HEMOGLOBINOPATHIES prt.2 Flashcards by Kyra Catedral

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

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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)

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Sickle cell

polychromasia???
______reticulocyte count
Increased_____

Moderate to marked

10% & 25%

RDW

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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.

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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

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Most common screening test for SCD

Capitalizes on the decreased solubility of deoxygenated Hb S in solution, producing TURBIDITY

Hemoglobin solubility test

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Hemoglobin solubility test reagents

BLOOD + Buffered salt Solution containing reducing agent (sodium hydrosulfite (dithionite)) + a detergent-based lysing agent (Saponin)

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Blood + Buffered salt Solution containing reducing agent (__________) + a detergent-based lysing agent (______)

sodium hydrosulfite (dithionite)

Saponin

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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

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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

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Hemoglobin Fraction and Quantification

AHIN

• Alkaline Hemoglobin electrophoresis
• HPLC (High Performance Liquid Chromatography)
• Isoelectric Focusing (IEF)
• Neonatal Screening

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• 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

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common 1st step in confirmation of hemoglobinopathies

Hgb molecules assume a negative charge and migrate toward the anode (positive charge)

Alkaline Hemoglobin electrophoresis

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It is BEST used for thalassemias

HPLC (High Performance Liquid Chromatography)

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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)

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• Uses electric current to push the hgb molecules across a ph gradient

Isoelectric Focusing (IEF)

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CONFIRMATORY TECHNIQUE that is expensive and complex

Requires well trained and experienced
laboratory personnel

Isoelectric Focusing (IEF)

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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

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Neonatal Screening
Requires a more sophisticated approach
Using 3 techniques:

adapted IEF
HPLC
Reversed-phase HPLC

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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.

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Assessment of Inflammation

good predictor of sickle cell complications and mortality

WBC Count

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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

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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