HEMOGLOBINOPATHIES prt.2 Flashcards
Incidence with Malaria
Greatest frequency in Central Africa, the Near East, the Mediterranean region, and parts of India.
•_______- offer some protection against______ in young patients
• Malaria used oxygen in the cell
Causes the cells to sickle
Sickle gene
cerebral falciparum malaria
Sickle cell
Observed on a wright-stained PBS
• (morphology)______
________- HALLMARK OF SCD
Normocytic, Normochromic
Long, curved cell with a pointed at each end (sickle cell)
Sickle cell
polychromasia???
______reticulocyte count
Increased_____
Moderate to marked
10% & 25%
RDW
Sickle cell
Leukocyte Alkaline Phosphatase (LAP) score - ???
Thrombocytosis -???
Immunoglobulin A -???
Serum ferritin -???
Chronic Hemolysis - ???
not elevated
Present
Elevated in all forms of SCD
normal in young patients but tend to be elevated later in life
Elevated levels of indirect and total bilirubin with accompanying jaundice.
Hemoglobin Solubility Test for the Presence of Hemo-globin S.
In a_______ test result, the solution is clear and the lines behind the tube are visible.
In a______ test result, the solution is turbid because of the polymerization of hemoglobin (Hb) S and the lines are not visible.
negative
positive
Most common screening test for SCD
Capitalizes on the decreased solubility of deoxygenated Hb S in solution, producing TURBIDITY
Hemoglobin solubility test
Hemoglobin solubility test reagents
BLOOD + Buffered salt Solution containing reducing agent (sodium hydrosulfite (dithionite)) + a detergent-based lysing agent (Saponin)
Blood + Buffered salt Solution containing reducing agent (__________) + a detergent-based lysing agent (______)
sodium hydrosulfite (dithionite)
Saponin
Hemoglobin solubility test
False Positive
(3)
False Negative
(2)
False Positive
→ Hyperlipidemia
→ Few rare
hemoglobinopathies
→ Too much blood added
False Negative
→ Infants younger than 6
mos
→ Low hematocrit
Other Hemoglobins that give POSITIVE result for hemoglobin solubility test
Hb C-Harlem (Georgetown)
Hb C- Ziguinchor
Hb S-Memphis
Hb S-Travis
Hb S-Antilles
Hb S-Providence
Hb S-Oman
Hb Alexander
Hb Porte-Alegre
Hemoglobin Fraction and Quantification
AHIN
• Alkaline Hemoglobin electrophoresis
• HPLC (High Performance Liquid Chromatography)
• Isoelectric Focusing (IEF)
• Neonatal Screening
• It was performed on cellulose acetate medium but it is being replaced by agarose medium -
Acid pH some hemoglobin assume a positive charge migrate toward the cathode (negative pole)
Alkaline Hemoglobin electrophoresis
common 1st step in confirmation of hemoglobinopathies
Hgb molecules assume a negative charge and migrate toward the anode (positive charge)
Alkaline Hemoglobin electrophoresis
It is BEST used for thalassemias
HPLC (High Performance Liquid Chromatography)
Separates hgb types in a cation exchnage column and usually requires only one sample injection
• Can identify and quantify low levels of Hb A2 and Hb F, but comigration of Hb A2 and Hb E occurs
HPLC (High Performance Liquid Chromatography)
• Uses electric current to push the hgb molecules across a ph gradient
Isoelectric Focusing (IEF)
CONFIRMATORY TECHNIQUE that is expensive and complex
Requires well trained and experienced
laboratory personnel
Isoelectric Focusing (IEF)
Requires a more sophisticated approach
Using 3 techniques: adapted IEF, HPLC &
Reversed-phase HPLC
• Distinguish not only the multitude of hemoglobin variants but also
numerous thalassemias
Neonatal Screening
Neonatal Screening
Requires a more sophisticated approach
Using 3 techniques:
adapted IEF
HPLC
Reversed-phase HPLC
Neonatal screening
Some reterence laboratories may use:
mass spectroscopy
matrix-assisted laser desorption- ionization time-of-flight (MALDI-TOF) mass spectrometry
lEF to separate hemoglobin types
nucleic acid identification of the genetic
mutation.
