Review of cases on hemoglobin capillary electrophoresis

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

http://globin.cse.psu.edu/

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


- 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


- 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




http://globin.cse.psu.edu/

Clinical Context

2 yrs old child with sickle cell

syndrom under oracillin treatment


- 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


- Hb 13.2 g/dL

- MCV 89.5 fL

- MCH 31.1 pg

Normal parameters

Clinical context

26-yrs old woman, not pregnant


- 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


- Hb 15.7 g/dL

- MCV 89 fL

Normal 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


- Hb 15.1 g/dL

- MCV 90 fL

Normal 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


- Hb 15 g/dL

- MCV 87 fL

Normal 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


- Hb 13.1 g/dL

- MCV 85.1 fL

- MCH 30.2 pg

Normal parameters

Clinical context

45-yrs old man


- 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%,


Composite heterozygote b-thalassemia/

b-thalassemia Hb F% ↑↑, HbA ↓↓


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)

Documents

Review of cases on hemoglobin capillary electrophoresis