Chemistry Flashcards
1
Q
Major analytical methods used in the clinical chemistry laboratory include:
A
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
2
Q
Detection Method - Various Assays/Analytical Instrument
- Spectrophotometric detection
- Chemical sensors
- Flame ionization detection
- Mass spectrometric detection
A
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)
3
Q
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 __
A
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
4
Q
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 __
A
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
5
Q
Inductively coupled plasma mass spectrometry (ICP-MS)
- Is it a spectrophotometric method?
- Used for analysis of __, especially __ found in small quantities in __ specimens
A
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
6
Q
Chemical sensors
- capable of detecting __ present in __ matrix
- those capable of detecting __ are classified under three broad categories:
A
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
7
Q
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 __
A
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)
8
Q
High-performance liquid chromatography (HPLC)
- Capable of analyzing __
- Common detectors include: __
- HPLC + __ is a superior technique
- __ ionization is commonly used in liquid chromatography + __ or __
A
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)
9
Q
Immunoassays
- __
- only one antibody is used
- for assays of __ such as __
- __
- two antibodies are used
- for assays of __
A
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
10
Q
- Homogenous immunoassay format:
- Heterogenous immunoassay format:
A
- 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.
11
Q
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 __
A
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
12
Q
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 __
A
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
13
Q
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)
A
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)
14
Q
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 __
A
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
15
Q
Bilirubin interference
- total bilirubin concentration > __ mg/dL may cause problems
- interference of bilirubin is mainly caused by its absorbance at __or __ nm
A
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
16
Q
Heterophilic antibodies
- May arise in a patient with:
- __, __, __
- Interference
- most commonly with __ used for measuring __
- rarely with __ assays
- causing mostly false __results
A
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
17
Q
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 __
A
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.
18
Q
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 __
A
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
19
Q
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 __
A
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
20
Q
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 __ (< __%)
A
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 (< 3.5%)
- Hemoglobin F Two alpha, two gamma (< 1%)
21
Q
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
A
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
22
Q
Location of Globin Genes
A
Location of Globin Genes
- Chromosome 16
- alpha chain
- Chromosome 11
- beta chain
- gamma chain
- delta chain
23
Q
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
A
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
24
Q
Hemoglobinopathies can be divided into 3 major categories:
A
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)
25
Q
Hemoglobinopathies
- Ineritance pattern
- Caused by inherent mutations of genes coding for __ synthesis
- Thalassemias:
- Hemoglobin variants:
A
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
26
Q
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:
A
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
27
Q
Alpha-Thalassemia Major (Hemoglobin H Disease):
- Genes?
- What is Hb H
- What are Heinz bodies?
- Clinical?
A
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
28
Q
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?
A
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
29
Q
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
A
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: < 3.5%
- HbF: 30-40%
- HbA: none
- Hgb: 11-14 g/dL; mild sickling disease
30
Q
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
HPFH is divided into two major groups:
A
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
31
Q
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
A
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
32
Q
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
A
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
33
Q
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
A
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
34
Q
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
A
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
42
Q
A
43
Q
A
44
Q
A
50
Q
HPLC
A
51
Q
HPLC
A
52
Q
Capillary Electrophoresis
A
54
Q
Urine Electrophoresis
A
55
Q
CSF Electrophoresis
A
56
Q
Protein Capillary Electrophoresis
A
57
Q
Cytochemical stains for typing blasts
- Myeloperoxidase (MPO)
- Sudan black B (SBB)Chloroacetate esterase (CAE)
- Nonspecific esterases (NSE)
- Monocyte NSE activity is inhibited by __
- Periodic acid Schiff (PAS)
- Oil red O
A
Cytochemical stains for typing blasts
- Myeloperoxidase (MPO)
- stains the primary (azurophilic) granules indicative of granulocytic differentiation
- negative in lymphoblasts, erythroblasts, monoblasts, and megakaryoblasts
- fine dusty positivity may be seen in monoblasts
- degrades quickly in wet specimens, but is stable in smears for up to a month
- Sudan black B (SBB)
- stains lipid material found in granulocytic and monocytic cells
- Chloroacetate esterase (CAE)
- found only in the granulocytic series
- monocytes and lymphocytes are negative
- Nonspecific esterases (NSE)
- include alpha naphthyl acetate esterase and alpha naphthyl butyrate esterase
- stain cells of the monocytic series and +/- megakaryocytic, lymphocytic, granulocytic, and erythroid series
- Monocyte NSE activity is inhibited by sodium fluoride (NaF)
- Periodic acid Schiff (PAS)
- positive in most lymphoid and some myeloid blasts
- In ALL, it shows “block” positivity, often encircling the nucleus in a “rosary bead” fashion
- In AML, when positive, it is usually a diffuse, granular hue of positivity
- The vacuoles in the blasts of L3 ALL stain positively with the oil red O stain
83
Q
Classical Complement Pathway
A
84
Q
Alternate Complement Pathway
A
85
Q
Complement Pathway
A
The complement pathways
- Classic pathway
- Activated by IgM and subclasses of IgG (IgG1, IgG2, IgG3)
- Fc portion of the Ig interacts with C1q to initiate this process
- Activated C1 catalyzes the association of C4 and C2 to make C4b2b (the C3 convertase of the classic pathway)
- C4b2b is capable of converting C3 to C3b + C3a
- C3b associates with C4b2b to make C4b2b3b (the C5 convertase of the classic pathway)
- Alternate pathway
- Can be activated in the absence of Ig by:
- bacterial cell walls, venoms, or endotoxin
- complexed IgA
- These substances bind to C3b and prevent its degradation
- C3b is present at low levels at all times in peripheral blood, minute amounts of C3 are constantly converted to C3a and C3b
- Normally C3b is rapidly inactivated by factor I, unless a suitable surface is present to prevent it and promote the association of C3b with factor B to makeC3bBb (the C3 convertase of the alternate pathway)
- C3bBb is capable of converting C3 to C3b + C3a
- Abundantly available C3b associates with C3bBb to make C3bBb3b (the C5 convertase of the alternate pathway)
- The whole point of both the alternate and classical pathways is to produce a C5 convertase (a molecule capable of converting C5 into C5b + C5a)
- Once C5b is formed, it quickly complexes (on the surface of adjacent cells such as bacteria, transfused red cells, etc.) with C6, C7, C8 and C9 to form C5b6789 (the membrane attack complex or MAC)
- Can be activated in the absence of Ig by:
- C3a and C5a are formed as by-products
- anaphylatoxins capable of increasing vascular permeability and causing vasodilation
- C3b alone, like IgG, when fixed to the surfaces of cells, acts as an opsonin
- interacts with receptors on phagocytic cells, leading to ingestion of the bound cell
