Hematology 1

Midterms notes part 2

Hemoglobin

-28% of the red cell mass

-4 globin chains & 4 heme groups
- holds 1.34 mL of oxygen
- Total adult red cell mass=600 g, capable of carrying 800 to 804 mL of oxygen
Main function:
: transport of gases
: Transport of nitric oxide to hypoxic sites to stimulate vasodilation to enable increase in oxygen
delivery
: Regulation of blood acid base balance

Heme
-ferroprotoporhyrin
-responsible for color
-1 heme molecule= 4 protoporphyrin rings with one iron (ferrous) atom per ring.
-synthesized by Basophilic normoblast and Polychromatic normoblast (peak of hgb synthesis)
-located in hydrophobic folds of the globe polypeptide chains

** Hydroxymethylbilane formation is in
between Porphobilinogen and
Uroporphyrinogen III

Prepared by: Hazel Hope Acosta HHMA’17

Heme synthesis

-Iniated by the condensation of Glycine and Succinyl CoA to form Delta Amino Levulinic Acid
(ALA) catalyzed by ALA synthetase with the coenzyme pyridoxal phosphate (Vit B6)
-2 molecules of ALA combine to form porphobilinogen or PBG (catalyzed by ALA dehydrase/
dehydratase)
-deamination of PBG will yield hydroxymethylbilane (catalyzed by PBG deaminase)
-Hydroxymethylbilane will be converted to uroporphyrinogen III which is a 4 ringed compound
(catalyzed by UPG III synthase)
-Uroporphyrinogen III upon the action of Uroporphyrinogen decarboxylase will be converted to
Coproporphyrinogen III (CPG III); CPG III will be oxidized to become Protoporphyrinogen IX that
will be further oxidized to Protoporphyrin IX
-Protoporphyrin IX will be coupled with iron (catalyzed by ferrochelatase) , with the aid of
ascorbic acid and reduced glutathione to form Heme.
- Incorporation of heme inside hydrophobic pockets between the E and F helices of the globin
Chains (beta) in
the ribosomes
constitutes the
final steps of
hemoglobin
synthesis.

Globin

-1 molecule of
hemoglobin
contains two
pairs of unlike
globin
polypeptide
chains.

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RBC destruction
-occurs in the Reticuloendothelial system (primarily in the spleen)
- Protoporphyrin is oxidized to form a green bile pigment known as biliverdin-globing (aka
verdohemoglobin)
- The central iron atom in the heme molecule attaches to a transport molecule known as
Transferrin (or siderophilin)
- The iron will be recycled for new hemoglobin production or may be stored first as Ferritin ( in
the ferric form)
- If there is an excess in iron, they will be stored as Hemosiderin pigments
- Biliverdin will be reduced to form free bilirubin which goes to the plasma (carrier: Albumin)
and will be conjugated in the liver to form bilirubin
- Bilirubin may go back to the plasma to be excreted in the urine or majorly, bilirubin will go to
the intestines where the normal intestinal flora will convert it to urobilinogen to be excreted in
the feces.

Hemoglobin compounds and dyshemoglobins

1. Oxyhemoglobin
-Arterial blood; oxygenated

-Blood: Bright red color / scarlet red

2. Deoxyhemoglobin
-Venous blood

-Blood: Purplish red color / dark red
** may pick up CO2 to form a molecule known as carbaminohemoglobin

3. Carboxyhemoglobin
-Carbon monoxide plus hemoglobin
-normal: 0.5%
-Blood: Cherry red color

-Reversible

-Cannot bind and carry O2

-CO has 200x or 210x (or 240x) greater affinity for Hgb compared to O2 Smoking, gas, etc.
Measured at 576 nm
-3%-15% (or 10%) in smokers

4. Methemoglobin
-Blood: Chocolate brown color
-Reversible

-Fe2+ is oxidized to Fe3+

- Hereditary methemoglobinemia: Methemoglobin reductase deficiency
-Toxic methemoglobinemia: Exposure to chemicals/drugs

