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Review of cases on hemoglobin capillary electrophoresis
technologies
By Youssef Maakaroun
SEBIA
CAPILLARYS 2/NEONAT FAST MINICAP
Hemoglobin analysis and Hb A2 quantification with capillary electrophoresis technologies
CAPILLARYS 2 Flex Piercing
MINICAP Flex Piercing
Anode
--
--
--
---
---
--- -
-
- Cathode
+
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
Detection Injection of
proteins
Electro-endosmotic flow Electrical field
The Electro-Osmotic Flow (EOF) is a stronger force than the Electrical Field. As a result, all proteins are carried towards the cathodic end of the capillary
Principle of hemoglobin electrophoresis using capillary electrophoresis technologies
Thermic bridge temperature
controlled by Peltier effect
Anode + Cathode -
Injection < 1nL
Capillary in silicate and
thermo-conductive resin
(25µm diameter)
Migration until 10.000 volts
Deuterium lamp
Detector
Hemolysis
+dilution
Direct
quantification
at 415 nm
Stored at 2-8°C during 7
days
(washed red blood cells
3 months
at -80°C)
MINICAP Flex Piercing
Sample cap piercing: Use capped tubes
Sample agitation by rotation: Use primary blood tubes
Minicap Flex Piercing: 9 samples/hr
• Hemoglobin electrophoresis profile must always be associated with other essential information to consider the clinical context in case of thalassemias, elevated HbF or hemoglobin variants:
– Hematological parameters (Hb, median cell volume (MCV), mean corpuscular hemoglobin (MCH)….) to relate anemia, microcytosis or hychromia
– Iron balance (in particular for reduced Hb A2 due to iron deficiency) – Patient’s age, geographical origin and any possible family history or
transfusion
Information required for interpretation
Age Hb (g/dl) RBC (T/l) MCV (fl) MCH (pg)
Child 2-6yrs 12.2 ± 0.7 4.67 ± 0.3 77.6 ± 3.3 26.3 ± 1.3
Child 6-12yrs 12.7 ± 0.8 4.68 ± 0.3 80.4 ± 3.4 27.3 ± 1.3
Child 12-16yrs 13.5 ± 1.1 4.74 ± 0.4 83.8 ± 4 29.2 ± 1.5
Adult woman 11.5-15 4-5 82-98 27-32
Adult man 13-17 4.5-5.5 82-98 27-32
Result at end of analysis :
- HbA, HbA2 and HbF are detected automatically
- Relative quantitation of the different fractions
- Centered pattern
- Variant separation in a given zone (Z1 à Z15)
Hb A2 normal control SEBIA
Hemoglobin electrophoresis on CE systems
180 Variants in the database
Z 15 : Hb H Z 14 : //
Z 13 : Hb-J Rovigo, Hb N-Baltimore
Z 12 : Hb Bart’s, Hb J-Providence, Hb J-Mexico, Hb J-baltimore….
Z 11 : Denaturated Hb A, Hb Kaoshiung
Z 10 : Hb Hope, Hb M-Iwate
Z (A) : Hb A, Hb Camperdown, Hb Phnom Penh……
Z 8 : Acetylated Hb F, Hb Altanta, Hb Athens-GA
Z (F) : Hb F, denaturated Hb S, Hb Porto-Alegre……..
Z (D) : Hb D-Punjab, denaturated Hb E, Hb Korle-Bu, Hb Lepore, Hb Köln….
Z (S) : Hb S, Hb Hasharon, Hb Handsworth, denaturated Hb O-Arab.
