Wk6.. Ch 1: write questions please, RBC & PLT preservation

In order to have a successful transfusion, we had to make sure to have these:

1. there are 5

A nontoxic anticoagulant
→ A safe chemical to prevent blood from clotting.

Appropriate devices to perform the transfusion from donor to patient
→ Proper tools to transfer blood safely between people.

Appropriate preservative solutions
→ Fluids that keep donated blood fresh and usable.

Avoiding circulatory overload
→ Preventing too much blood from entering the body at once.

Component therapy

• Using only the needed part of blood (like plasma or red cells) instead of whole blood.

Current Status of Blood Donations & Transfusions

• 3 private run organization collect blood which are:

• To donate blood you meet these qulifications

  • there are 5

Blood collection & distribution in the US is regulated by the FDA, and it is collected and standardized by 3 private peer-run organizations:

• - American Red Cross

• - American Association of Blood Banks (AABB)

• - College of American Pathologists (CAP)

To donate blood, you must meet these basic health and safety rules:

• Healthy & free of infectious disease

• Must be at least 16 years old.

• Hematocrit > 38%

  • Enough red blood cells to donate safely.

• Must weigh over 110 pounds.

• Wait at least 8 weeks between donations.

🩸 Current Status of Blood Donations & Transfusions

• unit is aka

• 1 unit is how many ml?

• How many unit in an average adult

• Whole blood can be separated in what parts

  • there are 5

1 Unit aka pint of whole blood collected:

• Each donation collects about 500 ml of blood.

• An average adult carries 10 to 12 pints of blood total.

Once donated, the whole blood can be separated into several useful parts:

• Packed Red Blood Cells (pRBCs)

  • Carries oxygen to tissues.

• Platelets (Plts)

  • Helps blood clot and stop bleeding.

• Fresh Frozen Plasma (FFP)

  • Contains clotting factors and proteins.

• Cryoprecipitate Anti-hemophilic Factor (VIII)

  • Rich in clotting factor VIII, used for hemophilia.

• Granulocytes (neutrophils)

  • White blood cells that fight infection.

Current Status of Blood Donations & Transfusions

• how much blood in mi is collected into the main bag

  • Anti-coagulant preservatives are added to the main bag and does what?

  • what does anti-coagulant do?

  • what do the preservatives provide for the cells in the blood?

500 ml of blood is collected in a main bag with anticoagulant-preservatives.

• Anticoagulant-preservatives minimize biochemical changes and increase shelf life.

  • Anticoagulant (stops blood from clotting) chemicals help keep blood fresh and usable longer.

• Anticoagulants prevent blood clotting.

• Preservatives provide nutrients for cells.

Red Blood Cell (RBC) Biology

• what is the normal lifespan of RBC in circulation?

• RBC survival depends on what? 

  • there are 3, if any of these 3 are defected then the RBC will survive fewer then the normal amount of days.

• RBC hold what protein inside of it?

  • what does this protein due

Key Areas for Red Blood Cell Survival ( Defects in any or all of these areas will result in RBC survival of fewer than the normal 120 days in circulation.)

• RBC membrane

  • The outer layer keeps the cell flexible and protects it.

• RBC metabolism

  • Internal processes give the cell energy and help it survive.

• Hemoglobin structure & function

  • Hemoglobin carries oxygen and must be shaped correctly to work well.

Red blood cells hold hemoglobin, which is the vital protein that picks up oxygen in the lungs and delivers it throughout your body

RBC Membrane

1. The RBC membrane is described as what?

2. What does the uneven layout of the membrane help with?

3. To much calcium does what to the RBC membrane?

4. Dehydrated, rigid, and dying RBC’s are sequestered & removed from peripheral circulation by what?

1. RBC membrane represents a semipermeable lipid bilayer supported by a protein mesh-like cytoskeleton structure.

   • The red blood cell (RBC) membrane is a flexible outer layer made of fats and proteins.

2. Asymmetrical organization – critical for deformability & permeability.

   • The uneven layout of molecules helps the cell bend and let substances in or out.

