Hemoglobin electrophoresis Flashcards
What is the purpose of the RBC? Hgb? O2?
Purpose of RBC is to carry Hgb.
O2 binds to heme on the hemoglobin molecule.
O2 carries electrons.
Electrons drives engine of mitochondria.
You are dead w/o electrons (and O2 and hgb, etc.)!
What comprises Hgb?
4heme molecules +4 globin molecules
normally 2 alpha and 2 beta globins
alpha globins come from genes on chromosome…
16
beta globins are transcribed from genes on chromosome…
11
What gene segments are usually not transcribed after 8 weeks gestation?
zeta and episolon
Gamma or “fetal” globin gene is usually not transcribed after…
the first year of life
how many pairs of alpha genes are on chromosome 16?
TWO!
they are redundant
what is hte difference between the detla globin and beta globin gene?
delta is not transcribed as efficiently and is a MINOR globin
beta is primarily an adult globin
What happens after transcription of chrom 16 and 11?
2 alpha and 2 beta globins pair up to form a tetramer which then combines with 4 heme molecules to make Hgb!
What are hte most favorable tetramers?
2 alpha and 2 betas because they are hte most stable
Major adult hgb
Hgb A
2 alpha + 2 beta
Minor adult Hgb
2 alpha + 2 delta (not as efficiently transcribed)
Fetal Hgb
Hgb F
2 alpha + 2 gamma
- gowers have episolon and are embryonic
What happens to normal hgb during the first year of life?
During first year of life, shut down transcription of gamma gene.
Quit making fetal Hgb under normal circumstances.
There are some abnormal circumstances where you continue to transcribe Hgb F.
How do we identify and quantitate these normal Hgbs?
• Hemoglobin electrophoresis (Hgb ELP)
— CAPILLARY ELP is very popular - most states use these
• High pressure liquid chromatography (HPLC)
thalassemia:
Inherited disorder where there is impaired or absent transcription of one or more globin genes.
You don’t have any mutated/abnormal globin genes. You have a normal globin gene that’s completely MISSING or impaired transcription.
In normal adults, how do you make hgb?
you get 2 betas from chromsoome 11 and 2 alpha globins from chromosome 16 so you have no trouble making adult hgb!
During fetal life and at birth how do you make Hgb F?
Gamma globin makes 2 gamma from chrom 11 + 4 alpha from chrom 16
Hgb F represents 99%!
Why should we switch Hgb types?
Hgb F can take more oxygen efficiently from mom. Becomes problematic when mom has hereditary persistent Hgb F, b/c then oxygen is not effectively transferred to baby. Worth knowing when in prenatal screening.
What happens if you knock out one alpha gene?
You are born missing 1/4 alpha genes….this is alpha thalassemia TRAIT.
If you’re missing ONE of these alpha genes you have 4 gammas transcribing and 3 alphas, so you have a mismatch with one excess gamma floating around.
Gammas can then form tetramers among themselves creating Hgb Barts.
If a newborn has alpha thalassemia trait what will its screen show?
Hgb Barts (tetramers of gamma globin)
What happens once you are born (or in adults) with alpha thalassemia trait?
Once born you reduce gamma production and incrase beta transcription so w/in a week beta is competing for alpha and you quit forming gamma tetramers so you’ll no longer see Hgb Barts.
W/ 2 beta genes and 3 alpha genes transcribing will you see anything abnormal on Hgb ELP?
NO!
What happens if you’re missing 2 alpha genes?
This is still alpha thalassemia trait, but you’re making significantly less globin. Less globin means less Hgb so your RBCs will be smaller.
2 alpha genes will give you microcytosis at birth and for your whole adult life.
This does NOT make you anemic. Since you have less hgb you will have incrased EPO production –> high RBC, normal hgb, low MCV which is their NORMAL CBC.
What’s the best way to pick up a thalassemia?
Barts @ birth w/ low MCV
** Key point: lifelong RBC microcytosis that you won’t pick up on adult Hgb ELP. Cheapest way to find this is newborn screen (find those Barts at birth)
What is hemoglobin H disease?
3 alpha genes knocked out which means alpha thalassemia MAJOR or Hgb H disease
There is so much excess beta that they will from tetramers called Hgb-H (extremely unstable).
What does it mean if you have Hgb H disease?
Lifelong severe microcytic anemia
**you will pick up Hgb H on adult Hgb ELP. At birth there are Barts + microcytic anemia
An unstable tetramer made up of four GAMMA globins; seen at birth when there are any (one, two, three or four) alpha genes deleted
Hemoglobin barts
An unstable tetramer made up of four beta globins; present in very small amounts in individuals with Hemoglobin H disease (three alpha genes deleted)
Hemoblogin H
Hemoglobin H disease
A severe, lifelong congenital hemolytic anemia
4 alpha genes
Normal
3 alphas
alpha trait
barts on newborn screen
2 alphas
alpha MINOR
barts on newborn screen
1 alpha
alpha MAJOR/Hgb H disease
barts on newborn screen
4 alpha deleted
hydrops fetalis/stillbirth
Anemia is so severe in utero as to cause fatal heart failure without transfusions.
