Chemistry Flashcards
Major analytical methods used in the clinical chemistry laboratory include:
Major analytical methods used in the clinical chemistry laboratory include:
- spectrophotometry
- chemical sensors
- gas chromatography with various detectors
- gas chromatography combined with mass spectrometry
- high-performance liquid chromatography
- liquid chromatography combined with mass spectrometry or tandem mass spectrometry
Detection Method - Various Assays/Analytical Instrument
- Spectrophotometric detection
- Chemical sensors
- Flame ionization detection
- Mass spectrometric detection
Detection Method - Various Assays/Analytical Instrument
- Spectrophotometric detection
- Colorimetric assays
- Atomic absorption
- Enzymatic assays
- Various immunoassays
- High-performance liquid chromatography with ultraviolet (HPLC-UV) or fluorescence detection
- Chemical sensors
- Various ion-selective electrodes and oxygen sensors
- Flame ionization detection
- Gas chromatography
- Mass spectrometric detection
- Gas chromatography/mass spectrometry (GC/MS)
- High-performance liquid chromatography (HPLC)/mass spectrometry (LC/MS)
- Handem mass spectrometry (LC/MS/MS)
- Inductively coupled plasma mass spectrometry (ICP-MS)
Spectrophotometric measurements
- based on __ Law
- __ measured as __ because there is a linear relationship between __ and concentration of the analyte in the solution
- scale of absorbance __ to __, __ = “no absorbance”
- __of light depends on the concentration of the analyte in the solvent and __
Spectrophotometric measurements
- based on Beer’s Law (BeerLambert Law)
- transmittance measured as absorption (“A”) because there is a linear relationship between absorbance and concentration of the analyte in the solution
- scale of absorbance 0 to 2, 0 = “no absorbance”
- Absorption of light depends on the concentration of the analyte in the solvent and length of the cell path
Atomic absorption spectrophotometry
- used for analysis of __
- components of gaseous samples are converted into __ by __ using a graphite chamber that can be heated after application of the sample
- hollow cathode lamp containing __ at a very __ pressure is used as a light source
- __ cathode contains the analyte of interest
- Atoms in the ground state then __ part of the light emitted by the __ to boost them into __
- part of the light beam is __ –> net __ in the intensity of the beam that arrives at the detector
- __ Law to measure concentration of the analyte of interest
- __ correction in flameless to correct for background noise
- __is vaporized at room temperature –> “cold vapor atomic absorption” can be used only for analysis of __
Atomic absorption spectrophotometry
- used for analysis of various elements, including heavy metals
- components of gaseous samples are converted into free atoms by flame or flameless manner using a graphite chamber that can be heated after application of the sample
- hollow cathode lamp containing an inert gas like argon or neon at a very low pressure is used as a light source
- metal cathode contains the analyte of interest
- Atoms in the ground state then absorb a part of the light emitted by the hollow cathode lamp to boost them into the excited state
- part of the light beam is absorbed –> net decrease in the intensity of the beam that arrives at the detector
- Beer’s Law to measure concentration of the analyte of interest
- Zimmerman’s correction in flameless to correct for background noise
- Mercury is vaporized at room temperature –> “cold vapor atomic absorption” can be used only for analysis of mercury
Inductively coupled plasma mass spectrometry (ICP-MS)
- Is it a spectrophotometric method?
