MODULE 3 Flashcards
(263 cards)
1
Q
is the most common complex organic molecule in vertebrates.
A
Hemoglobin (Hgb or Hb)
2
Q
It comprises approximately 95% of the cytoplasmic content of RBCs.
A
Hemoglobin (Hgb or Hb)
3
Q
provides protection from denaturation in the plasma and loss through the kidneys
A
hemoglobin in RBCs
4
Q
concentration of hemoglobin within RBCs
A
34 g/dL
5
Q
molecular weight of hemoglobin within RBCs
A
64,000 Daltons
6
Q
Hemoglobin’s main function is to transport oxygen from the (?) and transport carbon dioxide from the (?) for exhalation.
A
lungs to tissues
tissues to the lungs
7
Q
Hemoglobin also contributes to (?) by binding and releasing hydrogen ions and transports nitric oxide (NO)
A
acid-base balance
8
Q
a regulator of vascular tone
A
nitric oxide (NO),
9
Q
Components of Hemoglobin
A
- Four heme molecules each composed of:
a. The nitrogenous substance, protoporphyrin IX
b. Iron atom in the ferrous (Fe2+) state. - The protein component known as globin made up of two sets or dimers of two different polypeptide chains.
- The transient resident, 2,3-biphosphoglycerate (2,3-BPG) which regulates oxygen affinity to the hemoglobin molecule
10
Q
- Four heme molecules each composed of:
a. The nitrogenous substance, (?)
b. Iron atom in the (?) state.
A
protoporphyrin IX
ferrous (Fe2+)
11
Q
- The protein component known as (?) made up of two sets or dimers of two different polypeptide chains.
A
globin
12
Q
- The transient resident, (?) which regulates oxygen affinity to the hemoglobin molecule
A
2,3-biphosphoglycerate (2,3-BPG)
13
Q
Structure of the Hemoglobin Components
A
- Heme molecule
- Globin molecule
- The Complete Hemoglobin Molecule
14
Q
consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron (Fe2+)
A
Heme
15
Q
Each of the (?) is positioned in a pocket of the polypeptide chain near the surface of the hemoglobin molecule.
A
four heme groups
16
Q
The (?) in each heme molecule reversibly combines with one oxygen molecule.
A
ferrous iron
17
Q
When the ferrous irons are oxidized to the ferric state (Fe3+) the hemoglobin will become (?), which cannot bind oxygen.
A
methemoglobin
18
Q
The (?) comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each.
A
four globin chains
19
Q
Variations in amino acid sequences give rise to different types of
A
polypeptide chains.
20
Q
Each chain is designated by a
A
Greek letter
21
Q
The hemoglobin molecule can be described by its (?) structures.
A
primary, secondary, tertiary, and quaternary protein
22
Q
refers to the amino acid sequence of the polypeptide chains.
A
primary structure
23
Q
refers to chain arrangements in helices and non-helices
A
secondary structure
24
Q
refers to the arrangement of the helices into a pretzel-like configuration
A
tertiary structure
25
loop to form a cleft pocket for heme
Globin chains
26
Each globin chain contains a heme group that is suspended between the (?) of the polypeptide chain
E and F helices
27
The (?) at the center of the protoporphyrin IX ring of heme is positioned between two histidine radicals.
iron atom
28
Globin chain amino acids in the (?) are hydrophobic
cleft
29
Globin chain amino acids on the (?) are hydrophilic, which renders the molecule water soluble.
outside
30
This arrangement also helps iron remain in its (?) form regardless of whether it is oxygenated or deoxygenated
divalent ferrous
31
(carrying an oxygen molecule)
oxygenated
32
(not carrying an oxygen molecule).
deoxygenated
33
also called a tetramer; describes the complete hemoglobin molecule.
quaternary structure
34
The complete hemoglobin molecule is (?), has four heme groups attached to four polypeptide chains, and may carry up to four molecules of oxygen
spherical
35
The predominant adult hemoglobin, (?) (also known as Hb A), is composed of two α-globin chains and two β-globin chains.
Hb A1
36
hold the dimers in a stable form
Strong α1–β1 and α2–β2 bonds
37
are important for the stability of the quaternary structure in the oxygenated and deoxygenated forms
α1–β2 and α2–β1 bonds
38
Alpha
39
Beta
40
Gamma A
41
Gamma G
42
Delta
43
Epsilon
44
Seta
45
Theta
46
This is a substance produced in the anaerobic glycolytic (Embden-Meyerhof) pathway.
