DSA01 - Introduction to the Course, Laboratory Evaluation of Anemias, Blood Loss and Hemolysis

Deficiency in circulating RBC mass --> Decreased O2 carrying capacity in blood

Define Anemia

Proerythroblast: earliest identifiable stage of erythroid maturation; large cells + large nucleus + basophilic cytoplasm due to a high concentration of ribosomes.

-Hgb synthesis begins, and the cell is committed to the erythroid lineage

Identify stage of Erythroid Maturation Sequence

Basophilic Normoblast:

-Increased basophilia in the cytoplasm due to active ribosome production and hemoglobin synthesis

-Large nucleus + Prominent Nucleoli

-As the cell divides, its size decreases --> Ongoing preparation for Hgb accumulation

Identify stage of Erythroid Maturation Sequence

Polychromatic Normoblast:

-Intermediate erythroblast

-Basophilic and Eosinophilic --> grayish color

-Dual staining indicates that ribosome production continues while hemoglobin accumulation increase

-Nucleus becomes more condensed

-Undergo one or two more divisions before progressing

Identify stage of Erythroid Maturation Sequence

Orthochromatic Normoblast:

-Late erythroblast

-Dense, fully condensed nucleus that is about to be extruded from the cell.

-Cytoplasm is primarily eosinophilic due to high hemoglobin content

-Extrusion of the pyknotic nucleus marks the final preparation for the cell to become an enucleated reticulocyte, a characteristic of mature RBCs

Identify stage of Erythroid Maturation Sequence

Reticulocyte:

-After the nucleus is extruded

-Contain residual ribosomal RNA, which gives the cytoplasm a reticular (mesh-like) appearance when stained.

-Enter the bloodstream and continue maturing for about 1-2 days, during which they complete hemoglobin synthesis and lose remaining organelles

-LAST STAGE before full maturity and are capable of some oxygen transport

Identify stage of Erythroid Maturation Sequence

Polychromatophilic Red Cell (larger than Erythrocyte - still has ribosomes & mitochondria); Stains w/ New Methylene Blue/Cresyl Blue to show GRANULAR Material (instead of a mesh pattern)

Is this a reticulocyte or a Polychromatophilic Red Cell? How can you tell?

Erythrocyte:

-Fully mature red blood cell (biconcave, enucleated, and contains no organelles, maximizing space for hemoglobin and enhancing flexibility for travel through capillaries)

-Highly specialized for efficient oxygen and carbon dioxide transport and represent the final, functional stage of erythropoiesis

Identify stage of Erythroid Maturation Sequence

-Hgb
-Hematocrit (Hct)
-Mean Corpuscular Volume (MCV)
-Mean Corpuscular Hemoglobin (MCH)
-Mean Corpuscular Hgb Concentration (MCHC)
-RBC Distribution Width (RDW)

Name the RBC indices and morphological tests in CBCs for Anemia

Due to changes in EPO

Why do Hgb change (increases in 1st week then drops by 1-2 mo) in infancy?

Average volume per red cell, expressed in femtoliters (cubic microns)

Define Mean Cell Volume (MCV)

Microcytic; Iron Deficiency or Thalassemia

If MCV is smaller than normal, this means the cells are (microcytic/macrocytic), indicating what likely condition(s)?

Macrocytic; Vitamin B12 or Folate Deficiencies

If MCV is larger than normal, this means the cells are (microcytic/macrocytic), indicating what likely condition(s)?

Value can range as low as 78 FL in 1 yr olds to as high as 120 FL in premature infants

Why should MCV always be compared with age-appropriate norms in Pediatrics?

Average mass of hemoglobin per red cell, expressed in picograms

Define Mean Corpuscular Hgb (MCH)

Mirror MCV results (smaller = lower, bigger = higher)

How should MCH results be interpreted?

Average concentration of hemoglobin in a given volume of packed red cells, expressed in grams per deciliter

Define Mean Cell Hgb Concentration (MCHC)

When MCV is low

When might MCHC be low?

Conditions like Hereditary Spherocytosis (any conditions where Hgb is more concentrated inside RBCs)

When might MCHC be high?

