Hematology study of blood

Components:
- Plasma (55%)
- Formed Elements (45%)

Lab Tests:
- RBC count
- WBC count
- Platelet count
- Hematocrit
- Mean Corpuscular Volume (MCV)
- Mean Corpuscular Hemoglobin Concentration (MCHC)

Hematopoiesis - formation & development of blood cell

- begins as early as 19th day after fertilization of yolk sac of the embryo
- Fetal liver chief site
- Bone marrow chief site
- Minor site: Spleen, Kidney, Thymus, & Lymph Nodes
Types:
1. Medullary hematopoiesis in the bone marrow
2. Extramedullary hematopoiesis areas other than the bone

Hematopoietic tissue tissues involved in the proliferation, maturation, & destruction of

blood cells

Reticular Endothelial System (RES)

- also called mononuclear phagocytic system
- involved in cellular destruction bone marrow, liver, spleen & lymph nodes
Includes: Monocytes, Fixed & Free Macrophages

1. Spleen largest collection of lymphocytes & mononuclear phagocytes

Function:
a. Culling filtering & destruction of senescent(aged) RBCs
b. Pitting removal of particles

Pancytopenia decrease of blood

Thrombocytopenia decrease in platelet
Anemia decrease RBC
Leukopenia decrease WBC

Splenectomy removal of spleen

Hypersplenism (splenomegaly) enlarged spleen
Types:
1. Primary no underlying disease
2. Secondary caused by underlying disease

2. Lymph nodes
- composed of lymphocytes, macrophages & reticular framework
- filter to remove foreign substances
- Lymph formed from blood fluid that escapes the connective tissue
Structure:
a. Cortex inner area ; contains Plasma cells
b. Medulla outer area; contains follicles (B Lymphocytes, T Lypmhocytes,
Macrophage)

3. Thymus
- compartment for the maturation of T Lymphocytes
Structure:
a. Cortex
b. medulla

4. Bone Marrow inside the spongy bone

- Red Marrow - active
- Yellow Marrow inactive; fatty marrow
- Hyperplastic replaces yellow marrow with red marrow
- Hypoplastic replaces red marrow with yellow marrow

5. Liver
- Kupffer cell phagocytic cell ; less efficient in spleen

Hematopoietic cells:
1. Pluripotential Stem cell replace blood; differentiation into blood cell lines
2. Committed progenitor stem cells develop into distinct cell lines
3. Matured cells w/ specialized function
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Hemoglobin iron-bearing protein; main component of RBC

Gives the red color
Synthesis: Polychromatophilic normoblast stage
Stimulated: Hypoxia cause kidneys to increase production of EPO; increase Hgb
production
Function: buffering action, maintains blood pH, carry O2, remove CO2

Constitutes 1/3 of RBC

Proerythroblast
o 65% Erythroblast stage
o 35% Reticulocyte stage
Parts: (Produced)
o Heme - mitochondria
o Globin - polyribosomes
Mature red cells no nucleus & mitochondria
Arrangement Cluster:
o b- cluster- short arm chromosome 11
o a- cluster - short arm chromosome 16
Oxygenation
1 Hb = 4 O2
Hb-oxygen Dissociation Curve

Left Shift Right Shift

Low 2,3-DPG High 2,3-DPG

HbF High H+
High CO2
HbS
- Less readily give
up O2 - Easy O2 delivery

Oxyhemoglobin carrying O2
Deoxyhemoglobin without O2

Oxygen Affinity binding of O2 to Hgb

Increase O2 binds more, but it does not want to give it up
Decrease cause O2 to be released

Reference Range:
Adults:
o Male = 14-17.4 g/dL
o Female = 12-16.0 g/dL
Children:
o Birth = 13.5-20.0 g/mL
o 6-12 years = 11.5-15.5 g/mL

