Hematology (Purple/Gold) + Slides (no case studies)

GM-CSF, IL-3, EPO

CFU-E

Rubriblast → Prorubricyte → Rubricyte → Metarubricyte → Reticulocyte → Erythrocyte

Growth Factors:

Committed Progenitors:

GM-CSF, IL-3,5

CFU-GM [EO, BASO]

Myeloblast → Promyelocyte (→Basophil/Eosinophil) → Myelocyte → Metamyelocyte → Band Neutrophil → Segmented Neutrophil

Monoblast → Promonocyte → Monocyte [Macrophage (tissue)]

Growth factors:

Committed Progenitors:

GM-CSF, IL-3,6,11, TPO

CFU-MK

Megakaryoblast → Promegakaryocyte → Megakaryocyte → Platelet (thrombocyte)

Growth factors:

Committed Progenitors:

IL-1,2,4,5,6,7,12,15

CFU-TNK, CFU-B

CLP

Lymphoblast → Prolymphocyte → Lymphocyte → NK cell, T cell, B cell (→ Plasma Cell)

Growth Factors:

Committed Progenitors:

After HSC:

Left Shift

Increase in the number of immature white blood cells (particularly bands)

Routine Lab Test

Complete Blood Cell Counting & WBC differential
Peripheral Blood Smear/Film - preparation, staining, and examination

Advanced Lab Tests

Flow cytometry (for immunophenotyping), Molecular diagnostics (gene mutations), Cytogenetics (chromosomal changes/translocations), Cytochemical stains (MPO, SBB, NSE, etc.)

Additional Hema Lab Tests

G6PD phenotyping, SDS-PAGE (e.g. heriditary spherocytosis/vWD), HPLC (e.g. SCD), Body fluid analysis (cell count)

Romanowsky stains

Contains eosin Y and methylene blue for Peripheral Blood FIlms

Free methylene blue

Basic and stains acidic cellular components (RNA, DNA, basophil granules) from blue-grey to dark blue in color

Free eosin Y

Acidic and stains basic cellular components (Hemoglobin/Eosinophilic granules) from light to deep pink to red or orange in color

Neutrophils in Romanowsky Stain

Cytoplasmic granules with neutral pH and picks up staining characteristics from both stains (thiazine-eosinate complex). Appear pink/violet (lilac)

Basis of Romanowsky staining

Check staining quality, smear prep quality, sample integrity (PLT clumps, fibring strands, cell distribution), do WBC/PLT estimates (correlation w/ analyzer report), do WBC differential, abnormal WBCs (blasts)/reactive lymps, alterations in RBC/PLT morphology (shape, color, size, distribution pattern), parasites, bacteria

Metabolism

Embden-Meyerhof pathway, ATP production (i.e. Glycolysis)

Shunts in metabolism

Hexose monophosphate pathway (HMP), Rapoport-luebering pathway, Methemoglobin reductase pathway

Transmembrane (integral) proteins

Band 3, GLUT-1, Rh, ion pumps

Cytoskeletal (peripheral) proteins

Ankyrin, spectrin, actin, 4.1, junctional complexes, horizontal, and vertical interactions (HS/HE)

Hereditary Spherocytosis & Hereditary Elliptocytosis

Abnormal cytoskeletal proteins due to

Heme

Fe2+ & protoporphyrin IX

Hemoglobin function

Binds O2 readily in the lungs (high O2 affinity), unloads O2 easily in the tissues (low O2 affinity)

Oxygen dissociation curve (BAC)

Alkalosis, low 2,3-DPG, HbF → normal situation (steady state) → acidosis, high temperature, high 2,3-DPG

Basophilia

Due to allergic reaction and inflammation, possibly chronic inflammatory disorders/blood cancers

Iron Pathway

Intestines → Enterocyte (+Hepcidin) → Developing RBC → Circulating RBC → Splenic Macrophage (+Hepcidin) → Plasma Transferrin → Hepatocyte → Plasma Transferring → Circulating RBC (IEDCSPHPC)

Increase in hepcidin

If iron absorption is high in intestines and more iron is released, then the liver will notice a surplus pf iron levels and cause a ___ which blocks ferroportion from transporting iron out of Enterocytes, Macrophages, and Hepatocytes (EMH)

Decrease in hepcidin

If iron absorption is low in intestines and less iron is released, then the liver will notice depleted iron levels and cause a ___ which allows ferroportion to transport iron out of Enterocytes, Macrophages, and Hepatocytes (EMH)