3. Increased membrane calcium = membrane rigidity.

   • Too much calcium makes the membrane hard and less flexible.

4. Damaged or old RBCs are filtered out by the spleen to keep blood healthy.

RBC Membrane structure

• External layer of RBC membrane are made out of?

  • what does the external layer help with?

• The internal layer of the membrane is made up of what?

  • what does the internal layer help with?

External layer – glycolipids and choline phospholipids.
→ The outer surface contains sugars and fats that help with cell recognition and stability.

• Glycolipids are lipids (fats) with a a type of carbohydrate (sugar) attached.

• Choline phospholipids are phospholipids (a type of fat) with a choline-containing head group

Internal cytoplasmic layer – amino phospholipids.
→ The inner surface has fats with nitrogen groups that support cell shape and function.

RBC Membrane - Permeability

• RBC membrane is permeable to what?

• RBC membrane is not permeable to what

• What is pushed out of the RBC by what mechanism?

1. Permeable to water and anions (Cl⁻; HCO₃⁻).

   • Water and negatively charged ions (like chloride and bicarbonate) can pass through.

2. Impermeable to cations (Na⁺ ; K⁺).

   • Positive ions (like sodium and potassium) cannot pass through.

3. Calcium (Ca²⁺) is also actively pumped from the interior of the RBC.

   • Calcium is pushed out of the RBC by active transport

RBC Metabolic Pathways

1. RBC relies mostly on what to make ATP (what percentage)?

   • Does it use oxygen or without oxygen and use scientific name plz.

   • What do mature RBC lack?

2. RBC relies what other pathway beside the top one?

Red blood cells rely mostly on anaerobic metabolism to make ATP.

• RBCs make energy without using oxygen.

Mature RBCs lack a nucleus and mitochondria, so they can't perform oxidative metabolism.

• Mature RBC don’t have DNA or mitochondria, so they can't use oxygen to make energy.

Anaerobic glycolysis is the key energy-producing pathway (ATP) in RBCs.

• Glycolysis can be happen without oxygen, as it is an anaerobic process

About 90% of RBC ATP comes from glycolysis, and 10% from the pentose phosphate pathway (PPP).

• Most of their energy comes from glycolysis; a small part comes from another pathway called PPP

🧬 RBC Hemoglobin (Hb) – Characteristics & Structure

1. RBC hold what main protein?

   • what does this protein due?

2. What is this main protein made up of?

Red blood cells hold hemoglobin, which is the vital protein that picks up oxygen in the lungs and delivers it throughout your body

Hemoglobin occupies 33% of the RBC volume & 95% of the dry weight.

• Dry weight is your weight without extra fluid.

Normal Hemoglobin consist of Globin and 4 heme groups

• Globin is made of four protein chains—two alpha and two beta chains—linked together.

• 4 heme groups – Each heme contains a protoporphyrin ring plus iron.

  • Each hemoglobin has four heme parts, and each heme has a ring structure with an iron atom inside that grabs oxygen.

Define each

• O₂

• ß

• G

• Oxyhemoglobin

• Deoxyhemoglobin

• O₂

  • oxygen

• ß

  • beta chains

• G

  • globin

• Oxyhemoglobin

  • Hb with oxygen

• Deoxyhemoglobin

  • Hb without oxygen

Hemoglobin (Hb) – Function with O₂

1. Hemoglobin main job is what?

   • how can it due this job?

2. What is hemoglobin relax state?

   • another name for relax state is what

3. What is hemoglobin tense state?

   • another name for tense state is what?

Hemoglobin’s main job is to carry and release oxygen to tissues

• Hemoglobin flexible shape (having multiple chains) helps it change (allosteric) forms when picking up or dropping off oxygen

When Hemoglobin grabs oxygen, hemoglobin becomes oxy-hemoglobin, which is its relax state (R Form).

• In this relax state, the chains are pulled together and thus, pushes out 2,3-DPG.