Why is it important to diagnose alpha thal?
Low MCV of two gene deletions (alpha thal minor) can mimic iron deficiency or mask red cell macrocytosis on CBC. (normal MCV may be significant for macrocytosis)
What is sensitive and specific for diagnosing alpha thal on a newborn Hgb ELP?
detection of hgb barts
Detection of Barts Hemoglobin on the Newborn Hemoglobin Screen will alert you to need to follow up to determine whether there is one, two or three alpha genes deleted
In a patient with alpha thal, what does the adult Hgb ELP show?
NORMAL!
If you know that your patient has alpha thalassemia (two alpha genes deleted) you know that their “adult normal range” for MCV is
72-78 fl
A 67 year old woman originally from Vietnam is establishing care in your clinic at HCMC. She was previously followed by another HCMC resident who had prescribed iron for “anemia” for the past 3 yrs.
Hgb 13.4
Hct 41.7
RBC 5.28 H
MCV 79 L
Is she anemic?
This patient is NOT anemic!
She has erythrocytosis/polycythemia!!
Theoretically possible for someone w/polycythemia vera (myeloid neoplasm where clone of erythroid precursors that crank out more and more RBCs, w/o needing EPO) to become IDA from producing so many RBCs. When Dr. Dayton sees this CBC, she thinks it could be PV w/IDA or it could be alpha thalassemia (2 alpha genes deleted)—less globin (smaller RBCs à kidney produces more EPO). Recommend iron studies (Fe, transferrin, ferritin). But cheaper to look back at old labs.
Patient has MCV drop from 98 to 82 indicating microcytic so IDA. Fe study showed low ferritin (expected) but low transferrin (should be high for IDA). Low transferrin indicates…
underlying AOCD
in a postmenopausal women CRC is likely
What is beta thalassemia?
Deletion or decreased transcription of beta gene results in proportionate increase in delta globin. ααδδ tetramers are Hemoglobin A2
Interesting about beta: you don’t necessarily have a complete deletion of beta to have a beta thalassemia. Can have mutations that cause poor transcription of beta. So beta thalassemias are more variable. Impaired transcription of beta gene, missing gene, not transcribing gene all equal beta thalassemia. If making less beta, you see higher percent of Hgb A2 (minor Hgb). At birth or in fetal life, dependent on gamma so on newborn screen, you see nothing abnormal.
What is beta thal trait?
single beta gene deleted or poorly transcribed
–> microcytic slight anemia w/ increased red cell numbers (smaller RBCs have less O2 carrying capacity so kidneys produce more EPO leading to increased RBC production)
What is seen on newborn screen and on HgbELP in a patient w/ beta thal trait? (this is DIAGNOSIS)
Normal newborn screen
Elevated Hgb A2 (and sometimes Hgb F) detected after 6 mos to one yr
what is the Sensitivity/Specificity of Hgb ELP/HPLC in Beta Thalassemia Trait?
- Specificity - Only beta thalassemia syndromes (and some hemoglobinopathies) will increase the measured fraction of Hgb A2
- Sensitivity - Thalassemia trait can be masked by things that lower Hgb A2 such as IRON DEFICIENCY
What is the algorithm for diagnosing beta thal?
normal newborn screen >
neonatal cbc shows low MCV>
repeat CBC at 6 mos to 1 yr>
persistent low MCV w/ normal Fe studies >
order Hgb electrophoresis to rule out beta thal trait
What is beta thal MAJOR?
NO BETA genes so you can’t make Hgb A which leads to very SEVERE LIFE-LONG anemia
- No Hgb A detected on Newborn Screen
- Microcytic anemia on newborn CBC
- Should be followed by a Clinical Hematologist
“all F no A get a consultant right away!”
26 year old male HCMC Internal Medicine Resident
RBC 6.02 slightly elevated Hgb 12.2 Anemia MCV 68 microcytosis RDW 13.5 normal MCHC 29.8 LOW 93% Hgb A, 6% Hgb A2, 1% Hgb F
what are good ?s to ask?
etnicity? Previous CBC findings? family hx?
Resident didn’t feel well for a while and went to be seen in Rochester (didn’t want to be seen in the Cities). Did a bunch fo tests. Someone told him he had Polycythemia Vera and needed a bone marrow biopsy. So then he came back to HCMC. Elevated RBC, slightly low Hgb (so he’s anemic), low MCV. Could he have PV? Yes. Or he could have beta thalassemia trait. What would you do next? Not BM biopsy… they did LFTs, electrolytes. Tubes held in lab for 7 days available for add-ons. What would you add on to his sample? Check iron studies and blood smear. Did iron studies at HCMC, which were normal. For PV, with this CBC and low MCV, he would have to be iron deficient. Thus, it must be beta thal trait. Additionally, he’s of East Asian descent. Turns out multiple family members have beta thalassemia trait. Capillary ELP showed 6% Hgb A2 with detectable Hgb F—classic for beta thal trait. Felt crappy b/c he was exhausted from being a 3rd yr resident…not really b/c of his beta thal trait.
what is a hemoglobinopathy?
