Unit 1 Flashcards
1
Q
What are obstacles that need to be overcome transfusion therapy?
A
- a nontoxic anticoagulant
- appropriate devices to perform the transfusion
- appropriate preservation solutions
- avoiding circulatory overload
- component therapy
2
Q
What are pre-donation steps involved in the donation process?
A
- Educational history questionnaire
- Donor health questionnaire
- Abbreviated physical examination
3
Q
What are the three areas of RBC biology that are crucial for normal survival and function?
A
- RBC membrane
- hemoglobin structure and function
- RBC metabolism
- defects in any of these areas will result in RBC survival of fewer than the normal 120 days in circulation
4
Q
Describe current status
A
- efforts and standards of the American Association of Blood Banks (AABB)
- general requirements for collection of blood from volunteer donors
- components prepared from donated whole blood
5
Q
What is the amount of whole blood in a unit when donating?
A
Traditionally, 450 mL +/- 10% (1 pint)
Most recently 500 mL +/- 10%
- has to have minimum Hct of 38%
6
Q
How many pints does the average human have?
A
10-12 pints
7
Q
How long it take for donor RBCs to be replenished?
A
1-2 months
8
Q
How often can someone donate blood?
A
Every 8 weeks
9
Q
What are the 3 components of whole blood?
A
- pack RBC
- plasma
- platelets
10
Q
How long can a unit of whole blood (prepared) RBCs may be stored for?
A
21-42 days depending on the anticoagulant preservation solution
11
Q
Describe the abbreviated physical examination
A
- blood pressure
- pulse
- temperature
- hemoglobin
- Hct
- inspection of arms for skin lesions
12
Q
Describe the RBC membrane
A
- represents a semipermeable lipid bilayer supported by a protein mesh-like cytoskeleton structure
- phospholipids and their orientation
- integral and peripheral protieins
- membrane deformability
13
Q
Describe asymmetrical organization of the RBC memebrane
A
- external layer: glycoplipids and choline phospholipids
- internal cytoplasmic layer: amino phospholipids
14
Q
What is the biochemical composition of the RBC?
A
- 52% protein
- 40% lipid
- 8% carbohydrates
15
Q
Describe deformability of RBC
A
- loss of ATP: decreased phosphorylation of spectrin required for membrane deformability
- increases membrane calcium = membrane rigidity
- cells are sequestered by the spleen
- the loss of the RBC membrane, as is seen in spherocytes and bite cells, shortens the survival of these forms
16
Q
What are the 3 things that RBCs need to reman viable?
A
- Flexible
- Deformable
- Permeable
17
Q
Describe permeability of the RBC
A
- properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and control the volume of the RBCs
- permeable to water and anions (Cl-, HCO3-)
- impermeable to cations (Na+; K+)
- Calcium (Ca2+) is also actively pumped from the interior of the RBC
- ATPs are needed to keep Na+ and Ca+ out of the cell
- storage depletes ATPs: Na+ and Ca+ are allowed to accumulate intracellularly and K+ and water are lost
- cells becomes rigid
18
Q
How are RBC volume and water homeostasis maintained?
A
- by controlling the intracellularly concentrations of the sodium and potassium
19
Q
Describe metabolic pathways for RBCs
A
- pathways that produce ATP are mainly anaerobic
- RBC anucleate and have no mitchondrial apparatus for oxidative metabolism
- pathways;
—> anaerobic glycolytic pathway (90% of ATP)
—> 3 ancillary pathways that serve to maintain the structure and function of hemoglobin
20
Q
What are the 3 ancillary pathways of RBCs?
A
- Penrose pathway - 10% of ATP
- Methemoglobin pathway - affects the RBC survival and functions after transfusion
- Luebering-Rapaport Pathway - permits accumulation of 2,3 DPG
21
Q
Describe 2,3-diphosphoglycerate (2,3-DPG)
A
- important RBC organic phosphate
- amount in RBCs has a significant effect on the affinity of hemoglobin for oxygen and therefore affects how well RBCs function post-transfusion
22
Q
Describe Hemoglobin oxygen dissociation curve
A
- hemoglobin role in oxygen delivery to the tissues and carbon dioxide excretion (gas transport: most important function)
- a sigmoid-curve relationships
- allosteric changes occur as the hemoglobin loads and unloads oxygen
23
Q
What does it mean the Hemoglobin oxygen Dissociation curve shifts to the right?
A
- hypoxia
- increase in 2,3 DPG which increases the Hgb to rerelease more O2
24
Q
What does it mean when the Hemoglobin oxygen dissociation curve shifts to the left?
A
- alkalosis
- decrease in 2,3 DPG causes less O2 released
25
What are the 3 different ligands found within RBC?
- H+ ions
- CO2
- organic phosphate (2,3 DPG plays most important role)
26
What does normal hemoglobin function depend on?
Adequate 2,3-DPG levels in the RBC
27
Describe RBC preservation
- the goal of blood preservation is to provide viable and functional blood components for patients requiring blood transfusions
- viability of RBCs must be maintained during the storage time
28
What are the Food and Drug Administration requirements?
- average 24-hour post-transfusion RBC survival of more than 75%
- free hemoglobin less than 1% of total hemoglobin
29
Describe RBC viability
- assessment of post- transfusion RBC survival
- a measure of in vivo RBC survival following transfusion
- the loss of RBC viability has been correlated with the “lesion of storage”, which is associated with the various biochemical changes
- influences of 2,3-DPG levels
30
What occurs to 2,3-DPG levels in stored blood?
-As RBCs are stored, 2,3-DPG levels decrease
- DPG-depleted RBCs may have an impaired capacity to deliver oxygen to the tissues
- 2,3-DPG is reformed in stored RBCs after in vivo circulation depending on the recipients acid-base statues, phosphorus metabolism, degree of anemia, and the overall severity of the disorder
31
How is post-transfusion RBC survival determined?
1. RBCs are taken from a healthy individual and stored.
2. Labeled with radioisotopes
3. Reinfused to the original donor
4. Measure 24 hours after transfusion
32
What temperature does blood need to stored at the remain viable?
1-6 C
33
What are anticoagulant preservation solutions?
- incorporation of adenine and its effects on glycolysis and ATP levels
- Pathophysiologic effects of the the transfusion of RBCs with low 2,3- DPG level and increased affinity for oxygen
—> increased cardiac output
—> decreased in mixed venous (pO2) tension
—> combination of these
- effects of the plastic material used for PVC bags relates to the plasticized, di(ethylhexyl)-phthalate (DEHP), which is used in the manufacture of the bags
34
Describe additive solutions of RBCs
- preserving solutions that are added to the RBCs after removal of the plasma with/without platelets
—> effect on RBC viability
—> effects on viscosity of RBC concentrates
- all the additive solutions are approved for 42 days of storage for packed RBCs
- none of the solutions maintain 2,3 DPG throughout storage time
- 2,3-DPG is depleted by the second week of storage
35
What are the 3 additives that are licensed in the US?
- Adsol (AS-1) (Baxter Healthcare)
- Nutricel (AS-3) (Pall Corporation)
- Optisol (AS-5) (Terumo Corporation)
36
What are the RBC storage lesions (changes)?
- Viable cells —> Dec.
- glucose —> Dec.
- ATP —> Dec.
- pH —> Dec.
- 2,3-DPG —> Dec.
- Lactic acid —> Inc.
- Plasma K+ —> Inc.
- Plasma hemoglobin —> Inc.
- Oxygen dissociation curve —> shifts to left
37
What are the chemicals in anticoagulants?
- citrate (sodium citrate/acetic citrate) - chelate calcium
-monobasic sodium phosphate - maintains pH during storage, necessary for adequate levels of 2,3-DPG
- dextrose- substrate for ATP production
- adenine - production of ATP. Extends shelf from 21 days to 35 days. Only present in CPDA-1
38
What are 4 approved anticoagulant preservative solutions?
- acid citrate-dextrose (formula A) (ACD-A) (21 days)
- Citrate-phosphate dextrose (CPD) (21 days)
- citrate-phosphate- double dextrose (CP2D) (21 days)
- citrate-phosphate-dextrose-adenine (CPDA-1) (35 days)
39
Describe RBC freezing
- primarily used from autologous and the the storage of rare blood types
—> a cyroprotective agent is added to RBCs that are less than 6 days old
—> Glycerol (40% or 20% w/v) is used most commonly and is added to the RBCs slowly with vigorous shaking, enabling the glycerol to permeate the RBCs
- RBCs are then rapidly frozen and stored at 65C
- currently, the FDA licenses frozen RBCs for a period of 10 years from the date of freezing
40
What must be done after a transfusion of frozen cells?
- must be proceeded by a deglycerolization process
- removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline
- excessive hemolysis is monitored with the hemoglobin concentration the of the wash supernatant
- osmolarity of the unit should also be monitored to ensure adequate deglycerolization
41
What are the advantages of high concentration glycerol technique?
- initial freezing temp: -80 C
- No need to control freezing rate
- Mechanical freezer
- Max. Strorage temp: -65 C
- shipping requirements: Dry ice
- effects of changes in storage temperature: can be thawed and refrozen
42
What are disadvantages of the low concentration glycerol technique?
- initial freezing temp: = -196C
- Need to control freezing site
- liquid nitrogen freezer
- max. Storage temp: -120 C
- shipping requirements: liquid nitrogen
- effect of changes in storage and temperature: critical
43
What are advantages of RBC freezing?