Structure
- 4 polypeptide chain
o Heme group
Porphyrin ring
Ferrous iron
- Globin chain
o 2 Alpha chains
o 2 Beta chains
Synthesis
- occurs in mitochondria of developing Red cells as they mature on the bone marrow
- Necessary:
o Adequate iron supply & delivery
o Adequate synthesis of protoporphyrins
o Adequate globin synthesis
- Transferring delivers iron to the reticulocyte
- Mitochondria synthesis of protoporphyrins, mediated by EPO & vitamin B6
- Protoporphyrin + Iron = Heme
- Rate of globin synthesis = rate of porphyrin synthesis
- Chain Designations: a.) Alpha b.) Beta
c.) Delta d.) Epsilon
e.) Gamma f.) Zeta
Bohr Effect alteration in blood pH, shifts O2 dissociation curve
Acidic shift to the right ; enhanced capacity to release O2
Right Shift Hgb has less attraction to O2; more efficient to deliver O2
Ex. Anemia; Acidosis
Left Shift Hgb has more attraction to O2 ; less willing to release
Ex. Alkalosis

Carbon Dioxide Transport

Mechanism:
Dissolution in the plasma
Formation of bicarbonic acid
Binding to carbaminohemoglobin

Non-functional Hemoglobin
1. Hypoxia Inadequate amount of O2 in the blood
2. Cyanosis presence of >5 g/dL deoxyhemoglobin blood
- patient appears blue
3. Carboxyhemoglobin carbon monoxide instead of O2.
- Slightly increased levels - heavy smokers and environmental pollution.
- Can revert to oxyhemoglobin
4. Methemoglobin
- Ferric (Fe3) state instead of the ferrous (Fe2) state
- Cannot combine with oxygen
5. Sulfhemoglobin - Hemoglobin molecule contains sulfur.
- Sulfur-containing drugs or chronic constipation.
- Cannot revert and may cause death

Clinical Laboratory Test

RBC Count - no. of RBC found in mm3 of whole blood
- Indirect estimate of bloods Hgb content
Normal:
Men - 4.3 5.9 x 1012/L (4.3 5.9 x 106/mm3)
Women - 3.5 5.0 x 1012/L (3.5 5.0 x 106/mm3)

RBC (Erythrocyte)
- Produced in the bone marrow
- Released into the peripheral blood
- Circulation: 120 days
- Cleared by the reticuloendothelial system

Concentration:
Detect anemia
Calculate Hct
Calculate RBC indices
- For monitoring quantitative changes, Hct & Hgb concentration are generally used

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Hematocrit (Hct) / Packed Cell Volume (PCV) / Erythrocyte Volume Fraction (EVF)
- Commonly used clinically, simpler to perform
- Centrifugation of capillary tube
- Percentage comparison of height of the RBC to the whole blood
- Hct value is 3x the Hgb value
- Hct is in % or fraction of 1

Normal:
Men - 42% - 52% or 0.42 0.52
Women - 37% - 47% or 0.37 0.47

Low Hct Value High Hct Value

Conditio Anemia Polycythemia vera
n Over Hydration Dehydation
Blood Loss
Others Bone marrow suppressant Dengue Fever high Hct is a
Effects of drugs danger sign of increase risk of
Chronic disease dengue shock syndrome
Genetic alterations in RBC
morphology (Sickle Cell
Anemia)

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Hemoglobin
Normal:

Men - 14 18 g/dL
Women - 12 16 g/dL
- Low Hgb value indicates anemia
- Hgb determination directly reflects oxygen transport capability of blood

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Hgb Concentration

Estimated by:
o Spectrophotometer light intensity / color matching
- 540 nm wavelength

Methods:
o Hemoglobin Cyanide Method internationally recommended reference method
Principle:
Blood diluted in Drabkin solution (1:201) 20 uL in 4mL
Cyanmethemoglobin (HiCN) reaction occur standard

Calculation: A of test sample x concentration of STD x dilution factor

A of Standard 1000

Linearity of Standard Curve use 3 SD

 o Oxyhemoglobin Method

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RBC indices / Wintrobe indices