Exposure to nitrite, chlorine and nitrate , benzocaine, dapsone, primaquine
-normal: 1.5%
->30% = cyanosis
-Tx: Methylene blue

Prepared by: Hazel Hope Acosta HHMA’17

-Measured at 630 nm

5. Sulfhemoglobin
-inorganic sulfides+ hemoglobin= green hemochrome
-Blood: Mauve lavender

-Irreversible

-Cannot transport O2

-Can combine w/ CO forming carboxysulfhemoglobin

-Mixture of oxidized, partially denatured forms of hemoglobin that form during oxidative
hemolysis green hemochrome
-Phenacetin, acetanilide,phenylhydrazine, nitrites, sulfonamides, aromatic amine drugs
Bacteremia: Clostridium perfringens (Enterogenous cyanosis )
-measured at 618 to 620 nm

Laboratory Measurement of hemoglobin

A. Gasometric Method

- Also known as the Van Slyke Oxygen Capacity Method
- Principle: 1g Hb can carry 1.34 mL O2

- Measures oxygenated hemoglobin only 


B. Chemical Method

- Principle: Based on assumption of that the total iron content of blood is bound to
hemoglobin ; Iron content of whole blood not attached to hemoglobin is negligible
compared with hemoglobin iron. 


**ICSH Standard: 1 gram Hb= 3.47 mg (0.00347 g) Iron

- Methods:
a. Wong Test
Technique:

* Sulfuric Acid & Potassium persulfate: Liberation of iron
from hemoglobin

* Tungstic acid: Precipitates proteins

* Iron in protein-free filtrate is made to form ferric thiocyanate o Ferric thiocyanate is measured
spectrophotometrically

b. Assendelft Test
Technique:
*Iron is separated from hemoglobin, usually by acid or ashing
*Product of reaction is then titrated with TiCl or complexed with a reagent to develop color
* Color measured photometrically

C. Gravimetric Method (Copper Sulfate Method)


- Principle: Hemoglobin concentration is indirectly determined by assessing the specific gravity
of blood

- Method:
*40 copper sulfate solutions having specific gravity ranging from 1.035 to 1.075 are prepared
With Interval of 0.001

* Alternative: 16 copper sulfate solutions having specifc gravity
ranging from 1.015 to 1.075 with Interval of 0.004

Prepared by: Hazel Hope Acosta HHMA’17

* It is noted whether the drop sinks or rise in the solution

* If with same specific gravity: Stationary for 10-15 seconds
and then sinks

* If lighter, it will float o If heavier, it will sink
Normal: 1.055

D. Colorimetric Method - Methods:

a. Direct Matching Method

- Principle: Red color of fresh whole blood is compared to a colour standard

- Methods:

a1. Tallquist Hemoglobin Scale


- Utilizes a printed color scale graded from 10 to 100 percent.

- Method: Color of the scale is matched to the color of the patient’s blood on absorbent paper

- Highly erroneous
**Differences in the personal judgment of the colors 


** Percentage of the scales are not accurate

a2. Dare hemoglobinometer -Method: 


*Blood is drawn by capillary attraction between 2 glass plates

*One is transparent
* One is white and translucent

* Color is then matched with a rotating disk of tinted glass
varying in thickness and red colour intensity

a3. Spencer hemoglobinometer - Method:

* Determination of transmission of light through a layer of hemolyzed blood (oxyhemoglobin)
of constant depth 


* Compared with transmission of light of standardized glass wedge with a transmission of 540
nm
NOTE: Intensity of light is measured rather than the color

b. Acid Hematin 

- Principle: Formation of acid hematin produces a characteristic brownish yellow color

- Method:
*Red cells are laked with 0.1N hydrochloric acid, converting hemoglobin into acid hematin
*The brownish yellow color is diluted with distilled water until the color matches that of the glass
standard (present in the Sahli-Hellidge Hemoglobinometer) 


* Concentration of hemoglobin in grams (%) is read directly from the gram scale etched on the
tube 


c. Alkali denaturation 

c1. Betke Method

-Principle: Fetal hemoglobin resists alkali denaturation
- Method:
* A red blood cell hemolysate is prepared
* Hemolysate is added to cyanmethemoglobin reagent and then exposed to sodium hydroxide 