- Used for analysis of __, especially __ found in small quantities in __ specimens
Inductively coupled plasma mass spectrometry (ICP-MS)
- not a spectrophotometric method, but is a mass spectrometric method
- used for analysis of elements, especially trace elements found in small quantities in biological specimens
Chemical sensors
- capable of detecting __ present in __ matrix
- those capable of detecting __ are classified under three broad categories:
Chemical sensors
- capable of detecting various chemical species present in biological matrix
- those capable of detecting selective ions are classified under three broad categories:
- ion-selective electrodes
- redox electrodes
- carbon dioxide-sensing electrodes
Gas chromatography
- Used for separation of __
- Compounds identified by __
- __ depends on the flow rate of __ through the column, nature of the column, and __ of analytes
- After separation by GC, compounds can be detected by:
- __, __, __
- __ is the most specific detector for GC
- Drawback:
- only for analysis of __
Gas chromatography
- used for separation of relatively volatile small molecules
- compounds with higher vapor pressures (low boiling points) will elute faster than compounds with lower vapor pressures (high boiling points)
- Compounds identified by the retention time (RT)
- travel time needed to pass through the GC column
- RT depends on the flow rate of gas (helium or an inert gas) through the column, nature of the column, and boiling points of analytes
- After separation by GC, compounds can be detected by:
- flame-ionization detector (FID)
- electron-capture detector (ECD)
- nitrogen-phosphorus detector (NPD)
- Mass spectrometer is the most specific detector for GC
- Drawback:
- only for analysis of relatively volatile compounds or compounds that can be converted into volatile compounds using chemical modification of the structure (derivatization)
High-performance liquid chromatography (HPLC)
- Capable of analyzing __
- Common detectors include: __
- HPLC + __ is a superior technique
- __ ionization is commonly used in liquid chromatography + __ or __
High-performance liquid chromatography (HPLC)
- Capable of analyzing polar and non-polar compounds
- Common detectors include: ultraviolet (UV) detectors, fluorescence detectors, or electrochemical detectors
- HPLC + mass spectrometry is a superior technique
- Electrospray ionization is commonly used in liquid chromatography + mass spectrometry or tandem mass spec (MS/MS)
Immunoassays
- __
- only one antibody is used
- for assays of __ such as __
- __
- two antibodies are used
- for assays of __
Immunoassays
- Competitive immunoassays
- only one antibody is used
- for assays of small molecules such as a therapeutic drugs or drugs of abuse
- Immunometric (non-competitive, aka sandwich)
- two antibodies are used
- for assays of relative large molecules
- Homogenous immunoassay format:
- Heterogenous immunoassay format:
- Homogenous immunoassay format:
- After incubation, no separation between bound and free label is necessary.
- Heterogenous immunoassay format:
- The bound label must be separated from the free label before measuring the signal.
EMIT (enzyme multiplied immunoassay technique)
- __, __ immunoassay, __ molecules (__ Daltons)
- antigen is labeled with __
- enzyme that reduces __ to __
- __ = no signal at __ nm
- __ = absorbs at __ nm
- absorbance is monitored at __ nm
- labeled antigen binds with the antibody molecule –> enzyme label becomes __ and __ signal is generated
- signal intensity is proportional to analyte __
EMIT (enzyme multiplied immunoassay technique)
- homogenous competitive immunoassay, small molecules (<1000 Daltons)
- antigen is labeled with glucose 6-phosphate dehydrogenase
- enzyme that reduces NAD to NADH
- NAD = no signal at 340 nm
- NADH = absorbs at 340 nm
- absorbance is monitored at 340 nm
- labeled antigen binds with the antibody molecule –> enzyme label becomes inactive and no signal is generated
- signal intensity is proportional to analyte concentration
The Cloned Enzyme Donor Immunoassay (CEDIA)
- method is based on __ technology
- bacterial enzyme __ is genetically engineered into two inactive fragments
- when both fragments __, a signal is produced that is proportional to __
The Cloned Enzyme Donor Immunoassay (CEDIA)
- method is based on recombinant DNA technology
- bacterial enzyme beta-galactosidase is genetically engineered into two inactive fragments
- when both fragments combine, a signal is produced that is proportional to the analyte concentration
Kinetic interaction of microparticle in solution (KIMS)
- In the absence of __ molecules free antibodies bind to __ to form particle aggregates
- results in __ in absorption that is __ measured at various visible wavelengths (_-_ nm)
