Hematopoiesis

Hematopoiesis

RBC Inclusions

Inclusion	Composition	Seen in
Howell–Jolly bodies	• DNA remnants	• Post-splenectomy • Megaloblastic • Sickle cell disease (NOT THALASSEMIA) • Dyshematopoiesis • Hemolytic anemia
Reticulocytes	• Residual RNA (bluish network)	• Hemolysis • Blood loss • Response to therapy
Basophilic Stippling	• Ribosomal RNA	• Coarse → Lead poisoning • Fine → Thalassemia, Megaloblastic Anemia • Also seen in 5’ Pyrimidine nucleotidase
Heinz bodies	• Hb denaturation • Stained by Crystal Violet stain • (Supravital stain)	• G6PD
Pappenheimer Bodies	• Iron granules	• Sideroblastic anemia • Post-splenectomy
Cabot Rings	• Arginine-rich • Mitotic spindle remnants • Figure of eight/loop	• Severe anemia • Megaloblastic anemia • Sideroblastic anemia
Hemoglobin H inclusions	• β4 tetramers • (precipitated Hb H)	• α-thalassemia (Hb H disease)
Siderotic Granules	• Iron • In Prussian blue stain	• Sideroblastic states
Target cells	ㅤ	• Thalassemia • Iron deficiency anemia • Liver disease • Post-splenectomy • Hemoglobin C disease
Dohle bodies	• ER remnant	• Bacterial sepsis
Perinuclear hof	• Golgi apparatus	• Plasma cells

Sideroblastic anemia

• Causes
• B6 deficiency
• Alcohol
• ALA synthase (XLR)
• Lead poisoning

Sideroblasts

• Normal immature erythroid precursors in bone marrow

• Present in <30% of marrow cells

• Ringed Sideroblasts
• Bone marrow smear
• Formed when Protoporphyrin is absent
• Iron cannot bind → accumulates as granules around mitochondria

• Iron deposits appear scattered around the nucleus
• ≥5 granules
• ≥1/3rd nucleus

• Detected by:
• Pearl stain/ Prussian Blue Stain

Timelines of Hematopoiesis Sites

• From 5th month of Intrauterine life → Haematopoiesis is from Long Flat bones till 20 years

Erythropoiesis

Stages of Erythropoiesis

• Cells with "blast":
• In bone marrow, have nucleus.

• Cells without "blast"
• In peripheral blood, no nucleus.

Reticulocytes

• Normal count: 0.5 to 2%

• Contain Reticulum of RNA

• Requires Supra vital staining
• Stains RNA in reticulocytes
• To be viewed ≤ 2 hrs after staining.
• New methylene blue >>
• Brilliant Cresyl blue

• Super Vital () avanel → Brilliantil () padikkanam → New (New meth) Life kittum

Factors Regulating Erythropoiesis

• Promoting Factors:
1. EPO
2. Growth hormone
3. Cortisol
4. Thyroid hormone (T3)
5. Testosterone

• Inhibiting Factor:
• Estrogen
• causes ↓ RBC count in females

Clinical Significance of EPO

• Polycythemia vera:
• Gain of function mutation: In JAK 2 receptor.
• Effect: ↑ EPO → ↑↑ RBCs.
• Rx: Ruxolitinib (JAK 2 inhibitors).

• Drug forms:
• Epoetin alfa
• S/c twice or thrice weekly doses
• Darbepoetin alfa.
• Higher t1/2
• Once weekly or biweekly doses

• Indications:
1. Anemia of chronic disease (e.g., CKD).
2. Dialysis.

• Ensure before EPO administration
• S. Ferritin > 500ng/mL.
• PSAT (% Saturation of Transferrin) > 30%

• Blood doping:
• Misused by athletes to ↑ RBC count.

Reticulocyte Production Index (RPI)

Hematology and Red Blood Cells (RBCs)

• If Plt <20k → Transfuse and do BM
• NOT A C/I

Bone Marrow Studies

• Adult Bone Marrow Composition:
• Cells : Fat ratio = 1 : 1 (50% cells, 50% fat).
• Myeloid : Erythroid ratio = 3 : 1
• Cellularity = 100 - Age
• Eg, 30 year old → 70% Cellularity

• Bone Marrow Sites:

Notes	Sites
Most common	PSIS / iliac crest.
Obese individuals	ASIS
Children (preferred)	Anteromedial part of the tibia

• Bone marrow contains blast cells (have a nucleus).