2,3-Biphosphoglycerate (2,3-BPG)
47
This pathway generates energy for red blood cells.
Embden-Meyerhof
48
is specifically produced in the by-pass pathway within the Embden Meyerhof pathway which is known as the Luebering-Rapoport Shunt.
2,3-BPG
49
O2 affinity decreases
Hgb binds 2,3-BPG
50
oxygen affinity increases
Hgb releases the 2,3-BPG
51
This illustrates the reverse relationship between the amount of 2,3-BPG and the affinity of Hgb for O2.
Luebering-Rapoport Shunt
52
How is Hemoglobin synthesized by the red blood cells?
A. Heme Biosynthesis
B. Globin Biosynthesis
C. Hemoglobin Assembly (Heme + Globin)
D. Development of Hemoglobin from fetal to adult life (Switching of globin chains)
53
The biosynthesis of heme occurs mainly in the (?) of the bone marrow red cell precursors starting from the pronormoblast through the circulating reticulocytes.
mitochondria and the cytoplasm
54
As the red cell further matures and lose their (?), they lose their ability to further synthesize hemoglobin.
ribosomes and mitochondria
55
Heme biosynthesis begins in the mitochondria with the condensation of (?) catalyzed by aminolevulinate synthase to form (?)
glycine and succinyl coenzyme A (CoA)
aminolevulinic acid (ALA)
56
In the cytoplasm, ALA undergoes several transformations from (?) to (?), which, catalyzed by (?), becomes (?).
porphobilinogen
coproporphyrinogen III
coproporphyrinogen oxidase
protoporphyrinogen IX
57
In the mitochondria, (?) is converted to (?) by (?).
protoporphyrinogen IX
protoporphyrin IX
protoporphyrinogen oxidase
58
(?) is added, catalyzed by (?) to form heme.
Ferrous (Fe2+) ion
ferrochelatase
59
In the cytoplasm, heme assembles with an α chain and non-a chain, forming a dimer, and ultimately two dimers join to form the
hemoglobin tetramer
60
(?), a plasma protein, carries iron in the ferric (Fe3+) form to developing erythroid cells.
Transferrin
61
(?) binds to transferrin receptors on erythroid precursor cell membranes and the receptors and transferrin (with bound iron) are brought into the cell in an endosome.
Transferrin
62
releases the iron from transferrin
Acidification of the endosome
63
Iron is transported out of the endosome and into the mitochondria where it is reduced to the ferrous state, and is united with (?) to make heme.
protoporphyrin IX
64
Heme leaves the mitochondria and is joined to the
globin chains in the cytoplasm
65
code for six globin chains
Six structural genes
66
are on the short arm of chromosome 16
α- and ζ-globin genes
67
is on the short arm of chromosome 11
ε-, γ-, δ-, and β-globin gene cluster
68
In the human genome, there is one copy of each globin gene per chromatid, for a total of (?), with the exception of a and g.
two genes per diploid cell
69
There are two copies of the a- and γ-globin genes per chromatid, for a total of
four genes per diploid cell
70
The production of globin chains takes place in (?) from the pronormoblast through the circulating polychromatic erythrocyte, but not in the mature erythrocyte.
erythroid precursors
71
Transcription of the globin genes to messenger ribonucleic acid (mRNA) occurs in the
nucleus
72
translation of mRNA to the globin polypeptide chain occurs on
ribosomes in the cytoplasm
73
Although transcription of the a-globin genes produces more mRNA than the b-globin gene, there is less efficient translation of the
a-globin mRNA
74
are produced in approximately equal amounts
a and b chains
75
After translation is complete, the chains are released from the (?) in the cytoplasm.
ribosomes
76
After their release from ribosomes, each globin chain binds to a heme molecule, then forms a
heterodimer
77
have a charge difference that determines their affinity to bind to the α chains.
non-a chains
78
has a positive charge and has the highest affinity for a β chain due to its negative charge
α chain
79
has the next highest affinity, followed by the δ-globin chain
γ-globin chain
80
Two heterodimers then combine to form a (?)
This completes the hemoglobin molecule.
tetramer
81
Two a and two β chains form (?), the major hemoglobin present from 6 months of age through adulthood
Hb A
82
contains two a and two δ chains
Hb A2
83
Owing to a mutation in the promoter region of the δ-globin gene, production of the (?) is very low.