Coefficient of variation of red cell volume

Define Red Cell Distribution Width (RDW)

RBC is homogeneous in size = Normal; RBCs are HETEROGENEOUS in size (some small, some large) = Anisocytosis or High/Abn Variation (seen in Iron Deficiency Anemia or Pernicious Anemia)

How should RDW be interpreted?

Dacryocytes

What are these Teardrop Shaped RBCs?

Schishtocytes

What are these fragmented/torn/broken RBCs?

Target RBCs

What are these RBCs in which Hgb appears concentrated in center?

Spherocytes

What are these smaller spherical RBCs without central pallor?

Sickle Cells

What are these thin, curved, fusiform RBC cells w/ pointed ends?

Rouleaux formation

ID Pathology:

D/t increase in cathodal proteins (Igs and Fibrinogens)

-Hx:

> MUTLIPLE MYELOMA

> Macroglobulinemia

> Acute/Chronic Infex

> CTDs

> Chronic Liver Disease

Elliptocytes

What are these slightly egg-shaped to rod/pencil forms (Pencil = Iron Deficiency Anemia)?

Hereditary Elliptocytosis (HE)

If Elliptocytes > 25% of RBCs on smear, what is the probable condition?

Hemoglobin C Crystals (HbC) - from substitution of glutamic acid residue with lysine residue at position 6 mutation in Beta-globin chain ==> UNSTABLE HGB

What is this substance?

As red cells pass through splenic microvascular environment

When does precipitation and crystallization of HbC occur?

Homozygous HbC disease (NOT seen in HbC trait alone)

In what disease do HbC present as "cigar shaped", and usually accompany multiple target cells?

RBC (cells/L) x MCV (L/cell)

What is the formula to calculate Hct (%) from RBC & MCV?

Females

Between Males & Females, which generally has lower ranges for Hb, Hct, & Red Cell Count?

-WBC Count (+ automated differential count)
-Platelet Counts
-Immature platelet fraction

What are other tests for Anemia from the CBCs?

-Peripheral Smear (Manual or Automatic differential count & Microscopic RBC Abns)

-Reticulocyte count (manual or automated; separate order, NOT ROUTINE) - how well bone marrow compensates for anemia

What are other laboratory evaluations of Anemia besides CBC?

-RBC morphology
-Normal central pallor (if it's 1/2 cell diameter)
-Compare size of RBC to size of lymphocyte (8-9 micrometer --> RBC should be SMALLER, like 7-8 micrometer)
-Estimate platelet count

What exactly do Peripheral Blood Smears analyze?

Microcytic Hypochromic (smaller & increased central pallor)

Describe this blood cell

Normocytic Normochromic

Describe this blood cell

Macrocytic (usually normochromic --> NORMAL MCHC)

Describe this blood cell

W/ Anemia, may indicate BONE MARROW DISORDER or NUTRITIONAL (Folate/B12) DEFICIENCY due to issues producing RBCS

What does LOW Absolute Reticulocyte Count (ARC) mean?

Sickle Cell Anemia pts in Aplastic Crisis

In what condition might ARC dramatically drop?

W/ Hyperproliferative Anemia, may indicate PERIPHERAL CAUSE (BLEEDING OR HEMOLYSIS OR Response to Tx, like Iron supplementation of Iron Deficiency)

What does HIGH Absolute Reticulocyte Count (ARC) - ex: ARC > 100,000 - mean?

-Serum Iron Studies
-Bilirubin, Haptoglobin, LDH (Hemolysis)
-Vitamin B12
-Folate (Vitamin B9)
-Hgb electrophoresis
-Coombs test (autoimmune Ab mediated hemolysis)

What are lab tests for Anemia besides blood analyses?

Day 10

When is the best time to detect Hgb levels to monitor response to iron replacement therapy for Iron Deficiency Anemia?