Kinetic interaction of microparticle in solution (KIMS)
- In the absence of antigen molecules free antibodies bind to drug microparticle conjugates to form particle aggregates
- results in an increase in absorption that is optically measured at various visible wavelengths (500-650 nm)
Luminescent oxygen channeling immunoassays (LOCI)
- immunoassay reaction is __with light to generate __ molecules in microbeads (“Sensibead”) coupled to the __
- when bound to the respective antibody molecule, also coupled to another type of bead, it reacts with __
- __ signals are generated, proportional to the concentration of __
Luminescent oxygen channeling immunoassays (LOCI)
- immunoassay reaction is irradiated with light to generate singlet oxygen molecules in microbeads (“Sensibead”) coupled to the analyte
- when bound to the respective antibody molecule, also coupled to another type of bead, it reacts with singlet oxygen
- chemiluminescence signals are generated, proportional to the concentration of the analyte-antibody complex
Bilirubin interference
- total bilirubin concentration > __ mg/dL may cause problems
- interference of bilirubin is mainly caused by its absorbance at __or __ nm
Bilirubin interference
- total bilirubin concentration < 20 mg/dL does not cause interferences
- concentrations > 20 mg/dL may cause problems
- interference of bilirubin is mainly caused by its absorbance at 454 or 461 nm
Heterophilic antibodies
- May arise in a patient with:
- __, __, __
- Interference
- most commonly with __ used for measuring __
- rarely with __ assays
- causing mostly false __results
Heterophilic antibodies
- May arise in a patient with:
- exposure to certain animals or animal products
- infection by bacterial or viral agents
- use of murine monoclonal antibody products in therapy or imaging
- Interference
- most commonly with sandwich assays used for measuring large molecules
- rarely with competitive assays
- causing mostly false positive results
Heterophilic antibodies
- __in urine
- If a __ specimen is positive for an analyte, but it cannot be detected in the __ specimen, it indicates interference from a heterophilic antibody in the __
- Another way to investigate heterophilic antibody interference is __ dilution of a specimen
- if serial dilution produces a __ result, it indicates interference in the assay
- Interference can also be blocked by adding __
Heterophilic antibodies
- Absent in urine
- If a serum specimen is positive for an analyte (e.g. hCG), but beta-hCG cannot be detected in the urine specimen, it indicates interference from a heterophilic antibody in the serum
- Another way to investigate heterophilic antibody interference is serial dilution of a specimen
- if serial dilution produces a non-linear result, it indicates interference in the assay
- Interference can also be blocked by adding commercially available heterophilic antibody blocking agents to the specimen prior to analysis.
Autoantibodies
- interfere with an immunoassay to produce false __results
- __ will conjugate with immunoglobin or other antibodies to generate __, which can falsely __ a result
- example: __ and __ can produce falsely __ results in amylase and prolactin assays
- interference can be removed by __
Autoantibodies
- interfere with an immunoassay to produce false positive results (rarely false negative)
- endogenous analyte of interest will conjugate with immunoglobin or other antibodies to generate macro-analytes, which can falsely elevate a result
- Example: macro-amylasemia and macro-prolactinemia can produce falsely elevated results in amylase and prolactin assays
- interference can be removed by polyethylene glycol precipitation
Prozone (“hook”) effect
- __ levels of antigen can __ the concentrations of “sandwich” (Ab1:antigen:Ab2) complexes responsible for generating the signal by forming mostly __ complexes
- mostly causes __ interference (falsely __ results)
- best way to eliminate the hook effect is __
Prozone (“hook”) effect
- Very high levels of antigen can reduce the concentrations of “sandwich” (Ab1:antigen:Ab2) complexes responsible for generating the signal by forming mostly single Ab:antigen complexes
- negative interference (falsely lower results)
- best way to eliminate the hook effect is serial dilution
Hemoglobin
- 6 Globin Chains?
- Embryonic
- Gower-1 Two __, two __
- Gower-2 Two __, two __
- Portland-1 Two __, two __
- Portland-2 Two __, two __
- Fetal
- Hemoglobin F Two __, two __
- Adult
- Hemoglobin A Two __, two __ (__%)
- Hemoglobin A2 Two __, two __ (
- Hemoglobin F Two __, two __ (
Hemoglobin
- Globin Chains
- alpha chain (α-chain)
- beta chain (β-chain)
- gamma chain (γ-chain)
- delta chain (δ-chain)
- epsilon chain (ε-chain)
- zeta chain (ζ-chain).