Z (E) : Hb E, denaturated Hb C, Hb Köln, Hb A2 variants, M-Iwate Hb A2 variants
Z (A2) : Hb A2, Hb O-Arab
Z (C) : Hb C, Hb Constant Spring, Setif HbA2 variant
Z 1 : Hb dA2 Hb aA2, Hasharon Hb A2 variant, Winnipeg Hb A2 variant……
Hb identification for Capillarys/Minicap
Discrimination of normal curves from abnormal curves according to a color code
Normal profile
Abnormal profile
Known hemoglobin variants: > 1000
α chain variants
β chain variants
γ chain variants
δ chain variants
Others: double mutation, deletion, insertion, hybrids No current techniques cannot allow to separate all variants: Presence of rare variants co-migrating with normal hemoglobins or common variants (HbS, HbE, HbC…)
Result interpretation: Variants
Attentive observation of the pattern (variant α? β ? ? percentage of the abnormal fraction)
Use of complementary techniques to confirm the nature of the variant:
*HPLC
* Electrophoresis on acid agarose gel
* Isoelectrofocusing
* Denaturation by iso-propanol/heat to confirm unstable HB
* Itano test to confirm the presence of Hb S…
Hematological parameters
Ethnic origin
Family studies
Database of all known mutations of hemoglobin chains: http://globin.cse.psu.edu
• Definitive identification usually requires DNA analysis when a rare variant is observed by capillary electrophoresis
Result interpretation: Variants
Careful observation of the pattern ( HbA2 value? Increase HbF? Presence of additional fractions?)
Hematological parameters (Hb, MCV, morphological abnormalities…)
Iron status (to differentiate, alpha thalassemia and iron deficiency anemia : reduced Hb A2 is observed in case of iron deficiency)
Age and gender of the patient (HbF increased, HbA2 decreased for young children or pregnant women)
Ethnic origin
Family survey
Result interpretation: Thalassemias
How to interpret profiles with CE
Clinical context
9yrs child from Laos with
unknown family history
Hematological parameters
- WBC 5.62 G/L
- RBC 5.16 T/L
- Hb 8 g/dL
- Hct 27.9 %
- MCV 54.1 fL
- CCMH 15.5 pg
Iron status
- Iron 18 µmol/L (N: 10-30)
- Ferritin 115 µg/L (N: 10-200)
Hb H 4%
Hb A 94.7%
Hb A2 1.3%
Hb A
Hb A2
Hb H (zone 15)
Alpha-thalassemia on Capillarys/Minicap technologies
Presence of Hb H, reduced HbA2, anemia and
microcytosis indicate an alpha-thalassemia
Clinical context
11-yrs old palestinan child
Exploration of a subclavicular
adenopathy
Hematological parameters
- WBC 7.71 G/L
- RBC 5.18 T/L
- Hb 9.5 g/dL (11.5 - 15.5)
- Hct 29.6 % (35 - 45)
- MCV 57.1 fL (77 - 95)
Iron status
- Iron 15 µmol/L (N: 10-30)
- Ferritin 95 µg/L (N: 10-200)
Hb A2
Hb A
Beta-thalassemia on Capillarys/Minicap technologies
Elevated HbA2, anemia and microcytosis
indicate a beta-thalassemia
Hb A + PRESENCE OF A MAJOR VARIANT (Hb S, Hb C, Hb E)
Hb A + Hb E
Hb E = ~25 - 30%
Heterozygote A/E
Hb E < 25 %
Hb E > Hb A
Hb A + Hb C
Hb C = ~35 - 40%
Heterozygote A/C
Hb C < 35 %
Hb C > Hb A
Hb A + Hb S
Hb S = ~35 - 40%
Heterozygote A/S
Hb S < 35 %
Heterozygote A/S + a-thalassemia or
Iron deficiency
Hb S > Hb A
DoubleHeterozygote S/b+-thalassemia?
Vinatier I. CERBA recommandations (2010)
Heterozygote A/C + a-thalassemia or
Iron deficiency
Heterozygote A/E + a-thalassemia or
Iron deficiency
DoubleHeterozygote C/b+-thalassemia?
DoubleHeterozygote E/b+-thalassemia?
Variant in the S zone, what to do?
Heterozygous A/S
β6 Glu (negative) →Val (neutral)
Hb A2
Hb S
bm bm
a a
a a
b b
bm
b
Hb A
Heterozygous A/S
Heterozygote A/S on Capillarys/Minicap technologies
The HbX value is higher than 35%
(Hb S= ~35 to ~ 40% for heterozygote A/S)
Sickle cell test or Itano test positive
Variant in the S zone, what to do?