- long term storage
- maintenance of RBC viability and function
- low residual leukocyte and platelets
- removal of significant amounts of plasma proteins
44
What are disadvantages of RBC freezing?
- time consuming process
- higher cost of equipment and materials
- storage requirements (-65C)
- higher cost of product
45
Desribe RBC rejuvenation
- Rejuvenation of RBCs is the process by which ATP and 2,3-DPG levels are restored or enhanced by metabolic alterations
- RBCs stored in the liquid state can be rejuvenated at outdate or up to 3 days after outdate, depending on RBC preservative solutions used.
- must be transfused in 24 hours or frozen for long term storage
46
What does FDA-approved rejuvenation solution contain?
- phosphate, inosine and adenine
47
Describe the research and development in RBC preparation and preservation
- improved additive solutions
- procedures to reduce and inactivate pathogens
- procedures to convert A-, B- and AB_ type RBCs to O-type RBCs
- methods to produce RBCs through bioengineering (blood pharming)
- RBC substitutes - will provide safe and effective oxygen carrier that could eliminate many of the problems associated with blood transfusion
- ensuring product safety
- RBCs substitutes - hemoglobin-based oxygen carriers
- RBCs substitutes - perfluorocarbons
- Tissue engineering of RBCs
—>receiving more attention and funding than other blood substitutes
—>produced by culturing stem cells in the presence of the essential cytokines stem cell factor and erythropoietin
48
Describe platelet preservation `
- annually in the U.S. millions of platelets units are distributed and transfused
- the financial impact of outdated and returned platelet units is the primary reason to find a way to improve inventory management
- platelet preservation is one way to reduce the number of outdated, discarded platelet units
49
Why is platelet storage a major challenge to the blood bank?
Because of storage limitations:
- 5-day shelf life in the US
- Bacterial contamination at incubation of 22 C
- a varying degree of platelet activation/aggregation
- release of intracellular granules
- a decline in ATP and ADP levels
*platelet storage lesion
50
What are the quality control measurements typically required for platelet preservation?
- platelet concentrate volume
- platelet count
- pH of the unit
- residual leukocyte count if claims of leukoreduction are made
- platelet swirl assessment
51
What are clinical uses of platelets?
- treatment of bleeding associated with thrombocytopenia
- prophylactic treatment for hematology-oncology patients with thrombocytopenia
- today, platelets are prepared as concentrates from whole blood and increasing apheresis
- platelets concentrates prepared from the whole blood and apheresis components are routinely stored at 20-24, with continuous agitation for up to 5 days
- FDA standards define the expiration time as midnight of day 5
- in the US, platelets are being stored in a 100% plasma medium, unless a platelet additive solution used
52
Describe platelet viability
- maintaining pH was a key parameter for reacting platelet viability in vivo when platelets were stored at 20-24C
- pH 6.2 is the current standard for maintaining satisfactory platelet viablility
- second generation storage containers have increased gas transport properties
53
What are some platelet testing and quality control monitoring procedures?
- actual platelet yield
- weight/volume conversion is necessary to determine the volume of each platelet collection
- bacterial contamination testing
54
The efficacy of the transferred platelet concentrates is usually estimated from the _________________ of platelets measured after transfusion
- corrected count increment
55
What are advantages of Hemoglobin-based oxygen carriers?
- long shelf life
- very stable
- no antigenicity (unless bovine)
- no requirements for blood typing procedures
56
What are disadvantages for hemoglobin-based oxygen carriers?
- short intravascular half- life
- possible toxicity
- increased O2 affinity
- increased oncotic effect
57
What are advantages of perfluorechemicals?
- biological inertness
- lack of immunogenicity
- easily synthesized
58
What are disadvantages of perfluorochemicals?
- adverse clinical effects
- high O2 affinity
- Retention in tissues
- requirement for O2 administration when infused
- deep-freeze storage temperatures
59
What is the platelet storage lesion?
- pH —> Dec.
- ATP —> Dec.
- Morphology scores change from discoid to spherical —> Dec.
- platelet aggregation —> drop in response to some agonists
- Lactate —> Inc.
- Degranulation —> Inc.
- platelet activation marker —> Inc.
60
What is the corrected count increment (CCI) of platelets measured after transfusion?
- a calculated measure of patient response to platelet transfusion that adjusts for the number of platelets infused and the size of the recipient, based upon body surface area (BSA)
CCI = (postcount - pre count) x BSA/platelets transfused
61
Describe measurement of viability and functional properties of stored platelets
- pre-transfusion and post-transfusion platelet counts at 1 hour and/or 24 hours and expressing the difference base on the number of platelets transfused (corrected count increment) to assess platelets viability
- Room temp stored vs. cold-stored platelets
62
Describe platelet storage and bacterial contamination
- major concerns associated with storage of platelets at 20-24 C is the potential for bacterial growth
—> room temperature storage and the presence of oxygen can encourage bacterial proliferation
—> sepsis due to contaminated platelets is the most common infectious complication of transfusion
—> an estimated 10% to 40% of patients transfused with a bacterially contaminated platelet unit develop life-threatening sepsis
- in 2002, CAP added a requirement for laboratories to have a method to screen platelets for bacterial contamination
- AABB introduced a similar requirement in 2004
- samples are not taken until a after atleast 24 hours of storage to allow bacterial replication to detectable levels
-potentially effects availability of platelets
63
What are 3 FDA approved commercial systems for screening platelets for bacterial contamination?
1. BacT/ALERT (bioMerieux, Durham, NC)
2..eBDS (Pall corp., East Hills, NY)
3. Scansystem (Hemosystem, Marseilles, France)
64
Describe Pathogen Reduction for Platelets
- pathogen inactivation (PI) is the process of treating the blood component
- components are referred to as being pathogen reduced (PR)
- pathogen inactivation technology reduces the risk of transfusion-transmitted infections
65
What are the advantages of using platelet additive solutions?
- optimizes platelet storage in vitro
- saves plasma for other purposes
- facilitates ABO-incompatible platelet transfusion-related acute lung injury (TRALI)
- improves effectiveness of photochemical pathogen reduction technologies
- potentially improves bacterial detection
66
Describe BacT/ALERT
-measures bacteria by detecting a change in CO2 levels associated with bacterial growth
- provides continuous monitoring of platelet sample-containing culture bottles
67
Describe eBDS
- measures oxygen content of the air within the sample pouch for 18 to 30 hours following incubation
- decrease in oxygen level indicates the presence of bacteria
68
What are the majority of platelets transfused in the US today?
Apheresis platelet
69
What the storage time and storage temperature or platelet concentrates and apheresis platelet components?
5 days at 20-24 C
70
How long can RBCs be frozen for?
10 years
71
One criterion used by the FDA for approval of new preservation solutions and storage containers is an average 24 hour post-transfusion RBC survival of more than:
75%
72
What is the lowest allowable pH for a platelet component at outdate?
6.2
73
What test is approved for bacterial detection specific to extending the expiration of apheresed platelets to 7 days?
Pan Genera Detection (PGD) test
74
What is the most common causes of bacterial contamination of platelet products?
- entry of skin plugs into the collection bag
75
What method does the INTERCEPT pathogen reduction system use?
- Amotosalen and UV light
76
Describe classic genetics
Genetics is concerned with
- the biochemical and biophysical nature of nucleic acids
- population studies and epidemiology
- understanding of inheritance patterns
- provide insight on anitgen-typing discrepancies due to weakened or variant alleles
77
Where is genetic information carried?
- chromosomes
78
What is a gene?
A unit of inheritance coding
79
What are genes?
- Subunit of chromosome
- found along the chromosome at Loci
80
What is an allele?
Alternate forms of a gene at a given locus
81
What is amoph?
“Silent gene” (no gene product is produced
82
What is codominant?
Alleles that are both expressed in heterozygous state
(Example: AB blood group
83
What is genotype?
- total genetic makeup, both expressed and unexpressed genes
84
What is phenotype?
- observable produce of the gene at a given locus
(Example: A/O or A/A can be genotype, but both will be A as phenotype
85
What is Cis position?
- when the loci that the genres occupy are on the same chromosome
86
What is trans position?
- the location of two or more genres on opposite chromosomes of a homologous pair
- example DCe/DcE
—> C = trans position to E (opposite side of chromosome)
—> C = cis position to e (same side of chromosome)
87
What is heterozygous?
Different alleles at a given locus on a pair of chromosomes. Sometimes known as a single dose (AO or Kk)
88
What is homozygous?
Identical alleles at a given locus on a pair of chromosomes. Sometimes known as double dose (AA; KK or kk)
89
What is recessive?
Allele expressed in homozygous state, marked by a dominant allele
90
What is dosage?
Phenomenon where an antibody reacts more strongly with a red cell carrying a double dose rather than a single dose
91
What is public antigen?
- found on RBCs of greater than 98% of population
92
What is private antigen?
Found on RBCs of less than 1% of population
93
What is autosome?
Any chromosome other than than the sex chromosome (X and Y)
94
What are the major issues of population genetics of concern to blood banking?