- Embryonic
- Gower-1 Two zeta, two epsilon
- Gower-2 Two alpha, two epsilon
- Portland-1 Two zeta, two gamma
- Portland-2 Two zeta, two beta
- Fetal
- Hemoglobin F Two alpha, two gamma
- Adult
- Hemoglobin A Two alpha, two beta (92-95%)
- Hemoglobin A2 Two alpha, two delta (
- Hemoglobin F Two alpha, two gamma (
Newborn babies and infants up to __ old do not depend on HbA synthesis
- switch from Hb F (2__, 2__) to Hb A (2__, 2__) occurs around __
- disorders due to __chain defects manifest clinically after 6 mo
- diseases due to __chain defects are manifested in utero or following birth
Newborn babies and infants up to 6 months old do not depend on HbA synthesis
- switch from Hb F (2alpha, 2gamma) to Hb A (2alpha, 2beta) occurs around 3 mo
- disorders due to beta chain defects (SS) manifest clinically after 6 mo
- diseases due to alpha chain defects are manifested in utero or following birth
Location of Globin Genes
Location of Globin Genes
- Chromosome 16
- alpha chain
- Chromosome 11
- beta chain
- gamma chain
- delta chain
Heme synthesis
- Involves enzymes in both the mitochondrion and cytosol
- In mitochondria __+ __ –> delta-aminolevulinic acid –> cytoplasm –> __ –> coproporphyrinogen III –> mitochondria –> __–> protoporphyrin IX –> heme –> cytosol –> heme combines with __ –> hemoglobin molecule
Heme synthesis
- In mitochondria glycine + succinyl-CoA –> delta-aminolevulinic acid –> cytoplasm –> porphobilinogen –> coproporphyrinogen III –> mitochondria –> protoporphyrinogen III –> protoporphyrin IX –> heme –> cytosol –> heme combines with globulin –> hemoglobin molecule
Heme synthesis
-
mitochondria
- glycine + succinyl-CoA
- delta-aminolevulinic acid
-
cytoplasm
- porphobilinogen
- coproporphyrinogen III
-
mitochondria
- protoporphyrinogen III
- protoporphyrin IX
- heme
-
cytosol
- heme + globulin
- hemoglobin molecule
With enzymes
- In mitochondria glycine + succinyl-CoA –> delta-aminolevulinic acid –> cytoplasm –> converted into porphobilinogen (enzyme: aminolevulinic acid dehydrogenase) –> coproporphyrinogen III (multiple steps & enzymes) –> mitochondria –> protoporphyrinogen III (enzyme: coproporphyrinogen III oxidase) –> protoporphyrin IX (enzyme: protoporphyrinogen III oxidase) –> heme (enzyme: ferrochelatase) –> cytosol –> heme combines with globulin –> hemoglobin molecule
Hemoglobinopathies can be divided into 3 major categories:
Hemoglobinopathies can be divided into 3 major categories:
- Quantitative disorders of hemoglobin synthesis:
- Production of structurally normal globin chains, but in decreased amounts
- thalassemia syndrome
- Qualitative disorders of hemoglobin structure:
- Production of structurally abnormal globulin chains
- hemoglobin S (most common), C, O, or E
- Failure to switch globin chain synthesis after birth:
- Hereditary persistence of fetal hemoglobin (HbF)
- relatively benign condition
- can co-exist with thalassemia or sickle cell disease, result in decreased severity (protective effect)
- Hereditary persistence of fetal hemoglobin (HbF)
Hemoglobinopathies
- Ineritance pattern
- Caused by inherent mutations of genes coding for __ synthesis
- Thalassemias:
- Hemoglobin variants:
Hemoglobinopathies
- Autosomal recessive
- carriers with 1 affected and 1 normal chromosome are usually healthy/slightly anemic
- both parents are carriers, children have
- 25% chance of being normal
- 25% chance of being severely affected by the disease
- 50% chance of being mostly normal
- Caused by inherent mutations of genes coding for globin synthesis
- Thalassemias: mutations disrupt gene expression –> reduced production of alpha/beta globin chain
- Hemoglobin variants: point mutations of gene in coding region (exons) –> production of defective globin –> formation of abnormal hemoglobin
ALPHA-THALASSEMIA
- Chromosome _
- _ genetic loci for the alpha gene –> __ alleles for alpha-hemoglobin
- _ alleles inherited from each parent
- Alpha-thalassemia occurs when __
Alpha-thalassemia can be divided into four categories:
ALPHA-THALASSEMIA
- Chromosome 16
- 2 genetic loci for the alpha gene –> 4 alleles for alpha-hemoglobin (α/α, α/α)
- 2 alleles inherited from each parent
- Alpha-thalassemia occurs when there is a defect/deletion in one or more of the 4 alleles
Alpha-thalassemia can be divided into four categories:
- Silent Carriers:
- 1 defective/deleted gene, 3 functional genes
- (-/α,α/α)
- no health problems, +/- low MCV/MCH
- Alpha-Thalassemia Trait:
- 2 deleted/defective genes, 2 functional genes
- alpha thal 1 (-/-,α/α) or alpha thal 2 (-/α,-/α)
- +/- mild anemia
- Alpha-Thalassemia Major (Hemoglobin H Disease):
- 3 deleted/defective genes, 1 functional gene
- (-/-,-/α)
- persistent anemia & significant health problems
- Hb H disease + Hb Constant Spring = severity greater than Hb H disease alone
- Hydrops Fetalis:
- no functional alpha gene
- have hemoglobin Bart
- life-threatening unless an intrauterine transfusion is initiated
Alpha-Thalassemia Major (Hemoglobin H Disease):
- Genes?
