Blood Bank Flashcards
Tranfusion requirements for Sickle Cell patients
Matching for Rh and Kell ahead of time is associated with an a priori risk reduction from 30% rate of reactions to 10% rate of reactions.
So, always match a Sickle patient for Rh and Kell
Pasmin inhibitors
Prevent the generation of plasmin from plasminogen
Aminocaproate (Amicar) and Tranexamic acid (TXA)
Reducing the risk of HLA refractoriness
Leukoreduced platelets (filtration or UV radiation) are associated with a reduced risk of HLA/alloantibody-mediated platelet refractoriness development
[image: https://www.nejm.org/doi/10.1056/NEJM199712253372601?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0www.ncbi.nlm.nih.gov]
Reducing the risk of fever from platelet transfusions
Leukoreduction!
The leukocytes that make it into the product will produce cytokines otherwise.
Massive transfusion-associated complications
- Hypocalcemia (citrate-mediated chelation)
- Hyperkalemia (hemolyzed red cells)
- Hypothermia (blood products are cold!)
- Coagulopathy (dilutional)
Two mechanisms of TRALI
-
Donor antibody hypothesis
- Reaction of donor HLA or donor antibodies against recipient antigens causes cytokine release and leukocyte aggregation in the lungs.
-
Two-event hypothesis
- 1st event: Something happens to stimulate recipient neutrophils to accumulate in the lungs
- 2nd event: Transfusion of blood products that accumulate lipids that activate neutrophils
Platelet transfusion in bleeding patients on antiplatelets
A single dose of platelets is reasonable in patients on NON-ASPIRIN antiplatelets.
If the patient is on aspirin, more platelets won’t help. They will be immediately poisoned.
ddAVP and anti-fibrinolytics (aminocaproate, tranexamic acid) should be considered.
Platelet threshold: Prevent spontaneous bleeding
10,000
Platelet threshold: Prevent bleeding in a percutaneous or transbronchial biopsy
50,000
Platelet threshold: Prevent bleeding in a lumbar puncture
30,000
Platelet threshold: Prevent bleeding during epidural injection or catheter placement/removal
80,000
Platelet threshold: Treat active bleeding
50,000
Platelet threshold: Prevent bleeding during surgery
50,000
Note, this is the same as active bleeding, probably for a reason.
Platelet threshold: Prevent bleeding during central line placement
20,000
Platelet threshold: Prevent bleeding during neurosurgery
100,000
Biopsies in patients on antiplatelets
Biopsies can be performed safely on patients taking anti-platelets. This is not an indication for transfusion.
Treating bleeding in a patient with uremic platelet dysfunction
Transfusion here is obviously no good.
Instead, options include dialysis, ddAVP, and cryoprecipitate.
Contraindications for platelet transfusion
TTP, DIC, and/or newly diagnosed HIT are strong relative contraindications for platelet transfusion.
In TTP and HIT you can consider trying transfusion in cases of life threatening bleeding.
One unit of blood increases the Hgb and Hct by __.
One unit of platelets increases the platelet count by __.
One unit of blood increases the Hgb by 1.0 g/dL and Hct by 3%.
One unit of platelets increases the platelet count by 30,000 - 60,000 / uL.
These numbers are based on a 70 kg individual and will be lower in an individual greater than 70 kg.
Survival of transfused platelets
Generally 3-5 days
May be reduced by medications, anti-HLA antibodies, hypersplenism, DIC, and infection
HLA antibody-mediated refractoriness is unlikely if . . .
. . . there is an increase in platelet count > 15,000/uL in the 10 minutes to 1 hour following transfusion.
Indications for plasma transfusion
Correcting coagulopathy associated with multiple factor deficiencies or warfarin reversal in a patient with 1) clinically significant bleeding or 2) facing hemostatic challenge.
However, in immediately life threatening warfarin-induced bleeding, a 4-factor Prothrombin complex conentrate (Kcentra) is a superior first-line choice.
One unit of plasma contains ____
One unit of plasma contains 200-250 mL of plasma
For which conditions are specific clotting factor concentrates indicated?
- Hemophilia A (Factor VIII def)
- Hemophilia B (Factor IX def)
- von Willebrand’s (vWF def)
- Congenital Antithrombin-III deficiency
Crypoprecipitate contents
- Factor VIII
- Factor XIII
- vWF
- Fibrinogen
- Fibronectin
Indications for cryoprecipitate
- Hypofibeinogenemia (< 100 mg/dL)
- Platelet dysfunction or uremia that is refractory to ddAVP and dialysis or when dialysis is unavailable
Standard dose of cryoprecipitate to correect for hypofibrinogenemia at BIDMC
10 units
In an average sized adult, this should increase the circulating fibrinogen concentration by 50-100 mg/dL
All cellular blood products at BIDMC are . . .