δ chain polypeptide
84
comprises less than 3.5% of total hemoglobin in adults
Hb A2
85
contains two a and two γ chains
Hb F
86
In healthy adults, (?) comprises 1% to 2% of total hemoglobin, and it is present only in a small proportion of the RBCs (uneven distribution).
Hb F
87
These RBCs with Hb F are called
F or A/F cells
88
The various amino acids that comprise the globin chains affect the net charge of the
hemoglobin tetramer
89
are used for fractionation, presumptive identification, and quantification of normal hemoglobin and hemoglobin variants
Electrophoresis and high-performance liquid chromatography (HPLC)
90
(?) of globin gene DNA provides definitive identification of variant hemoglobin.
Molecular genetic testing
91
Hemoglobin composition differs with
prenatal gestation time and postnatal age
92
Hemoglobin changes reflect the sequential activation and inactivation (or switching) of the globin genes, progressing from the (?) on chromosome 16 and from the (?) on chromosome 11.
ζ- to the a-globin gene
ε- to the γ-, δ-, and β-globin genes
93
normally appear only during the first 3 months of embryonic development.
ζ- and ε-globin chains
94
These two chains, when paired with the a and γ chains, form the
embryonic hemoglobins
95
During the second and third trimesters of fetal life and at birth, (?) is the predominant hemoglobin.
Hb F (a2γ2)
96
By 6 months of age and through adulthood, (?) is the predominant hemoglobin, with small amounts of Hb A2 (a2δ2) and Hb F.
Hb A (a2β2)
97
In utero, (?) predominates.
fetal hemoglobin
98
When compared with adult hemoglobin, fetal hemoglobin has (?), a characteristic that allows sufficient oxygen transfer to the fetus in the absence of gas exchange with the external environment due to the relatively hypoxic environment in utero.
very high oxygen-binding capacity
99
As a result, the hemoglobin level in a near-term fetus or term infant is relatively high and remains elevated up to around the (?) to compensate for the high oxygen affinity of hemoglobin.
8th to the 12th week post-partum
100
reference intervals for hemoglobin concentration
Men:
Men: 14 to 18 g/dL (140 to 180 g/L)
101
reference intervals for hemoglobin concentration
Women:
Women: 12 to 15 g/dL (120 to 150 g/L)
102
reference intervals for hemoglobin concentration
Newborns:
Newborns: 16.5 to 21.5 g/dL (165 to 215 g/L)
103
Reference intervals for infants and children vary according to
age group
104
Individuals living at high altitudes have (?) as a compensatory mechanism to provide more oxygen to the tissues in the oxygen-thin air.
slightly higher levels of hemoglobin
105
Hemoglobin variants are a part of the
normal embryonic and fetal development
106
They may also be (?) of hemoglobin in a population, caused by variations in genetics.
pathologic mutant forms
107
Some well-known hemoglobin variants, such as in sickle-cell anemia, are responsible for diseases and are considered (?).
hemoglobinopathies
108
Other variants cause no detectable pathology, and are thus considered
non-pathological variants
109
1. In the embryo (products of yolk sac erythroblasts)
• Gower 1 (ζ2ε2)
• Gower 2 (α2ε2)
• Hemoglobin Portland I (ζ2γ2)
• Hemoglobin Portland II (ζ2β2)
110
2. In the fetus: (begins in early embryogenesis, peaks during third trimester and declines just before birth)
• Hemoglobin F (α2γ2).
111
3. Right after birth up to before the first year of life:
• Hemoglobin F (α2γ2) at 60 – 90% of total Hb
• Hemoglobin A (adult hemoglobin) (α2β2) at 10 – 40% of total Hb.
112
4. Two years through adulthood:
• Hemoglobin A (adult hemoglobin) (α2β2)
• Hemoglobin A2 (α2δ2)
• Hemoglobin F (fetal hemoglobin) (α2γ2)
113
– 90% of total Hb
• Hemoglobin F (α2γ2) at 60
114
– 40% of total Hb.