- What is Hb H
- What are Heinz bodies?
- Clinical?
Alpha-Thalassemia Major (Hemoglobin H Disease):
- 3 deleted/defective genes, 1 functional gene, (-/-,-/α)
- only 1 alpha gene –> decreased production of alpha-globin
- high β-globin-to-α-globin ratio (a 2- to 5-fold increase in β-globin production)
- Hb H formation: tetramer containing only 4 β chains
- cannot deliver oxygen in peripheral tissues 2/2 very high oxygen affinity
- microcytic hypochromic anemia with target cells and Heinz bodies (precipitated HbH)
- Chronic hemolytic anemia (variable)
- red cells that contain hemoglobin H are sensitive to oxidative stress and more susceptible to hemolysis, especially with oxidants such as sulfonamides
- mature erythrocytes contain more precipitated hemoglobin H and are removed from the circulation prematurely
- Subsequent increase in erythropoiesis –> erythroid hyperplasia
- bone structure abnormalities with marrow hyperplasia
- bone thinning
- maxillary hyperplasia
- pathologic fractures
BETA-THALASSEMIA
- Chromosome __
- __ genetic locus for the beta gene –> __ alleles for beta-hemoglobin
- due to deficit/absent production of beta-globin –> excess production of __
Beta-thalassemia can be broadly divided into 3 categories:
- What’s decreased?
- What’s increasd?
BETA-THALASSEMIA
- Chromosome 11
- 1 genetic locus for beta gene –> 2 alleles for beta-hemoglobin
- β0 - a defective gene is incapable of producing any beta-globin
- β+ - mutated gene can retain some function
- β0 or β+ does not predict the severity of disease
- due to deficit/absent production of beta-globin –> excess production of alpha-globin
- >200 point mutations have been reported
- deletion of both genes is rare
Beta-thalassemia can be broadly divided into 3 categories:
- Beta-Thalassemia Trait:
- 1 defective and 1 normal gene, β0/β or β+/β
- mild anemia, not be transfusion-dependent
- low MCV/MCH
- HbA2 increased
- HbF +/- elevated
- Beta-Thalassemia Intermedia:
- 2 defective genes, β+/β+ or β+/β0
- some beta-globin production is still observed
- +/- significant health problems that require intermittent transfusion
- Beta-Thalassemia Major (Cooley’s Anemia):
- 2 defective genes, β0/β0 or β+/β0
- no synthesis of beta-globin
- severe form of disease, requires lifelong transfusions, +/- shortened lifespan
- elevated HbA2 and HbF
- excess alpha-globin chain precipitates, leading to hemolytic anemia
- due to HbF, no symptoms prior to 6 months of age
Sickle Cell Diseases
- HbAS
- HbS:
- HbA2:
- Hgb:
- HbSS
- HbS:
- HbA2:
- HbA:
- Hgb: g/dL
- HbSβ0
- HbS:
- HbA2:
- HbA:
- Hgb: g/dL
- HbSβ+
- HbS:
- HbA2:
- HbA:
- Hgb: g/dL
- HbS/α-thalassemia
- HbS:
- HbA2:
- HbA:
- Hgb:
- HbSC
- HbS:
- HbC:
- Hgb:
- HbS/HPFH
- HbS:
- HbA2:
- HbF:
- HbA:
- Hgb: g/dL
Sickle Cell Diseases
- HbAS
- HbS: 35-40%
- HbA2: < 3.5%
- normal Hgb; no apparent illness
- HbSS
- HbS: >90%
- HbA2: +/-3.5%
- HbA: none
- Hgb: 6-8 g/dL; severe disease, chronic hemolysis
- HbSβ0
- HbS: >80%
- HbA2: >3.5
- HbA: none
- Hgb: 7-9 g/dL; severe sickle cell disease
- HbSβ+
- HbS: >60%
- HbA2: >3.5%
- HbA: 5-30%
- Hgb: 9-12 g/dL; variable mild-moderate sickle cell disease