. . . leukoreduced at the time of collection
CMV-reduced risk cellular products
Either negative for antibodies to CMV or leukoreduced at collection. These methods are equivalent in reducing risk of CMV infection.
Indications:
- Hematopoietic stem cell transplant
- Solid organ transplant in the severely immunocompromised
- Premature or low birth weight infants
Transfusion-associated GVHD
A rare, but fatal occurrence. May be prevented by irradiating cellular components to inactive lymphocytes, in patients who are at elevated risk.
Indications for irradiation:
- Patients who have undergone HSCT
- Patients receiving severely immunosuppressive chemotherapy (leukemia, lymphoma, MDS, etc)
- Patients on antimetabolite-type chemotherapy for any reason
- Recipients of HLA-matched platelet products
- Patients with congenital T-cell immunodeficiencies
- Neonates
What to do if you suspect an acute hemolytic transfusion reaction
Stop the transfusion
Send the entire blood sample with line to the blood bank for analysis, including evidence of hemoglobinemia and a repeat direct antibody test (direct Coombs).
Treat with fluids and diuresis with monitoring of urine output and blood component therapy if there is evidence of DIC.
Proposed etiology of febrile nonhemolytic tranfusion reaction vs TRALI
Febrile nonhemolytic: Presence of donor white blood cells, anti-white blood cell antibodies, or cytokines within a tranfused product.
TRALI: Presence of donor anti-HLA or anti-granulocyte antibodies.
If someone has been tranfused in the last 3 months, their phenotype. . .
. . . may not be their own.
They may have a preponderance of donor cells which affect their response to novel tranfusions
When IDing antibodies with crossouts, when can you rule out an antigen as being the target?
When there is no reaction with a cell homozygous for the antigen.
Heterozygotes do NOT count. This is considered a “half dose.”
“Type, screen, and crossmatch”
“Cold reactive” antibodies
Typically react in initial spin (IS) +/- 37 oC
Generally clinically insignificant IgM with carbohydrate antigens (ABO is the exception and is VERY important): ABO, Le, I, i, P, M, and N
“Warm” reactive antibodies
Typically react in anti-human globulin phase (AHG) +/- 37 oC
Most commonly significant IgG antibodies with protein antigens.
Common antigens: Rh, K, Jk, Fy
How do we promote agglutination and antibody crosslinking in the lab for ID and diagnostic purposes?
- Centrifugation
- LISS reaction buffer (low ionic saline solution, less ions to interfere with interaction) or other potentiator (Albumin, PEG)
- Acidic pH (~7.0)
Zeta potential
The mechanical potential of the repulsive force between two red cell membranes, which are negatively charged due to the abundance of sialate in the red cell membrane.
On average keeps red cells 14 nm apart
Which, go figure, is exactly the diameter of an IgG
Why does IgG have a harder time than IgM agglutinating red cells?
The IgG’s diameter is 14 nm, exactly equivalent to the Zeta potential that keeps red cells apart.
An IgM’s diameter is 30 nm, making the Zeta potential less of an issue.
So, even though IgG has higher affinity, IgM is often the better agglutinator.
Phases of a Tube Coomb’s
First direct, then indirect
The first antibody is in the patient serum, the second is the anti-human globulin.
The first read is after the first arrow (direct), the second read is after the second arrow (indirect)
0-4+ scale for tube agglutination
Note that 0+ and 1+ have a “turbid background”, while 2+ does not.
This is a key differentiator of 1+ and 2+.
0-4+ scale for well agglutination
Use of proteolytic enzymes to remove or reveal antigens on a red cell
Papain and ficin are the most used.
These enzymes will basically cleave “tall” proteins, removing them as antigens in order to reveal the “short” proteins that were previously hidden under the foliage of other proteins.
“Adsorption” in blood banking
A way to remove antbibodies from the sample by incubating with antigen-positive control red cells.
Types include autoadsorption and alloadsorption.
Autoadsorption
Useful in scenarios where a patient’s autoantibody may be masking the presence of another antibody; let’s say anti-K.
Notably, the patient’s own red cells will be able to adsorb these autoantibodies, but will not have the K antigen and so cannot adsorb the anti-K. Thus, adding the patient’s own red cells will selectively sequester nonspecific autoantibodies.
