Welcome to my talk

Hemoglobinopathies: The how, why and what

James D. Hoyer, MD Associate Professor Mayo Medical School Mayo Clinic Rochester, MN, USA

Dr Hoyer has no relevant financial relationships to disclose

Goals
To compare and contrast the various
methods used in the workup of hemoglobin disorders (advantages, limitations)

To use a series of cases to illustrate

the appropriate work up of these disorders

Outline
First Half

Established methods Hemoglobin (alkaline/acid)

Electrophoresis Isoelectric Focusing (IEF) High Performance Liquid Chromatography (HPLC)

Outline
First Half

Emerging Methods Capillary Electrophoresis (CE) Mass Spectrometry Specialized Methods Functional: Sickle solubility, Hb

Stability, O2 Dissociation curves Molecular methods, Amino acid sequencing

Outline
Second Half

Case Studies

Ill Be Back

CAP HG Survey

1 9 9 9 2 0 1 1

1 9 9 9

2 0 1 1

Established methods

Alkaline/Acid Electrophoresis Isoelectric Focusing (IEF) High Performance Liquid

Chromatography (HPLC)

Color Atlas of Hb Disorders, CAP Press, used with permission

Alkaline Electrophoresis
pH 8.6 Cellulose acetate is
support medium
A2 S A

Hemoglobin
molecule is negatively charged and will migrate toward anode

Method is based on
charge differences

Acid Electrophoresis
Citrate bufferbuffer- pH 6.2 Agar is support
medium, Citrate Agar
C S A + F

Agaropectin combines
with some variants to alter mobility compared to Hb A

Useful for
confirmation of Hb S, C, E

A2 S A

C S A

Alkaline Electrophoresis

Acid Electrophoresis

Hemoglobin Electrophoresis
Advantages Inexpensive, user friendly systems
available Satisfactory to confirm Hbs S, C and E

Disadvantages Specimens must be batched, long run

times Hb A2 quantitation is imprecise Many variants show similar mobility, or do not separate from Hb A

Isoelectric Focusing
Utilizes carrier
ampholytes to establish a pH gradient through out the medium (agarose)
A2 S A

pH range usually 66-8 Hb fractions will travel

to their isoelectric point and stop

Isoelectric Focusing
Advantages
Better separation of Hb variants that
show similar mobilities on alkaline electrophoresis, but still many variants have similar mobilities Better separation of rarer variants from Hb A Minor bands may be seen more easily (Hb H, Hb Barts, and delta chain variants)

IEF

Isoelectric Focusing

Disadvantages
Long run times, samples must be
run in batches Many minor bands (degradation, aging, glycosylated) Difficult to quantitate off IEF

High Performance Liquid Chromatography (HPLC)

Cation exchange column Separates Hb variants
based on charge differences

The ionic strength of the

eluting buffer eventually overcomes the attraction to the column

Hb variants come off the

column at a specific retention time

HPLC

Hb S trait

Hb C trait

Hb E trait

HPLC
Advantages Precise quantitiation of Hb A2, Hb F Retention time of a variant is a
reproducible number

Compact, useruser-friendly, specimens

can be run singly, short run lengths

HPLC
Disadvantages Hb H and Hb Barts are difficult to
identify, and cannot quantitate Minor peaks due to aging, glycosylation Hb A2 cannot be quantitated in the presence of Hb E Many variants show similar retention times

All of the methods discussed so far all have limitations because many Hb variants can have similar mobilities

Hb Variants CoCo-migrating with Common Variants*

Alkaline electrophoresis Hb S: 52 variants (20 (20 ,30 ,30, 2 2 )

Hb C: 5 variants (5 (5 ) Hb E: 5 variants (5 (5 ) Hb DD-Punjab: 52 variants (20 (20 ,30 ,30,2 ,2 ) )

* Within .5 Schneider-Barwick units

Hb Variants CoCo-migrating with Common Variants*

Isoelectric focusing (14 ,13 ,13) Hb S: 27 variants (14

Hb C: 1 variant ( () Hb E: 3 variants (3 (3) Hb DD-Punjab: 15 variants (6 (6 ,9 ,9 )

* Within .4 mm

Variants in each window (Bio(Bio -Rad Variant Variant Classic)

Many variants have similar RTs to one another and fall into the same HPLC windows
Peak (in order of RT)
P1 UNKNOWN F P2 UNKNOWN P3 UNKNOWN A UNKNOWN A2 D S UNKNOWN C

# of variants
1 0 8 8 4 50 24 58 22 25 29 34 33 12

Rare Variant Confusion Hb E

Hb E

Hb Osu-Christiansborg

Rare Variant Confusion Hb C

Hb C

Hb G-Siriraj

Rare Variant Confusion Hb S

Hb S

Hb Richmond

Rare Variant Confusion Hb S/S

Homozygous Hb S

Hb S and Hb Richmond

5 variants account for the vast majority of Hbs that are seen
On HPLC, Hb S accounts for 94% of
Hb variants seen in S window