- HbS/α-thalassemia
- HbS: 35-40%
- HbA2:
- HbA: 60%
- Hgb: microcytosis
- HbSC
- HbS: 50%
- HbC: 50%
- Hgb: 10-12 g/dL; moderate sickling disease, +/- chronic hemolytic anemia
- HbS/HPFH
- HbS: 60%
- HbA2:
- HbF: 30-40%
- HbA: none
- Hgb: 11-14 g/dL; mild sickling disease
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
HPFH is divided into two major groups:
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
HPFH is divided into two major groups:
- Deletional HPFH
- caused by a variable length deletion in the beta-globin gene cluster
- leads to decreased (or absent) beta-globin synthesis and a compensatory increase in gamma-globin synthesis
- pancellular/homogenous distribution of HbF in red blood cells
- Non-deletional HPFH
- broad category of related disorders with increased HbF
- Heterocellular distribution of HbF in RBCs
- also seen in beta-thalassemia and deltabeta-thalassemia
- HbA2 is normal
- Both homozygous and heterozygous HPFH are asymptomatic with no clinical or significant hematological change
- homozygous HPFH, up to 100% HbF
- heterozygous, 20-28% HbF
- If HPFH is associated with sickle cell, it can reduce the severity of the disease.
- HbS/HPFH: high levels of HbF, few (if any) sickle cell disease-related complications
- If HPFH is associated with thalassemia, less severe disease complications
HbS
- __% prevalence of S trait in African Americans
- Genetics
- Under deoxy conditions
- shortened RBC survival, average lifespan __days (normal __days)
- Cells that remain sickled despite re-oxygenation
- Splenic sequestration crisis
- Hyperhemolytic crisis
- Infections are a major source of morbidity/mortality, made worse by __
- __most common
- Others:
- Neurologic complications are frequent
- 1 in 3 patients will have angiographic appearance of __ disease
- Acute hepatic cell crisis (right upper quadrant syndrome)
- Pregnancy
- 7 classic sickle cell nephropathies
- Priapism in up to __% of male
- Ocular complications
HbS
- 10% prevalence of S trait in African Americans
- Genetics
- Chromosome 11
- sub thymine for adenine in the sixth codon of the beta-chain gene, GAG to GTG
- (β6glu→val) coding of valine instead of glutamate in position 6 of the Hb beta chain
- Under deoxy conditions
- decreased solubility, increased viscosity, and polymer formation at concentrations exceeding 30 g/dL
- forms a gel-like substance containing Hb crystals called tactoids
- gel-like form of Hb is in equilibrium with its liquid-soluble form
- factors influence equilibrium: oxygen tension, concentration of Hb S, presence of other hemoglobins
- shortened RBC survival, average lifespan 17 days (normal 120 days)
- Cells that remain sickled despite re-oxygenation—irreversibly sickled cells (ISCs) in PB smears
- sickled cells should return to their normal shape upon exposure to atmospheric oxygen
- sickled forms on the PB smear are by definition ISCs
- ISC percent is more or less constant in an individual and does not appear to predict or reflect episodic crises
- seem to be correlated inversely with that patient’s red cell survival.