• The relax state also has a higher affinity (attraction) for O₂ and thus, binds to oxygen.

When hemoglobin lets go of oxygen, the chains widen, and 2,3-DPG binds. 

• When unloading, this form is called T (tense) form of Hb and is called deoxy-hemoglobin. 

• Deoxy Hb has lower affinity (attraction)  to oxygen and thus, releases it. 

1. How tightly hemoglobin holds oxygen depends on what?

2. R forms means what

3. T forms means what

1. How tightly hemoglobin holds oxygen depends on its shape: 

   • 2,3-DPG (2,3-diphosphoglycerate) is a phosphate containing molecule.

2. Oxy-hemoglobin (R form = Relaxed)

   • Think of it like a hug:

     • hemoglobin hugs oxygen tightly and pushes 2,3-DPG away

3. Deoxyhemoglobin (T form = Tense)

   • Now it’s like hemoglobin is hugging 2,3-DPG’s and ignoring oxygen.
     

Hemoglobin - Oxygen Dissociation Curve

1. What curve does hemoglobin follow base on oxygen levels?

2. When does allosteric occur for hemoglobin

   • what does allosteric mean?

3. The curve shifts to the right when?

   • what does shifting right make hemoglobin do with oxygen?

4. The curve shifts to the left when?

   • what does having low 2,3-DPG due?

   • what does shifting left make hemoglobin do with oxygen?

1. Hemoglobin doesn’t grab or release oxygen in a straight-line way. Instead, it follows an S-shaped (Sigmoid-Curve) based on oxygen levels.

2. Allosteric (changes) occur as the hemoglobin loads and unloads oxygen.

3. The curve shifts to the right when:

   • oxygen is low (hypoxia), blood is acidic (acidosis), or body temperature is high

   • All of this this helps hemoglobin release oxygen more easily.

4. The curve shifts to the left when:

   • blood is more basic ( alkalosis), body temperature is low (colder), or after transfusions

   • Transfused blood that’s low in 2,3-DPG makes hemoglobin hold onto oxygen too tightly, reducing oxygen delivery.

   • All of this makes hemoglobin hold onto oxygen more tightly, so tissues get less oxygen. Red blood cells become less effective at oxygen delivery.

If someone loses blood and becomes anemic, their body may adjust by making hemoglobin release oxygen more easily to help tissues get enough (shift the oxygen dissociation curve to the right means to let go of oxygen easily)

• Anemia is a condition in which the body does not have enough red blood cells (RBCs) to carry oxygen effectively throughout the body

Po2: how much oxygen there is

Hb saturation is the percentage of hemoglobin saturated with oxygen

Cooperative binding for hemoglobin is the process where the binding of one oxygen molecule makes it easier for the next oxygen molecule to bind.

Carbon dioxide travels from tissues to lungs in three ways:

• Indirectly by RBC, mostly (75%) by reacting inside red blood cells, indirectly 

• Some (20%) attaches directly to red blood cells

• A small amount (5%) floats freely in the plasma

              H₂O + CO₂ → H₂CO₃

In the main method (indirect), CO₂ enters red blood cells and mixes with water. An enzyme called carbonic anhydrase turns it into carbonic acid.

               H₂CO₃ → H⁺ + HCO₃⁻

Still the indirect method: Carbonic acid breaks into hydrogen and bicarbonate. The hydrogen sticks to deoxy-hemoglobin, and the bicarbonate goes back into the plasma.

RBC Preservation

The goal of blood preservation is to provide viable & functional blood components for patients requiring blood transfusion.

• The purpose of storing blood is to keep red cells alive and working for patients who need transfusions.

RBC Viability (life)

• RBC storage lesion

  • Is the loss of RBC viability over time due to various biochemical changes.

• We need enough 2,3-DPG to help them deliver oxygen properly

As RBCs are stored, 2,3-DPG levels decrease. 2-DPG-depleted RBCs may have an impaired capacity to deliver oxygen to the tissues, which will then adversely affect the recipient

• As RBC are stored, 2,3-DPG is loss over time, making it harder for them to release oxygen. This can harm the patient receiving the transfusion.