Can occur during infancy OR during pregnancy; disproportionate increase in plasma volume, RBC volume and Hgb mass (overall not as many RBCs)

Define Physiologic Anemia

NOT hereditary/congenital disorder

ID Potential Anemia Etiology:

Pt always had normal CBC count before

Inherited/Congenital Hemolytic Anemia (less likely = Bone Marrow Hypoplasia)

ID Potential Anemia Etiology:

Anemia since childhood

Chronic Hemolytic Anemia

ID Potential Anemia Etiology:

PMHx of Splenectomy, Gallstones, Jaundice a/w Anemia

Hereditary Hemolytic Anemia (Ex = RBC enzyme/membrane disorder, Thalassemia, Hgb-opathy)

ID Potential Anemia Etiology:

FHx of Splenectomy, Gallstones, Jaundice a/w Anemia

Hemolytic Anemia (intravascular hemolysis)

ID Potential Anemia Etiology:

Dark Urine

Pappenheimer Bodies (iron deposits in RBCs --> (+) Prussian Blue Staining)

What are these small blue-grey inclusions at the edge of RBCs called and what is their significance?

-False elevation in platelet count
-A/w condtions such as:
> Asplenia/Postsplenectomy
> Iron Overload
> Sideroblastic Anemia
> Thalassemia
> Megaloblastic Anemia
> Hemolytic Anemia
> Congenital Dyserythropoietic anemia

What are the issues a/w Pappenheimer Bodies?

Howell Jolly Bodies (nuclear remnants from karyorrhexis/chromosomal fragments from abnormal cell division --> (-) Prussian Blue Staining)

What are these large inclusions at the center of RBCs called and what is their significance?

Normally removed by spleen, so a/w...
> Asplenia/Hyposplenia
> Megaloblastic Anemia
> Myelodysplastic Syndromes (MDS)

What are the issues a/w Howell Jolly Bodies?

Basophilic Stippling - aggregated ribosomal RNA

What are these small coarse, deep blue to blue-gray granules throughout the RBC?

-Anemias (Severe anemia, Thalassemia)
-Lead poisoning
-Hemoglobinopathies (ex: Thalassemia)
-Sideroblastic Anemina
-Megaloblastoc Anemia
-Sickle Cell Anemia
-MDS

What are the issues a/w Basophilic Stippling?

Heinz bodies - often seen with supravital blue dye; considered denatured or unstable Hgb

What are these large, blue-purple intracytoplasmic inclusions (usually found in the inner cell membrane of "bite cells")?

-G6PD Deficiency
-Congenital Heinz Body Anemia (amino acid substitution in Hgb)
-Hemolytic Anemia
-Thalassemia

What are the issues a/w Heinz bodies?

Nucleated RBCs (nRBCs/Normoblasts) - usually created d/t increased erythropoietic activity OR sudden release from marrow storage

What are these cells that are commonly seen in newborn blood (in 1st day of life, but should disappear within 3-5 days - may be longer than week in immature infants)?

-Severe Hemolysis
-Profound Stress
-Acute Infection
-Hypoexmia
-Bone Marrow Disease

What issues are nRBCs usually a/w?

Compares relative proportions of granulocytic (myeloid) and erythroid cells in the bone marrow (normal = 3:1)

What does the Myeloid: Erythroid (M:E) ratio in Bone Marrow studies tell you?

Ratio of hematopoietic tissue to fat in the bone marrow (more fat = HYPOCELLULAR)

What does "cellularity" in a Bone Marrow Biopsy tell you?

From birth to 5 y/o

At what ages does ALL bone marrow produce hematopoietic cells?

Long bones lose ability for hematopoiesis --> replace red marrow with fatty yellow marrow

Between 5 to 20 y/o, what happens to the sites of hematopoiesis?

-Pelvis
-Ribs
-Sternum
-Vertebrae

After 20 y/o, where is most hematopoietic tissue produced (hint - think PRSV)?

Presence of NORMAL IRON STORES (Ferritin in RBCs, Hemosiderin in Macrophages)

What does blue staining of bone marrow biopsy cells indicate?

Presence of immature white blood cells (leukocytes) and nucleated red blood cells (nRBCs) in the peripheral blood smear, as well as misshapen RBCs & certain precursors; indicates the release of immature precursors from the bone marrow into the bloodstream, often due to disruption or stress on the bone marrow environment

Define Leukoerythroblastic Reaction

When bone marrow is replaced by fibrosis or other non-hematolymphoid processes (Carcinomas) --> impairs the bone marrow's ability to produce and release normal blood cells

How might a Myelopthisic Process cause a Leukoerythroblastic Reaction?