Hb C accounts for 92.4% of variants

in C window

Hb E accounts for 84.3% of variants

in A2 window

D window: Hb DD-Punjab (52.5%), Hb

G-Philadelphia (32.8%)

A Window
66 total variants in this window 44 variants show totally normal
chromatogram; most others will show either a shoulder but could look totally normal

38 with functional abnormalities (abnormal

p50, abnormal Hb stability test, microcytosis)

24 have clinical symptoms: erythrocytosis

(13), hemolytic anemia (11)

Emerging Methods

Capillary Electrophoresis (CE) Mass Spectrometry

Capillary Electrophoresis

Instrumentation has been around

since early 1990s, with mixed acceptance

Capillary zone electrophoresis Utilizes very long thin capillary (100

m diameter)

Excellent dissipation of heat so can

use very high voltages (10,000V)

This reduces run times, multiple

capillaries can be run in parallel (8)

Capillary Electrophoresis
Capillary Detector

Anode

Cathode

Data display and storage

High voltage

Sample

Buffer

Buffer

CP1304830-1

Capillary Electrophoresis
The silica capillary has a negative
charge on its inner surface

Electro Electro-osmotic force (EOF) flow of

the buffer solution towards the cathode

Hemoglobin fractions will separate

out based on their affinity for the positive or negative pole, but overall are still carried towards the cathode due to the EOF

Sebia, used with permission

Capillary Electrophoresis

Hb E Trait

Capillary Electrophoresis
Advantages Good quantitation of Hb A2 and Hb F Hb E separates from Hb A2 Detects minor variants very well Easy to use system, specimens may
be run singly

Capillary Electrophoresis
Disadvantages Many variants have similar mobility, but

fewer are similar to Hbs S, C and E Rare variants may not separate from Hb A If Hb A is not present (e.g. Homozygous Hb S, C, or E), no zones will be produced. Must mix specimen with normal in order to see zones

Homozygous Hb C

Unmixed

Mixed with Normal

Number of Hb Variants in Each CE Zone

Zone 15 Zone 14/15 Zone 14 Zone 13 Zone 12/13 Zone 12 Zone 11 Zone 10 Zone 9 Zone 8/9 Zone 8 Zone 7/8

2 1 1 3 1 18 12 4 68 2 11 5

Zone 7 Zone 6/7 Zone 6 Zone 5/6 Zone 5 Zone 4/5 Zone 4 Zone 3 Zone 2/3 Zone 2 Zone 1

19 6 33 5 10 2 5 5 1 3 1

Zone 9 (Hb A) 64 Hb variants within +/+/- 2 units of Hb A 40 variants variants, , 24 variants 5 Hb M (Boston, Hyde Park, Iwate,
MilwuakeeMilwuakee -1, Saskatoon)

21 Hb variants with altered oxygen

affinity (16 clinically significant)

15 unstable Hb variants (8 clinically

significant)

Hb A and San Diego @ 150

Hb A2 @ 241

Hb San Diego

Hb M-Saskatoon

Zone 5 (Hb S) Hb Evanston Hb Russ Hb Hamadan Hb OO-Indonesia Hb Shimonoseki Hb Belfast Hb GG-Copenhagen

14 TrpArg 51 GlyArg 56 GlyArg 116 GluLys 54 GlnArg 15 TrpArg 47 AspAsn

Hb G Copenhagen - Z5 - 213

Hb S - Z5 - 212

Hb S Trait

Hb G-Copenhagen

Zone 4 (Hb E)
Hb A2- Babinga Hb AgenogiAgenogi- Japanese, AfricanAfricanAmerican, Hungarian

Hb E Trait

Hb Agenogi

Hb E trait, zone 4 @ 225

Hb Agenogi, zone 4 @223

Zone 2/3 (Hb C)

Warning!

Hb C and Hb CC-Harlem migrate almost identical on CE. All Hb Cs must be confirmed by another method

The only other variant is Hb Constant

Spring

Capillary Electrophoresis

Hb C-Harlem Trait

Hb C Trait

Homozygous Hb C

Capillary Electrophoresis

HPLC

Zone 6
33 Hb variants in this zone Within 12 units of each other In this zone are Hb DD-Punjab, Hb
KorleKorle -Bu, and Hb G G-Philadelphia

Hb A-Z9-150

Hb A2 Z1-279

A2-Z3-244

Hb G-Philadelphia Hb A2'
Hb G PhiladelphiaZ6-204

Hb A-Z9-150

Hb A2-Z3-241 Hb G2 Z1-267

Hb A-Z9-150

Hb H-Z15-36
A2-Z3-241

Hb Barts

Hb H disease

Hb F-Z7-178

Hb A-Z9-150

Barts-Z12-99
A2-Z3-243

Complementary nature of methods

Combinations of are useful to

confirm the common variants; do not need to rely on acid electrophoresis. help to narrow down the ID of unusual variants, as these typically show varying mobilities on different methods.