Then, you simply spin down and remove patient RBCs and collect the superatant, which will be serum depleted for autoantibody but still containing the anti-K.
Alloadsorption
Useful in the situation where a patient has multiple antibodies.
A stock RBC containing certain known positive antigens for which the patient has known antibodies can help deplete these known antibodies.
Then, by spinning these cells down and collecting the supernatant, we obtain serum depleted of known antibodies but still containing unknown antibodies.
Alternatively, the collected cells may be separated from the serum and eluted to isolate the bound antibodies.
Eluting antibodies from a red cell
Glycine (most effective)
Temperature manipulation
DTT treatment
Dithiothreitol, aka Cleland’s reagent. Can be used to destroy certain proteins.
On RBCs: K, Lu, Do, Yt, and LW antigens are destroyed.
In serum: deactivates IgM.
Beta-mercaptoethanol treatment
Deactivates IgM in serum samples
Chloroquine treatment
Strips IgG from DAT+ RBCs
Removes Bg antigens from RBCs.
FDA requirements for an antibody screen
- Patient serum must be compared against at least 2 type O donor controls
- Must be read at AHG (IS is optional, but usually done)
- Must cover these antigens:
- D, C, c, E, Fya, Fyb, Jka, Jkb, K, k, Lea, Leb, M, N, P1, S, s
- Note: Lea, Leb, M, N, P1 are usually insiginificant clinically, but are required
Any RhD-negative pre-menopausal female must get. . .
. . . D-negative blood, unless there is an emergency with no other option.
RhD matching in RhD+ post-menopausal females
Treated the same as males.
Generally should be given RhD negative, but RhD positive may be given as long as they do not have anti-D.
Only about 22% of these patients will form an anti-D.
Most common etiologies of an incompatible crossmatch in the setting of a pan-negative screen
-
IS-phase positivity:
- ABO incompatible donor RBCs
- Presence of Anti-A1
- Presence of cold antibodies (IgM)
-
AHG-phase positivity:
- Warm antibody (IgG) against a low-frequency antigen
- False negative antibody screen
When can we do electronic crossmatch?
- When the antibody screen is negative
- Patients have been ABO and Rh typed twice, on two independent blood draws
- When the system for electronic crossmatch has been validated/liscensed on site.
Cryoprecipitate matching
Cryoprecipitate is the only blood product that requires NO ABO matching
Whole blood tranfusions must be ___, while components must be ___.
Whole blood tranfusions must be ABO identical, while components must be ABO compatible.
This is because whole blood contains both the antigens and the antibodies in the plasma and cellular components.
Crossmatch autocontrol positivity interpretation
If the autocontrol (patient’s plasma plus patient’s cells) is positive, it means that the patient has antibodies against their own cells. This could mean one of a few things:
- Warm autoantibodies
- Cold autoantibodies
- Antibody against recently transfused RBCs
- Passive transmitted alloantibodies (IVIG, plasma transfusion, Rhogam)
Lectins in blood banking
Plant-derived (or salivary!) lectins can be used similarly to antibodies to crosslink red cells on the basis of their carbohydrate antigen expression.
Remember, lectins are carbohydrate-binding proteins.
Especially useful in the case of polyagglutination.
ABO-related systems
- ABO/H system
- Lewis system
- I system
- P system
All are enzyme enhanced systems
H antigen
Fucose attached to the terminal galactose of on a type 1 or 2 chain. Created by different mechanisms on type 1 and type 2 chains.
On a type 1 chain, it is created by the action of the Se gene, aka fucosyltransferase 2 (FUT2). This is known as the H2 antigen and is present in 80% of people.
On a type 2 chain, it is created by the action of the H gene, aka fucosyltransferase 1 (FUT1). This is known as the H1 antigen and is present in 99.9% of people.
The H antigen is the “base” for the A or B antigen.
A and B blood groups
A gene product: GalNAc transferase. Adds a terminal GalNAc to the galactose in a type 1 or 2 chain.
B gene product: Gal transferase. Adds a terminal Gal to the galactose in a type 1 or 2 chain.
When we add A or B, we effectively “lose” the H antigen on those chains. However, not every chain is modified by the A and B enzymes, so we all retain some form of H antigen.
Locus of the A, B, O coding genes
Located on chr 9
Relative amount of H antigen among different ABO types
From most to least:
O > A2 > B > A2B > A1 > A1B
Antibodies in the ABO system
Type A and B: IgM antibodies against the other blood group antigen.
Type O: IgG (and some IgM) antibodies against A and B, as well as a combined IgG anti-A,B antibody which recognizes both antigens.