-Decreased RBC production
-Increased RBC destruction
-Acute blood loss

What does the KINETIC approach of Anemia Classification address?

-Microcytic
-Normocytic
-Macrocytic

How does the MORPHOLOGIC approach of Anemia classify conditions (based on MCV)?

White Pulp = 25%, Red Pulp = 75%

Describe the pulp composition of the Spleen

•Chief vascular component of the red pulp

•Venous structure unique to the spleen

•Composed of an incomplete lining of endothelial cells surrounded by a highly fenestrated basement membrane

•Supported externally by reticulin fibers which regulate the porosity of the sinus

Describe the Splenic Sinus (part of Red Pulp)

•Intervening reticulum is arranged in cords aka cords of Billroth

•Densely packed elements (fibroblasts, collagen fibers, and cells of the mononuclear phagocytic cells)

•Terminal arterioles and capillaries deliver circulating blood to the cords that percolates toward the sinuses

•Circulating elements of blood in transit are all packed within the cords

Describe the Splenic Cords (part of Red Pulp)

RBC flows from cords to sinus fenestrations --> Goes through sinus, but inclusion body stays in cords ==> macrophage takes away chopped off inclusion body --> RBC in sinus returns to VENOUS circulation

Describe the "pitting" process of the Spleen (removes undesirable elements from circulation)

Diverse group of blood disorders caused by accelerated RBC destruction; can be intrinsic (within RBC) or extrinsic OR intravascular VS extravascular

Define Hemolytic Anemia

-Ab mediated (Isohemagluttinins from transfusion or Rh Newborn Disease, AutoAbs)

-Mechanical Trauma to RBCs (TTP/DIC, Defective valves)

What are causes of extrinsic (extracorpuscular/outside the RBC) hemolysis?

-RBC membrane defects (hereditary spherocytosis, elliptocytosis, abetalipoproteinemia)

-Enzyme deficiencies (G6PD, Glutathione synthestase, PK, Hexokinase)

-Hgb Synthesis Disorders (Hemoglobinopathies = Sickle Cell, Unstable Hgb; Deficient globin synthesis = Thalassemia)

-Paroxysmal Nocturnal Hemoglobinuria (PNH)

What are causes of intrinsic (intracorpuscular/within the RBC) hemolysis?

Occurs PHYSIOLOGICALLY (when Spleen recycles amino acids and iron from old RBCs after they've been degraded by bone marrow macrophages) --> Haptoglobin is NOT saturated normally --> becomes PATHOLOGIC when it's accompanied by intravascular hemolysis

Does extravascular hemolysis occur physiologically - why or why not?

-RBC membrane defects (hereditary spherocytosis, elliptocytosis, abetalipoproteinemia)

-Parasitic Infection (Malaria = destroys infected RBCs in spleen)

-Structurally abnormal globins (Hemoglobinopathies = Sickle Cell)

-Immune Mediated Hemolysis

What are examples of PATHOLOGIC Extravascular Hemolysis?

Usually from RUPTURE or LYSIS of RBC w/n circulation (not supposed to be senescent yet)

How does Intravascular Hemolysis occur?

LOW HAPTOGLOBIN in Intravascular Hemolysis (too much Hgb in blood --> overwhlems Haptoglobin production)

What is a key difference between Pathologic Extravascular Hemolysis and Intravascular Hemolysis?

-Darker red plasma (increased Hgb concentration)

-Excess Hgb dimers in blood

-Hemoglobinuria (positive Heme Rxn on Dipstick)

What are signs of Intravascular Hemoylsis?

-Enzyme deficiencies (G6PD, Glutathione Synthetase, PK, Hexokinase)

-Parasitic Infex (Babesiosis --> RBC Rupture)

-Paroxysmal Nocturnal/Cold Hemoglobinuria (PNH)

-Mech Trauma to RBCs (TTP/DIC, Defective Cardiac Valves)

-Ab Mediated (Isohemagglutunins - Transfusion Rxns, Rh Newborn Disease)

What are causes of Intravascular Hemolysis?