- pioneering work of Linnaeus and Darwin
- Mendels law of inheritance
- Hardy-Weinberg principle
- inheritance patterns
95
Describe Mendel’s First law of inheritance
- shows that alleles of genes have no permanent effect on one another when present in the same plant but segregate unchanged by passing into different gametes
- unlike the flower color of many types of plants, most blood group genes are inherited in a codominant manner
- in codominace, both alleles are expressed, and their gene products are seen at the phenotypic level
—> in the MNSs blood group system, a heterzygous MN individual would type as both M and N antigen positive
96
Describe sweet pea flower experiment
* Mendel’s first law
- parental: bred all one color flower
—> homozygous for either red (RR) or white (rr)
- first filial: cross-bred two plants; obtained second generation
—> heterozygous (Rr)
- second filial: produced red and white flower in 3:1 ratio
97
Describe the Mendel’s second law of inheritance
- law of independent assortment
- genes from different traits are inherited separately from each other
- this allow for all possible combinations of gene to occur in the offspring
- if genes for separate traits are closely linked on a chromosome, they can be inherited together as a single unit (linkage)
98
Describe the law of independent assortment experiment
- Seeds: Round, wrinkled, yellow or green
- parental genotypes: RRYY and rryy
- results are di-hybrid giving following phenotypes:
—> round/yellow
—> round/green
—> wrinkled/ yellow
—> wrinkled/green
—> ratio of 3:3:3:1
99
Describe Hardy-Weinberg principle
- this principle allows the study of Mendelian inheritance in great detail
- this principle specifically addresses questions about recessive traits and how they can be persistent in populations
- the Hardy-Weinberg formula states (p+q)(p+q) = 1
—> p = the gene frequency of the dominant allele
—> q = the frequency of the recessive allele
—> This can also be stated as p^2 + 2pq + q^2 = 1
100
What is the criteria for the use of the Hardy-Weinberg formula?
- the population studied must be large.
- mating among individuals must be random
- mutations must not occur in parents or offspring
- there must be no migration, differential fertility or mortality of genotypes studied
101
Describe inheritance patterns
- describe how a disease is transmitted in families
- these patterns help to predict the recurrence risk for relatives
- in general, inheritance patterns for single gene disorders are classified based on whether the are autosomal or X-linked and whether they have a dominant or recessive pattern of inheritance
102
Describe autosomal dominant inheritance
- mode of genetic inheritance by which only one copy of a disease allele is necessary for an individual to be susceptible to expressing the phenotype
- autosomal dominant inheritance is often called vertical inheritance because of the transmission from parent to offspring
103
Describe autosomal recessive
- In autosomal recessive inheritance, two copies of a disease allele are required for an individual to be susceptible to expressing the phenotype
- typically, the parents of an affects individual are not affected but are genes carriers
104
Describe X-linked dominant
- only one copy of a disease allele on the X chromosome is required for an individual to be susceptible to an X-linked dominant disease
- both males and females can be affected, although males may be more severely affected because they only carry one copy of genes found on the X chromosome
105
Describe X-linked recessive inheritance
- two copies of a disease allele on the X chromosome are required for an individual with two X chromosomes (female) to be affected with an X-linked recessive disease
- since males only have one X chromosome, any male with one copy an X-linked recessive disease allele is affected.
106
Describe Cellular genetics
- human possess 46 chromosomes
—> 22 autosomes and 1 set of sex chromosomes
107
What is chromatin?
Nucleic acids an structural proteins called histones
108
What are heterochromatin?
Stains as dark bands
109
What is Achromatin?
- stains as light bands: consists of highly condensed regions that are usually not transcriptionally active
110
What is euchromatin?
- swollen form of chromatin in cells, which is considered to be more active in the synthesis of RNA for transcription
111
What is a locus?
- The specific location of a gene on a chromosome
- at each locus, there may be only one or several different forms of the gene, which are called alleles
112
What is hemizygous?
- refers to the condition when one chromosome has a copy of the gene and the other chromosome has that gene deleted or absent
113
Describe mitosis
- the process by which somatic cells divide to create identical daughter cells
- chromosomes are duplicated and one of each pair is passed to the daughter cells.
- quantitatively and qualitatively identical DNA is delivered to daughter cells formed by cell division
- Steps
1. Interphase: restring stage. No cells are dividing
2. Prophase: chromatin condenses to form visible chromosomes and the nuclear envelope starts to breakdown
3. Metaphase: the chromosomes are lined up along the middle of the nucleus and paired with the corresponding chromosome.
4. Anaphase: the cellular spindle apparatus is formed and the chromosomes are pulled to opposite ends of the cell. Cell becomes pinched in the middle and cell division starts to take place
5: telophase: the cell is pulled apart, division is complete, and the chromosomes and cytoplasm are separated into two new identical daughter cells.
114
Describe Meiosis
- process used to produce gametes or sex cells.
- results in 4 unique daughter cells.
- allows for great genetic diversity in organisms and controls the number of chromosomes within dividing cells
- gametes carry a haploid number of chromsomes
- occurs only in germinal tissues
115
Describe molecular genetics
- chromosomes are composed of long, linear strands of DNA tightly coiled around highly basic protein called histones
116
Describe DNA
- purines and pyrimidine
- helical structures of DNA which are formed by double stranded material
- codons and stop codons
117
Describe purines and pyrimidines
- nitrogenous bases that make up the two different kinds of nucleotide bases in DNA and RNA
118
What are codons?
- sequence of three nucleotides in the DNA strand for the genetic code for a specific amino acid
119
What are stop codons?
- sequence of three nucleotides that stop peptide from being translated form mRNA
120
What is the generative cell cycle?
- G0 - temporarily resting period, no cell division (2N)
- G1 - Cells produce RNA and synthesize protein (2N)
- S: DNA replication occurs (4N)
- G2: during the gap between DNA synthesis and mitosis; the cell continues to synthesize RNA and produce new proteins (4N)
- M cell division (2N)
121
Describe DNA replication
- complex process involving numerous enzymes, nucleic acid primers, various small molecules and the DNA helix molecule that serve as it own template for the replication process
- bidirectional manner
- semiconservative in nature
122
What are the steps of DNA replication?
1. Sections of DNA must be uncoiled and two strands must be separated and kept apart. This is done by the enzyme DNA gyrase (opens coils) and DNA helicase (separates two strand of duplex DNA)
2. DNA polymerase III can synthesize a new strand in the 5’ and 3’ direction on the leading strand
3. Proteins (single-stranded binding proteins) interact with the open strands of DNA to prevent hydrogen bonding when it is not needed during replication
4. DNA polymerase III also proofreads the addition of new bases to the growing DNA strands and can remove an incorrectly incorporated based, such as G paired to T
123
What is required for DNA replication to take place?
- there must be a short oligonucleotide (primers) that binds to the begging of the region to be replicated
124
Describe DNA repair
- mechanisms can detect the mistakes or changes and correct the actual DNA sequence
- most important mechanism is: proofreading ability of DNA polymerases
- occurs in both 5’ to 3’ and 3’ and 5’ directions and allows the polymerase to backtrack on a recently copied DNA strand and remove an incorrect nucleotide and inserting the correct on in place
- systems can recognize mismatched base pairs, missing nucleotides and altered nucleotides in DNA sequences
125
What are the major DNA repair systems?
- photo reactivation (PR)
- excision repair ( also called cut and patch repair)
- recombinational repair
- mismatch repair
- SOS repair
126
Describe photoreactivation of DNA
- thymine dimmers are formed after exposure to UV lights, the photoreactivation enzyme becomes active and enzymatically cleaves the thymine dimers.
127
Describe excision repair of DNA
- thymine dimers can be removed by this complex process
- disrupted section of DNA is removed
- cut is made on side of dimer that bulges out from rest of duplex DNA
- DNA polymerase I synthesizes a short replacement strand for the damaged DNA section
- old strand is removed by DNA polymerase I as it moves along the DNA and the newly formed DNA segment is ligated into place
128
Describe recombinant repairs of DNA
- uses correct strand of DNA to fill in strand where the error was deleted.
- Polymerase I and DNA ligase then fill in other strand
- when lost sections of DNA have been lost, the double strand breaks can be handled this method.
129
Describe mismatch repair
- activated when base pairing is incorrect and a bulge occurs in the duplex DNA.
- Mismatch repair enzymes are able to remove incorrect nucleotides and insert the correct ones.
- Methyl groups on adenine are used by mismatch enzyme systems to determine nucleotide is correct and which is a mistake
130
Describe SOS repairs of DNA
- used when DNA and cell damage occurs
- damage can occur through UV radiation, chemical mutagens and excessive heat
- gens of SOS response system must work in a coordinated manner to repair the damaged DNA through recombination events that remove the damage sections and replace them with the correct sequences
131
What are factors that affect the DNA replication?
- errors in the primary nucleotide sequence
- chemical and environmental factors
- ionizing radiation and strong oxidants
- ultraviolet (UV) radiation
- medications
132
Describe mutations in DNA
- any changes in the structure or sequences of DNA (physical or biochemical)
133
What is wild type?
- the original form of the DNA sequence, and the organism in which it occurs
134
What are mutagens?
The various chemicals and conditions that can cause mutations
135
What are the types of mutations?
- point mutation
- silent mutation
- transition mutation
- missense point mutation
- nonsense mutation
- insertion/deletion of of one more nucleotides
- frameshift mutation
136
Describe point mutation
- only one nucleotide in DNA sequence is changed
- included substitutions, insertions, and deletions
137
Describe the silent mutation
- occurs when a mutation happens that causes a change in the peptide sequence
- no mutation is seen at the phenotypical level
138
Describe transition mutations
- one purine is substituted for another purine, or one pyrimidine is substituted for another pyrimidine
139
What is a transversion?