Blood group O
- Carry the H antigen, no A or B
- Ulex europaeus lectin can interact with the H antigen to identify type O blood
- Carry anti-A, anti-B, and anti-A,B
-
Most common form of HDFN, but more mild than RhD
- Group O mom, Group A or B baby
- Transfer of IgG anti-A, anti-B, anti-A,B via the placenta
- Hemolysis
How common different ABO blood groups are
From most common to least common:
O > A > B > AB
Group A subtypes
Divided into A1 and A2
Bloodgroup A1 has 5x more A antigen on its surface than A2. Conversely, A2 has more H antigen than A1.
Monoclonal anti-A antibodies cannot distinguish the two, but Dolichos biflorus lectin binds selectively to A1.
Acquired B phenotype
- Occurs when A1 red cells come into contact with enteric gram negative organisms
- May occur in colon cancer, GI obstruction, or gram negative sepsis
- Due to deacetylation of GalNAc to produce GalN
- Manifests as a:
- strong positivity for A antigen,
- a weak positivity for B antigen,
- and presence of only an anti-B antibody (with complete negativity for Anti-A)
Bombay phenotype
- Also called the Oh phenotype
- Homozygous loss of both Se and H, and thus negative for all H antigen on both type 1 and type 2 chains
- Strong anti-A, anti-B, and anti-H
- Require transfusion from other Bombay phenotype individuals (H-negative)
Para-Bombay phenotype
- Homozygous loss of H, but have at least one copy of Se
- Phenotypes: Ah, Bh, ABh
- Also carry anti-H antibody and therefore require tranfusion from another Bombay or Para-Bombay phenotype donor (H-negative).
Lewis system
Le gene, aka fucosyltranferase 3 (FUT3)
Only acts on type 1 chains and adds fucose to the subterminal GalNAc (instead of the terminal Gal).
Le in the absence of Se gives the phenotype Lea, which has only the Le fucose. In this case it is impossible to make Leb.
Le in the presence of Se gives the phenotype Leb, which has two fucoses. However, there will also be a small amount of Lea.
Net phenotypes are Le(a-b+), Le(a+b-), and Le(a-b-). Only Le(a-b-) make IgM antibodies, but these are generally clinically insignificant.
Rosette test
A rule in/rule out test for fetal cells in maternal circulation that is done prior to the KB
Principal: RhD- maternal RBCs and RhD+ fetal cells.
Process: Incubate 3-5% RBCs from maternal blood at 37oC with Rhogam (IgG anti-D). Wash, then add “indicator cells” (ficin-treated R2R2 cells) that bind the antibody-coated infant RBCs. Readout: Look under microscopy. A certain number of RBCs per HPF is designated a positive test, indicating a minimum of ~30 mL of fetal blood in maternal circulation. This indicates a KB test and additional Rhogam administration.
KB test
Test to determine the amount of fetal blood in maternal circulation in a RhD- mother / RhD+ fetus situation with concern for HDFN. Used to determine the appropriate dose of Rhogam.
Principal: Maternal cells have HbA1, fetal cells have HbF
Process: Treat RBCs from maternal blood with citrate buffer pH 3.3. This will dissolve HbA1, but HbF will remain intact. Shepard’s stain is then applied to highlight the intact Hb as bright pink.
Readout: Look under microscopy. Fetal cells will appear bright pink while maternal cells will appear like pale “ghost cells.” Caveat: Adults do have some small portion of fetal Hb, so there is a background. You must calculate the % of fetal blood mathematically.
Rhogam dosing: 1% HbF = 50 mL of fetal blood in maternal circulation. 300 micrograms Rhogam prevents sensitization of up to 15 mL of RhD+ red cells.
Minimum Hb for a donor to be able to donate
Male: 13 g/dL
Female: 12.5 g/dL
Autologous (either gender): 11 g/dL
How often can one person donate blood?
1 unit of RBCs every 8 weeks for an average weight person
2 units of RBCs every 16 weeks for a larger person
How often can one person donate platelets?
2 days per week (rolling 7 day period), at least 2 days apart
Maximum of 24 times per year (rolling 12 month period)
Temperature and blood donation
If you’re temperature is over 99.5, you can’t donate blood components.
“Good health” requirement
To donate blood, you must “appear to be in good health”
This is subjective and determined by whoever is overseeing the donation.
But if someone walks in on an oxygen tank, they probably shouldn’t be giving blood.