- Provide information regarding RBC size, Hgb concentration, & Hgb weight
- classification of anemia
- Include:
MCV
MCHb
MCHbC
- Peripheral blood smear provide most information
- Poikilocytosis variation in RBC shape
- Anisocytosis variation in RBC size

1. Mean Cell Volume (MCV) ratio of Hct to RBC count

- assess average RBC size, reflects anisocytosis
MCV = (Hct % X 10) / RBC (millions)
Normal: MCV = 90 10
Low MCV Microcytic (undersize) RBC Iron Deficiency
Anemia
High MCV Macrocytic (oversize) RBC Vit. B12 or Folic Acid
Deficiency
MCV can be normal in mixed anemia

2. Mean Cell Hemoglobin (MCHb) assess amount (wt.) of Hb in an average cell

MCHb = (Hb x 10) / RBC (millions)
Normal: MCHb = 30 4

3. Mean Cell Hemoglobin Concentration (MCHbC) average concentration of Hb in an

average RBC
- More reliable index of RBC Hb than MCHb
- Normochromic anemia: change in size due to weight of Hb;
concentration of Hb remains normal
MCHbC = (Hb x 100) / Hct
Normal: MCHbC = 34 3
Low MCHbC Hypochromia (pale RBC) Iron Deficiency
Anemia

Anemia reduction in Hgb concentration, Hematocrit concentration or RBC count

- Iron Deficiency
- 2 SD below the mean
- Malignancy: WHO criteria
o Men - Hg < 14
o Women - Hg < 12
 - Physical Manifestation: Spoon Nails

Symptoms:
Exertional dyspnea and Dyspnea at Exertion
Headaches
Fatigue
Bounding pulses and Roaring in the Ears
Palpitations
PICA

Kinetic Approach Morphological Approach

Decreased RBC production Microcytic - MCV < 80
Lack of nutrients (B12, folate, Reduced iron availability
iron) Reduced heme synthesis
Bone Marrow Disorder Reduced globin production
Bone Marrow Suppression Normocytic - 80 < MCV < 100
Increased RBC destruction Macrocytic - MCV > 100
Inherited and Acquired Liver disease, B12, folate
Hemolytic Anemias
Blood Loss

Laboratory:

- History taking & Physical Examination

- CBC w/ diff:
a. Aplastic anemia leukopenia w/ anemia
b. Infection increased neutrophil
c. Myelodysplasia increased monocytes
d. Thrombocytopenia hypersplenism, marrow involvement w/ malignancy,
autoimmune
destruction, folate deficiency
- Reticulocyte Count
- Peripheral Smear

Iron Deficiency Anemia

High: RDW (red cell distribution width) ; TIBC (total iron-binding capacity)
Low: Retic Count ; Iron level ; Ferritin

Bone Marrow Biopsy gold standard for diagnosis

- Invasive & costly
- Prussian Blue Staining shows lack of iron in erythroid precursors &
macrophages

Anemia of Chronic Disease

- EPO (erythropoetin) production inadequate
 - Erythroid marrow responds inadequately to stimulation
- Low serum iron, increased red cell porphyrin, transferrin 15-20%, normal to
increased ferritin
Causes:
Inflammation
Infection
Tissue injury
Cancer

AICD (activation induced cell death) vs. Iron deficiency

- Soluble Transferrin Receptor - elevated in ID
- Ferritin elevated in anemia of CD
- Bone Marrow Biopsy: Anemia CD
o Macrophages contain
normal/increased iron & erythroid
precursor;
o Show decreased /absent amount of iron

Treatment treat the underlying CAUSE

- Consider co-existent iron deficiency
- If underlying disease require, consider EPO injection

Reticulocyte Count percent of immature RBC (released earlier in anemia from the
marrow)