Combinations of methods also

Zone 5 (Hb S)
HPLC Retention Time (min)*

Hb Evanston Hb Russ Hb Hamadan Hb OO-Indonesia Hb Shimonoseki Hb Belfast Hb GG-Copenhagen

* Bio-Rad Variant beta thal program

4.53 4.38 3.66 4.84 4.44 4.18 3.74

Hb S = 4.4 min

Isoelectric Focusing
HPLC Retention Time (min)*

Hb E HbE HbE-Sasakatoon Hb CC-Harlem Hb OO-Arab

* Bio-Rad Variant beta thal program

3.60 4.34 4.82 4.80

Capillary Electrophoresis
Zone 6, 206 Zone 6, 202

Hb D-Punjab

Hb Korle-Bu

HPLC

4.08 min

3.88 min

Hb D-Punjab

Hb Korle-Bu

Complementary methods are also useful because Hb variants that do not separate on one method may show separation on another method

Hb Austin

CE

A2 S A

+ H P L C

IEF

Hb S/Richmond

Capillary Electrophoresis

HPLC

Capillary Electrophoresis

Hb S Trait

Hb Richmond Trait

Caveat
All of the methods discussed so far will have some degree of variability in the migration or elution depending on lots of reagents.

S trait - Z5 - 212

Capillary Electrophoresis

Mass Spectrometry

Around for 3535-40 years in clinical

laboratories

Drug identification Hormones Metabolites inborn errors of

metabolism

Only recently applied to analysis of

proteins

MS detects mass to charge ratios (m/z) of ionized molecules

Chromatography

Ionization Source

Mass Analyzer

Detector

Data analysis

Electrospray Ionization Mass Spectrum of Normal Hemoglobin

Multiply charged ions for both Alpha and Beta chains

Deconvoluted Spectrum of Normal Hemoglobin

Normal Alpha m/z = 15,126 Normal Beta m/z = 15,867

MS Applications to Hemoglobin Analysis

Determine mass difference of affected intact globin chains compared to normal globin chains

Electrospray Ionization Mass Spectrum of Hemoglobin S

Normal Alpha m/z = 15,126

Abnormal Beta m/z = 15,837

Normal Beta m/z = 15,867

Predicted mass change for glu to val substitution is -30 Daltons

A2 S

C S A F

+ +

Alkaline

A2 S A

Acid

HPLC

IEF

Acet Mobility -4.7 Tolerance 1 Unknowns N

Agar PH9 Mass Stable O2 Affinity 0 8.1 * S * 4 2 3 N Y Y

IEF -8 1 Y

Short Long 4.26 11.71 6 10 Y Y

NAME RESIDUE CASAMANCE A4 A2FLATBUSH D22 AVICENNA B47 EVANSTON A14 EDMONTON B50 MAPUTO B47 G WAIMANALO A64 WATTS A74 G PEST A74 ATAGO A85 ETOBICOKE A84 SAN ANTONIO A113 L PERSIAN GULF A57 STANLEYVILLE-2 A78 OLEANDER A116 INKSTER A85 SHIMONOSEKI A54 G NORFOLK A85 PARK RIDGE A9 BEILINSON A47 ARYA A47 HANDSWORTH A18 SAVARIA A49 MIZUSH A75 PERSPOLIS A64 MONTGOMERY A48 BIBBA A136 RIVERDALE BRONX B24 BOYLE HEIGHTS A6 CRANSTON B145 FORT DE FRANCE A45

SUBST PRO-ARG ALA-GLU ASP-ALA TRP-ARG THR-LYS ASP-TYR ASP=ASN DELETION ASP-ASN ASP-TYR SER-ARG LEU-ARG GLY-ARG ASN-LYS GLU-GLN ASP-VAL GLN-ARG ASP-ASN ASN-LYS ASP-GLY ASP-ASN GLY-ARG SER-ARG ASP-GLY ASP-TYR LEU-ARG LEU-PRO GLY-ARG ASP-0 DELE ADD HIS-ARG

HEL A2 B4 CD6 A12 D1 CD6 E13 EF3 F6 F5 GH1 E6 EF7 GH4 F6 E3 F6 CE5 CE5 A16 CD7 EF4 E13 CD6 H19 B6 A4 HC2 CD3

Mass ACET AGAR PH9 PH6 -5 4 8.3 15185 -4.5 0 -99 16244 -5 0 -99 15823 -4.6 0 8.1 15096 -5.2 0 -99 15894 -5.2 0 -99 15915 -5.1 0 8 15125 -4.6 0 7.9 15011 -4.7 0 7.8 15125 -4.2 0 8.1 15174 -4.3 0 7.7 15195 -4.6 0 8 15169 -5 0 7.5 15225 -5.3 0 7.6 15140 -4.5 0 7.3 15125 -5 0 7.9 15110 -5.2 0 8.1 15154 -5.4 0 7.6 15125 -4.1 0 8 15140 -5.3 0 8.2 15068 -5.2 0 7.7 15125 -4.9 0 7.7 15225 -5.6 1 8 15195 -4.7 1.9 7.9 15068 -4.5 2 8 15174 -4.6 3.5 7.9 15169 -5 0 -99 15110 -5 0 -99 15966 -4.2 0 -99 15011 -5.2 0 -99 17064 -5.2 0 7.4 15145