Reasons for a 3 month deferral of blood component donation
- Blood transfusion
- Solid organ transplant
- Accidental needle stab
- Transactional sex
- Graft (skin, bone)
- Tattoo at an uncertified location
- Ear or body piercing at an uncertified location
- Male donor who had sex with another male they do not know the health status of
- Treatment for syphilis or gonorrhea
Pregnant patients who want to donate blood
To others: Defer until 6 weeks after delivery
Autologous: Left to the discretion of the blood bank manager. For some patients with high risk for PPH and rare blood type, this may be reasonable.
What diseases do we test donated bloof for, and how?
- Babesia: NAT
- HIV-1, -2: Antibody, NAT
- HBV: NAT, HBsAg, anti-HBc
- HCV: Antibody, NAT
- WNV: NAT
- HTLV-1, -2: Antibody
- Syphilis: Antibody
- T. cruzi: Antibody
What things don’t we test donated blood for that we are still worried about
- Zika (we used to, but do not test anymore)
- CMV (leukoreduction usually takes care of this, and so many patients are positive that it would be prohibitive)
- Anaplasma
- Ehrlichia
- Lyme
- Ricketsia
- Dengue (no good screening)
- Chikungunya fever (no good screening)
- Malaria (no good screening – we do try to rule out via questionaire and travel history)
Medications that buy you an “indefinite” deferral from ever donating blood
- Etretinate (anti-psoriasis medication, 2nd generation retinoid – mostly removed from US market by now, high risk of birth defects)
- Antiretroviral therapy (ART)
Dental procedures and blood donation
“Unexpected” root canal buys you a deferral for 72 hours
“Expected” root canals are not indications for deferral (presumably because it was low-risk and the patient would have had preoperative antibiotics)
Vaccines that buy you a deferral from blood donation
-
4 week group -- the live attenuated strains (except intranasal infulenza):
- Varicella
- Rubella
- MMR (because of Rubella)
-
2 week group – viral vaccines with a strong response:
- Oral polio (Sabin)
- Oral thyphoid
- Measles
- Mumps
- Yellow fever
-
No deferral:
- Everything else (all bacterial vaccines, all other viral vaccines not listed above – including the injectable Polio vaccine aka Salk vaccine)
- Intranasal influenza, despite being live attenuated, also falls into this group
Reactions that someone might have to blood donation
- Vasovagal syncope
- Hematoma / compartment syndrome
-
Hypocalcemia
- Give this person tums
- Iron deficiency anemia (chronic donation)
I system
I antigen (adults), i antigen (babies). Related to “chain complexity” (i less complex, I more complex)
ABO-related, found on type II chains
Autoimmune associations are important here!
- Auto-anti-I is associated with Mycoplasma pneumonia and is the target of cold agglutinin hemolytic anemia.
- Auto-anti-i is associated with infectious mononucleosis in younger children
Paroxysmal cold hemoglobinuria (PCH)
-
Biphasic IgG that reacts against P antigens on RBCs
- known as the “Donath-Landsteiner biphasic hemolysin”
- Biphasic means binds at cold temperatures and fixes complement and dissociates at warm temperatures leaving complement behind
- Therefore, DAT with anti-IgG will be negative, but anti-C3b will be positive
- Etiologies:
- Post-infectious following viral infections in children
- Sometimes seen in syphilis
- Smear findings: Agglutination, hemophagocytosis, rosetting around phagocytes
- Treatment: Methylprednisone pulse, possibly rituximab if this fails. Dialysis may be indicated if renal injury is great.
Rh system
- Enzyme enhanced system
- Two genes: RHD gene and RHCE gene
- Broken into haploytpes:
- If D ⇒ R, if d ⇒ r
- Letter ⇒ CE
- R0 or r ⇒ ce
- 1 or ‘ ⇒ Ce
- 2 or ‘’ = cE
- Only four are common: R1, R2, R0, and r
- anti-Rh antibodies are exposure-requiring warm IgGs
- Major causes of HDFN
- anti-D is most common
- anti-c gives the most severe HDFN
- There are 50 total Rh antigens, however most others are rare of not clinically significant
Kidd system
-
Jka and Jkb
- Jka is more common
- Enzyme enhanced system
-
Exposure-requiring warm IgG, often with some IgM component
- 50% fix complement
- Often react negatively against heterozygous Kidd antigen, but very strongly against homozygous Kidd antigen
-
Variable expression of host Kidd antigen with time
- For this reason, often cause significant delayed hemolytic transfusion reactions
- May cause mild HDFN, but never severe
All patients with benign constitutional neutropenia have which blood group?
Duffy null (neither A nor B)