To keep blood usable, it’s stored cold (1–6°C) in its liquid state. It is stored for a set time based on the type of preservative used.

An increase in 2,3-DPG lowers hemoglobin's affinity for oxygen, which promotes its release to tissues, especially in conditions like high altitude or chronic anemia.

On the other hand, a low level of 2,3-DPG increases hemoglobin's affinity for oxygen, causing it to be held more tightly and not released as readily to the tissues. 

Food and Drug Administration (FDA) rules:

• At least 75% of RBCs must survive for 24 hours post-transfusion

• Free hemoglobin less than 1% of total hemoglobin (In other words: Hemoglobin should be in your RBCs and Free hemoglobin (outside RBCs) should be less than 1%.)

  • Free hemoglobin is hemoglobin that has leaked out into the blood plasma (the liquid part of blood), usually when red blood cells break down.

RBC Preservation – Anticoagulant Preservative Solutions

Approved anticoagulant preservative solutions for whole blood and RBC storage at 1°C to 6°C and includes 

• ACD-A, CPD, CP2D, CPDA-1.

Adenine in CPDA-1 helps make more ADP, which pushes cells to make more ATP (energy) through glycolysis

RBC Preservation: Additive Solutions

Additive solutions (AS) are preserving solutions that are added to the RBCs after removal of the plasma with/without platelets.

• Additive solutions helps with red blood cells staying alive and healthy during storage

• They also make the red blood cell mixture thinner and easier to transfuse.

Currently, four additive solutions are licensed in the United States.

• Adsol (AS-1) (Fenwal Inc)

• Nutricel (AS-3) (Haemonetics Corporation)

• Optisol (AS-5) (Terumo Corporation)

• SOLX (AS-7) (Haemonetics Corporation)

Benefits of additive solution:

• Extends storage of packed RBC’s (pRBC’s) to 42 days.

• Allows for harvesting of more plasma & plts from the unit.

  • The additive supports the red blood cells, and thus, we can take out more of the plasma (platelets are in the plasma), and the RBCs will still be fine

• Produces a pRBC unit of lower viscosity that is easier to infuse.

All additives have saline, adenine, and glucose.

• AS-1, AS-5, and AS-7 also use mannitol to prevent hemolysis.

• AS-3 uses citrate and phosphate to prevent hemolysis

RBC Freezing

Freezing is mainly used for:

• Autologous units (person donates their own blood to later use on themselves, such as during surgery)

• Rare blood types

• Military or civilian natural disasters

A cryo-protective agent is added to RBCs that are less than 6 days old.

• Glycerol (20–40%) is the most used cryo-protective chemical. It’s mixed in slowly with shaking so it enters the RBCs.

• After adding glycerol, the red cells are quickly frozen and kept at –65°C.

• FDA licenses frozen RBCs for a period of 10 years from the date of freezing.

RBC Freezing – Deglycerolization

Before transfusion of frozen RBC’s, they must be deglycerolized.

• Deglycerolized means to remove glycerol

Removal of glycerol is achieved by replacing the cryo-protectant with decreasing concentrations of saline.

Hemolysis is monitored with the hemoglobin concentration of the wash supernatant.

• Hemolysis = red blood cells breaking open.

• When RBCs break, hemoglobin leaks out into the surrounding liquid

Osmolality of the unit should also be monitored to ensure adequate deglycerolization.

• We also check the salt concentration to make sure the glycerol was removed properly.

RBC Rejuvenation

Rejuvenation is the process where energy (ATP) and oxygen-releasing ability (2,3-DPG) are restored or enhanced by changing the metabolism

• Rejuvesol is the only solution officially approved in the U.S. to rejuvenate red blood cells.

• Rejuvenating blood is costly and slow, so it’s rarely done but it’s super useful for saving rare or auto-logous (self-donated) blood for future use.
  