Assessment of Inflammation
good predictor of sickle cell complications and mortality
WBC Count
Assessment of Inflammation
Common indicators of inflammation
1.White Blood Cell count
- Erythrocyte Sedimentation Rate & C-Reactive Protein level
- C-Reactive Protein & Secretory Phospholipase A2
- Interleukin-6, Interleukin-10, and Protein S
- Annexin A5
- Elevated levels of Malondialdehyde (a marker of oxidative stress) & depleted levels of Alpha-Tocopherol (Vitamin E)
- Alpha-Tocopherol (Vitamin E) & C-Reactive Protein
exhibit variability too great to reliably predict episodes
Erythrocyte Sedimentation Rate & C-Reactive Protein level
both elevated during VOC and acute chest syndrome
C-Reactive Protein & Secretory Phospholipase A2
indicators in clinical practice
Interleukin-6, Interleukin-10, and Protein S
elevated before and during VOC
Annexin A5
in lipid damage from oxidative stress
Elevated levels of Malondialdehyde (a marker of oxidative stress) & depleted levels of Alpha-Tocopherol (Vitamin E)
rises during bouts of inflammation
Alpha-Tocopherol (Vitamin E) & C-Reactive Protein
Sickle Cell Trait
Heterozygous state
Hemoglobin AS: (Hb AS)
• Benign condition
• Generally asymptomatic
• No significant clinical or hematologic manifestations
• Under extremely hypoxic conditions
Hemoglobin AS: Hb AS
• Systemic sickling and vascular occlusion with pooling of sickled cells in the spleen, focal necrosis in the brain, rhabdomyolysis, and even death can occur
• Patient may develop splenic infarcts and cause sickling
• Severe respiratory infection, unpressurized flight at high altitudes and anesthesia
• Failure to concentrate urine - CONSISTENT abnormality in SCT
• Strenuous exercise and Military Recruits
Hb AS
• Peripheral Blood Smear in Sickle Cell Trait
• Normal RBC morphology with few target cells
• No abnormalities in leukocytes
and thrombocytes
• Hemoglobin Solubility - POSITIVE
• Electrophoresis or HPLC - presence of Hb S and Hb A
Hb AS
Most common non sickling variant encountered in the US
• 3RD most common in the world
Hb C
• Structural formula: a2B2 6Glu→Lys
• Result of this substitution is a net change in charge of +2
• Form a short, thick crystal within the RBC
• It is less soluble than Hb A
Hb C
• Crystalizes in the oxygenated state
• Shorter Hb C crystal does not alter RBC
Less splenic sequestration and hemolysis
Hb C
Clinical Features of Hemoglobin C
• Homozygous Hb C disease (____)
• Milder disease compared to_____
• Mild___ and ____
• No Vasoocclusive crises Heterozygous hemoglobin C trait (_____)
• Asymptomatic
Hb CC
SCD
splenomegaly and hemolysis
Hb AC
Laboratory Diagnosis for…
Normocytic, normochromic anemia
Marked increase of target cells
Slight to moderate increase in the number of reticulocytes (2%-3%)
Nucleated RBCs may be present
Hemoglobin C
Homozygous Hb C disease
Hexagonal Crystals within RBCs
Seen in PBS
Seen on wet preparation
Washing RBCs and resuspending in sodium citrate or hypertonic saline
HGB is concentrated within boundary of the crystal
Densely stained and vary in size
Appear oblong with pyramid-shaped or
pointed ends
Hb C
Hb C
Hgb solubility test
Definitive Diagnosis
NEGATIVE
using electrophoresis, HPLC, or nucleic acid testing
First Describe in 1954
• Hb E is a Beta chain variant in which lysine is substituted for glutamic acid in position 26
Hemoglobin E
Hemoglobin polymerization does notoccr
• Amino acid substitution at codon 26 inserts a cryptic splice site at the junction of exon 1 and intron 1 that causes abnormal alternative splicing and decreased transcription of function mRNA for the Hb E globin chain
Hemoglobin E
Mild anemia with microcytes and target cells
Shortened RBC survival
Icterus, hemolysis or splenomegaly
P. falciparum multiplies more slowly
Homozygous state (Hb EE)
Hb E
Hemoglobin solubility test -
NEGATIVE
Hb E
•______ state - > 90% Hb E
Mild anemia (hgb between 11.0 to 13.0 g/dL)
Very low MCV (55 to 65 fL)
Few to many target cells
Normal reticulocyte count
Homozygous
Hb E
•_____ state
Normal hemoglobin
MCV of 65 fL
Slight erythrocytosis and target cells
Approximately 25% to 30% Hb E
Heterozygous
Beta Chain variant
Substitution of lysine for glutamic acid at amino acid position 121
No clinical-symptoms are exhibited
Homozygotes - mild splenomegaly
Hemoglobin O - Arab
is inherited with Hb S - severe symptoms similar to those in Hb SS result
• only hemoglobin to move just slightly away from the point of application toward the cathode on citrate agar at an acid ph
Hb O-Arab
A group of atleast 16 B chain variants (Hb D) and 6 alpha chain variants (Hb G) that migrate in an alkaline pH at the same electrophoretic position as Hb S
They do not sickle
•(named after the place they discovered)
Hb D-Punjab and Hb D-Los Angeles
• Alpha chain variant of G hemoglobin
• Lysine replaces asparagine at 68 position
• The most common G variant
Hb G - Philadelphia
Hb D and G
Laboratory Diagnosis
• Hemoglobin Solubility test -_____
• Alkaline Electrophoresis
• Hb D and Hb G have same mobility as_____
• Hb D and Hb G can be separated from HbS on______
• Hb DD can be confused with heterozygous state for Hb D and BO-thalassemia
• Can be differentiated based in the MCV, levels of Hb A2 and family studies
Negative
Hb S
citrate at pH 6.0
Most common compound heterozygous syndrome
• Position 6 - glutamic acid is replaced by valine (Hb S) on one B-globin chain by lysine (Hb C) on other B-globin chain
Hemoglobin SC
CLINICAL FEATURES
• Milder form of SCD
• Growth and development are delayed compared with normal children
• Usually does not produce significant symptoms until the teenage years.
• Vasoocclusive complications - less frequest
• Moderate hemolytic anemia and splenomegaly
• Proliterative retinopathy is more common
Respiratory tract infection with S. pneumoniae are common
Hemoglobin SC
Hb SC
Hemoglobin solubility test -___
POSITIVE
Are rare compound heterozygous hemoglobinopathies
• Cause severe chronic hemolytic anemia with vasoocclusive episodes
• Both mutations replace glutamic acid at position_____;
______substitutes lysine and _____substitutes glutamine
Hemoglobin SO-Arab and Hb SD-Punjab
121
O-Arab
D- Punjab
Rare hemoglobin variant with substitution of aspartic acid for aspagine at position 73 of the B chain
Heterozygous
conditions Hb S-Korle Bu -
Asymptomatic
Hemoglobin S-Korle Bu
• 2 substitutions on the Beta chain: the sickle mutation and the Korle Bu mutation
• Heterozygous Hb C- Harlem are Asymptomatic
Hemoglobin C- Harlem