STB N S S S S S S S S S S S S S S S S S S S S S S S S S U U U U U

O2A U U N I U N N U U I I U U U N I U U U U U U N N U U U I I I I

IEF SHRT LNG -7 -99 -99 -99 -99 -99 -8.21 -99 -99 -8.3 4.53 11.92 -99 -99 -99 -99 -99 -99 -8.01 4.4 11.99 -8.5 4.36 11.66 -7.6 4.51 11.89 -7.7 4.18 11.71 -8.2 4.09 11.47 -7.4 4.05 11.86 -99 -99 -99 -8.2 4.36 11.67 -99 -99 -99 -8.7 3.95 11.18 -8.9 4.44 11.96 -8.7 4.23 11.7 -8.3 3.94 10.88 -8.6 4.5 12.45 -8.9 4.31 12.08 -7.1 4.98 12.62 -9 4.54 12.28 -99 -99 -99 -99 -99 -99 -8 4.51 12.92 -99 -99 -99 -99 -99 -99 -99 -99 -99 -99 -99 -99 -9 -99 -99

Mass Spectrometry Data

15140

List of Possible Variants after MS Data

Acet -4.7 1 N Agar PH9 Mass Stable O2 Affinity IEF Short Long 0 * 15140 S * -8 4.26 11.71 4 2 3 1 6 10 N Y Y Y Y Y

Mobility Tolerance Unknowns

NAME RESIDUE STANLEYVILLE-2 A78 PARK RIDGE A9

SUBST ASN-LYS ASN-LYS

HEL EF7

Mass ACET AGAR PH9 PH6 -5.3 0 7.6 15140 -4.1 0 8 15140

STB S S

O2A U U

IEF SHRT LNG -8.2 4.36 11.67 -8.3 3.94 10.88

MS Applications to Hemoglobin Analysis

Trypsin cleaves peptides on the CCterminal side of lysine and arginine amino acid residues

Determine mass of tryptic peptides;

look for fragment with mass shift

Tryptic peptides average 10 amino acids in length

 Globin Chain Tryptic Peptides

Tryptic Peptide T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 Amino Acid # 1-8 9-17 18-30 31-40 41-59 60-61 62-65 66 67-82 83-95 96-104 105-120 121-132 133-144 145-146 AA Sequence VHLTPEEK SAVTALWGK VNVDEVGGEALGR LLVVYPWTQR FFESFGDLSTPDAVMGNPK VK AHGK K VLGAFSDGLAHLDNLK GTFATLSELHCDK LVHDPENFR LLGNVLVCVLAHHFGK EFTPPVQAAYQK VVAGVANALAHK YH

MS Applications to Hemoglobin Analysis

Tryptic peptides can be further
analyzed by tandem MS to gain additional information

Thus, in most instances amino acid

substitution can be determined

Mass Spectometry
Advantages Advantages-In most cases definitive identification of Hb variants by a single method -Very fast analysis times, can analyze very small amounts -Can screen for silent variants

Mass Spectometry

Disadvantages
-Instruments are very expensive, require high degree of expertise -Difficult to quantitate Hb A2, Hb H (Daniel YA,et al Clin Chem 2007, 53:1448) -Variants with small mass shift may be difficult to detect (Hb E, Hb C, Hb DD-Punjab, Hb OO-Arab) - Cant detect Leu vs Ile -? , chain variants

Ancillary Methods

Functional: Sickle solubility,

Hb Stability, OxygenOxygendissociation curve (p50) Globin Chain Electrophoresis Reverse Phase HPLC Amino Acid Sequencing

Functional Studies

Useful for deciding whether further

workwork -up needs to performed

Abnormal p50 or abnormal unstable

Hb stabilty test likely indicates the presence of a silent Hb variant

Sickle Solubility Test

Lysate of RBCs is placed in a high phosphate buffer solution Dithionate lowers oxygen tension of solution Hemoglobin S, if present will precipitate from solution Does not distinguish Hb S trait from homozygous S, or Hb S with another Hb variant

Molecular methods
DNA sequencingsequencing- useful to confirm point
mutations, Hb variants or most beta thalassemia mutations

MLPA (Multiplex LigationLigation-dependent

Probe AmplificationAmplification- useful to detect large deletional alpha or beta mutations

DNA Sequencing: Globin Gene

DNA Sequencing of Globin Gene

4 5 6 7 8

Hb S 6 GAG

GTG (Glu to Val)

MLPA Probe Design

Region of interest

Exonic Sequence 5 Exon Specific Probe Universal Primer 3 Exon Specific Probe Stuffer Sequence Ligation Site Universal Primer

Denature DNA

Hybridize probes Ligate probes PCR w/universal

primers

5 3

Probe

Probe

3 5 Stuffer DNA Universal primer

Hybridize Probes Ligate/Denature

5

Amplify with Universal Primers

Fragment analysis
by Capillary Electrophoresis

Separate probes by size using CE

Data analysis

Deletion of Exon 3
1 1 Ratio to Control Marker 1.0 2 2 3 4 4 5 5 6 6 7 7

0.5

Ctrl

HPFH & Thalassemia Deletions

Gene Cluster
HS5-1

HPFH

Black-USA Black-Ghana

Spanish

-Thalassemia
Sicilian

Summary
All methods available for hemoglobin analysis are acceptable, although a single method is not sufficient for definitive identifcation of a Hb variant, particularly for more uncommon Hb variants

Summary
Multiple combinations of methods are
possible to confirm the three most common variants, and help to narrow possibilities for uncommon Hb variants

The choice of instrumentation and

degree of complexity depends on the laboratory

Questions?