A mutation in a globin gene results in production of mutant globin protein which can form an abnormal tetramer (example: mutant βs gene transcribes mutant S-globin which forms tetramers to create sickle hemoglobin).
- mutations in beta are much more common than in alpha and more obvious that those are the major hemoglobinopathies that we are aware of.
clinically significant hemoglobinopathies are essentially ONLY …
beta globin mutations
A single mutated beta gene when doubly heterozygous with a beta-zero or beta plus thalassemia gene will produce a phenotype similar to or more severe as compared to a homozygous genotype
Beta S hemoglobinopathy
ααβSβS
African
Hetro: normal CBC, Hgb S on ELP
Homo: Sickle cell disease
Beta C hemoglobinopathy
ααβCβC
African
Hetero: mild hemolytic anemia
Homo: mild hemolytic anemia
Beta E
ααβEβE
SE asian
Hetero: low MCV, not anemic
Homo: low MCV, slight anemia
How do we identify and quantitate these abnormal hemoglobins?
- Hemoglobin electrophoresis (Hgb ELP)
- High pressure liquid chromatography (HPLC)
- Capillary electrophoresis
Hemoglobin electrophoresis IS NOT interpretable within 3 months of red cell transfusion!
FAS
Single beta S, normal beta, 4 normal alphas
Sickle trait; repeat ELP at one year to rule out double heterozygosity for beta-plus thalassemia
FS
2 beta S genes, 4 normal alphas
Sickling Disease; Repeat ELP at one year to rule out double heterozygosity for beta zero thalassemia
If missing one alpha gene and 2 beta S genes, you’ll see Barts in addition to sickling anemia.
If all alphas intact, gammas all intact, one beta S gene, and missing other beta gene, you’ll see FS. You wouldn’t know difference but would know on baby’s CBC b/c all they’re making is beta S and not a whole lot of Hgb so likely will be microcytic.
What are examples of some sickling diseases?
- Homozygous βS βS
- Double heterozygous βS βC
- Double heterozygous βS βE
- Double heterozygous βS βzero thalassemia • Double heterozygous βS βplus thalassemia • Double heterozygous βS βO-Arab
How do you diagnose sickling disease?
• Identification of sickled red cells on blood smear
• Confirmation of hemoglobin S
– Precipitation Test (Sickledex, Sicklequik, etc.) – Latex bead immunoassay (Hemocard)
– Hgb ELP, Capillary electrophoresis or HPLC
Hemoglobin electrophoresis IS NOT interpretable within 3 months of red cell transfusion!
how do you diagnose sickle cell crisis?
- THIS IS A CLINICAL DIAGNOSIS!
* THERE IS NO LABORATORY TEST FOR THE DIAGNOSIS OF SICKLE CRISIS
- First HCMC admission for 27 year old African female presenting to Labor and Delivery at full term with what she reports are labor pains.
- She reports that she is in excellent health, that she has had a completely normal, well monitored pregnancy and that she is visiting the US and has received all prenatal care in Nigeria but has not brought records with her.
- Clinical Hematology is consulted because of abnormal admission CBC findings. Consult request reads: Atypical HELLP Syndrome?
Hgb 13 NOT anemic HCT 36.6 normal RBC 5.32 high POLYCYTHEMIA MCV 68 Microcytic MCHC normal RDW 19 elevated PLTs 82 LOW
High Ferritin
High IBC
do not admit pt to L&D w/o getting auto CBC w/diff. she’s not anemic. Has polycythemia. Low platelets. Low MCV—microcytic.
o Useful trick: when looking at CBC, always multiply HGb X 3 and subtract Hct. 13X3=39 – 36.6 = +2.4. If >+2, has to be RBCs sticking together in machine, free Hgb in specimen (don’t know if in pt circulation or in draw or in test tube), or something mistaken for Hgb by machine (typically TGs or bilirubin (if all indirect—thinking hemolysis).
High ferritin so NOT IDA
Pt who is NOT anemic but has erythrocytosis and low MCV and normal Fe studies think….
thallasemia or hemoglobinopathy or combo of two
What is important to keep in mind re haptoglobin in pregnancy?
haptoglobin is hard to interpret when in labor. Acute phase reactant. Especially if liver is functioning really well. If HELLP syndrome, would likely have low haptoglobin.
What is the take home points from the pregnant pt’s case…
This pt had infarcted her spleen—has hx of crises. Big clue on her CBC was why does she have erythrocytosis and microcytosis w/o anemia—clue that something else was going on. L&D residents saw plt count and automatically thought HELLP syndrome. They didn’t look at MCV.
o When you’re pregnant and have SC, functioning parts in spleen significantly enlarge. Substantial splenomegaly. She had platelet sequestration from having enough functioning spleen.
o Completely changed strategy: put her on oxygen and monitoring her. Decided abdominal pain was not contractions but likely splenic crisis. Hydrated her. Gave her 24-36 hours and then induced labor for vaginal delivery. Was successful. Spared her a C-section and rushed delivery by getting a smear and identifying SC.
Hemoglobinopathy
mutation in globin gene (usually beta)
thalassemia
deletion or FAILURE to transcribe globin geme