Iron deficiency anemia caused by

Inadequate intake, increased need (insufficient stores), impaired absorption, and chronic blood loss

Stage 1 Iron Deficiency anemia (brief desc)

No evidence of anemia, RBC production normal, storage iron depletion, common stage

Stage 2 of Iron Deficiency anemia (brief disc)

Latent iron deficiency, relies on transport iron, restricted erythropoeisis starts, FEP increased CBC still normal (question Prussian blue results on BM biopsy)

Stage 3 of Iron Deficiency anemia (brief disc)

Overt anemia, storage depleted, functional iron low, low Hb and HCT, hypochromic, microcytic picture, anisocytosis (the first abnormality to show up on PBS) — usually with mild ovalocytosis, high rDW

Anisocytosis

The first abnormality to show up on PBS of Stage 3 Iron Deficiency anemia

Laboratory Test Values of IDA

Hemoglobin, serum iron, TIBC, ferritin, sTIR, hemoglobin content of reticulocytes (HSTFSH)

Normal Iron Status

N | Hemoglobin

N | Serum Iron

N | TIBC

N | Ferritin

N | sTfR

N | Hemoglobin content of reticulocytes

Stage 1 IDA Status (Latent)

Decreased | Ferritin

Increased | sTfR

Stage 2 IDA Status (Latent)

Decreased | Serum Iron

Increased | TIBC

Decreased | Ferritin

Increased | sTfR

Decreased | Hemoglobin content of reticulocytes

Stage 3 IDA Status (IDA)

Decreased | Hemoglobin

Decreased | Serum Iron

Increased | TIBC

Decreased | Ferritin

Increased | sTfR

Decreased | Hemoglobin content of reticulocytes

Screening tests of IDA

CBC: Hb, HCT, RBC indices, retic count, PBS examination [CHHRRP]

Diagnostic tests of IDA

Serum iron (SI) level, Total-Iron binding capacity (TIBC), Transferrin saturation (SI/TIBC*100), Serum ferritin level, Soluble transferrin receptor (sTFR) level, Hemoglobin content of reticulocytes [STTSSH]

Specialized tests of IDA

Bone marrow or liver biopsy with Prussian blue staining

RBC free/zinc protoporphyrin level (FEP/ZPP)

Initial response after iron therapy

Increase in Hb content of reticulocytes, within 2 days post-treatment

Increase in reticulocyte number

Within 5-10 days post-treatment

Definite increase in hemoglobin

Within 2-3 weeks, full normalization by 2 months with iron therapy

Dimorphism apparent

Microcytic picture on PBS of IDA may stay for several months

Infections associated with anemia of chronic inflammation

Human immunodeficiency virus, Tuberculosis

Autoimmune diseases associated with anemia of chronic inflammation

Rheumatoid arthritis, Systemic lupus erythematosus

Malignancies

A severe type of condition that may cause anemia of chronic inflammation

In anemia of chronic inflammation, the main mechanism is

Impaired ferrokinetics: iron-restricted erythropoeisis in spite of abundant iron in the body, increased hepcidin inhibits iron absorption

Increased hepcidin

Inhibits iron absorption from intestines and iron release from macrophages/hepatocytes

Peripheral blood smear findings of anemia of chronic inflammation

Usually normochromic, normocytic, if hypo/micro is due to existing iron deficiency

Iron studies of anemia of chronic inflammation

Decreased | Hemoglobin

Low | Serum Iron

Low | TIBC

High | Ferritin

Normal | sTfR

Decreased | Hemoglobin content of reticulocytes

Normal/low | Transferrin saturation

Sideroblastic anemia

Heme production disorder caused by disrupted protophyrin production pathway

Peripheral blood smear of sideroblastic anemia

Iron is abundant in the bone marrow, Prussian blue staining shows “ringed” sideroblasts (iron deposits in the mitochrondria surrounding the nucleus)

Hereditary | Acquired forms of sideroblastic anemia

Porphyria | Lead Poisoning

Lead poisoning (Sideroblastic Anemia)

Growing children will have impaired mental development with anemia

Effects of lead on heme synthesis pathway (Sideroblastic Anemia)

Increased erythroroid protoporphyin (EFP) or zinc protoporphyin (ZPP)

Lead inhibition in the heme synthesis pathway

ALA (aminolevulinic acid) dehydratase - leading to accumulation of ALA (aminolevulinic acid)