The HbX value is below 35%
(Hb S= ~35 to ~ 40% for heterozygote A/S)
Heterozygous A/non S beta
variant
Hb X non S
bm bm
a a
a a
b b
bm
b
Hb A
Heterozygous A/X
Heterozygote A/ X (in S zone) on Capillarys/Minicap technologies
Sickle cell test or Itano test negative
Minor fraction of alpha variant
Beta variant (15% heterozygote)
Alpha variant (10-15% heterozygote, major fraction)
Alpha variant (17% heterozygote, major fraction)
Beta variant (15-25% heterozygote)
Database of known mutations for hemoglobin variants: http://globin.cse.psu.edu
Alpha variant (14-19% heterozygote, major fraction)
Alpha variant (9-32% heterozygote, major fraction)
Alpha variant (15-25% heterozygote, major fraction)
Clinical Context
2 yrs old child with sickle cell
syndrom under oracillin treatment
Hematological parameters
- WBC 10.48 G/L
- RBC 5.53 T/L
- Hb 10.6 g/dL
- Hct 30.8 %
- MCV 55.7 fL
Biochemical parameters
Ferritin 177 µg/L
Itano test: Positive
Hb A 15.6%
Hb F 14%
Hb S 64.5%
Hb A2 5.9%
Hb A
Hb S (zone S)
Hb A2 Hb F
Heterozygote A/S + beta thalassemia on Capillarys/Minicap technologies
Elevated HbA2, Hb S > HbA, anemia and
microcytosis indicate a beta-thalassemia
Variant in the D zone, what to do?
The HbX value is below 35%
(Hb D= ~ 35 to ~ 40% for heterozygote A/D)
Heterozygous A/non D beta
variant
Hb X non D
bm bm
a a
a a
b b
bm
b
Hb A
Heterozygous A/X
Heterozygote A/ X (in D zone) on Capillarys/Minicap technologies
b et chains recombination by crossing over. Alkalin buffer: Decrease of the total charge → Migration slowed down
like Hb S on agarose gel, more anodic than S (D Zone) on Capillarys/Minicap
Heterozygous form:
Hb Lepore fraction: 5 – 15 %
Clinical signs of minor b thalassemia
Homozygous form:
Hb Lepore fraction: about 30 %
Clinical signs of homozygous b
thalassemia
Frequency – localization:
Lepore (- Boston – Washington): Found mainly in Italian families; it has also been observed in families from Rumania, Australia, Mexico.
Characterization:
Hemoglobin Lepore (in D zone) on Capillarys/Minicap technologies
2 beta variant and HbA, what to do?
3 beta globin chains Hb A, Hb S, Hb C
but only 2 genes beta on chromosomes
Check for a blood transfusion for a
suspected heterozygote S/C. Request another sample before transfusion if
transfused patient
Hb S
Hb A
Hb C
Heterozygote S/C transfused with normal blood on Capillarys/Minicap technologies
Result must not be reported
Phenotype HbA2 HbE
Heterozygote A/E 3.5%±0.4% 25.6%±1.4%
Elevated HbA2 in
presence of HbE but
without anemia and
microcytosis
Heterozygote A/E and HbA2 value on Capillarys/Minicap technologies
Clinical context
30-yrs old asian women
Hematological parameters
- Hb 13.2 g/dL
- MCV 89.5 fL
- MCH 31.1 pg
Normal parameters
Clinical context
26-yrs old woman, not pregnant
Hematological parameters
- Hb 10 g/dL
- MCV 71 fL
Anemia, microcytosis
Iron balance parameters
- Iron 8 µmol/L (N: 10-30)
- Ferritin 8 µg/L (N: 10-200)
Iron imbalance
Low HbA2 with iron deficiency on Capillarys/Minicap technologies
Reduced HbA2 must be rechecked after treatment
of iron deficiency
Low HbA2 in
presence anemia
and microcytosis.