When a purine is substituted for a pyrimidine or pyrimidine for a purine
140
Describe the missense point mutation
- results in a change in a codon, which alters the amino acid in the corresponding peptide.
- changes cannot be accommodated by the peptide while still maintaining its function
141
Describe a nonsense mutation
- results when a point change in one of the nucleotides of a DNA sequences causes one of the three possible stop codons to be formed
142
What are the 3 stop codes?
- amber (UAG)
- opal (UGA)
- ochre (UAA)
143
Describe insertion/deletion of one or more nucleotides in the DNA sequences mutation
- result of this type of mutation is a change in the triplet codon sequence and a subsequent alteration in the frameshift reading so that a large change in the amino acid sequence occurs
144
Describe insertion/deletion of one or more nucleotides in the DNA sequences mutation
- result of this type of mutation is a change in the triplet codon sequence and a subsequent alteration in the frameshift reading so that a large change in the amino acid sequence occurs
145
Describe the frameshift mutation
- results in a nonfunctional transferase protein that is seen phenotypically as the O blood group
146
What are the types of RNA?
- Ribosomal RNA (rRNA)
- Messenger RNA (mRNA)
- Transfer RNA (tRNA)
- small RNA molecules which have other various functions within the cell
147
Describe RNA
- single stranded structure
- uracil pairs with guanine
- substitutes deoxyribose with sugar ribose
- used to transmit genetic information from nucleus to cytoplasm in translated into peptides and proteins
- synthesized in a 5’ to 3’ direction, starts at 3’ end of coding strand
148
What are proteins the product of?
Transcription and translation
149
What is central dogma of molecular biology ?
The flow of genetic information from DNA to RNA to proteins
150
Describe rRNA
- RNA is translated
- RNA polymerase I transcibes rRNA
- most abundant and consistent form of the RNA in the cell
151
Describe mRNA
- the initial link between the information stored in DNA and the translation of that information into amino acids.
- it is this form that is transcribed from DNA that encodes specific genes
- RNA polymerase II transcribe mRNA
152
Describe tRNA
- involved in delivering amino acids to the mRNA bound on the ribosome
- functional areas
1. The anticodon: it consists of 3 nucleotides that hydrogen-bond to the correct site on the corresponding codon on the mRNA
2. 3’ hydroxyl end and binds an amino acid
153
Describe transcription
- the cellular process by which one strand of duplex DNA is copied into RNA
- begins when RNA polymerase II binds to the region upstream of a gene
- consensus sequence: used to position RNA polymerase properly for transcription of a gene starts in the correct position. This is referred to as promoter
154
Describe translation
- is the cellular process by which RNA transcipts are turned into proteins and peptides, the structural and functional molecules of the cell
- the ribosomes move down the codons on the mRNA one at a time, adding a correct amino acid to the growing peptide chain
- after translation is complete, the mRNA is rapidly degraded by enzymes ( helps control gene expression), or attaches to another ribosome, and the entire translation process starts all over again
155
What is a mutation?
- change in a gene potentially capable of being transmitted to offspring
156
What is deletion?
- the loss of a portion of chromosome
157
What is inversion?
- the breaking of a chromosome during division with subsequent reattachment occurring in an inverted or upside down position
158
What is translocation?
Transfer of a portion of one chromosome to its allele
159
What are some common approaches used in Modern Genetics techniques?
- isolation of material
- visualization
- amplification
- separation of species and subspecies
- quantification
160
Describe roles of genetics in the future?
- DNA-based applicable to transfusion medicine
- genetic variation can impact response to many types of treatment
- personalized medicine: treatment and prevention of disease taking into account individual variability in genes, environment, and lifestyle for each person
161
What is an intron?
- the noncoding region of a gene
162
What is the purpose of transcription?
- synthesize RNA using DNA as a template
163
When a male possesses a phenotypic trait that he passed to all his daughters and non of this sons, what trait is this?
X-linked dominant
164
When a female possess a phenotypic trait that she passes to all of her sons and none of her daughters, the trait is said to be what?
X-linked recessive
165
How is tRNA different from other types of RNA?
- it recognizes amino acids and nucleic acids
166
Describes translation of proteins
- occurs on ribosomes in the cytoplasm
- post-translation processing can include glycosylation
- the codon UAA, UGA, or UAG would terminate translation
167
What is the purpose of meiosis?
- generate daughter cells that contain half the number of chromosomes of somatic cells that contain new DNA sequences
168
What is the pattern of inheritance most commonly expressed by blood group genes?
- autosomal codominant
169
Describe cellular defense mechanism
- mediated by macrophages, T cells and dendritic cells
170
Describe humoral defense mechanism
- B cells produce specific antibodies
- complement binds to immunoglobulin molecules that have specific complement receptor site
171
Describe detection of allo-antibodies and auto-antibodies
- describe upon binding forces between antigens and antibodies, properties of the antibody itself and individual host characteristics
- antigen-antibody reactions are influenced by a number of factors, including distance, antigen-antibody ratio, pH, temperature and immunoglobulin type
172
Describe innate immune response
- consists of physical barrier, biochemical effectors and immune cells. (Skin)
- second line of defense is internal, can recognize common invaders with a nonspecific response
- last line of defense: acquired immune response
- does not function in specfic manner
- recognizes complex repeating patterns present on common invading pathogens
173
Describe the acquired immune response
- relies of formation of specific antigen-antibody complexes and specific response and IS memory allows resistance to a pathogen that was previously encountered
- highly evolved
- has memory
- specific
174
What are the two major cells that can use phagocytosis to remove pathogens?
1. The polymorphonuclear cells (neutrophils, basophils, and eosinophils)
2. Mononuclear cells (monocytes in plasma and rheumatoid macrophages in tissues)
175
What are the 3 major roles of complement in immunity?
1. Final lysis of abnormal and pathogenic cells via the binding of antibody
2. Opsonization and phagocytosis
3. Mediation of inflammation
176
What are the types of antibodies?
- autoantibodies: directed against self antigens
- alloantibodies: directed vs. non-self antigens
- antigen: molecules found on the surface of foreign cells or on damaged internal cells
177
What are types of B cells?
- B memory cells
- plasma cells
178
What are types of T cells>?
- Th- turns on immune system
- Tc- kills tumor cells and turns off immune system
- T-memory - remembers
179
What are the types of APCs?
- macrophages
- neutrophils
- dendritic cells
- some B cells
180
Describe NK cells
- kill tumor cells
181
Describe MHC class and class II antigens
- cell membrane proteins that help T-cells recognize foreign antigens
- MHC I helps T cytotoxic cells (CD8)
- MHC II helps T cytotoxic cells (CD4)
182
What are the CD markers of pluripotent stem cell?
CD34
183
What are the CD markers of granulocytes?
- CD11b
- CD16
- CD35
184
What are the CD markers for T cells?
- CD2
- CD3
- CD4
- CD8
185
What are the CD markers of B cells?
- CD19
- CD20
- CD21
- CD22
- CD35
186
What are the CD markers for NK cells?
- CD16
- CD56
187
Describe T cells
- matures in thymus
- responsible for making cytokines and destroying virally infected host cells.
188
Describe B cells
- Mature in bone marrow
- undergo gene rearrangement in order to have the correct antibody made that can react with the correct antigen
189
Describe plasma cell
- derives from B cell
- secrete antibodies
190
Describe dendritic cells
- are present throughout many systems of the body and are responsible for antigen processing and presenting
191
Describe effector cells
- B and T cells both mature into this
- functional units of immune system
192
Describe MHC class I
- code for HLA-A, HLA-B and HLA-C antigens
193
Describe MHC class II
- codes for HLA-DR, HLA-DQ, and HLA-DC antigens
194
Why are MHC classes important?
- recognition of foreign substances and the immune reactions against them.
195
What are the two major functions of T cells?
- produce immune mediating substances such as cytokines
- to kill cells that contain foreign matter
196
Describe Th cells
- CD4
- further grouped into Th1 and Th2
- have ability to recognize antigen, along with MHC class II molecules, and provide help to B cells to evolve into plasma cells and make antibodies
- determines which antigens become immune system targets
- aid in proliferation of immune cells after the encounter antigen
197
Describe Tc cells
- CD8
198
Describe Th1 cells
- ability to recognize antigen, along with MHC class II molecules, and provide help to B cells to evolve into plasma cells and make antibodies
199
Desribe latency
- also called pre-seroconversion or window period
- lag phase
- antibody can be detected with serological testing in this “window” of time
200
Describe memory cells
- stored in immune organs of host and wait for contact with previous antigen.
- activate and produce a stronger and more rapid response
201
What are super antigens?
- can stimulate multiple T cells, causing them to release large amounts of cytokines.
202
What are the two main cytokine types?
- lymphokines - produced by lymphocytes
- monokines - produced my monocytes and macrophages
203
What is the role of MHC class I?
- play key role in cytotoxic T-cell function
- found on all nucleated cells
204
What is the role of MHC class II role?
- essential for presenting processed antigen to CD4 T cellss and are necessary for T-cell functions and B-cell help
205
describe the Ig structure
- 4 polypeptide chains: two identical light chains and two identical heavy chains
- disulfide bonding hold the light and heavy chains together
- have amino and carboxyl terminal
- each heavy and light chain have a constant region
- variable region = amino terminals of light and heavy chains
- domains = regions of light and heavy chains that are folded into compact globular loop structures
206
Describe papain
- splits the antibody molecule at hinge region to give three fragments: one crystallizable Fc fragment and 2 antigen binding site.
