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1

Erythroid Maturation process

-Cell size decreases
-Cytoplasm ratio increases
-nuclear size decreases and disappears
-cytoplasm staining changes from blue to pinkish red
-chromatin pattern matures and disappears with nucleus

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main cytoplasmic component of RBC

Hemoglobin

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What does hemoglobin consist of

Consists of ring of carbon, hydrogen and nitrogen atoms with an atom of Fe attached in the center
Globular protein consisting of 4 heme portion has series of tetrapyrrole ring structure with iron in the centre
Globin portion is a protein, consisting of 4 chain of amino acids ( 2 β and 2 α)
Each of the 4-globin chains attached to heme to form a single haemoglobin molecule
Each heme molecule carries one molecule of oxygen
Hb = 4 heme rings + 4 globin chains

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Function of Hemoglobin:

Hgb binds oxygen molecules in the lungs and carries oxygen from the lungs to the tissue bound to the ferrous iron in Heme.

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• Methhemoglobin

Iron in the oxidized form (brownish in colour).

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• Carboxyhemoglobin-

Hgb bound to carbon monoxide. Carbon monoxide has an affinity 200 times greater for hemoglobin than oxygen

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• Sulfhemoglobin

irreversible oxidation of hgb with sulfonamides (green pigment)

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Normal Adult Hemoglobin:

Hb A normal adult Hb (2 alpha and 2 beta globin chains)
Hb A2 another form of adult haemoglobin (2 alpha and 2 delta chains)
Normal Adult Composition: (Hb A & A2 =95% HBF <5%)

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Hb F in the fetus/newborn

The haemoglobin in the unborn baby is HbF.
At six months of age, HbF contribute less than 1% to the total Hb of a normal infant.
At 6 months of age, over 95% of Hb is HbA, <4% is HbA 2 and <1% is HbF. Haem F starts to decrease.

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Hemoglobinopathies

(Defects in Hemoglobin)
disorders affecting the structure, function, or production of hemoglobin.

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Hb S

Amino acid substitution in the beta chain (valine for glutamic acid)
If 2 copies of gene: ‘sickle cell disease’- Severe haemolytic anemia and sickling
If 1 copy of gene: ‘sickle cell trait’- asymptomatic
Found in Blacks, Middle Eastern, Indians and Mediterranean’s . Not getting enough oxygen , rips walls of blood vessel, how you can be anemic because RBC are being destroyed and the bone marrow cannot compensate. High amount of nucleated RBC. Can be seen if smears are not made properly

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Hb C

(substitution of lysine for glutamic acid)
May be inherited along with Hb S
Mild haemolytic anemia
Forms crystals
Found in Blacks of West African heritage . Always on the globin and not the haem. Mild anemia, shape of cells not changed , but haem crystalizes (could also be made from bad smear). Cells look like targets – diagnostic for liver disease

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Hb E

Common in people of South East Asian descent
Mutation on beta chain from a glutamic acid to lysine
If 2 copies of gene: ‘E disease’
If 1 copy of gene: ‘E trait’
Mild microcytic anemia. Cells are empty indicative for anemia

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Hb H

(beta chains- no alpha chain) -Alpha thalassemia (α-thalassemia)
Hereditary disorder found in African, Asian, Mediterranean, or Middle Eastern descent
Moderate anemia
Golf ball like inclusions in RBC

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Beta Thalassemia

failure to produce beta chains
Hereditary disorder found in Mediterranean, Middle East, India, Southeast Asia, South to Northern Africa, Europe
Decreased Haemoglobin
RBC-hypo and micro
Target Cells
Jaundice (because their RBC are being destroyed, bone issues. Liver issues

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Hb D Punjab (D Los Angeles)

Asiatic Indian heritage and those of European descent
Glutamine substituted for glutamic acid
DD- mild haemolytic anemia. Hb S and Hb D genes together react as sickle cell

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Hb O Arab

Balkans (Bosnia, Bulgaria, Croatia, Serbia) and the Middle East, and occasionally in African-Americans.
Substitution of lysine for glutamic acid
Mild anemia with many target cells

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HbA1c

glycosylated Hb measures the blood plasma glucose attach to the globin chain
Average over 2 to 4 weeks prior to test)
Increased glucose= increased HbA1c levels (diabetes) in blood and urine
A1c has an affinity for glucose

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Decreased Hemoglobin Level

Anemia’s
Iron Deficiency
Vitamin B12 or Folic Acid Deficiency
blood loss by destruction or haemorrhage
decreased RBC production (aplastic anaemia, leukemia) –a- without plastic – plasia (decrease)
abnormal production of globin chain

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Hemoglobin Normal Values SI units

Women 120-160 g/L -- 7.62 - 9.48 mmol/L
Men 140-180 g/L ------ 8.67 - 10.8 mmol/L
Newborn 15.2 - 23.5 g/dL 9.42 - 14.6 mmol/L
infant 160-230 g/L - -----6.69 - 7.93 mmol/L

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Critical Values: --- Low Hemoglobin :

<60 g/L (cause bleeding and heart failure) – low oxygen levels, heart is pumping faster to provide you move oxygen, strains heart. Bleeding occurs spontaneously

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Critical Values: --- High Hemoglobin :

> 200 g/L (Conditions: polycythemia vera , severe burns (lost plasma), chronic obstructive pulmonary disease, and congestive heart failure. May cause clogging of capillaries due to hemoconcentration Lots of RBC but they don’t function properly. Lots of RBC causes them to get sticky because they are concentrated

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Types of Blood Sample:

• EDTA specimen
• Venous or capillary

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Cyan-methemoglobin Hemoglobin Method

Spectroscopy:
Principle: Blood( Hb) + Drabkin’s solution (K Cyanide + K ferricyanide ) RBCs lyses. The ferricyanide converts the hemoglobin to methemoglobin. The cyanide then reacts with the methemoglobin to form the stable cyanmethaemoglobin which absorbs light at 540 nm. Always put an expiry date on the standard graph

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Digital Hemoglobin Photometer (Point of Care)

Principle: The reaction in the microcuvette is a modified azidemethemoglobin reaction.
Reagent on microcuvette mixes with blood
RBC lysed by sodium deoxycholate, releasing the hemoglobin.
Sodium nitrite mixes with the blood and converts the hemoglobin iron from the ferrous to the ferric state to form methemoglobin
Methemoglobin, combines with azide to form azidmethemoglobin.
Measures absorbance at 570 nm and 880 nm, for compensation of turbidity.

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Portable Haemoglobin Analyzer

Portable
Battery operated
Electronic
Reagents on strip
Methodology: Reflectance photometry
• Blood and chemicals react on the strip.
• Narrow beam of mono chromatic light from LED is focused on the strip.
• The amount of light reflected is directly proportional to the hemoglobin concentration
• The reflected light is detected by a photo-detector
Principle: Azidemethemoglobin
Sample Type: Whole blood
Sample Size: 1 drop (approximately 12 μL)

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Point of Care Testing (POCT)

CLIA (Clinical Laboratory Improvement Amendments) waived
Technician or user must be trained
Uses capillary sample
Reagent on strip or microcuvette mixes with blood
Instrument measures absorbance or reflected light

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Quality Control:

• record the lot number of reagent used
• check expiry date of reagent
• run controls (commercially prepared samples of known value) and check that results fall within ± 2SD

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Safety:

• standard precautions
• protective clothing, gloves
• devices to prevent aerosol formation
• read protocols for disposal of waste