• Hemoglobin A (adult hemoglobin) (α2β2) at 10
115
– The most common with a normal amount over 95%
• Hemoglobin A (adult hemoglobin) (α2β2)
116
– δ chain synthesis begins late in the third trimester and, in adults, it has a normal range of 1.5–3.5%
• Hemoglobin A2 (α2δ2)
117
– In adults Hemoglobin F is restricted to a limited population of red cells called F-cells (1 – 2%)
• Hemoglobin F (fetal hemoglobin) (α2γ2)
118
B. Variant forms that may cause disease:
1. Hemoglobin D-Punjab (α2βD2)
2. Hemoglobin H (β4)
3. Hemoglobin Barts (γ4)
4. Hemoglobin S (α2βS2)
5. Hemoglobin C (α2βC2)
6. Hemoglobin E (α2βE2)
7. Hemoglobin AS
8. Hemoglobin SC
119
A. Normal Hemoglobins
120
is one of the sub-variants of Hemoglobin D, a variant of hemoglobin found in human blood
Hemoglobin D-Punjab (α2βD2)
121
It is so named because of its higher prevalence in the Punjab region of India and Pakistan.
Hemoglobin D-Punjab (α2βD2)
122
accounts for over 55% of the total hemoglobin variants there
Hemoglobin D-Punjab
123
Hemoglobin D-Punjab was first discovered in the early 1950s in a mixed British and American family of Indian origin from the Los Angeles area; hence it is also sometimes called
“D Los Angeles”
124
A variant form of hemoglobin, formed by a tetramer of β chains, which may be present in variants of α thalassemia.
Hemoglobin H (β4)
125
Although each of the beta (β) globin chains is normal, the (?) does not function normally.
tetramer of 4 beta chains
126
It has an increased affinity for oxygen, holding onto it instead of releasing it to the tissues and cells.
tetramer of 4 beta chains
127
is also associated with significant breakdown of red blood cells (hemolysis) as it is unstable and tends to form solid structures within red blood cells
Hemoglobin H
128
Serious medical problems are not common in people with (?), though they often have anemia.
hemoglobin H disease
129
– A variant form of hemoglobin, formed by a tetramer of γ chains, which may be present in variants of α thalassemia.
Hemoglobin Barts (γ4)
130
If a small amount of Hb Barts is detected, it usually disappears shortly after birth due to
dwindling gamma chain production
131
These children have one or two alpha gene deletions and are silent carriers or have the alpha thalassemia trait.
Hemoglobin Barts (γ4)
132
If a child has a large amount of Hb Barts, he or she usually has
hemoglobin H disease and a three-gene deletion
133
have hydrops fetalis and usually do not survive without blood transfusions and bone marrow transplants
Fetuses with four-gene deletions
134
– A variant form of hemoglobin found in people with sickle cell disease.
Hemoglobin S (α2βS2)
135
There is a variation in the β-chain gene, causing a change in the properties of hemoglobin, which results in sickling of red blood cells.
Hemoglobin S (α2βS2)
136
The gene defect is a single nucleotide mutation of the β-globin gene, which results in (?) being substituted by (?) at position 6 (E6V) substitution.
glutamic acid (E/Glu)
valine (V/Val)
137
This is normally a benign mutation, causing no apparent effects on the secondary, tertiary, or quaternary structures of hemoglobin in conditions of normal oxygen concentration.
Hemoglobin S (α2βS2)
138
However, under low oxygen concentration, (?) polymerizes and forms fibrous precipitates because the deoxy form of hemoglobin exposes a hydrophobic patch on the protein between the E and F helices.
HbS
139
However, under low oxygen concentration, HbS polymerizes and forms fibrous precipitates because the deoxy form of hemoglobin exposes a hydrophobic patch on the protein between the E and F helices.
140
– Another variant due to a variation in the β-chain gene
Hemoglobin C (α2βC2)
141
Hb C or HbC is an abnormal hemoglobin in which substitution of a (?) residue with a (?) residue at the 6th position of the β-globin chain has occurred (E6K substitution).
glutamic acid
lysine
142
This variant causes a mild chronic hemolytic anemia.
Hemoglobin C (α2βC2)
143
E6V substitution
Hemoglobin S (α2βS2)
144
E6K substitution
Hemoglobin C (α2βC2)
145
E26K substitution
Hemoglobin E (α2βE2)
146
– is an abnormal hemoglobin with a single point mutation in the β chain.
Hemoglobin E (α2βE2)
147
At position 26 there is a change in the amino acid, from glutamic acid to lysine (E26K).
Hemoglobin E (α2βE2)
148
is very common among people of Southeast Asian including Northeast Indian, East Asian descent
Hemoglobin E (α2βE2)
149
This variant causes a mild chronic hemolytic anemia.
Hemoglobin E (α2βE2)
150
– A heterozygous form causing sickle cell trait with one adult gene and one sickle cell disease gene.