207
How many C1 and C3 domains are there on heavy chains of IgA, IgG, and IgD?
3
208
How many C2 and C4 domains are on the heavy chains of IgE and IgM?
4
209
What are the types of types of heavy chains?
- IgG - gamma
- IgM - Mu
- IgD - delta
- IgA - alpha
- IgE - epsilon
210
What are the types of light chains?
- kappa
- lambda
211
What do the FC and Fab regions bind?
FC- complement
Fab - antigen
212
What Immunoglobulins are significant for blood banking?
- most significant: IgG, IgM and IgA
- reaction temperatures
- naturally occurring antibodies
- commonly encountered IgM and IgG antibodies
- IgG subclasses
- Role of IgE
- Role of IgD
213
Describe IgG of blood banking
- reacts at body temperature (37C)
- size
—> sedimentation coefficient - 6.7/ molecular weight 150,000
- crosses placenta
- fixes complement
- found only in monomer form
- opsonizes foreign material
- transfusion reactions and HDN
214
Desribe IgM of blood banking
- size
—> sedimentation coefficient - 19/ molecular weight 900,000
- fixes complement
- found in pentameter form
- opsonization foreign material
- testing problems: mask reactivity of IgGs
- found in:
—> ABO system
—> Lewis structure
—> Ii system
—> MNS system
—> P system
215
Describe IgA of blood banking
- size
—> sedimentation coefficient 7-15/ molecular weight 160,000 and 500,000
- found in body fluids
- does not activate complement
- found in both monomer and dimer forms
- has secretory components
216
Describe IgE of blood banking
- size
—> sedimentation coefficient = 8/ molecular weight 196,000
- does not activate complement
- found only in monomer form
- associated with hypersensitivity
217
Describe IgD of blood banking
- size
—> sedimentation coefficient 7/ molecular weight is 160,000
- does not activate complementi
- found only in monomer form
218
What are the 3 types of immunoglobulin variations?
1. Isotypes
2. Allotypes
3. Idiotype
219
Describe isotype
- antibody variants present in all members of a species
220
Describe allotype
Antibody variant in constant region that does not occur in all memebers of species
221
Describe idiotypes
Antibody variant in the variable region and is specific for each antibody molecule
222
Describe immunoglobulin Fc receptors
- IgG subclasses involved
—> IgG1 and IgG3 can attach to phagocytic receptors
—> allows for removal of incompatible RBCs
- cells involved: macrophages and monocytes
223
Describe complement system
- biological roles: direct lysis of cells, bacteria and viruses
- mediation of inflammation
- circulating and cell membrane proteins
—> inactive forms as proenzymes except factor D
—> once activated they in a cascade of events
224
What are the activation pathways?
- classical
- alternative
- lectin
225
Describe classical complement pathway
- activation when antibody binds to antigen
- activation of components
- fragments with ananphylatoxin activity
- membrane attack complex formation
- cell lysis
226
Describe alternative complement pathway
- activation of alternative pathway: surface contacts with complex molecules
-
227
What are important protein factors of the alternative pathway?
- factor D
- factor B
- factor P
- C3
228
Describe lectin pathway
- activation: attachment of mannose binding lectin to microbes
- elements common with classical pathway
229
Describe the Membrane Attack Complex (MAC)
- binding of complement by RBC antibodies
- activation by IgG
—> binds less efficiently due to small size
—> 2 molecules are needed in close proximity to bind
- activation by IgM: can activate complement
- IgG Rh antibodies: do not usually bind complement
- IgM Lewis antibodies: can bind complement but rarely cause HTR
- ABO antibodies: can activate complement and cause HTR
230
Describe antigens
- initiate formation of and reaction to antibodies
- different blood group antigens differ in their immunogenicity
- antigen characteristics that are influencing immune response:
—> size
—> complexity
—> conformation
—> charge
—> accessibility
—> solubility
—> digestibility
—> chemical composition
231
What are the type of blood group antibodies?
- polyclonal
- monoclonal
- naturally occurring
- immune
- unexpected
- naturally occurring ABO
- allo-
- auto-
232
Describe naturally occurring antibodies
- produced without transfusion, injection, pregnancy
- IgM, RT or lower, activate complement, may be hemolytic at 37C
- ABH, Hh, Ii, Lewis, MN, P
233
Describe immune antibodies
- transfusion or pregnancy
- IgG, 37C
- requires AHG for detection
- Rh, Kell, Duffy, Kidd, Ss
234
Describe unexpected antibodies
- all antibodies directed against red cell antigens except for the ABO group are considered UNEXPECTED
- detection techniques
- importance in pretransfusion testing
235
Describe Naturally occurring ABO antibodies
- Isoagglutinins
236
Describe alloantibody production
- produced after exposure to genetically different (non self-antigens)
237
Describe autoantibody production
- produced in response to self antigens
- cold auto or warm auto antibodies
- associated with autoimmune disease
- special transfusion techniques needed
238
What are some characteristics of Antigen-Antibody reaction?
- intermolecular binding forces
- properties
—> affinity - first attraction
—> avidity - strength of binding
- specificity
—> specific reaction
—> cross-reaction
—> no reaction
- host factors influencing immune response (age, health, immune status)
- Duffy system and malaria: those who don’t inherit the Duffy Blood system antigens and resistant to malarial invasion
- immune tolerance effects: lack of immune response or an active immunosuppressive response
239
What are the blood samples required for RBC antigen-antibody reactions?
- serum: ensures amount of complement are available
- plasma (EDTA): most common.
—> EDTA binds calcium and will obstruct complement activation
- plasma (sodium heparin)
—> inhibits the cleavage of C4 and obstructs compliment activation
240
What are traditional methods used in blood bank?
- hemagglutination
- precipitation
- agglutination inhibition
- hemolysis
- ELISA (EIA), IF, WB
241
Describe hemagglutination method of blood bank
- major technique used in blood banking
- red cell agglutination reactions
242
Describe precipitation method of blood bank
- soluble antigen + soluble antibody to create an insoluble antigen-antibody complex
243
Describe hemolysis method of blood bank
- antigen-antibody reaction has occurred and complement has been fixed and RBC lysis has occurred
244
Describe Red cell agglutination reactions
1 sensitization: antigen binding to the antibody occurs
2. Lattice formation: multiple antigen-antibody bridges between RBC antigens and antibodies is formed. This allows for agglutination to form
245
What are factors that influence agglutination reactions?
- centrifugation
- zeta potential
- antigen-antibody ratio
- pH
- temperature
- immunoglobulin type
- different techniques for IgG and IgM
- enhancement media
- protein media
- zeta potential, sialic acid in red cells
- low ironic strength media (LISS)
- polyethylene Glycol (PEG) and polybrene
- proteolytic globulin (AHG) reagents
- chemical reduction of IgG and IgM
246
How does centrifugation influence agglutination?
- decreases reaction time by increasing the gravitational forces on the reactants and brings them closer together
247
Describe Zeta potential
RBCs natural repulsive effect
248
What phases are in the antigen-antibody ratio?
- prozone
- equivalence
- postzone
249
What is the ideal pH for Ag/Ab complexes?
6.5 - 7.5
250
Describe how temperature influences agglutination
- IgM reacts best at 22C or below and are seen at the immediate spin phase of testing
- IgG reacts at 37C require incubation and anti-human globulin reagent to be seen
251
What are IgM antibodies of importance ?
- ABH
- Ii
- MN Lewis
- Lutheran
- P
252
What are the IgG antibodies of imporance?
- Ss
- Kell (Kk, Jsa, Jsa, Kpa, Kpb)
- Rh (DCcEe)
- Luthern (Lub)
- Duffy (Fay, Fyb)
- Kidd (Jka, Jkb)
253
Describe the different techniques for IgG and IgM that influence agglutination reactions
- IgM: immediate spin reaction ; ABO testing
- IgG: 37C incubation; antibody screening cross match testing
- IgG: AHG reagent; antibody screening, antibody identification and crossmatch testing
254
How does enhancement media influence agglutination?
* also known as potentiators
* especially IgG
- AHG: cross-links sensitized cells
- LISS: causes RBCs to take up antibody more rapidly
- enzymes: reduces surface charge; can destroy or enhance different RBC antigens
255
How does protein media influence agglutination?
- 22% albumin - adjusts the zeta potential between RBCs
- PEG: increases test sensitivity by causing closer proximity of RBC
256
What are disadvantages of monoclonal reagents?
- overspecificity
- complement may not be fixed in antigen-antibody reaction
- oversensitivity
257
Describe the non traditional method used in blood banking
Flow cytometry
- used to Obama in quantitative and qualitative data on cell populations to sort different cell populations
- used for (in blood bank):
—> quantify fetomaternal hemorrhage
—> study transfused cells
—> distinguish heterozygous and homozygous antigen expression
258
What diseases are important in serological testing?
- immunodeficiency
- hypersensitivity
- monoclonal and poly clonal gammopathies
- Autoimmune disease
- hemolytic disease of the newborn (HDN)
259
Describe Transfusion-Related Immunomodulation (TRIM)
- immune system may undergo transient depression following a blood transfusion
- causes increased risk of developing an infection
- cells, cytokines involved in TRIM
—> alloreactive lymphocytes are inactivated and removed
—> immune system unresponsive due to failure of T cells to respond
—> cells are inhibited by cytokines
- effects of leukoreduction
—> transfusion of leukoreduced packed cells has decreased the risk of TRIM
260
What are the steps of the classical pathway?