Urine measurement for lead poisoning

Aminolevulinic acid

Lab picture of lead poisoning

Coarse basophilic stippling (Classic finding), anemia mostly normochromic/normocytic but will be hypo/micro if exposure is chronic

Coarse basophilic stippling (punctate basophilia)

Inhibition of pyrimidine 5’-nucleotidase

Iron Deficiency Anemia Iron Studies

Serum ferritin

Serum iron

TIBC

Transferrin saturation

FEP/ZPP

sTfR

Hemoglobin content of reticuloytes

BM iron (Prussian blue reaction)

Sideroblasts in BM

Other special tests

Decreased

Decreased/N

Increased

Decreased

Increased

Increased

Decreased

No stainable iron

None

None

B-thalassemia minor Iron Studies

Serum ferritin

Serum iron

TIBC

Transferrin saturation

FEP/ZPP

sTfR

Hemoglobin content of reticuloytes

BM iron (Prussian blue reaction)

Sideroblasts in BM

Other special tests

Increased/N

Increased/N

Normal

Increased/N

Normal

Normal

Decreased

Increased/Normal

Normal

Increased Hb A2

Anemia of Chronic Inflammation Iron Studies

Serum ferritin

Serum iron

TIBC

Transferrin saturation

FEP/ZPP

sTfR

Hemoglobin content of reticuloytes

BM iron (Prussian blue reaction)

Sideroblasts in BM

Other special tests

Increased/Normal

Decreased

Decreased

Decreased/Normal

Increased

Normal

Decreased

Increased/Normal

None/very few

Specific tests for inflammatory disorders or malignancy

Sideroblastic Anemia Iron Studies

Serum ferritin

Serum iron

TIBC

Transferrin saturation

FEP/ZPP

sTfR

Hemoglobin content of reticuloytes

BM iron (Prussian blue reaction)

Sideroblasts in BM

Other special tests

Increased

Increased

Decreased/N

Increased

Increased

Normal

Normal

Increased

Increased (ring)

N/A

Lead poisoning Iron Studies

Serum ferritin

Serum iron

TIBC

Transferrin saturation

FEP/ZPP

sTfR

Hemoglobin content of reticuloytes

BM iron (Prussian blue reaction)

Sideroblasts in BM

Other special tests

Normal

Variable

Normal

Increased

Marked Increased

Normal

Normal

Normal

Normal (ring)

Increased Amino-Levulinic Acid (ALA) in urine and Increased Blood lead levels

Other special tests

Iron Deficiency Anemia

B-thalassemia minor

Anemia of chronic inflammation

Sideroblastic anemia

Lead poisoning

N/A

Increased Hb A2

Specific tests for inflammatory disorders or malignancy

N/A

Increased Amino-Levulinic Acid in urine & Increased blood lead levels

Deficiency of spectrin/ankyrin & band 3/protein 4.2

Leads to release of microvesicles and formation of spherocytes

Spherocyte formation

Involves splenic trapping/erythrostasis, decreased pH/increased macrophage contact, continuous loss of membrane (microspherocytes) and hemolysis

Megaloblastic characteristics

Oval macrocytes, Hypersegmented neutrophils, Pancytopenia, MCV > 100

Non-megaloblastic macrocytic anemia may be seen in

Normal newborns, reticulocytosis (hemolytic anemia/thalassemia), liver disease, chronic alcoholism, bone marrow failure

Confirmation for megaloblastic macrocytic anemia

Serum Folate and Vitamin B12 Levels

Vitamin B12 deficiency due to

Inadequate intake, increased need, and impaired absorption

Folate Deficiency due to

Inadequate intake, increased need, impaired absorption, impaired use, and excessive loss (renal dialysis)

No deficiencies, but indications of megaloblastic macrocytic anemia

Myelodysplastic syndrome, acute erythroid leukemia, congenital dyserythropoetic anemia, reverse transcriptase inhibitors (MACR)

Lack of intrinsic factor (functional parietal stomach cells) in Vitamin B12 deficiency due to

Pernicious anemia, H. pylori infection, gastrectomy, and hereditary intrinsic factor deficiency

Competition for B12 due to

D. latum infection, blind loop syndrome

Cobalamin

i.e. Vitamin B12. Tetrapyrrole ring, cobalt in the middle, attached to a ribonucleotide (5,6-dimethylbenzimidazolyl)

Forms of Cobalamin

Hydroxy-cobalamin & cyano-cobalamin (foods)

Methyl-cobalamin (co-enzyme)

5’-deoxyadenosylcobalamin (co-enzyme)

Impaired absorption may occur in any of these steps

P separation from CBL, R separatoin from CBL, IF-CBL binding (lack of intrinsic factor), malabsorption, competition for available vitamin B12 [PRIMC]

Neurologic symptoms

In Vit B12 deficiency, ___ are more prominent, such as memory loss, numbness and tingling in fingers, loss of vibratory sense.