Bone Marrow Findings in Various Disorders

Peripheral Smear

• Components: 
• RBC, WBC, Platelets

Stains:

• General: Romanowsky family.
• Members: Gimsa, Leishman, Wright, Jenner, Field.
• Most used (99% cases): Gimsa and Leishman.

• Field stain:
• For Plasmodium falciparum

Red Blood Cell Formation (Erythropoiesis)

RBC Indices

Mentzer Index

• Formula: (MCV / RBC count) x 100.

• Interpretation:
• <13: Thalassemia minor
• Mnemonic: Tha Lessemia → < 13
• >13: Iron Deficiency Anemia

Measurement of PCV and ESR:

ㅤ	Wintrobe tube	Westergren pipet	Automated ESR
Tube	Small	Long	ㅤ
Measures	PCV and ESR	Only ESR	ㅤ
ㅤ	Rob both PCV and ESR	WEST → ESR → Only ESR	ㅤ
Vacuntainer	Lavender	Light blue	Black
ㅤ	Lovely Wind	Western blue	Black Auto

• Light Blue
• ESR → 4:1
• APTT/PT/INR → 9:1

RDW

 

Microcytic Anemia

• Low MCV, MCH, MCHC.
• MCV < 80 fL

• Causes:
• Iron Deficiency
• Thalassemia
• Anemia of Chronic Disease
• Sideroblastic Anemia

• Mnemonic:
• for causes: SITAL
• Iron deficiency- Truely poor, Don't have anything in hand as well as in Bank
• Anemia of chronic disease - Varsha - Behave as echi( Rest all low ), But have money in bank ( Ferritin high )
• Sideroblastic anemia - Nott nirodhanam - High money ( high fe ferritin ) But cant use, So capacity (TIBC) low. Just opposite to IDA
• Mnemonic: TIBC
  • Top → I
  • Bottom → C, S

Macrocytic Anemia

• MCV > 100 fL

• Megaloblastic Anemia
• Associated with hypersegmented neutrophils.
• B12 Deficiency:
• Neurological problems.
• Subacute combined degeneration of the cord.
• Serum B12 ⬇.
• Folate Deficiency:
• Serum Folate ⬇.

• Normoblastic (Non-megaloblastic) Anemia
• Causes:
• Alcohol
• Liver disease
• Hypothyroidism
• Pregnancy
• Reticulocytosis
• Myelodysplasia
• Drugs: cytotoxic

Normocytic Anemia

• Normal MCV (80-100 fL)

• Often seen in Hemolytic Anemias.

Specific Hemolytic Anemia Types

• Sickle Cell
• Blood smear shows sickle cells.
• Sickle solubility test:
• Detects hemoglobin S.
• Does not differentiate disease from trait.
• Hb Electrophoresis: For asymptomatic patients to detect trait.

• Autoimmune Hemolysis
• Diagnosis: Coombs test.
• Treatment: Steroids.

• Hereditary Spherocytosis
• Blood film shows spherocytes.
• Diagnosis: Osmotic fragility test.
• Treatment: Splenectomy.

• G6PD Deficiency
• Blood film shows Heinz bodies.
• Diagnosis: Check G6PD levels.
• Treatment: Stop offending drugs.

Sideroblastic anemia

Sideroblastic anemia

• Causes
• B6 deficiency
• Alcohol
• ALA synthase (XLR)
• Lead poisoning

Sideroblasts

• Normal immature erythroid precursors in bone marrow

• Present in <30% of marrow cells

• Ringed Sideroblasts
• Bone marrow smear
• Formed when Protoporphyrin is absent
• Iron cannot bind → accumulates as granules around mitochondria

• Iron deposits appear scattered around the nucleus
• ≥5 granules
• ≥1/3rd nucleus

• Detected by:
• Pearl stain/ Prussian Blue Stain

Heme Synthesis

• In marrow:
• Iron + Protoporphyrin = Heme
• Heme + Globin = Hemoglobin

• Succinyl CoA + Glycine → Protoporphyrin (in mitochondria)
• Enzyme: Delta ALA dehydratase

Peripheral smear

• Pappenheimer

Types of Sideroblastic Anemia

Anemia of chronic disease (AOCD)

• Cause → ↓ sed activity of hepcidin
• Hepcidin:
• Master regulator of iron,
• ⛔ Ferropotin (iron entry/release).
• ⛔ release of iron from Ferritin

Macrocytic Megaloblastic Anemias

• Large RBCs.