1. C1r and C1s are converted into active enzymes as binding of C1q occur
2. C1r is activated when C1q is bound
3. When activated, C1r cleaves thioster bond on C1s which activates it. (Ends recognition stage)
4. C1s cleaves C4 to create C4b and cleaves C2 to make C2a
5. C3 convertase (C4b2a) is formed
6. C3b binds to C4b2a to form C5 convertase (C4b2a3b) (ends activation stage)
7. C5 convertase splits C5 into C5a and C5b
8. C5b attaches to the cell membrane to initiate formation of the MAC (C5b6789)
261
What are the steps of the lectin pathway?
1. Mannan lectin binding to mannose sugar
2. MASP-2 cleaves C4 and C2 to make C4b and C2a
3. C3 convertase is formed
4. C3b binds to C4b2a to form C5 convertase (C4b2a3b)
5. C5 convertase splits into C5b and C5a
6. C5b attaches to cell membrane to initiate formation of MAC (C5b6789)
262
What are the steps of the alternative pathway?
1. C3 is hydrolzyed b water to produce C3b, AMD binds to Factor B and they attach to target cell surface
2. B is cleaved by Factor D into Ba and Bb. Bb combines with C3b to form C3bBb
3. More C3is cleaved, forming more C3bBb. This enzyme is stabilized by properdin and cleaves additional C3
4. If C3 remains attached to C3bBbP, convertase now has ability to cleave C5 (C3bBbp3b) into C5a and C5b
5. C5b attaches to cell membrane to initiate formation of MAC
263
What are the host factors that influence the immune response?
- nutritional status
- hormones
- genetics
- age
- Race
- exercise level
- disease
- injury
264
What are specimen requirements for blood bank?
- collected in 6 mL pink top tube (EDTA) with blood bank band ID label
- tubes should be transported to at room temperature to lab
- if there is a delay in testing, specimens should be refrigerated (1-6C)
- specimen can be used for routine testing for 3 days following collection
265
What are causes for rejection of a specimen?
- hemolyzed
- clotted specimen
- wrong tube or container
- tube not labeled or mislabeled
- inadequate quantity
266
What are the 3 phases of laboratory testing?
- pre-analytical: specimen collection, transport and processing
- analytical: testing
- post-analytical: testing result transmission, interpretation, follow-up, retesting
267
What are pre-analytical errors?
- patient ID: tube not labeled, wrong person drawn from
- phlebotomy technique: venous access difficulties, vein collapse
- test collection procedures order of draw/ wrong tube
- specimen transport: timing, temperature
- specimen processing: clotting, mixing incorrectly
268
What are some analytical errors in the laboratory?
- test systems not calibrated
- results reported when control results out of range
- reagents stored inappropriately or used after expiration date
- contamination of cells or saline used in washing
269
What are post analytical errors?
- interpretation of results
- critical value not called
- transcription errors in reporting
- report sent to the wrong location
- report illegible
- report not sent
270
Describe the Coombs test
- also called antihuman globulin test (AHG)
- obtained from immunized nonhuman species to bind human globulin (IgG and complement
- two major blood group antibodies (IgM and IgG)
- IgM bind to corresponding antigen and directly agglutinate RBCs suspend in saline
- IgG will not bind agglutinate sensitized RBCs directly
— Coombs contain anti-IgG and this will binge RBCs sensitized with IgG antibodies allowing for agglutination
- some blood group antibodies have ability to bind complement to the RBC membrane
271
Describe AHG reagents
- polyspecific AHG
- IgG
- C3d component of complement
- other antibodies may be present (anti-C3b, anti-C4b, Anti-C4d)
- little if any activity against IgA and IgM heavy chains
- May contain antibody activity to kappa and lambda light chains on all immunoglobulins
272
What are the polyspecific AHG reagents?
- Rabbit polyclonal: contains anti-IgG and anti-C3d
- Rabbit/murine monoclonal blend: contains a blend of rabbit polyclonal antihuman IgG and anti-C3d is a murine monoclonal IgM antibody
273
What are the monospecific AHG reagents?
- Anti-IgG (rabbit polyclonal): contains anti-IgG with no anti complement activity
- Anti-IgG (gamma-clone AHG): murine monoclonal IgM antibody secreted by a hybridoma cell line
- Anti- C3d: main component of reagent is murine monoclonal antibody to C3d. Will cause agglutination of RBCs coated with C3d and/or C3b complement components
274
Describe monospecific AHG
- contain only one antibody specificity
—> IgG
—> C3b
—> C3d
275
Describe anti-IgG
- contains no anti-complement activity
- contains no anti-IgG specific for Fc fragment of IgG molecule
- if not labeled “gamma heavy chains specific, may contain anti-light chain specificity and react with cell sensitized with IgM and IgA as well.
276
Describe anti- complement
- reactive against designated complement components only
- no activity against human immunoglobulins
277
Describe preparation of AHG
- involves injecting human serum into animal
- Human IgG injected into a rabbit results in anti-IgG production (anti-complement)
278
Describe preparation of Polyspecific AHG
- can be made using polyclonal or monoclonal antibodies
- rabbits, goats, and sheep are common animals used
- immunize with IgG and C3
- hyperimmunized to produce high-titer, high avidity antibodies
- absorbed with A1, B and O cells
- purified
279
Describe preparation of monoclonal AHG reagents
- immunization of mice with purified human globulin
- spleen cells (lymphocytes as fused with myeloma cells
- results in hybridomas
- screened for specificity and affinity
- clones are propagated in tissue culture
- inoculated into mice -antibody collected in ascites
- no need for absorption
280
What antibodies are required in AHG?
- Anti-IgG
- nonagglutinating IgM
- rare IgA alloantibodies
- anti-complement
281
Describe Anti-IgG of AHG reagents
- antibody activity to nonagglutinating blood group antibodies
—> IgG1
—> IgG3
282
Describe anti-complement of AHG
- some antibodies fix complement to RBC after complexing of antibody with antigen
- can be detected by anti-complement activity in AHG
- not clinically significant: anti-Lea and anti P1
- clinically significant: anti-Jka
283
Describe AHG and DAT
- DAT = direct antiglobulin test
- detects in vivo sensitization of RBCs with IgG and/or complement
- C3 and C4 split into C3b and C4b, bind to RBC membrane
- further degradation of membrane bound C3b and C4b occurs by removal of C3c and C4c leaving C3d and C4d firmly attached
- degradation occurs in vitro with both warm and cold autoimmune hemolytic anemias if incubation is greater than 1 hour
284
What is the principle of AHG test?
- antibody molecules and complement components are globulins
- injecting an animal with human globulin stimulates the animal to produce antibody
- antihuman globulin reacts with human globulin molecules either bound or free in serum
- washed RBCs coated with human globulin are agglutinated with AHG
285
What are clinical conditions resulting in vivo coating in DAT?
- hemolytic disease of Newborn (HDN)
- hemolytic transfusion reaction (HTR)
- autoimmune and drug induced hemolytic anemias (AIHA)
286
Describe DAT panel
- test one drop of 3-5% suspension of washed RBCs with polyspecific AHG (anti-G and anti-C3d reagent)
- testing with monospecific AHG can help differentiage
—> Anti-IgG + and Anti-C3d + = WAHA (67%)
—> Anti-IgG + and Anti-C3d - = WAHA (30%)
—> Anti-IgG - and Anti-C3d + = CHD, PCH, WAHA (13%)
287
Describe evaluation of positive DAT results
- interpreting significance of positive DAT
—> patients diagnosis
—> drug therapy
—> recent transformation history
—> may occur without clinical manifestations pf immune mediate hemolysis
- DAT does not require incubation phase — Ag/Ab complexes in vivo
288
What does the AABB manual state about positive DAT results?
- results of serological tests are not diagnostic of their significance, they can only be assessed in relationship to the patients clinical condition
289
What are the following questions that should be asked before examining a positive DAT to determine further testing?
1. Is there evidence of in vivo hemolysis?
2. Has patient been transfused recently?
3. Does the patients serum contain unexpected antibodies?
4. Is the patient receiving any drugs?
5. Has patient received blood products containing ABO-incompatible plasma?
6. Is the patient receiving any anti-lymphocyte globulin or anti-thymocyte globulin?
290
What is the principle of IAT?
- performed to determine in vitro sensitization of red cells and used:
1. Detection of incomplete antibodies to potential donor RBCs (compatibility tests) or screening cells in serum
2. ID of antibody specificity using a panel of RBC’s with known antigens
3. Determination of RBC phenotype using know antisera (Kell typing or weak D testing)
4. Titration of incomplete antibodies
291
What are tasks that are performed while running an IAT?
1. Incubate RBCs with antisera
—> allows time for antibody molecule attachment to RBC antigen
2. perform at least 3 saline washings
—> removes free globulin molecules
3. Add anti-globulin (AHG) reagent
—> forms RBC agglutinates
4. Centrifuge
—> accelerates agglutination by bringing cells closer
5. Examine for agglutination
—> interprets test as positive or negative
6. Grade agglutination reactions
—> determines the strength of reaction
7. Add antibody coated RBCs to negative reactions
—> checks for neutralization of antisera by free globulin molecules (Coombs control cells are D-positive RBCs that are coated with anti-D
292
What are application types of IAT?
