or more lobes or 5% Neutrophil with 5 lobes Hemolytic Anemias (normal - 3 to 5 lobes)

Haemolytic anaemia

• Intravascular Hemolysis (inside blood vessel)

• High LDH

• Hemoglobinuria

• Decreased Haptoglobin levels

• Decreased Hemopexin levels

• Extravascular Hemolysis (in spleen, liver):

• Splenomegaly

• Gall stones

Rbc membrane

Rbc Membrane

• Protein responsible for the biconcave shape of RBC: Spectrin

• Most abundant protein: Glycophorin

• Band-3 defect: Pincer cells (mushroom)

Heriditary spherocytosis

Hereditary Spherocytosis

• Most common defect: Ankyrin gene defect (AD)

• Most severe defect: Spectrin gene mutation (AR)

• Defect never seen: Glycophorin

Hereditary sperocytosis. Pathology

Pathophysiology

Normal shape of RBC: Biconcave

Ankyrin gene defect

个

Spherical RBC due to water loss

￩

Increased MCHC

Lifespan decreases to 10-20 days

RBCs lysed by the spleen

(Extravascular Hemolysis)

Her spherocytosis c/ f

Clinical Features

• Jaundice

• Splenomegaly

• Gall stones

Diagnosis and management her spherocytosis

Diagnosis & Management

• Peripheral smear

• Spherocytes

￫ MCC: Auto-immune hemolytic anemia

￫ 2° MCC: Hereditary Spherocytosis

￫ 3" MCC: G6PD deficiency anemia

• Increased Reticulocyte count

• Decreased MCV

• Increased MCHC

• Increased RDW

• Aplastic Crisis in Parvovirus B19 Infection

• Screening test: Osmotic Fragility Test

• Normal RBC: Lysis starts at 0.5% NaCl and is completed by 0.3%

o In HS: Lysis starts before 0.7% NaCl (right shift) due to

increased fragility

• Confirmatory test: Eosin-5-Maleimide Test

• Flow cytometry using Eosin-5-Maleimide dye

• Treatment

• Splenectomy

￫ Give prophylactic vaccination for capsulated organisms like

Neisseria meningitidis, Hemophilus influenzae, and

Pneumococcus before splenectomy

Rbc other shapes

Other Shapes

Acanthocytes Spurr Cell. Echinocytes

Also known as: Target cells or Codocytes. Acanthocyte/spike cell Burr cells

Thallasemua. Alkaptonurea. Burns and uremia

G6 po4 deficiency. Causes

Causes

• X-linked recessive (XLR) disorder

• Seen more commonly in males

• Intermittent disorder

• Triggers:

• Infections (MC)

• Anti-malarial drugs

• Fava beans consumption

• No splenomegaly

• No gallston

G6 po4 deficiency diagnosis

Diagnosis

• Blood Findings:

• Heinz Bodies (Denatured hemoglobin)

￫ Crystal Violet stain

• Bite cells/Degmacytes on Romanowsky stain

• Screening test:

• Fluorescent spot test

• Methemoglobin reduction test

• Confirmatory test: ELISA (G6PD levels)

• Romanowsky stain

• Howell-Jolly Bodies (single dot) -

Vitamin B12 Deficiency

• Pappenheimer Bodies (multiple dots) -

Sideroblastic Anemia

• Crystal Violet stain:

• Heinz-bodies - G6PD Deficiency

Pnh causes

Paroxysmal Nocturnal Hemoglobinuria

-

Causes

• Only acquired defect causing hemolytic anemia due to

Intravascular hemolysis

• PIGA gene defect:

• GPI anchors not formed, leading to the absence of:

￫ CD55 (DAF)

￫ CD59 (MIRL)

• Decreased Leukocyte alkaline phosphate (LAP) enzy

Pnh diagnosis

Diagnosis

• Screening test:

• Ham'sacidified serum test

• Sucrose lysis test

o Gel card test

• Confirmatory test:

o Flow Cytometry: Dual Population of CD markers

￫ CD 55 Negative, CD 59 Negative

￫ CD 55 Positive, CD 59 Positive

• FLAER: Measures GPI anchors

Pnh complication

Complications

• Thrombosis

• Cerebral vein thrombosis

• Hepatic vein thrombosis (Budd-Chiari syndrome)

• Aplastic Anemia

• Acute Leukemia

Pnh treatment

Treatment

• Eculizumab: Acts like CD 55 & CD 59

• Curative treatment: Hematopoietic Stem Cell Transplant

Haemoglobinopathy sickle cell anaemia causes

Types

HEMOGLOBINOPATHIES

Sickle Cell Anemia

Causes

• Autosomal Recessive condition

• Previously, it was thought to be due to the replacement of Glutamate by Valine at the Beta-6 position

• Latest advance: Replacement of Glutamate by Valine at Beta 7 position

• Types:

• Sickle cell Trait (some Hb is sickled)

• Sickle cell Anemia (all Hb is sickled)