JDH

Case Studies

Hb analysis is an intrepretation of not

only the Hb studies, but also incorporation of many other pieces of information. examination of the peripheral blood smear, clinical presentation, physical exam, correlation with other family members (esp parents) and possibly the ethnic background.

This includes other laboratory data,

Lets run that through the mass spec

And the diagnosis is..

Anyone? Anyone?

Case 1

You are called by the ER late at night

about a 26 yo AfricanAfrican-American female.

She indicates she is having severe

abdominal pain, and needs narcotics for pain control.

She indicates that has a history of

sickle cell disease.

Case 1
Here is the testing that is available at this time of night.

RBC Hgb MCV RDW WBC Plt

4.33 X 1012/L 12.4 g/dL 81.2 fL 12.5 4.7 X 109/L 320 X 109/L

Sickle solubility test is positive

Case 1

The patient is admitted for

observation and pain control.

Anything concerning you yet?

The CBC is normal

Does she have sickle cell anemia?

There are insufficient studies at this point to say either way

Case 1
Hb studies are performed the next day
A2 S A C S A F

Alkaline Electrophoresis, pH 8.6

Acid Electrophoresis, pH 6.2

Case 1

Hb A Hb S Hb A2

60.3% 36% 3.7%

HPLC

Case 1

Capillary Electrophoresis

Case 1
What other history would be critical?
Any recent transfusions

Case 1

What is your interpretation?

Sickle Cell Trait

Is there any other test that might

prove helpful? Examination of the peripheral blood

Case 1- PB Smear

Normal!

Sickle Cell Anemia

1) Systemic manifestations 2) Chronic intravascular hemolysis 3) VasoVaso-occlusive phenomena

VasoVaso -occlusive Crises

Triggers: Hypoxia, acidosis,
dehydration, infection, or unknown

Acute chest syndrome Musculoskeletal crises Abdominal crises Neurologic crises Stroke Hematuria, papillary necrosis

Chronic VasoVaso-occlusive Effects Autosplenectomy Pulmonary Pulmonary- Fibrosis Chronic Renal failure CNS CNS- Neurologic deficits Ocular Ocular- Proliferative retinopathy Skeletal Skeletal- Osteonecrosis (Femoral head) Skin ulcers

PB Findings in SSA

Drepanocytes Hyposplenic
changes (target cells, HowellHowellJolly bodies, acanthocytes

Case 1

Diagnosis: Hb S Trait

Hemoglobin S Trait
Found in 88-9% of AfricanAfrican-Americans Asymptomatic Normal growth and development Possibly some hyposthenuria and hematuria Sudden death in military recruits Hb S = 3535-40% of total Hb No abnormalities on PB smear

Case 2
This is a 10 year old AfricanAmerican male whose hemoglobin studies are being repeated for a second opinion. He has a previous history of thalassemia intermedia. However, the child is clinically well, and there is no organomegaly. He has never needed a blood transfusion.

Case 2
CBC
Hgb RBC MCV WBC Plt 12.1 g/dL 6.09 X 1012/ 62.0 fL 5.9 X 109/L 401 X 109/L

Case 2

Hb A Hb F Hb A2

21.3% 75.5% 3.2 %

HPLC

Case 2

Capillary Electrophoresis

Case 2
Is the HPLC and CE compatible with
thalassemia intermedia? Yes

What further could be done?

Either family studies or molecular studies

Case 2
Father
HPLC

Mother

Hgb RBC Hct MCV RDW

14.3 5.05 44.0 87.1 15.5

Hgb

12.2 5.40 39.9 73.9 15.4

Hb A Hb F Hb A2

62.6% 35.2% 2.2%

RBC Hct MCV RDW

Hb A Hb F Hb A2

95.5 % 0.3% 4.2%

Case 2
Father Mother

Capillary Electrophoresis

Case 2

Interpret the findings seen in both

parents Father Hereditary Persistence of Fetal Hemoglobin Trait MotherMother - Beta Thalassemia Trait

Case 2
Brother, 18 y.o

Sister, 17 y.o.