- antibody detection
- antibody identification
- antibody titration
- red cell phenotype
293
Describe Antibody detection IAT
Tests:
- comparability testing
- antibody screens
In vitro sensitization
- recipient antibody reacting with donor cells. Antibody reacting with screening cells
294
Describe antibody identification IAT
Tests:
- antibody panel
In vitro sensitization:
- antibody reacting with panel cells
295
Describe antibody titration IAT
Tests: Rh antibody titer
In vitro sensitization: antibody and selected Rh cells
296
Describe Red cell phenotype
Tests: RBC antigen detection (weak D, K, and Fy)
In vitro sensitization: specific antisera + RBCs to detect antigen
297
What are factors that affect the antiglobulin test?
- detects 150 to 500 IgG molecules per RBC
- Ratio of serum to cells
- reaction medium
- temperature
- incubation time
- washing of cells required for DAT and IAT
- saline
- addition of antihuman globulin
- centrifugation and resuspending cells for reading
298
Describe ratio of serum cells affect on antiblobuin test
- ratio of serum to cells increases sensitivity
- 40:1 ratio minimum
- 1 drop of serum and 1 drop of 4% of RBCs
299
Describe reaction medium affects on the antiglobulin test
- 2 drops of albumin — zeta potential
- 2 drops of LISS — enhances antibody uptake
- 2 drops of PEG — increase antibody update — removes water
—> anti-IgG is AHG of choice
300
Describe incubation time of antiglobulin test
- between 30 to 120 minutes
- with LISS - 15 minute
301
Describe washing of cells required for DAT or IAT
- minimum of three saline washings
- removes unbound globulins
- inadequate results in false-negative reactions \
- performed in as short a time as possible
- cell pellet should be completely resuspended before next wash
302
Describe saline of antiglobulin test
- fresh
- buffered to a pH f 7.2 to 7.4
- bacterial contamination can cause false positives
303
Describe addition of antihuman globulin of antiglobulin tests
- added immediately after washing cells to minimize the chance of antibodies eluting from cells and neutralizing the AHG reagent
- Add amount of recommended by manufacturer
304
Describe centrifugation of antiglobulin test
- resuspending cells for reading
- crucial step
- 500 to 100 RCF - higher RCF yields more sensitive results depending on how resuspended
- for 20 seconds
- weak false-positive if inadequately resuspended
- negative results is too vigorous resuspended
305
What could cause a false positive of an antiglobulin test?
- improper specimen (refrigerated, clotted specimen) may cause in vitro complement attachment
- autoagglutinable cells
- bacterial contamination of cells or saline used in washing
- cells with a positive DAT used for IAT
- dirty glassware
- over centrifugation and over reading
- polyagglutinable cells
- preservation-dependent antibody in LISS reagent
- contamination antibodies in AHG reagent
306
What causes false negatives in antiglobulin test?
- inadequate or improper washing of cells
- AHG reagent nonreactive - deterioration of neutralization
- AHG reagent not added
- serum not added in the IAT
- serum nonreactive because of deterioration of complement
- inadequate incubation conditions in the indirect test
- cells suspension too weak or too heavy
- undercentrifuged or overcentrifuged cells
- poor reading technique
307
Describe modified and automated AHG techniques
- LISS, PEG, Albumin
- Enzymes
- LIP (low ionic polybrene)
- microplates
- gel test
308
Describe the gel test of AHG
- RBCs are centrifuged through a gel contained in micro tube
- easy to ready
- can save to be reviewed
- gel acts as a trap
—> free unagglutinated RBCs form pellet in bottom
—> agglutinated RBCs trapped in gel
309
What are the advantages of saline test tubing?
- no additives
- reduced cost
- avoids reactivity with auto Abs
- ability to assess multiple phases of reactivity
310
What are disadvantages of saline-tube testing?
- long incubation
- least sensitive
- requires highly trained staff
- most procedural steps
- fewer method-dependent Abs detected
311
What are advantages of LISS-tube testing?
- reduced cost
- avoids reactivity with auto Abs
- shortest incubation time
- increased Ab uptake
- most common tube method
- ability to assess multiple phases of reactivity
312
What are the disadvantages of LISS tube testing?
- inability to be automated
- requires highly trained staff
- many highly production steps
- fewer method-dependent Abs detected
313
What are advantages of PEG tube testing?
- reduced cost
- decreased incubation time
- increased Ab update
- enhances Ab uptake
- most common tube method
- ability to assess multiple phases of reactivity (not 37C)
314
What are disadvantages of PEG tube testing ?
- requires highly trained staff
- many procedural steps
- detects more unwanted Abs
- inability to be automated
- fewer method-dependent Abs detected
315
What are the advantages of Gel testing?
- more sensitive DAT method
- no washing steps
- no need for check cells
- stable endpoints
- small test volume
- enhanced anti-D detection
- ability to be automated
316
What are disadvantages of Gel test>
- warm auto Abs enchanced
- mixed-cell agglutination with cold Abs
- increased costs
- increased need for additional instrumentation
- increased chances of detected unwanted Abs
317
What are the advantages of solid phase testing?
- no need from check cells
- stable endpoints
- small test volume
- enhanced anti-D
- increased sensitivity for all Abs
- ability to be automated
318
What are the disadvantages of solid phase testing?
- increased sensitivity for all Abs
- detects unwanted Abs
- warm auto Abs enhanced
- increased costs
- increased need for additional instrumentation
319
Polyspecific AHG sera contains what?
- antibodies to human IgG and C3d components of human complement
320
Monospecific AHG sera contain what?
- only one antibody specificity: either anit-IgG or antibody to anti-C3b-C3d
321
Describe solids phase technology
- used for performing antiglobulin tests
- can be fully automated
- if no specificity occurs, RBCs will settle to the bottom of the well.
322
Describe Gel test
- detects RBC antigen-antibody reactions by means of a chamber filled with polyarcylamide gel
- gel acts as a trap
- negative reactions appear as buttons in the bottom of tube
- positive reaction are fixed in gel
323
What is the significance of ABO?
- most important blood group systems
- causes major transfusion problems with Cell incompatibility
- the only blood group system that has antibodies to antigens that are ABSENT for the their RBCs
324
Describe forward grouping ABO testing
- using known sources of reagent antisera to detect antigens on a persons RBCs
—> Anti-A and Anti-B
- known antisera + unknown patient cells
325
Describe reverse grouping ABO testing
- using known sources of reagent RNC antigens to test for the person’s ABO group antibodies
—> Reagent Cells A1 and B
- known RBC’s + unknown patient serum
326
Describe the Grading results of ABO testing
- 4+ = one solid buttons/clear background
- 3+ = 2-3 large buttons/clear background
- 2+ = multiple smaller buttons
- 1+ = small clumps
- hemolysis: positive reaction-complement fixation
- negative = sea of red cells
327
Who discovered the first human blood group system?
Karl Landsteiner
328
What are the frequency of blood groups with O phenotype?
White = 45%
Black = 49%
Mexican = 56%
Asian = 43%
329
What are the frequency of blood groups for A1 phenotype?
White = 33%
Black = 19%
Mexican = 22%
Asian = 27%
330
What are the frequency of blood group with A2 phenotype?
White = 8%
Black = 8%
Mexican = 6%
Asian = Rare
331
What are the frequency of blood group with B phenotype?
White = 10%
Black = 19%
Mexican = 13%
Asian = 25%
332
What are the frequency of blood groups with A1B phenotype
White = 3%
Black = 3%
Mexican = 4%
Asian = 5%
333
What are the frequencies of blood group with A2B phenotype?
White = 1%
Black = 1%
Mexican = Rare
Asian = Rare
334
Describe ABO antibody production
- naturally occurring in misnomer
- sources of stimulation found in nature
—> Bacteria
—> seeds from plant
—> pollinating plants
—> foods
335
Describe ABO antibodies
- unique in that it does not require introduction of foreign red cells by pregnancy or transfusion
- Not present at birth
—> titers not detectable until 3-6 months
- Antibody production peaks at 5-10 years of age
- Declines with advanced age
—> after 65 have low titers that may be undetectable in reverse typing
336
Describe “other” blood groups `
- some blood groups other than ABO can occasionally cause the production of antibodies without transfusion or pregnancy
- these antibodies are usually in all patients that have the recessive antigen `
337
Describe testing for ABO
- easy to perform
- lacking the corresponding antigen serves as confirmation of the forward grouping results
- very rare (0.01% or random population) to see complete absence of anti-A and/or anti-B in normal healthy individuals unless of course the person has AB blood type
338
Describe ABO typing
- treacherous if wrong ABO group is given
—> severe if not fatal complications
- Forward and Reverse typing on all patients, not reciprocal relationship of antigens and antibody (discrepancy)
- O is universal donor
- AB is universal recipient
339
What is the relationship between ABO antibodies and IgM ?
- cold reacting
- bind complement (causing lysis)
- does not cross placenta
- group A and/or B individuals contain
—> Anti-B and/or Anti-A - IgM only (Majority)
—> Anti-B and/or Anti-A - IgM and IgG
—> Anti-B and/or Anti-A - IgM and IgA
—> Anti-B and/or Anti-A- IgM, IgG and IgA
- IgM is always predominant
340
What does Group O individuals contain?