Hemoglobin AS
151
– A compound heterozygous form with one sickle gene and another encoding Hemoglobin C.
Hemoglobin SC
152
These individuals have a mild hemolytic anemia and moderate enlargement of the spleen.
Hemoglobin SC
153
Persons with Hb SC disease may develop the same (?) complications as seen in sickle cell anemia, but most cases are less severe.
vaso-occlusive (blood vessel-blocking)
154
Patients with sickle cell trait (?) are protected from malaria.
(heterozygote for the sickle cell gene, Hb AS)
155
- the process of measuring the concentration of hemoglobin in blood.
Hemoglobinometry
156
- Determination of the concentration of hemoglobin in blood may be done using any of the following principles:
a. Methods based on the development of color when blood is mixed with a reagent.
b. A method based on specific gravity of the blood.
c. Measurement of oxygen combining capacity.
d. Indirect measurement using iron content.
e. By converting hemoglobin into one of several compounds and comparing the resulting compound with a known standard either visually or photoelectrically.
f. Electrical impedance or light absorption using automated hematology analyzers.
157
Laboratory determination of hemoglobin concentration
1. Gravimetric Method (Specific gravity method or Copper Sulfate method)
2. Gasometric method (Oxygen capacity method)
3. Chemical method
4. Colorimetric method
5. Hemoglobin estimation using Automated Hematology Analyzers
6. Hemoglobin Electrophoresis
158
– based on the estimation of specific gravity of blood, assuming that the patient has normal protein levels.
Gravimetric Method (Specific gravity method or Copper Sulfate method)
159
Specific gravity of copper sulfate of 1.053 corresponds to an Hb level of
12.5 g/dL
160
A drop of blood, allowed to fall into a copper sulfate solution of specific gravity 1.053, becomes encased in a +?), which prevents dispersion of fluid for 15 seconds
sac of copper proteinate
161
a. If the drop of blood falls in a few seconds,
it has a greater specific gravity than the solution
162
b. If the drop of blood rises in a few second,
it has a lower specific gravity than the solution
163
c. If the drop of blood remains suspended for about 15 seconds and then falls,
more or less it has the same specific gravity as the solution
164
This method is used by blood banks as a screening test for blood donors.
Gravimetric Method (Specific gravity method or Copper Sulfate method)
165
In most cases, this method is capable of estimating Hb within ~0.5 g/dL, which is comparable to a coefficient of variation (CV) of 2%
Gravimetric Method (Specific gravity method or Copper Sulfate method)
166
Based on the amount of oxygen in a given sample of blood, considering that hemoglobin will combine with and liberate a fixed quantity of oxygen.
Gasometric method (Oxygen capacity method)
167
(?)
The blood is hemolyzed with (?) and the oxygen is collected and measured in a (?)
Gasometric method (Oxygen capacity method)
saponin
Van Slyke apparatus
168
The oxygen combining capacity of blood is
1.34 ml O2 per gram of hemoglobin.
169
The volume of oxygen is corrected for
temperature and pressure
170
the hemoglobin concentration is determined with the use of the following formula:
171
An indirect measure of hemoglobin based on the amount of iron for a given sample of blood.
Chemical method
172
Based on the molecular structure, the iron content of hemoglobin is
0.347%
173
Thus, 1 gram or 1000 mg of Hb contains (?) of iron.
3.47 mg
174
The concentration of hemoglobin in blood is calculated by
dividing the iron content (mg/dl) by 3.47
175
(?)
Iron is detached from the hemoglobin by treating the blood with (?) in the presence of (?).
Wong’s method
concentrated sulfuric acid
potassium persulfate
176
(?)
The protein is precipitated with (?) and filtered out.
Wong’s method
tungstic acid
177
The iron content of the filtrate is determined in a colorimeter and the Hb value is calculated with the following formula:
178
The color of fresh blood is compared with a series of colored standards representing known quantities of hemoglobin.
Visual colorimetric method: Direct matching method
179
- In this method drop of blood is placed on filter paper and the color is matched with standard (Fig. 3-5[A]).
▪ Tallquist method
180
- In this method small glass chamber is filled with whole blood by capillary action. Then the glass chamber is illuminated by battery bulb. Color of the blood is matched with standard after seeing through eye piece.
▪ Dare’s method
181
- In this method the color of diluted oxyhemoglobin is matched visually.
▪ Spencer’s method
182
This method is less accurate than Sahli’s method. It is more difficult for the human eye to accurately grade and match small differences in red color than brown color of acid hematin.