Sickle cell anemia

Causes

Clinical Features

• Vaso-occlusion

• Skull: Crew-cut appearance/ hair on end

• Fish mouth vertebrae

• Auto-splenectomY

Sickle cell anaemia

Diagnosis

Diagnosis

• Peripheral Smear:

• Sickle cells or Drepanocytes

￫ Seen in sickle cell anemia

￫ Not seen in sickle cell trait patients

• Sickling Test:

• Blood reacts with 2% sodium metabisulfite ￫ Induces hypoxia ￫

Sickling occurs

• Hemoglobin Electrophoresis

ｰ

• H: Hemoglobin

• A: Adult Hb

• F: Fetal Hb

• S: Sickled Hb

• A: A, H

• HPLC (Gold standard investigation)

Sikle cell anaemia treatment

Treatment

• Hydroxyurea

• Increases fetal hemoglobin (Increased oxygen affinity)

Hb c disease

Hbc Disease

01:13:10

• At the beta-6 position, Glutamate is replaced by Lysine

• HbC crystals are seen

Thalassemia intro

Thalassemia

• Normal adult hemoglobin: a,B,

• 2 alphachains from 4 alphagenes on chromosome 16

• 2 beta chains from 2 beta genes on chromosome 11

• Alphagene deletion: a-Thalassemia

• Betagene mutation: B-Thalassemi

Alpha thallasemia

Alpha Thalassemia

01:15:35

aa/aa

aa/a-

aa/--

a-/-- with B-4 tetramers

Alpha (a) Gene Condition

Normal

Asymptomatic

Alpha Thalassemia Trait (asymptomatic)

HbH Disease

Golf ball inclusions

- - / -- with y- y-4 tetramers Hb Bart's Disease

• Results in intra-uterine death or Hydrops fetalis

Eta thallasemia

Beta Thalassemia

• Splicing Mutations (MC)

• MCC in India

• In Introns (IVS), at codon 1-5, there is a change of Gto C

• Frameshift Mutations

• Codon 8/9

• Codon 41/42

• Only Deletion

• 619 base pair deletion

Beta thalasemia

Types

01:18:08

NEET PG 2025

FMGE Jan 2023

Features B -thalassemia major/cooley's

anemia

B-thalassemia

intermedia

B-thalassemia

minor/trait

Clinical Features • Require lifetime blood transfusion

• Pallor

• Jaundice

• Splenomegaly

• Chipmunkfacies

• Crew-cut/Hair-on-end appearance

• Target cells/Codocytes onPBS

• Pallor

• Jaundice

• Splenomegaly

Asymptomatic

Hemoglobin Levels 3-5 5-8 >8

Iron Profile Increased Normal Normal

Hb Electrophoresis & HPLC HbF >90%

(Gold Standard) HbA, >3.5%

Ida v/ s thallasemia

Iron deficiency anemia Features Beta-thalassemia minor

>13 Mentzer Index (MCV/RBC) ‹13

Increases RDW Normal

‹3.5% HbA, confirmation >3.5%

Nestrof test

Nestrof Test

• Naked Eye Single Tube Red Cell Osmotic Fragility (NESTROF) Test

• Normal saline is added to red blood cells

• Normal RBCs break down - Line visible

• Thalassemia RBCs do not break down - Line not visible

Autoimmune haemolytic anaemai

• Type II hypersensitivity disorder

• Two types of antibodies:

• Warm antibodies (37°C): IgG

￫ CLL

• Cold antibodies (4°C): IgM

￫ Mycoplasma infection

• Cold Hemolysin disease/Paroxysmal Cold Hemoglobinuria:

• Occurs at 4°C

• Due to IgG: Donath Landsteiner antibodies

Schtocytic angiopathic

Systocytic angiopathic. CausesSCHISTOCYTIC/ANGIOPATHIC HEMOLYTIC ANEMIA

• Schistocyte/ fragmented RBCs

Causes

01:25:56

01:29:50

1. Prosthetic heart valves

RBC lysis

Macro-angiopathic hemolytic anem

2:: Hemolytic Uremic Syndrome, TTP & DIC

Micro-angiopathic hemolytic anemia

Harmolytic anaemias

HEMOLYTIC ANEMIAS

01:31:22

Intravascular Hemolysis

High LDH

Hemoglobinuria

Decreased Haptoglobin

Decreased Hemopexin

PNH

PCH

G6PD Deficiency

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Page 177

Red Blood Cells

Extra-vascular Hemolysis (Spleen, Liver) Hereditary Spherocytosis

Sickle Cell Anemia

Thalassemia

GOPD Deficiency

Intra-corpuscular Defect Hereditary Spherocytosis

G6PD Deficiency

Hemoglobinopathies

Extra-Corpuscular Defect

AIHA

MIHA

Dog ear projections

• RBC precursors in bone marrow show dog-ear projections are seen in:

• Parvovirus B19 infection

Beta thallasemia types