Hb A Hb F Hb A2

95.3% 0.3% 4.4%

Hgb 13.7 g/dL RBC 6.3x1012/L MCV 69.4 fL

Hb A Hb F Hb A2

68.1% 30.1% 1.8%

Hgb RBC MCV

12.8 g/dL 4.9x1012/L 80.6 fL

 Thalassemia Trait

Defined by an elevation in Hb A2 (4.0 (4.08.0%)

Usually associated with microcytosis

(MCV=65 fL), elevated RBC, and slight if any anemia

o - No production of chains + - Reduced production of chains

Hereditary Persistence of Fetal Hemoglobin

Elevation of Hb F into adulthood, but no
other problems

Multiple genetic lesions in several different

ethnic groups

Most are large deletions that involve both

the and genes

Hereditary Persistence of Fetal Hemoglobin

In HPFH trait, Hb F is 3030-35% of total
hemoglobin

The genetic switch to chains does not

occur, so chains remain expressed at high levels

Therefore, no globin chain imbalance

 Thalassemia

F thalassemia

thalassemia trait: 1010-15% Hb F, low

or normal Hb A2, low MCV

Also due to large deletions involving

and genes

Case 2

Does this combination explain the

clinical findings seen in the child? Yes done?
This could be molecular analysis (both DNA sequencing and MLPA assay)

Why?

What further testing could be

DNA Sequencing of Globin Gene

Mother

Case 3

Poly A AATAAA

AACAAA, +

Classification of Thalassemia According to Clinical Severity

Subtype Configuration Clinical Manifestation
Severe anemia, RBC transfusions, H/Smegaly

thal major

o/o or +/o

thal intermedia +/o or +/ +

Moderate anemia, occ RBC transfusions

thal minor

o/ or +/

Slight if any anemia,microcytosis

Case 3

You are evaluating a Hb

electrophoresis on a 19 day old.

This follow up specimen was sent

because of an abnormal newborn screen from the state lab.

Case 3
A2 S A C S A F

Alkaline electrophoresis

Acid electrophoresis

Case 3

Capillary Electrophoresis

Case 3

Capillary Electrophoresis - Mix

Case 3

Hb A Hb S Hb F Hb A2

0% 15% 84.5% 0.5%

HPLC

Case 3

What are the possibilities for this

childs condition? Homozygous Hb S, Hb S/ S/o thalassemia and Hb S/HPFH are all possibilities

What further testing should be done?

Molecular studies are possible, but easiest is to repeat studies at about one year of age

Hb S/ HPFH

Hereditary Persistence of Fetal

Hemoglobin

Multiple molecular lesions found in

several ethnic groups, including AfricanAfrican -Americans

Hb S = 6060-65%, Hb F= 3030-35% Hb S/ HPFH is not a sickling syndrome,

high levels of Hb F prevent sickling

Case 3
A followfollow-up specimen was again submitted at 13 months of age.

Case 3
A2 S A + + C S A F

Alkaline electrophoresis, pH 8.6

Acid electrophoresis, pH 6.2

Case 3

Capillary Electrophoresis

Case 3
Hb Hct RBC 10.3 g/dL 30.8 % 3.58 X 1012/L

MCV 85.8 fL

HPLC

Case 3
What could this pattern be
compatible with? Homozygous Hb S, and Hb S/HPFH are the main possibilities. Hb S/ S/o thalassemia is unlikely with the normal MCV.

What further testing could be done?

Family Studies or molecular studies.

Case 3
Mother
A2 S A C S A F

**

**

Alkaline electrophoresis

Acid electrophoresis

HPLC

Case 3
Mother

35.4%

Capillary Electrophoresis

Case 3
Father
A2 S A C S A F

Alkaline electrophoresis

Acid electrophoresis

HPLC

Case 3
Father

Capillary Electrophoresis

Case 3

Interpret the parents Hb

electrophoresis MotherMother - Hb S trait Father - Normal

What you do now?

Ouch. Maybe wish we hadnt done family studies.

Case 3
In an ambiguous clinical
presentation, it would certainly be appropriate to consider molecular studies

Both DNA sequencing (for point

mutations), as well as MLPA (for large deletions) would be useful.

Case 3
DNA Sequencing of Globin Gene
4 5 6 7 8

Hb S 6 GAG

GTG (Glu to Val)

Case 3
DNA sequencing revealed no
thalassemia mutations

MLPA assays detected no deletional

mutations in the globin gene

Therefore, molecular studies are

most consistent with Homozygous Hb S

Why the high Hb F?

Mainly related to haplotype, but may

be other as yet undiscovered modifying factors

Hb S haplotypes first identified by

restriction enzyme analysis

At least 4 different haplotypes,

suggesting that Hb S mutation has arisen independently several times

Hb S Haplotypes

Senegal (SEN) Benin (BEN) Bantu (CAR) Cameroon (CAM) Saudi/Indian

SEN and Saudi /Indian may be the same and are associated with the highest Hb F levels, BEN with the lowest

Case 4

This a 23 yo female for presenting

her first prenatal visit. She is of Cambodian heritage.

