Whats New in Blood Banking

Terry Kotrla, MS, MT(ASCP)BB Adjunct Professor ACC March 2013

Objectives

State weight requirements for different types of donors. State 3 infectious diseases and the methodology used to detect in blood donors. State 3 testing techniques used for type, screen and antibody identification. Describe 3 types of artificial platelet and red blood cell components . Describe the advantage for having a massive transfusion protocol.

Donors

Physical exam no real changes Weight requirement depends on amount removed and size of bag
Double red cells 130 males 150 females 500 mL bag 123 lbs. 450 mL bag or apheresis platelets still 110 lbs.

Testing Donor Blood

Many blood collection centers now send blood out for testing. Want safest blood possible. Requires very sensitive and specific tests. Molecular and chemiluminescent techniques are very expensive.

Testing of Donor Blood Formats

Chemiluminescent Immunoassay Enzyme Immunoassay (EIA) Immunofluorescent assay (IFA) Nucleic Acid Testing (NAT)
Polymerase chair reaction (PCR) Transcription Mediated Amplification (TMA)

Western Blot Rapid Immunoassay (kit tests such as OraSure)

Infectious Agents Tested For

Hepatitis B NAT pool up to 24 donors Hepatitis C NAT - pool up to 24 donors HIV 1 NAT pool up to 24 donors HIV 1/2 - antibody test - EIA HTLV Types I & II antibody test Trypanosoma cruzi - EIA West Nile Virus NAT individual Syphilis RPR riskier donor CMV antibody test - optional - EIA

Type, Screen, Crossmatch

Three methods for performing testing:

Traditional tube method. Solid phase Gel

Most sites that use solid phase and gel use tube testing for problem solving.

Tube Testing
Simple, versatile and reliable. Disadvantage is lack of an objective endpoint which severely hinders attempt to automate.

Solid Phase Antibody Screen

RBCs or membranes are immobilized to microplate wells to detect antibody-antigen interaction. Coat wells with intact RBCs or membranes Add patient serum or plasma and LISS Incubate at 37C, antibody, if present, will attach. Wash free of serum or plasma proteins Add IgG coated RBCs Centrifuge, forces indicator cell to contact immobilized reagent RBCs. Positive tests show adherence of indicator cells to well.

Solid Phase Adherence

Test

1 Antibody screen Test 2 Antibody detection top, ABO grouping bottom

Solid Phase Results

Solid Phase Adherence

Solid Phase
Endpoint is red cell adherence, easily interpreted visually or spectrophotometrically. Computer interface permits interpretation and recording of results. Easily automated. Immucor Galileo Echo or Capture-R

Gel Testing
Based on principle of controlled centrifugation of RBCs through a dextran-acrylamide gel. Strip or card of several microtubes of reactants allows for performance of several tests simultaneously. Can be used for detection of direct agglutination (ABO, Rh, RBC phenotyping)and for IAT and DAT. RBCs allowed to interact with antibodies in chambers at TOP of column. Centrifuged to force RBCs into column to separate agglutinated from nonagglutinated.

Testing Techniques - Gel

Nonagglutinated

cells pass freely through gel and pellet at bottom of microtube. Agglutinated cells are to large to enter the gel matrix and remain at top of column. A= 4+ thru E=negative

Ortho MTS and ABO/D Typing

Testing Techniques - Gel

Microliters

of plasma added, CANNOT USE SERUM. Incubate, spin and read, NO washing or tube shaking. Sesnitivity of 98% compared to LISS tube method. Cards can be saved for peer review or documented with photo. Simplifies cross training, improves productivity and workflow efficiency. Ortho MTS and automated Provue

Antigen Genotyping
Antigen testing coming into 21st century with the advent of molecular genotyping. Most blood group antigens inherited in straightforward Mendelian fashion. Availability of single-nucleotide polymorphism (SNP) genotyping, the blood bank is poised to become one of the primary laboratory disciplines to benefit from the PCR-technology revolution. Many of the approximately 270 serologically determined RBC antigens are related to single nucleotide polymorphisms (SNPs) which lead to a single amino acid difference in RBC antigens.

Antigen Genotyping
ABO more complicated Multiple alleles may encode same phenotype Molecular events other than SNPs in the antigen coding region of the gene may also give rise to blood group antigens, or affect antigen expression.

Antigen Genotyping
High-throughput SNP genotyping platforms enables rapid, cost-effective screening for multiple clinically significant blood groups in single assay. Can now consider individualized transfusion treatment, matching donors with patients at multiple blood group loci. Holds promise for reducing or potentially eliminating alloimmunization.

Antigen Genotyping The future is NOW!