Check iron status
Heterozygote Hope variant affecting Hb A
quantification on Capillarys/Minicap
technologies
Presence of an additional peak in zone Z10
Incomplete quantification of Hb A and Hb X peaks
Overestimation of HbA2 peak
Requantification of Hb A and Hb X peaks using Phoresis manual
quantification
HbA
Elevated
Hb A2
Hb Hope
Hb Hope
+ Hb A Normal
Hb A2
Heterozygote δ chain globin hemoglobin variant
α
β
α2β2: HbA
α22: HbA2
α22: HbF
+
α2*2
Clinical context
42-yrs old man
Hematological parameters
- Hb 15.7 g/dL
- MCV 89 fL
Normal parameters
Iron balance parameters
- Iron 19 µmol/L (N: 10-30)
- Ferritin 156 µg/L(N: 30-300)
Normal iron status
Presence of an additional peak in zone Z1
Reduced HbA2 with normal hematological and iron parameters
True HbA2 value = HbA2 value + peak in Z1
Delta
variant
Heterozygote delta variant on Capillarys/Minicap technologies
m m
a a
m
Hb A2
Delta-thalassemia and heterozygote delta variant on Capillarys/Minicap technologies
Presence of an additional peak in zone Z1 and reduced HbA2 indicated
the presence of delta variant
HA2 value on ZA2 (Z3) < Delta variant value on Z1 with normal
hematological parameters indicated the presence of delta-thalassemia
Delta
variant
m m
a a
m
Decreased
Hb A2
Clinical context
38-yrs old man
Hematological parameters
- Hb 15.1 g/dL
- MCV 90 fL
Normal parameters
Iron balance parameters
- Iron 22 µmol/L (N: 10-30)
- Ferritin 180 µg/L (N: 30-300)
Normal iron status
Heterozygote α chain globin hemoglobin variant
α
β
α2β2: HbA
α22: HbA2
α22: HbF
α2*β2
+
+
+
α2*2
α2*2
Clinical context
27-yrs old man
Hematological parameters
- Hb 15 g/dL
- MCV 87 fL
Normal parameters
Iron balance parameters
- Iron 27 µmol/L (N: 10-30)
- Ferritin 203 µg/L(N: 30-300)
Normal iron status
Heterozygote alpha variant on Capillarys/Minicap technologies
Presence of 2 additional peaks in zones D and Z1
Reduced HbA2 with normal hematological and iron parameters
True HbA2 value = HbA2 value + peak in Z1
Alpha variant
Hb A2
Search between zones if a similar name is found
= Major fraction of an alpha variant
= Major fraction of an alpha variant
= Minor fraction of an alpha variant
= Minor fraction of an alpha variant
1<Hb F <5% Normal HbA2
MCH >27pg
5<Hb F <35% Normal or ↓ HbA2
MCH >27pg
Heterozygote hereditary persistance of Hb F (HPFH)
Hb F% (15-35%), normal MCH
Heterozygote b-thalassemia Hb F% (5-15%), MCH<27pg
Moderated hereditary persistance of Hb F
Diabetes Pregnancy
Hyperthyroidy Chemotherapy
Dyserythropoiesis Anemia stress
Elevated Hb F on Capillarys/Minicap technologies
Elevated Hb F due to pregnancy
Hb F Hb A2
Hb A
Clinical context
21-yrs old pregnant woman
Hematological parameters
- Hb 13.1 g/dL
- MCV 85.1 fL
- MCH 30.2 pg
Normal parameters
Clinical context
45-yrs old man
Hematological parameters
- Hb 14 g/dL
- MCV 86.2 fL
- MCH 29.2 pg
Normal parameters
Elevated Hb F on Capillarys/Minicap technologies
Heterozygote hereditary
persistance of Hb F (HPFH)
Hb F
Hb A2
Hb A
Hb F >35% MCH <27pg
Homozygote hereditary persistance of Hb F
No HbA, HbF 100%, Microcytosis ,hypochromia
Composite heterozygote HPFH/ b-thalassemia
Hb F% (70%), normal HbA2 Anemia, microcytosis
Homozygote b-thalassemia No HbA, HbF 100%,
Anemia, microcytosis
Composite heterozygote b-thalassemia/
b-thalassemia Hb F% ↑↑, HbA ↓↓
Anemia, microcytosis
Intermediate b-thalassemia Hb F% ↑, HbA2↑, HbA ↓
Anemia (6-9g/dl), microcytosis
Major b-thalassemia HbA ↓↓ or absent
Hb F% ↑↑, HbA2↑ o N,
Anemia (7< g/dl), microcytosis, hypochromia
Heterozygote Camperdown migrating on zone A
on Capillarys/Minicap technologies
Normal sample on Capillarys/Minicap
technologies
Unsual shape
of HbA
Hematology Normal in heterozygote
Electrophoresis Moves slightly faster than Hb A on alkalin gel.At acidic pH,
moves between Hb A and Hb F, close to Hb F.
Moves on HbA on capillary electrophoresis
Function studies Normal
Occurrence Quantity in the heterozygote about 50%
Hb Camperdown
(b104 Arg→Ser)