▪ Spencer’s method
183
- This technique of estimating hemoglobin is based on comparing the color of a drop of blood absorbed on a particular type of chromatography paper against a printed scale of color corresponding to different levels of hemoglobin ranging from 4 to 14 g/dl.
▪ WHO hemoglobin color scale method
184
(?)
Blood is mixed with (?). This hemolyzes the red cells and converts the hemoglobin to a brownish yellow solution of acid hematin.
Acid hematin method
0.1 N HCl
185
is then compared with a colored glass standard (Comparator Block)
acid hematin
186
The procedures employed in the following are based on the principle of Acid Hematin method:
▪ Sahli – Hellige method
▪ Haiden – Hausser method
▪ Sahli – Adams method
▪ Osgood – Haskin method
▪ Haldane method
▪ Newcomer method
187
▪ Sahli –
Hellige method
Adams method
188
▪ Haiden –
Hausser method
189
▪ Osgood –
Haskin method
190
(?)
Blood is mixed with (?).
The solution is then boiled.
The hemoglobin is then converted to a blue–green solution of (?).
The color of the solution is then compared with a known standard or in a colorimeter
Alkali hematin method
0.1 N NaOH
alkaline hematin
191
Alkali hematin method will not accurately measure the hemoglobin of an infant, because infant’s blood contains
alkali resistant fetal hemoglobin (HbF)
192
The principle of Alkali – hematin method is used in the following:
▪ Standard method using Gibson and Harrison’s standard solution
▪ Clegg and King method
193
Principle:
Blood is mixed with either 0.1% sodium carbonate or 0.007 N Ammonium hydroxide solution. This converts the Hb to oxyhemoglobin. The depth of the resulting color is then measured in a photometer with a green filter (540 nm) and 0.007 N ammonium hydroxide as a blank
Oxyhemoglobin method
194
Principle:
Blood is diluted with Drabkin’s solution which contains ferricyanide and Cyanide. The potassium ferricyanide oxidizes hemoglobin to hemiglobin and potassium cyanide provides cyanide ions to form hemiglobincyanide, which has a broad absorption maximum at a wavelength of 540 nm. The absorbance of the solution is measured in a Photometer or spectrophotometer at 540 nm and compared with that of a standard HiCN solution.
Cyanmethemoglobin method (MHbCN method) or Hemiglobincyanide (HiCN) method
195
The concentration of hemoglobin in MHbCN method is computed using the following formula:
196
Hb determination is done by HiCN or the oxy-hemoglobin method. In the former, the blood specimen is diluted with a reagent containing ferricyanide and cyanide, which converts Hb to HiCN. The absorbance of the HiCN at 540 nm wavelength is then used for quantitation. In the latter, the blood specimen is diluted with an aqueous solution tetrasodium salt of ethylenediaminetetraacetic acid (EDTA) and mixed with air to convert Hb to oxyhemoglobin. The absorbance of oxyhemoglobin at 540 nm is then measured. A typical analyzer working on venous blood has a CV of ≤1.2% for Hb measurement.
Hemoglobin estimation using Automated Hematology Analyzers
197
These analyzers have become increasingly sophisticated in the last few decades with the incorporation of noncyanide methods. Hb determination is done using sodium lauryl sulfate (SLS), a surfactant that dissolves lipoproteins of the cell membrane of the red blood cells to release Hb and converts it into SLS-Hb. Concentration of SLS-Hb is measured as light absorbance and is calculated by comparison with the absorbance of the diluent measured before the sample is added
Hemoglobin estimation using Automated Hematology Analyzers
198
– Test for abnormal hemoglobins. This is generally considered the best method for separating and identifying hemoglobinopathies.
Hemoglobin Electrophoresis
199
One protocol for hemoglobin electrophoresis involves the use of two systems:
Cellulose acetate and agarose medium
Citrate agar
200
Initial electrophoresis is performed in alkaline buffers.
Cellulose acetate and agarose medium
201
are the major support media used because they yield rapid separation of HbA, F, S and C and many other mutants with minimal preparation time.
Cellulose acetate and agarose medium
202
because of the electrophoretic similarity of many structurally different hemoglobins, the evaluation must be supplemented by a procedure that measures some other property
Cellulose acetate and agarose medium
203
A simple procedure which confirms the identification of both HbS and HbC, as well as HbA, HbF and many other mutants
Citrate agar electrophoresis
204
This method is based on the complex interactions of the hemoglobin with the electrophoretic buffer (acid pH) and the agar support.