* Other hemoglobin variants are destroyed while

Prepared by: Hazel Hope Acosta HHMA’17

fetal hemoglobin remains intact
** Ammonium sulfate is added
-Halt the denaturation process and precipitate the denatured hemoglobin
** Solution is filtered and measured spectrophotometrically.
- Compared with the spectrophotometric readings of the original cyanmethemoglobin solution
to determine the % Hb F present

c2. Singer method


-Principle: Fetal hemoglobin resists alkali denaturation - Method:
* A red blood cell hemolysate is prepared
* Hemolysate is incubated with potassium hydroxide. 

***Other hemoglobin variants are destroyed while fetal hemoglobin remains intact
* Ammonium sulfate is added

-Halt the denaturation process and precipitate the denatured hemoglobin

- Non-altered Hb F is measured spectrophotometrically

d. Oxyhemoglobin method
- Method:
*A 1:251 dilution of blood is made by adding 0.02 mL of blood into 5 mL aqueous NH4OH in a
stoppered cuvette
* Shake to ensure oxygenation
* Read spectrophotometrically at 540 nm against a blank 

made up of 0.007 N NH4OH


e. Cyanmethemoglobin (HiCN) method

* Principle: Dilution of blood is prepared in a solution of potassium ferricyanide and potassium
cyanide Potassium ferricyanide converts hemoglobin to hemoglobin while potassium
cyanide provides cyanide ions to form hemiglobincyanide (cyanmethemoglobin) 

Hemiglobincyanide has a broad absorption maximum at a wavelength of 540 nm 


* Reagent: Drabkin’s reagent

*Potassium ferricyanide: Oxidizes Fe2+ to Fe3+; Converts hemoglobin to hemoglobin
(methemoglobin)
* Potassium cyanide: Provides cyanide to form hemoglobincyanide (cyanmethemoglobin)
* Non-ionic detergent (Triton X-100): Improves lysis of RBCs; Decreases turbidity
* Dihydrogen potassium phosphate (KH2PO4): Allows test solution to be read for 3 minutes;
Original solution contains sodium bicarbonate which allows reading after 15 minutes incubation
- Method:
- Twenty microliters (0.02 mL) of blood is added to 5.0 mL of diluent
- Mix preparation and allowed to stand at room temperature for at least 3 minutes
- Absorbance is read at 540 nm against a reagent blank. 


f. Acid Elution Test


- Principle: In an acid medium (pH 3.2 to 3.3), hemoglobin F is resistant to elution from the red
blood cell, while other types are removed from the red cells

- Method:
- Blood smears are fixed with ethyl alcohol
- Incubated in a citric acid-buffer solution
- The slides are stained with: 

* Hematoxylin (stains the white cell nuclei with blue color)
*Erythrosine B (stains the red cells with Hb F with a pink color)

Prepared by: Hazel Hope Acosta HHMA’17

** Smears are viewed microscopically to determine presence and percentage of RBCs with Hb
F
- Hb F: Pink cells

- Hb A & Other Hb Variants: Ghost cells

Oxygen Hemoglobin Dissociation curve

- Dissociation of oxygen to hemoglobin is dependent on factors:
A. Heterogenous structure of hemoglobin
B. 2,3 DPG binding
C. Heme-heme interaction
-normal curve : Sigmoidal
-p50 value: Partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen.
**** SHIFT TO THE RIGHT, WON’T HOLD TIGHT (to oxygen) !! , SHIFT TO THE LEFT, WON’T
LET GO (of oxygen)

Heme to heme interaction

* Two conformations of hemoglobin due to the binding of oxygen to the heme molecule:
T (Taut form/ Tensed form)
-lower affinity to oxygen, higher affinity to carbon dioxide; with 2,3-DPG in between beta global
chains , attached to the salt bridges
R (Relaxed form)
-With bound oxygen ; w/o 2,3 DPG; Higher affinity to oxygen

Prepared by: Hazel Hope Acosta HHMA’17