Cardiovascular disease

In folate deficiency, CNS involvement symptoms are not common, but risk of ___ has been reported due to high serum homocysteine.

CBC Folate Deficiency/Vitamin B12 Deficiency

Decreased

Increased

Manual Diff

HGB, HCT, RBCs, WBCs, PLTs, Absolute Retic [HHWARP]

MCV, MCH, Serum total/indirect bilirubin, Serum LDH [MMBS]

Hypersegmented neutrophils, oval macrocytes, anisocytosis, poikilocytosis, RBC inclusions

Specific Diagnostic Tests of both Folate/Vit B12 Deficiency

Erythroid hyperplasia and increased serum/plasma homocysteine (folate indicator)

Specific Diagnostic Tests of Folate Deficiency

EH, decreased serum/RBC folate, absent antibodies to IF/gastric parietal cells, increased serum/plasma homocysteine, and rest normal

Specific Diagnostic Tests of Vitamin B12 Deficiency

EH, increased Methylmalonic acid (MMA), decreased vitamin B12, variable folate, antibodies to IF/gastric parietal cells (PA), elevated serum gastrin (PA), achloyhdria (PA), decreased Holotranscobalamin, and Diphyllobothrium latum [EMBFAGAHD]

Normalized MCV with megaloblastic anemia

Coexisting IDA, chronic inflammation, or beta-thalassemia minor

Key highlighted indicator of megaloblastic anemia

Dacrocyte, RBC fragments, microspherocytes (high RDW), oval macrocyte, hypersegment4ed neutrophils

Rare indicators seen in megaloblastic anemia

nRBC, HJ bodies, basophilic stippling, Cabot ring

Low retic count in megaloblastic anemia

DNA synthesis is impaired due to Vitamin B12/Folate deficiency which translates to ineffective hematopoiesis, fewer reticulocytes enter blood stream and die early

Vitamin B12 deficiency Flow Chart

Increased MMA, normal Homocysteine

Serum Vit B₁₂ <150 pg/mL, Serum folate > 4ng/mL

Vitamin B₁₂ deficiency combined with folate deficiency Flow Chart

Increased MMA, increased Homocysteine

Serum vitamin B12 = 150-300 pg/mL, Serum Folate = 2-4 ng/mL

Vitamin B12 and folate deficiency excluded Flow Chart

Normal MMA, normal Homocysteine

Serum Vitamin B₁₂ = 150-300 pg/mL, Serum Folate = 2-4 ng/mL

Expected Folate deficiency Flow Chart

Normal MMA, increased Homocysteine

Serum Vitamin B12 = 150-300 pg/mL, Serum Folate = 2-4 ng/mL

Definite Folate Deficiency Flow Chart

Vitamin B₁₂ > 300 pg/mL, Serum Folate < 2 ng/mL

Vitamin B12 deficiency and folate deficiency unlikely

Serum Vitamin B₁₂ > 300 pg/mL, Serum Folate > 4 ng/mL

Autoantibody

Pernicious anemia, an autoimmune disease in which an — is raised against intrinsic factor or gastric parietal cells

T CD4-mediated response, H+/K-ATPase pump, achlohydria

Through — — response, parietal cells will be destroyed over time (significant decrease in IF secretion in stomach), and these pathologic T cells will also attack and destroy — on parietal cell membrane leading to reduction in HCL production (—)

Diagnosis of pernicious anemia

Schilling test for absence of IF

Serum gastrin level (highly increased in achlohydria)

Detection of Abs against IF or parietal cells (blocking antibody)

Parietal cell destruction due to bacteria

Helicobacter pylori

Reporting of neutrophil hypersegmentation

>5 five-lobed per 100 WBCs OR >SINGLE 6-lobed

Schilling test

A definitive test useful in distinguishing cobalamin deficiency due to malabsorption, dietary deficiency, or absence of IF.

Radioactively labeled crystalline vitamin B12

The Schilling test measures the amount of an oral dose of this, that is absorbed in the gut and excreted in the urine.