Citrate agar electrophoresis
205
Because some hemoglobins have the same charge and, therefore, the same electrophoretic mobility patterns, hemoglobins that exhibit an (?) may be subjected to electrophoresis at an acid pH for definitive separation.
abnormal electrophoretic pattern at an alkaline pH
206
In an acid pH some hemoglobins assume a negative charge and migrate toward the
anode
207
while others are positively charged and migrate toward the
cathode (negative pole)
208
Hb S migrates with Hb D and Hb G on (?) but separates from Hb D and Hb G on (?).
alkaline electrophoresis
acid electrophoresis
209
Similarly, Hb C migrates with (?) on alkaline electrophoresis but separates on acid electrophoresis.
Hb E and Hb O
210
The relative amount of hemoglobin is not proportional to the size of the band
211
in sickle cell trait (Hb AS), the bands may appear equal, but the amount of HbA
exceeds that of HbS
212
The main function of hemoglobin is to
transport oxygen.
213
since oxygen is (?), it has to depend on hemoglobin found in red blood cells for its transport to the different organs and tissues of the human body
non-water soluble
214
hemoglobin increases (?() in blood by about a hundredfold. this means that without hemoglobin, in order to provide sufficient oxygen to the tissues, blood would have to make a complete circuit through the body in less than a second, instead of the minute that it actually takes.
o2 solubility
215
That would take a mighty powerful heart, which in normal circumstances cannot be maintained by the human heart leading to increased (?) that may result to heart failure.
cardiac output
216
During oxygenation, each of the (?) in a hemoglobin molecule can reversibly bind one oxygen molecule.
four heme iron atoms
217
Approximately (?) of oxygen is bound by each gram of hemoglobin.
1.34 mL
218
Let's follow the path of oxygen from the lungs to the peripheral tissues. Oxygen diffuses from the (?) of the lungs, little sacs at the end of the finely divided air passageways in the lung into the (?) of the bloodstream and then into the (?), where it binds to hemoglobin.
alveoli
capillaries
red blood cells
219
The concentration of oxygen is relatively high in the alveoli, about (?) which means that Hb is virtually 100% saturated in the lungs and all four heme molecules have an O2 molecule bound to them.
100 mmHg
220
The reference interval for arterial oxygen saturation is
96% to 100%
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The affinity of hemoglobin for oxygen relates to the (?)
partial pressure of oxygen (PO2)
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often defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin, called the
P50 value
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The relationship is described by the (?), which plots the percent oxygen saturation of hemoglobin versus the PO2 (Figure 3-8).
oxygen dissociation curve of hemoglobin
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The curve is (?), which indicates low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension.
sigmoidal
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among hemoglobin subunits contributes to the shape of the curve.
Cooperation
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Hemoglobin that is completely (?) has little affinity for oxygen.
deoxygenated
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The secret to hemoglobin’s success as an oxygen delivery molecule is the fact that it has (?) that communicate to each other.
four subunits
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Evidence for this is provided by hemoglobin’s (?) in oxygen binding.
“cooperativity”
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In other words, the binding of one O2 molecule affects the binding of others, as we can see by the following:
• In order to achieve 25% saturation (an average of 1 O2 molecule per hemoglobin), the amount of O2 needs to be about (?).
• In order to achieve 50% saturation (an average of 2 O2 molecules per hemoglobin), the amount of O2 needs to be about (?).
18 mm Hg
27 mm Hg
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Therefore, it is easier to bind the than
second molecule of O2
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Nobel Prize winners for Chemistry for their studies of the structures of hemoglobin and myoglobin.
Max Perutz and John Kendrew
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Using (?), hemoglobin was found to have two different forms or shapes.
X-ray diffraction
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The (?) is dependent on the presence or absence of oxygen.
conformation or shape
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The experiments revealed that (?) has a relatively low attraction for oxygen, but when one molecule of oxygen binds to a heme group, the structure changes to the oxygenated form, which has a greater attraction for oxygen.
Therefore, the second molecule of O2 binds more easily, and the third, and fourth even more easily.
deoxyhemoglobin
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The oxygen affinity of (?) is many times greater than that of (?).
oxy-hemoglobin
deoxy-hemoglobin
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illustrates the relationship between oxygen saturation of hemoglobin and the partial pressure of oxygen.
oxygen dissociation curve
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In the Normal (N) hemoglobin-oxygen dissociation curve, P50 is the partial pressure of oxygen (O2) needed for
50% O2 saturation of hemoglobin
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In the Left-shifted (L) curve with reduced P50, it can be caused by decreases in (?) (e.g., multiple transfusion of stored blood), (?) (raised pH), (?) (PCO2), and/or (?).