- Splenic sequestration crisis presents as worsening of anemia a/w enlarged, tender spleen
- often occur during a viral illness
- Children (whose spleens have not yet undergone fibrosis) and adults with SC disease or sickle cell-β+-thalassemia are most susceptible
- Hyperhemolytic crisis
- sudden exacerbation of anemia in association with profound reticulocytosis and hyperbilirubinemi
- has been a/w concomitant G6PD deficiency
- Infections are a major source of morbidity/mortality, made worse by functional asplenia
- S. pneumoniae most common
- Others: Salmonella, Haemophilus (HITB), and M pneumoniae
- Neurologic complications are frequent
- 1 in 3 patients will have angiographic appearance of moyamoya disease (segmental arterial stenoses with ‘puff of smoke’ collaterals)
- Acute hepatic cell crisis (right upper quadrant syndrome)
- progressive jaundice, elevated LFTs, and a tender, enlarged liver
- usually resolves within 2 weeks, but it may progress to liver failure
- Pregnancy
- increased rate of both maternal and fetal death
- increased risk of pregnancy-induced hypertension (preeclampsia)
- increased incidence of intrauterine growth retardation, intrauterine fetal demise, and prematurity
- 7 classic sickle cell nephropathies: gross hematuria, papillary necrosis, nephrotic syndrome, renal infarction, isosthenuria, pyelonephritis, and renal medullary carcinoma (also increased in AS and SC)
- Priapism in up to 40% of male
- Ocular complications (proliferative retinopathy)
- more common in SC and S/β+ than SS
- Osteonecrosis
HbA2’ (hemoglobin A2 prime)
- clinically __, __-chain variant
- __% of African Americans
- heterozygotes gel electrophoresis, A2’ __
- may underestimate __ –> underdiagnosis of _
- __ and HbA2’ levels must be added
- easily detectable by __, minor peak in __area
HbC (β6glu→lys)
- AC
- __% of hemoglobin in the in C band (__ + HbC)
- generally asymptomatic, __ target cells
- CC
- __% HbC, __% HbF, __% HbA2, __% HbA
- __ hemolytic anemia, splenomegaly, and __target cells
- __-shaped crystals in the red cells, esp after __
HbE (β26glu→lys)
- common in __
- __indices, __target cells
HbD & G
- clinically __
- cellulose acetate - runs with __
- citrate - runs with __
- D & G distinguished
- HbD is a __-chain defect, HbG is an __-chain defect
- HbG: two __ bands (1 normal, 1 abnormal) separated by a distance equal to that separating __ from HbG
HbLepore
- Common in __
- Suspect if < __% (~__%) HbS on electrophoresis
- Actual HbS rarely this low (RBC exchange)
- result of fusion between _ and _ genes
- cellulose acetate - runs with __
- inefficiently produced, __% of total Hb
- HbF up to __%
Hb Constant Spring (CS)
- __indices
- mutation in _ gene stop codon
- produces __ transcript, unstable
- __ gene is inefficient –> __
- heterozygote hemoglobins produced:
- __
- adult: cellulose acetate - _ bands
- newborn: also have __
High oxygen affinity hemoglobins
- A group of hemoglobins with __
- Ex:
- Most cannot be resolved on __
- Clue to their Dx is __ on the CBC
- __ is diagnostic
Unstable hemoglobins
- A group of hemoglobins a/w __ on PB
- __ may precipitate hemolytic crisis
- Screening: incubating __ with 17% __–> precipitation of __
- Ex: __, only Hb __ a/w severe hemolysis
HbA2’ (hemoglobin A2 prime)
- clinically insignificant δ-chain variant
- 1–2% of African Americans
- heterozygotes gel electrophoresis, A2’ barely detectable
- may underestimate A2 –> underdiagnosis of β-thalassemia trait
- HbA2 and HbA2’ levels must be added
- easily detectable by HPLC, minor peak in S area
HbC (β6glu→lys)
- AC
- 40-50% of hemoglobin in the in C band (HbA2 + HbC)
- generally asymptomatic, scattered target cells
- CC
- 90% HbC, 7% HbF, 3% HbA2, 0% HbA
- mild hemolytic anemia, splenomegaly, and numerous target cells
- hexagonal/rod-shaped crystals in the red cells, esp after splenectomy
HbE (β26glu→lys)
- common in Southeast Asia
- thalassemic indices, numerous target cells
HbD & G
- clinically normal
- cellulose acetate - runs with HbS
- citrate - runs with HbA
- D & G distinguished
- HbD is a β-chain defect, while HbG is an α chain defect
- HbG: two HbA2 bands (1 normal, 1 abnormal) separated by a distance equal to that separating HbA from HbG
HbLepore
- Common in Mediterranean, esp Italy
- Suspect if < 30% (~15%) HbS on electrophoresis
- Actual HbS rarely this low (RBC exchange)
- result of fusion between δ and β genes
- cellulose acetate - runs with HbS
- inefficiently produced, 8 - 15% of total Hb
- HbF up to 20%
Hb Constant Spring (CS)
- thalassemic indices
- mutation in α gene stop codon
- produces abnormally long transcript, unstable
- αcs gene is inefficient –> thalassemia
- heterozygote hemoglobins produced:
- α-β (HbA), αcs-β (HbCS), α-δ (HbA2), αcs-δ
- adult: cellulose acetate - 4 bands
- newborn: also have α-γ (HbF), αcs-γ
High oxygen affinity hemoglobins
- A group of hemoglobins with left-shifted oxygen dissociation curves
- Ex: HbChesapeake and HbDenver
- Most cannot be resolved on gel electrophoresis or HPLC
- Clue to their Dx is erythrocytosis on the CBC
- HbO2 dissociation curve (P50) is diagnostic
Unstable hemoglobins
- A group of hemoglobins a/w Heinz bodies and bite cells on PB
- Oxidative stresses may precipitate hemolytic crisis
- Screening: incubating lysed red cells with 17% isopropanol –> precipitation of unstable Hbs
- Ex: Hbs Hasharon, Koln, & Zurich, only Hb Hammersmith a/w severe hemolysis
Methemoglobin (Hi, hemiglobin)
- form of hemoglobin in which iron is in the __ ___ (Fe?) state instead of __ (Fe?)