Prenatal testing was performed

Case 4
CBC

RBC Hgb MCV RDW WBC Plt

5.78 X 1012/L 12.1 g/dL 65.9 fL 15.1 5.2 X 109/L 253 X 109/L

Case 4
A2 S A C S A F

* *

Alkaline electrophoresis

Acid electrophoresis

HPLC

Case 4

Capillary Electrophoresis

Case 4

CE-Mix

Case 4

Interpret the Hb electrophoresis

Homozygous Hb E

Is there anything further that needs

to be done? The husband must be tested as well

Case 4
Husband-26 yo

RBC Hgb MCV RDW WBC Plt

6.52 X 1012/L 13.2 g/dL 63.7 fL 13.8 6.0 X 109/L 312 X 109/L

Case 4
Husband-26 yo
A2 S A

5.2% Husband-26 yoHb A2

5.2%

Alkaline electrophoresis

HPLC

Husband-26 yo

Capillary Electrophoresis

Case 4

Interpret the husbands Hb studies

thalassemia trait

What do you counsel this couple

about future offspring? There is a 50% chance of a child with Hb E trait, and a 50% chance of a child with Hb E/ thalassemia

Hb E
26 GAG AAG (glu
to lys)

Found in several
ethnic groups in SE Asia, rarely in Caucasians and AfricanAfrican -Americans

From: Winter WP: Hemoglobin Variants in Human Populations, V2:p118

Prevalence of Hb E in U.S. Khmer Tai Dam Lowland Lao Hmong Vietnamese 31% 11% 36% 4% 2%

Hb E Trait

Clinically benign Unstable in vitro Associated with a thalassemic

blood picture - low MCV (75 fL), target cells on PB smear

Hb E accounts for 3030-35% of total

hemoglobin

Hb E Trait
A2 S A C S A F

+ *

*
Alkaline electrophoresis

Acid electrophoresis

HPLC

Hb E Trait

Capillary Electrophoresis

Hb E Trait
Peripheral Blood

Homozygous Hb E
Benign disorder, possibly slight
anemia in women.

RBCs are more microcytic than Hb E

trait, mimicking thalassemia trait.

PB smear shows target cells with

minimal anisocytosis.

Hb E/ Thalassemia
Usually Hb E with a o mutation ( 3 most
common thalassemia mutations in SE Asia are o)

Clinically either thalassemia intermedia or

thalassemia major (splenomegaly).

Moderate anemia, more microcytic than

homozygous Hb E.

Hb F is elevated. PB smear shows targets cells with

prominent anisopoikilocytosis.

 Thalassemia Mutations
In SE Asia

Type Frameshift 4141-42 IVS IVS-2 nt 654 Stop codon 17 -28 A G

0/+ 0 0 0 +

% 28 20 10 13

Hb E/o Thalassemia
A2 S A + C S + A F

Alkaline Electrophoresis

Acid electrophoresis

HPLC

Hb E/o Thalassemia

Capillary Electrophoresis

Hb E/o Thalassemia

Homozygous Hb E

Hb E/0 Thalassemia

DNA Sequencing: Exon 1

16

17

18

19

20

21

22

23

24

25

26

27

17 AAG lys stop

TAG

26 GAG Hb E

AAG

Case 5
This is a cord blood specimen from a fetal demise at 32 weeks gestation. Both parents are of Laotian descent.

Case 5
A2 S A
+ +

F
-

Alkaline Electrophoresis

Acid Electrophoresis

Case 5
A2
-

A
+

Isoelectric Focusing

HPLC

Case 5

Capillary Electrophoresis

Case 5

Capillary Electrophoresis

Case 5
What is the diagnosis?
Barts Hydrops Fetalis

Is there any further testing that needs

to be done? Molecular testing would be informative, but best done before pregnancy

Hb Barts Hydrops Fetalis

Two gene deletion is inherited from

each parent (/)

No Hb A or Hb F can be produced, only

Hb Barts ( (4)

Incompatible with life, unless

extraordinary measures are taken

Significant morbidity possible for mother

as well

Hb Barts Hydrops Fetalis

Peripheral Blood Smear

Case 5

Hb Barts

Hb Portland II (22) Hb Gower I (22)

Hb H

Hb Portland I (22)

Capillary Electrophoresis

Combinations of Thalassemias
# of genes deleted Config 0 1 2 2 3 4 / / / / / / Subtype Normal thal 2 hetero thal 2 homo thal 1 hetero Hb H disease (thal 1 homo) Thalassemia intermedia Lethal Barts hydrops fetalis Microcytosis Clinical Manifestations Normal Clinically silent Microcytosis

Hb H Disease

Hb H disease is a form of hemolytic

anemia

Usually associated with SE Asia ,

but can occur in other geographic areas

Thalassemia intermediaintermedia- mild to

moderate anemia, splenomegaly, erythroid hyperplasia

Hb H Disease
A2 S A

+ *

Alkaline Electrophoresis

Acid Electrophoresis

Hb H/Constant Spring

A2

S A

+ *
IEF

HPLC

Capillary Electrophoresis

Hb H/ Hb Constant Spring
Normal stop codon (TAA) is changed to CAA, which
codes for glutamine.

31 additional amino acids are added to the chain

until the next stop codon is reached.

Hb Constant Spring found in very low amounts (1(12%).

Hb Constant Spring acts as a nondeletional

thalassemia mutation.

When combined with a two gene deletion on the

opposite chromosome 16 (such as --SEA), will produce Hb H disease.