- Normal: 0.5 1.5 % (Non-anemic)

a. < 1% - Inadequate production
b. >= 1 - Increased production (hemolysis)
- Corrected Retoculocyte Count compares anemic to non-anemic counterpart
(response)
CRC = (% Retic X HCT) /45

Reticulocyte Correction Factor

RPI = % reticulocytes X HCT/45 X 1/Correction Factor

Normal RPI =1
RPI < 2 - Hypoproliferative
RPI >= 2 - Hyperproliferative Disorder

Hematocrit Correction Factor

40-45 1
35-39 1.5
25-34 2
15-24 2.5

Causes of Iron Deficiency

Blood Loss (occult or overt): PUD, Diverticulosis, Colon Cancer
Decreased Iron Absorption: achlorhydria, atrophic gastritis, celiac disease
 Foods and Medications: phytate, calcium, soy protein, polyphenols decrease
iron absorption
Uncommon causes: intravascular hemolysis, pulmonary hemosiderosis, EPO,
gastric bypass
Decreased Intake (rare)

Porphyria accumulation of porphyrins ; genetic & metabolic disorder

- Does not convert porphyrins to heme

Common Symptoms:

1. Neurological Symptoms
2. Photosensitivity skin damage
3. Porphyrin in feces & urine excreted excess
o High level port wine color in urine

* King George III Mad King George

Group:

1. Erythropoietic porphyria
2. Hepatic porphyria
a. Acute
b. Chronic

Porphyria cutanea tarda Chronic hepatic porphyria

- Acquired porphyria
- Hypertrichosis(red teeth) locally as Monkey Children

Hemoglobin - 2 Alpha (141 AA) & 2 Beta chains (146 AA) -> Helical Structure
Hemoglobinopathies - 400 defined

- Heterozygotes mild effect

- Homozygotes moderate to severe effect
Hemolytic Anemia
Erythrocytosis High oxygen affinity
Cyanosis Low oxygen affinity
1. Thalassemias quantitative defect in globin synthesis involving gene mutation
2. Sickle cell disease & Hgbs C, D, & E
qualitative defect in amino acid substitution in globin chain

Most Common:

1. Hgbs S, C, D & E
- Homozygote form severe disease
- Heterozygote form mild disease
2. Hemoglobin electrophoresis
Thalassemia
- abnormal production of globin chain
- quantitative failure
- missing 1 to 4 genes
Diagnosis: (Normal)
Hgb A = 97.5 % = alpha (2) + beta (2)
Hgb A2 = 2.0 % = alpha (2) + delta (2)
Hgb F = 1.0 % = alpha (2) + gamma (2)

1. Alpha Thalassemia Diagnosis: AT

Missing 1 alpha silent carrier ; normal phenotype ; normal MCV
Minor
Missing 2 alpha mild anemia ; decreased MCV - Hgb 11.5
African/American Alpha+/alpha- // alpha+/alpha- - MCV 70
Asian Alpha- /alpha- // alpha+/alpha+ - RBC = 5.85 M
- Hgb A = 97
Ex. Holy Land Patient *Hgb A =
2%
a. AT Minor missing 2 alpha genes *Hgb F =
b. Hemoglobin H disease missing 3 alpha genes
1%
- Mild to moderate anemia (Hgb 7 to 10)
*diagnosis by
- Hgb H (4 betas) = 5-30% - electrophoresis
- Reticulocytosis, microcytosis, hypochromia & exclusion
targeting.hemolysis.splenomegaly
c. Hydrops Fetalis missing 4 alpha genes
o More common in Asians ; less common in Blacks ; stillborn

2. Beta Thalassemia Minor

- Gene deletion (Beta 0) / Mutation (Beta +)
- Heterozygous form Increase in Hgb A2 = 4-7% ; Slight increase in Hgb F
- Confused w/ Iron deficiency

Differential:

Thalassemia Alpha or Beta Iron Deficiency

RDW not increased (uniform red cells) Increase
- Pencil cells
- Targets present - Anisocytosis
- Decreased MCV (75) - Poikilocytosis