- results from __of hemoglobin
- incapable of combining with __
- Under normal circumstances, small degree of hemoglobin oxidation
- Hi up to __% of total Hb
- reduced in the erythrocyte by the __
- Cyanosis results when Hi reaches __% of total Hb or around __g/dL
- blood is grossly __
- __ is capable of measuring methemoglobin directly
- pulse oximetry and arterial blood gas analyzers
- estimate O2 sat by emitting a __light (wavelength of __nm) absorbed mainly by reduced hemoglobin and an __light (wavelength of __nm) absorbed by oxyhemoglobin
- Hi absorbs _
- increasing levels of methemoglobin result in __ measured oxygen saturation towards __%, form of oxygen saturation gap
- Hereditary methemoglobinemia can result from either
- __ or __
- Hb M: __
- Cyanosis appears at __ age, unless there is M fetal hemoglobin in which case cyanosis abates at __
- Most M hemoglobins run with __ on routine gels.
- Acquired methemoglobinemia results from __ that increase formation of Hi
- Ex:
- Hi has a very high affinity for __, Tx for __toxicity involves giving __to generate Hi to chelate __
- Treatment: methylene blue, reduces Hi to Hb
Methemoglobin (Hi, hemiglobin)
- hemoglobin in which iron is in the oxidized ferric (Fe+++) state instead of ferrous (Fe++)
- results from oxidation of hemoglobin
- incapable of combining with oxygen
- Under normal circumstances, small degree of hemoglobin oxidation
- Hi up to 1.5% of total Hb
- reduced in the erythrocyte by the NADH-dependent methemoglobin reductase system
- Cyanosis results when Hi reaches 10% of total Hb or 1.5 g/dL
- blood is grossly chocolate brown
- co-oximeter is capable of measuring methemoglobin directly
- pulse oximetry and arterial blood gas analyzers
- estimate O2 sat by emitting a red light (660 nm) absorbed by reduced hemoglobin and an infrared light (940 nm) absorbed by oxyhemoglobin
- Hi absorbs equally at both wavelengths, essentially undetectable
- increasing levels of methemoglobin result in decreased measured oxygen saturation towards 85%, form of oxygen saturation gap
- Hereditary methemoglobinemia can result from either
- deficiency in the reductase system
- abnormal Hbs (HbM) upon which this enzyme cannot act
- Hb M: group of Hbs that prefer the ferric (methemoglobin) state
- 2/2 various aa subs
- cyanosis appears at 6 mo
- M fetal Hb, cyanosis abates at 6 mo
- Most M hemoglobins run with A on routine gels
- Acquired methemoglobinemia results from exposure to drugs/chemicals that increase formation of Hi
- Ex: nitrites, quinones, phenacetin, and sulfonamides
- Hi has a very high affinity for cyanide, Tx for cyanide toxicity involves giving nitrites to generate Hi to chelate cyanide
- Treatment: methylene blue, reduces Hi to Hb
HbA2’ (hemoglobin A2 prime)
- clinically __, __-chain variant
- __% of African Americans
- heterozygotes gel electrophoresis, A2’ __
- may underestimate __ –> underdiagnosis of _
- __ and HbA2’ levels must be added
- easily detectable by __, minor peak in __area
HbA2’ (hemoglobin A2 prime)
- clinically insignificant δ-chain variant
- 1–2% of African Americans
- heterozygotes gel electrophoresis, A2’ barely detectable
- may underestimate A2 –> underdiagnosis of β-thalassemia trait
- HbA2 and HbA2’ levels must be added
- easily detectable by HPLC, minor peak in S area