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79
79
79
Goals
To compare and contrast the various
methods used in the workup of hemoglobin disorders (advantages, limitations)
Outline
First Half
Outline
First Half
Emerging Methods Capillary Electrophoresis (CE) Mass Spectrometry Specialized Methods Functional: Sickle solubility, Hb
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 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
Isoelectric Focusing
Utilizes carrier
ampholytes to establish a pH gradient through out the medium (agarose)
A2 S A
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
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
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
# of variants
1 0 8 8 4 50 24 58 22 25 29 34 33 12
Hb E
Hb Osu-Christiansborg
Hb C
Hb G-Siriraj
Hb S
Hb Richmond
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
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
Emerging Methods
Capillary Electrophoresis
Capillary Electrophoresis
Capillary Detector
Anode
Cathode
High voltage
Sample
Buffer
Buffer
CP1304830-1
Capillary Electrophoresis
The silica capillary has a negative
charge on its inner surface
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
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)
Hb A2 @ 241
Hb San Diego
Hb M-Saskatoon
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
Warning!
Hb C and Hb CC-Harlem migrate almost identical on CE. All Hb Cs must be confirmed by another method
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
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.
Zone 5 (Hb S)
HPLC Retention Time (min)*
Isoelectric Focusing
HPLC Retention Time (min)*
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
Chromatography
Ionization Source
Mass Analyzer
Detector
Data analysis
A2 S
C S A F
+ +
Alkaline
A2 S A
Acid
HPLC
IEF
IEF -8 1 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
15140
HEL EF7
Mass ACET AGAR PH9 PH6 -5.3 0 7.6 15140 -4.1 0 8 15140
STB S S
O2A U U
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 Studies
Molecular methods
DNA sequencingsequencing- useful to confirm point
mutations, Hb variants or most beta thalassemia mutations
Hb S 6 GAG
Region of interest
Exonic Sequence 5 Exon Specific Probe Universal Primer 3 Exon Specific Probe Stuffer Sequence Ligation Site Universal Primer
Denature DNA
5 3
Probe
Probe
Fragment analysis
by Capillary Electrophoresis
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
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
Questions?
JDH
Case Studies
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.
Anyone? Anyone?
Case 1
Case 1
Here is the testing that is available at this time of night.
4.33 X 1012/L 12.4 g/dL 81.2 fL 12.5 4.7 X 109/L 320 X 109/L
Case 1
Case 1
Hb studies are performed the next day
A2 S A C S A F
Case 1
Hb A Hb S Hb A2
HPLC
Case 1
Capillary Electrophoresis
Case 1
What other history would be critical?
Any recent transfusions
Case 1
Case 1- PB Smear
Normal!
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
HPLC
Case 2
Capillary Electrophoresis
Case 2
Is the HPLC and CE compatible with
thalassemia intermedia? Yes
Case 2
Father
HPLC
Mother
Hgb
Hb A Hb F Hb A2
Hb A Hb F Hb A2
Case 2
Father Mother
Capillary Electrophoresis
Case 2
Case 2
Brother, 18 y.o
Sister, 17 y.o.
Hb A Hb F Hb A2
Hb A Hb F Hb A2
Thalassemia Trait
Thalassemia
F thalassemia
Case 2
Why?
Mother
Case 3
Poly A AATAAA
AACAAA, +
thal major
o/o or +/o
thal minor
o/ or +/
Case 3
Case 3
A2 S A C S A F
Alkaline electrophoresis
Acid electrophoresis
Case 3
Capillary Electrophoresis
Case 3
Case 3
Hb A Hb S Hb F Hb A2
HPLC
Case 3
Hb S/ HPFH
Case 3
A followfollow-up specimen was again submitted at 13 months of age.
Case 3
A2 S A + + C S A F
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.
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
Case 3
In an ambiguous clinical
presentation, it would certainly be appropriate to consider molecular studies
Case 3
DNA Sequencing of Globin Gene
4 5 6 7 8
Hb S 6 GAG
Case 3
DNA sequencing revealed no
thalassemia mutations
Hb S Haplotypes
Case 4
Case 4
CBC
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
Case 4
Husband-26 yo
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%
Alkaline electrophoresis
HPLC
Husband-26 yo
Capillary Electrophoresis
Case 4
Hb E
26 GAG AAG (glu
to lys)
Found in several
ethnic groups in SE Asia, rarely in Caucasians and AfricanAfrican -Americans
Prevalence of Hb E in U.S. Khmer Tai Dam Lowland Lao Hmong Vietnamese 31% 11% 36% 4% 2%
Hb E Trait
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.
Hb E/ Thalassemia
Usually Hb E with a o mutation ( 3 most
common thalassemia mutations in SE Asia are o)
Thalassemia Mutations
In SE Asia
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
16
17
18
19
20
21
22
23
24
25
26
27
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
Case 5
Hb Barts
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
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.
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
Case 6
Case 6
A2 S A C S A F
*
Alkaline Electrophoresis
*
Acid Electrophoresis
Case 6
A2 S A
*
Isoelectric Focusing
HPLC
Case 6
Case 6
Hb CC-Harlem
Two substitutions:
1) 6 glu to val (Hb S) 2) 73 asp to asn (Hb KorleKorle-Bu)
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
Homozygous Hb C
1/5000 AfricanAfrican-Americans are
homozygous for Hb C (.02%)
Homozygous Hb C is a mild to
moderate hemolytic anemia
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 S/C Disease
PB Findings
Increased target
cells
clam or boat
cells
Sickling Syndromes
Case 7
52 y.o. white male referred for
opinion about recently diagnosed polycythemia vera.
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
Case 7
Physical exam was normal. There was
no hepatosplenomegaly.
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.
Case 7
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
Case 7
Case 7
Case 7
Case 7
Case 7
Is this actually polycythemia vera?
Highly unlikely
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
High O2 Affinity Hb Variants Epo Receptor mutations VHL mutations Hif 2 2 Mutations PHD2 mutations BPG Mutase Deficiency