• Type of macrocytic anemia with MCV >90 fL.

• Due to VitB12 or folic acid deficiency.

Differences between B12 deficiency and Folate deficiency

Laboratory Test	B12 Deficiency	Folic Acid Deficiency
Absorption Site	Terminal ileum  (requires Intrinsic Factor)	Jejunum, Duodenum?
Intrinsic Factor	Released by Parietal cells	ㅤ
Pernicious Anemia	Due to ↳ ↓ Intrinsic Factor ↳ Parietal cell issues	ㅤ
Cause	• Veganism	• Inadequate vegetable intake
Serum B12 level	↓↓↓	Normal
Serum folic acid level	Normal	↓↓↓
Serum LDH, Bilirubin	↑↑↑↑	Normal
Achlorhydria	++	ㅤ
Methyl Malonic aciduria	+++	ㅤ
Specific Test	Schilling test  • Determine cause of megaloblastic anemia • Positive ⇒ No absorption problem	Figlu test  (Formiminoglutamic acid) • After Histidine Load Test
Neurological signs	Present (SACD)	Absent

B12 Deficiency

• B12 needed for:
• Methylmalonyl CoA → Succinyl CoA (myelin production).
• Homocysteine → Methionine.

• Deficiency → ↑ Methylmalonyl CoA → ↓Succinyl CoA → ↓myelin.

Causes

• Nutritional (strict vegans)

• M/c: Alcohol

• Gastric
• ↓ intrinsic factor:
• Autoimmune pernicious anemia,
• Gastrectomy
• Gastric bypass surgery

• Intestinal
• Crohn's disease
• Diphyllobothrium latum (Fish tapeworm)
• Stagnant loop syndrome
• SIBO
• Terminal ileum resection
• Malabsorption

• Measure Methylmalonic acid level in urine after fasting

NOTE:

• In Orotic aciduria, PS shows
• Hypochromic Megaloblastic anemia
• Seen in
• Type 2 hyperammonemia
• No anemia
• Allopurinol

Clinical Features:

• Pale, fatigued.

• Reversible dementia in old age.

• Pure vegetarian diet

• Hyperpigmentation of knuckles and phalanges.

• Subacute Combined Degeneration of Spinal Cord
• Dorsal column + UMN

Peripheral Smear Findings (Megaloblastic Anemia):

• Howell-Jolly bodies

• Cabot ring

• Fine Basophilic stippling

• Macroovalocytes (Macrocytes),

• Hypersegmented neutrophils
• ≥ 5% neutrophils with ≥ 5 lobes /
• ≥ 1 neutrophil with ≥ 6 lobes.

RBC Inclusions

Inclusion	Composition	Seen in
Howell–Jolly bodies	• DNA remnants	• Post-splenectomy • Megaloblastic • Sickle cell disease (NOT THALASSEMIA) • Dyshematopoiesis • Hemolytic anemia
Reticulocytes	• Residual RNA (bluish network)	• Hemolysis • Blood loss • Response to therapy
Basophilic Stippling	• Ribosomal RNA	• Coarse → Lead poisoning • Fine → Thalassemia, Megaloblastic Anemia • Also seen in 5’ Pyrimidine nucleotidase
Heinz bodies	• Hb denaturation • Stained by Crystal Violet stain • (Supravital stain)	• G6PD
Pappenheimer Bodies	• Iron granules	• Sideroblastic anemia • Post-splenectomy
Cabot Rings	• Arginine-rich • Mitotic spindle remnants • Figure of eight/loop	• Severe anemia • Megaloblastic anemia • Sideroblastic anemia
Hemoglobin H inclusions	• β4 tetramers • (precipitated Hb H)	• α-thalassemia (Hb H disease)
Siderotic Granules	• Iron • In Prussian blue stain	• Sideroblastic states
Target cells	ㅤ	• Thalassemia • Iron deficiency anemia • Liver disease • Post-splenectomy • Hemoglobin C disease
Dohle bodies	• ER remnant	• Bacterial sepsis
Perinuclear hof	• Golgi apparatus	• Plasma cells