- Anti-A
- Anti-B
- Anti-AB
341
Describe Anti-AB
- separate cross-reacting antibody that is a mixture of IgG and IgM or IgG, IgM and IgA
- crosses the placenta more frequently than anti-A or anti-B
- produced in response to exposure to A or B cells by transfusion or pregnancy
- knowing the amount of IgG anti-A, anti-B and anti-AB allows prediction of diagnosis of hemolytic disease of the newborn (HDN) caused by ABO incompatibility
342
Describe inheritance of ABO blood groups
- inherit one gene from ABO group from each parent
- Chromosome 9 has one position for the ABO group
- A or B refer to phenotypes
—> what is detectable by testing (EXPRESSED)
- AA, AO, BB. BO, OO denote genotypes-
—> what genes are on each chromosome (EXPRESSED AND NON EXPRESSED)
343
If the parent phenotype is A X A, what are the genotypes and offspring possibility?
- genotype =
—> AA X AA. O.S. = A (AA)
—> AA X AO. O.S. = A (AA X AO)
—> AO X AO. O.S. = A (AA X AO) or O(OO)
344
If the parent phenotype is B X B, what is the genotype and offspring possibility?
- gentype =
BB X BB. O.S. = B (BB)
BB X BO. O.S. = B (BB or BO)
BO X BO. O.S = B (BB or BO) or O (OO)
345
If the parent phentype is A X B, what is the genotype and Offspring possibility?
- genotype
AA X BB. O.S. = AB (AB)
AO X BB. O.S. = AB (AB) or B (BO)
AA X BO O.S. = AB (AB) or A (AO)
AO X BO. O.S. = AB (AB) or A(AO) or B(BO) or O (OO)
346
Describe formations of A, B, H, antigens
- A, B, and H antigens are not fully developed at birth
—> development begins at 6 weeks of fetal life
—> not fully developed until 2-4 years old
- ABO genes do not actually code for the production of ABH
- Code for enzyme (glycosyltransferases) that add sugars to basica precursor substances on the RBC
347
Describe the H gene
- inherited independently from ABO genes
- is the backbone of which other genes are expressed
- found on chromosome 19
- Known as FUT-I gene
- 99.9% of population has the H gene
- “h” amorph is very rare - known as Bombay phenotype.
- H substances must be formed for others sugars to be attached to the RBC (regardless t the inherited A or B gene)
348
Describe interaction of HH and ABO genes
- inheritance of at least one H genes cause production of enzyme (alpha-2-L-fucosyl-transferase) that transfers a sugar
- genotype HH or Hh is possible ( h is an amorph of H)
- gene is found on chromosome 19
349
Describe Bombay phenotype (OH)
- lacks normal expression of the ABH antigens because of the inheritance of the hh genotype
- does not elicit production of alpha-2-L-fusoslytransferase
- devoid of all ABO antigens
- types contain Anti-A, -B, -AB & -H
- they can only be transfused with another Bombay type
- would phenotype as O blood group
350
What is age is antbodies production is at its peak?
5-10 years, declines with age
351
Describe the A gene
- codes for production of alpha-3-N-acetylgalatosaminyltransferase
- elicits high concentrations of transferase than the B gene, leading to conversion of nearly all of the H antigen on the RNCs to an A antigen sites
352
Describe the B gene
- codes for production of alpha-2-D-galacttosylatransferase and attaches to D-galactose (Gal) sugar to the H substance previously placed on type 2 precursors
- this sugar is responsible for B specificity
353
Describe when both A and B genres are inherited
- B enzyme seems to compete more efficiently for the H substance than the A enzyme
354
Describe ABO antigens
- blood group is determined by the terminal sugars
- if neither the A or B gene is present “the O condition” the antigen is H
- in the presence of the B gene, the sugar galactose is added
- in the presence of the A gene, the sugar N-acetylgalactosmine is added
355
Describe formation of A, B, and H SOLUBLE antigens
- the presence of ABH soluble antigens can also be going in all Boyd secretions IF they inherit the Se gene
- those who inherit the SeSe or SeSe gene will secrete these soluble antigens in body fluids, IF they inherit the sese gene, they will not be secretors
356
Describe A1 and A2
- makes up 99% of all A groups (both)
- A1 reacts with both Anti-A and anti-A1 sera
- A2 only reacts with Anti-A1 sera
- 80% of all Group A (or AB) are A
- note: Anti-A1 sera is NOT the same as A1 cells
357
What are the two ways antigens present on RBCs surface of A1 and A2 are presented?
- group A1: A and A1 antigens
- group A2: A antigen only
358
What are additional weak A subgroups?
- A3 - demonstrate a mixed field pattern with Anti-A and most of Anti-B
- Ax - will not be agglutinated by anti-A reagent by will agglutinate with anti-AB reagent
359
Describe Weak B subgroups
- very rare, less frequent
- recognized by variations in the strength of reaction using anti-B and anti-A, B
- B3, Bx, Bm, and Bel
360
Describe
361
Describe ABO discrepancies
- unexpected reactions in the forward and reverse results of an ABO blood group typing
- the forward grouping does not correspond to the results of the reverse grouping
OR
- there is abnormal reactivity present (mixed field reaction)
362
Describe ABO discrepancies due to technical error?
- cell suspension too heavy or too light
- clerical errors
- missed hemolysis
- failure to add reagents
363
Describe ABO discrepancies due to other factors
- patients age
- diagnosis
- previous transfusion
- medication
- previous pregnancy
364
What are the groups of ABO discrepancy?
- Group 1
- Group 2
- Group 3
- Group 4
365
Describe group 1 of ABO discrepancy
- associated with unexpected reactions in the reverse grouping due to weakly reacting or missing antibodies
366
Describe group 2 of ABO discrepancy
- associated with unexpected reactions in the forward grouping due to weakly reacting or missing antigens
367
Describe Group 3 of ABO discrepancy
- associated with discrepancies between forward and reverse grouping caused by protein or plasma abnormalities resulting in a rouleaux formation
368
Describe Group 4 of ABO discrepancy
- Associated with discrepancies between forward and reverse grouping due to miscellaneous problems
369
What are causes of Group 1 ABO discrepancy?
- newborns: lack antibodies
- elderly: depressed antibodies
- leukemia
- immunosuppressive Drugs
- ABO subgroups
- bone marrow transplant
- plasma transfusion
370
How is a Group 1 ABO discrepancy resolved?
Goal: enhance weak or missing reaction
- 1. Incubate at room temperature for 15-20 minutes ad centrifuge
2. If no reaction, then incubate at 4C for 13-30 minutes and centrifuge
3. Always remember to use an auto and O cell control with reverse typing
371
What are some causes of Group 2 discrepancy?
- subgroups of A or B
- leukemia weakened A or B antigens
- Hodgkin disease
- acquired B phenomenon
372
How is a Group 2 discrepancy resolved?
GOAL: enchant weak or missing reaction
1. Incubate at room temperature for 15-30 minutes and centrifuge
2. If there is still no reaction, then incubate at 4C for 15-30 minutes and centrifuge
3. RBCs can treated with enzymess and retested with antisera. In the case of suspected “Acquired B”, treat RBCs with acetic anhydride reacertylates and retest
373
What are the causes for Group 3 ABO discrepancy?
- elevated levels of globulin-multiple myeloma, Waldenstroms macroglobinemia
- elevated levels of fibrinogen
- plasma expanders such as dextran and polyvinylpyrrolidone
- Wharton’s jelly in cord blood samples
374
How is a Group 3 ABO discrepancy resolved?
GOAL: remove interfering substance
1. Perform a saline dilution or saline replacement to free cells
2. Wash cells 6-8 times with saline to alleviate the rouleaux due to Wharton’s Jelly
375
What are causes of Group 4 ABO discrepancy?
- cold reactive autoantibodies
- circulating RBCs of more than one ABO group due to transfusion or transplant
- unexpected ABO isoagglutinins
- unexpected non-ABO alloantibodies
376
How is Group 4 ABO discrepancy resolved?
GOAL: eliminate spontaneous agglutination
1. Forward: incubate at 37C for a short time and then wash at 37C three times and retype
2. Reverse: warm to 37C mix and test at 37C
* if forward doesn’t resolve with 1, then treat pateitns cells with DTT
* If reverse doesn’t resolve with 2, try an auto absorption form the serum and then use the absorbed serum to repeat the typing at room temperature
377
Describe Ulex europaeus
- closely parallels the reactions of human anti-H
- both antisera agglurinate RBCs of group O and A2 and reach very weakly or not at all with groups A1 and A1B
- Group B cells give reactions of variable strength
378
Describe the effects of disease on the expression of ABH antigens and antibodies
- various disease states seem to alter RBC antigens, resulting in weaker reactions or additional acquired pseudo antigens
- Leukemia, Chromosome 9 translocations and hemolytic disease that induce stress hematopoiesis have shown to depress antigen strength
- Hodgkin disease has been reported to weaken or depress ABH RBC antigens, resulting in variable reactions during forward grouping similar to follow the course of the disease.
- antigen strength increases again as the patient enters remission
- a lack or detectable ABO antigens can occur in patients with carcinomas of the stomach or pancreas
379
Describe the quantitative difference of subgroups A1 and A2
- decreased number of antigen sites
- decreased amount of transferase enzyme
- decreased amount of branching
380
Describe the Qualitative differences A1 and A2 subgroups
- differences in the precursor oligosaccharide chains
- subtle differences in transferase enzymes
- formation of anti-A1, in a percentage of some subgroups
381
What is the immunodominant sugar responsible for H specificity?
L-fucose
382
What is the immunodominant sugar for A specificity?
- N-acetylgalactosamine
383
What is the immunodominant sugar responsible for B specificity?
D-galactose
384
What does the Se gene code for?
The production of L-fucosyltransferase