Antigen Genotyping
Chronically transfused patients or patients with AIHA can be antigen typed. Prenatal testing

Type father for antigens mother has antibodies to, if homozygous fetus will be affected. Maternal weak D determination. Fetal D determination.

Massive Transfusion Protocols

Resuscitation of patients with massive hemorrage has advanced from reactive, supportive treatment to standardized massive transfusion protocols (MTP). Damage control resuscitation in MTP

Allow more permissive hypotension. Reduce large volume IV fluid therapy. Transfuse preemptively using balanced ratio of plasma, platelet and RBCs.

Massive Transfusion
Massive hemorrhage in trauma is a major cause of morbidity and responsible for 50% of deaths within 24 hours of injury and 80% of intraoperative trauma mortalities. Massive transfusion greater than 10 units of RBCs within 24 hours. Does not address patients who would benefit from blood component therapy.

Massive Transfusion Protocols

When blood products are transfused early overall number of blood products needed in first 24 hours is decreased. With fewer units of exposure use of an MTP reduced risk of organ failure and post injury complications.

Massive Transfusion Protocols

MTPs vary as to ratio of RBCs:FFP:PLT

6:6:1 6:4:1 5:5:1

Perform lab testing to evaluate. Treatment does not totally rely on lab results, additional MTP packages are requested for patients who continue to hemorrhage and other blood products may be requested (eg, cryo or pharmaceuticals)

Massive Transfusion Complications

Blood volume replacement

inadequate or excessive, aim at Hgb of 10g/dL. Should be based on physiologic needs based on oxygen demand.

Thrombocytopenia inevitable Coagulation factor depletion O2 affinity changes stored blood high Hypocalcemia Hyperkalemia

Artificial Blood

The attempt to develop a viable blood substitute spans more than 7 decades. Focus on ability of RBCs to carry oxygen. Artificial oxygen carriers (AOCs) are synthetic solutions with the ability to bind, transport and deliver oxygen. Most products in advanced-phase clinical trials are derivatives of hemoglobin-based oxygen carriers (HBOCs). To date no substitute has been approved by FDA.

Artificial Blood

Two viable categories

The two have dramatically different structures. Both work through passive diffusion, gasses tendency to move from areas of greater concentration to less until equilibrium is reached, O2 moves from blood to tissues.

Hemoglobin based oxygen carriers (HBOCs) Perfleurocarbons (PFCs)

Artificial Blood -HBOC

Artificial Blood -HBOC

Uses purified human, bovine or recombinant hemoglobin as raw material. Purified hemoglobin is either altered chemically or microencapsulated. Modified in 3 different ways

Cross-linking with O2 carrying Hgb derivative Polymerizing by binding Hgb molecules to each other Conjugating by bonding to a polymer

Artificial Blood -HBOC

Float in plasma, pick up oxygen from lungs deliver to capillaries. Molecules are much smaller than RBCs so can fit into spaces RBCs cannot, swollen tissues or abnormal vessels around tumors. Stay in circulation for 1 day.

Artificial Blood - PFCs

Liquid fluorinated hydrocarbon compounds capable of carrying dissolved oxygen and delivering oxygen under physiologic conditions. Extremely small and can fit into spaces that are inaccessible to RBCs. Extremely good at carrying dissolved gasses.

Artificial Blood - PFCs

Require emulsification as they do not readily mix in aqueous systems such as blood, they are oily and slippery. Emulsifiers eventually break down as they circulate from the blood. Liver and kidneys remove them from the blood. Lungs exhale the PFCs the way they would carbon dioxide.

Artificial Blood
Still working on it. Next generation may look more like red cells and carry enzymes and antioxidants.

Artificial Platelets
Made from tiny structures known as hydrogels. Circulate until activated by clotting process. Once activated, change shape, converting to a thin film to seal wound.

Synthetic Platelets
Polymeric template core upon which layers of proteins and polyelectrolytes are deposited, layered and crosslinked to create stable platelet shaped particle. Particle coated with proteins found on activated platelets. Perform platelet function but can also be used to carry imaging agents to ID damaged blood vessels or deliver drugs to dissolve clots.

Artificial Platelets

Synthetic Platelets

Major use in military.

The artificial platelets can be freeze-dried and could be put in an injector device the size of a smartphone. Soldiers wounded on battlefield could device to help control bleeding and stabilize the injury while waiting for clinical treatment. Soldier extends needle and injects in abdomen. Circulate inactive until initiation of clotting is activated.

Summary
The field of transfusion medicine changes constantly. It is YOUR responsibility to keep up with current practices and gain the knowledge to do your job well.

http://www.smartbrief.com/ http://www.mlo-online.com/ http://laboratorian.advanceweb.com/

This makes your job fun!