2,3-bisphosphoglycerate
H+ ions
partial pressure of carbon dioxide
body temperature
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A left shifted curve is also seen with hemoglobin F and hemoglobin variants that have (?) and in alkalosis.
increased oxygen affinity
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In the (?) with increased P50 can be caused by elevations in 2,3-BPG (e.g., in response to hypoxic conditions such as in high altitudes), H+ ions (lowered pH), PCO2, and/or temperature.
Right-shifted (R) curve
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A right-shifted curve is also seen with (?) and in the presence of hemoglobin variants that have decreased oxygen affinity.
pulmonary insufficiency, congestive heart failure, sever anemia
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Factors that affect Hemoglobin affinity for Oxygen
1. Partial pressure of oxygen
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patient with arterial and venous PO2 levels in the reference intervals (80 to 100 mm Hg arterial and 30 to 50 mm Hg venous):
higher percent oxygen saturation
higher affinity for oxygen than a patient for whom the curve is normal
Shift to the left
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patient with arterial and venous PO2 levels in the reference intervals (80 to 100 mm Hg arterial and 30 to 50 mm Hg venous):
a lower oxygen affinity
shift to the right
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explains the lower affinity of hemoglobin for oxygen due to increases in the partial pressure of carbon dioxide (CO2) which eventually decreases the blood pH
Bohr effect
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Whenever the human body undergoes increased cellular respiration, such as in strenuous physical activities, there is also an increase in metabolic activity within the tissues involved resulting in the production of CO2 as a metabolic waste product.
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To transport CO2 through the venous blood, it diffuses into the red blood cells combining with water to form carbonic acid (H2CO3). This reaction is facilitated by the enzyme, carbonic anhydrase. The carbonic acid will then dissociate to release H+ and bicarbonate (HCO3-). The increase in H+ due to this reaction decreases the blood pH as explained by the Bohr effect.
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In addition to hydrogen ions and carbon dioxide, a key allosteric effector of hemoglobin is
2,3-Biphosphoglycerate (2,3-BPG)
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a small molecule made in red blood cells
2,3-Biphosphoglycerate
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affects oxygen-binding affinity by binding in a small central cavity of deoxygenated hemoglobin. This shifts the equilibrium towards deoxy-hemoglobin
2,3-BPG
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The presence of acids leads to:
• ↑ H+
• ↓ pH
• ↑ O2
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This promotes formation of the deoxy form of hemoglobin, shifting the oxygen dissociation curve to the right, promoting oxygen release to actively respiring tissues.
presence of acids
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At high altitude, when oxygen in the atmosphere is scarce because the air is “thinner,”:
• ↑ 2,3- BPG
• ↑ CO2
• ↓ O2
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(helping hemoglobin to release more of its bound oxygen = ↑ aerobic capacity)
• ↑ 2,3- BPG
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It takes about 24 hours forits levels to rise, and over longer periods of time, the levels continue to increase as part of the acclimation effect.
2,3-BPG
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2,3-BPG does not bind to fetal hemoglobin
• ↑ metabolic rates
• ↑ thermal energy
• ↑ average kinetic energy
• ↑ temperature
• ↓ affinity for oxygen
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Giving the developing fetus better access to oxygen from the mother's bloodstream:
• ↑ 2,3- BPG
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This results in tighter binding of oxygen relative to maternal hemoglobin.
2,3-BPG does not bind to fetal hemoglobin
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.
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Hb decrease its affinity for oxygen to facilitate delivery to the tissues; shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
• ↑ temperature
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Carbon dioxide
↑ cellular respiration
↑ metabolic activity
↑ CO2
↓ O2
↑ H+ ions
↑ 2,3-BPG
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In response to higher temperature, the Hb decrease its affinity for oxygen to facilitate delivery to the tissues. Thus, increased temperature in the blood shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
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In response to higher temperature, the Hb decrease its affinity for oxygen to facilitate delivery to the tissues. Thus, increased temperature in the blood shifts the oxygen dissociation curve to the right such as when tissues are actively engaged in physical activity, these tissues would require and eventually receive more O2.