Case 6
There is no information available on the following specimen at the time of Hb electrophoresis

Case 6
A2 S A

*
Capillary Electrophoresis Alkaline Electrophoresis

Case 6

Capillary Electrophoresis- Mix

Case 6

Are these studies sufficient for

confirmation of Homozygous Hb C? No!

Case 6
A2 S A C S A F

*
Alkaline Electrophoresis

*
Acid Electrophoresis

Case 6
A2 S A

*
Isoelectric Focusing

HPLC

Case 6

Peripheral Blood Smear

Case 6

What is the diagnosis?

Hb C/ Hb CC-Harlem (Georgetown)

Does the distinction from

Homozygous Hb C from Hb C/Hb C-Harlem matter? Yes!

Hb CC-Harlem

Two substitutions:
1) 6 glu to val (Hb S) 2) 73 asp to asn (Hb KorleKorle-Bu)

Therefore, Hb C/Hb CC-Harlem is

equivalent to Hb S/C disease

Hb S/C Disease
Differences from Homozygous Hb S

Usually milder in severity (ie fewer vasovasoocclusive events, milder anemia) than Homozygous Hb S, but still a sickling disorder

Autosplenectomy is uncommon More often aseptic necrosis of the humeral

head

Renal papillary necrosis and proliferative

retinopathy are more common

Homozygous Hb C
1/5000 AfricanAfrican-Americans are
homozygous for Hb C (.02%)

Hb C is less soluble than Hb A and

will crystallize

Homozygous Hb C is a mild to
moderate hemolytic anemia

Mild splenomegaly Microcytic indices

Hb S/C Disease
A2 S A C S A F

+ *

+ *

Alkaline Electrophoresis

Acid Electrophoresis

Hb S/C Disease

CE

HPLC

CE-Mix

Homozygous Hb C
PB Findings

Numerous target
cells

Hb C crystals Pseudospherocytes Usually minimal

anisocytosis

Hb S/C Disease
PB Findings

Increased target
cells

Rare sickle cells Few true Hb C

crystals

SC poikilocytespoikilocytescontain irregular crystals

clam or boat
cells

Sickling Syndromes

Homozygous Hb S Hb S/ S/ thalassemia Hb S/C Disease Hb S/DS/D- Los Angeles (Punjab) Hb S/OS/O-Arab

Not all that sickles is Homozygous Hb S

Case 7
52 y.o. white male referred for
opinion about recently diagnosed polycythemia vera.

Presented to his family physician in

May 2002 for evaluation of lipoma of shoulder.

A CBC was done prepre-operatively.

Case 7
CBC Hgb Hct WBC Plt 21.3 g/dL 64.0% 8.5 x 109/L 168 x 109/L

Case 7
He was referred to a local hematologist. At this time the patient complained of mild
fatigue, but no other symptoms such as headaches, dizziness, pruritis, visual difficulties or paresthesias

There was no history obtained of bleeding

or thrombotic events.

Erythropoetin level at this time was 12

mU/ml

Case 7
Physical exam was normal. There was
no hepatosplenomegaly.

PMH was significant only for an

extensive smoking history, over 75 packpack -years. the hematologist noted the patient had had hematocrits in the 50s, sometimes as high as 59%, dating back at least 20 years.

On further review of his medical history,

Case 7

He was diagnosed with polycythemia

vera and begun on a program of weekly phlebotomies.

This brought his hematocrit down to

47.9% after three months.

Case 7
CBC Hgb Hct RBC MCV RDW WBC Plt 14.9 g/dL 45% 5.90 x 1012/L 76.2 fL 17.3 6.7 x 109/L 238 x 109/L

Case 7
At presentation to MC, still no
significant complaints

No hepatosplenomegaly, JAK2 V617F

testing was normal

A BM examination was performed

Case 7

PB, 40X Wright-Giemsa

Case 7

BM Asp, 40X Wright-Giemsa

Case 7

BM Biopsy, 20X, Hematoxylin + Eosin

Case 7

BM Biopsy 20X, reticulin

Case 7
Is this actually polycythemia vera?
Highly unlikely

What further testing could be done?

Many further tests, but start with oxygen dissociation curve

Oxygen Dissociation Curve

p50 = 14 mm Hg

Case 7
A2 S A J +

control

Alkaline Electrophoresis

Case 7

HPLC

Case 7

Capillary Electrophoresis

Case 7
A2 S
A
+

Isoelectric Focusing

Case 7
Mass Spectrometry, chains

15899, +32

Case 7
DNA Sequencing of the Globin Gene
107 108 109 110 111 112

109 GTG

ATG

Case 7
109 val GTG ATG = met (+32) =

Hb San Diego

Case 7 Diagnosis
Erythrocytosis due to Hb San Diego, a high oxygen affinity hemoglobin variant

Secondary (Congenital) Erythrocytosis

High O2 Affinity Hb Variants Epo Receptor mutations VHL mutations Hif 2 2 Mutations PHD2 mutations BPG Mutase Deficiency

Thank you for your attention