RBC Substitutes

RBC substitutes aim to safely carry oxygen and solve blood transfusion problems like  short shelf-life, compatibility (matching issues), immunogenicity, transmission of infectious agents, and shortages.

Scientists are studying two main types of RBC substitutes

• Hemoglobin-based carriers (use real oxygen-carrying protein)

• Perfluorocarbons (synthetic chemicals that carry oxygen)
  

Despite years of research, RBC substitutes are still not in use today.

Stem cells are like the “master cells” of your body. They have two special abilities:

1. They can make more of themselves (self-renewal).

2. They can turn into different types of cells (like blood cells, nerve cells, or muscle cells).

Platelet Preservation

A platelet, or thrombocyte, is a tiny blood component that forms blood clots to stop bleeding when a blood vessel is damaged

Since 20–30% of donated platelets expire or get returned, we need to find a way to improve inventory management.

Storing platelets is tricky because:

• They only last 5 days

• Bacterial contamination risk due to incubation at 22°C (room temp)

• Platelets can clump (aggregate) or activate early

• They release internal chemicals

  • Release of intracellular granules

• Their energy molecules (ATP and ADP) drop

Platelet Storage lesion means platelets lose quality over time because of chemical changes

When pH falls below 6.2, the platelets swell, and there is a disk-to-sphere transformation in morphology that is associated with a loss of membrane integrity. The platelets then become irreversibly swollen, aggregate together, or lyse, and when infused, will not circulate or function.

• If pH drops below 6.2, platelets change shape, swell, and break down—making them useless for transfusion.

Once pH goes below 6.2, damage can’t be undone. But pH stays stable if lactic acid doesn’t overpower the storage solution’s buffering power.

Clinical Use of Platelets

Platelets function in blood coagulation.

• Platelets help blood clot to stop bleeding.
  

Platelets are given to stop bleeding or to prevent bleeding (prophylactic) before it starts.

• Prophylactic use means taking preventive measures to stop a disease or other unwanted outcome from happening

• Thrombocytopenia is a condition characterized by a low platelet count in the blood
  

Most platelets (92%) come from apheresis donation

• Apheresis means taking blood, spinning it to separate parts, keeping the platelets or other components, and returning the rest.

Only 8% of platelets come from regular whole blood donations.

The effectiveness of a platelet transfusion is usually checked by counting how much the platelet count increases afterward, called the corrected count increment (CCI)

• CCI takes into account how many platelets were given and the patient’s body size (body surface area (BSA).

Formula for CCI:

• CCI = (postcount – precount) × BSA / platelets transfused

  • aka

• CCI = (platelet count after – before) × body size/platelets given.
  

Platelet Viability means platelets stay active in the body after transfusion.

• Even if the platelet count is enough, they may not function well if they are damaged during storage, which is why keeping the right pH and preventing storage problems is important

Before use of platelet for transfusion, the unit is observed for aggregation. There should be no visible aggregates, as aggregation indicates a loss of membrane integrity, so poor viability & function

• Before use, platelets are checked for clumping. Clumps mean the platelets are damaged and won’t work properly.

Platelet Storage & Quality Monitoring

Platelets from whole blood or apheresis are stored at 20°–24°C (room temp) and gently shaken for 5 days.

• Platelets must have a pH ≥ 6.2 to stay healthy and usable.

• One unit from whole blood has at least 55 billion platelets (≥5.5 × 10^10 plts/uL) in 40–70 mL of plasma.

• One apheresis unit has at least 300 billion platelets (≥3.0 ×10¹¹) —equal to 4–6 whole blood units—and comes in 250–300 mL of plasma, depending on whether collected in a 5d or 7d bag

• In the US, platelets are stored in either a 100% plasma medium, or a platelet additive solution (PAS).
  

The FDA has approved two PAS types: PAS-C (Intersol) and PAS-F (Isoplate).

• PAS helps keep platelets healthy even when less plasma is used during storage.

Sepsis is a dangerous, whole-body reaction to infection. Instead of just fighting germs, the immune system starts damaging healthy tissues and organs

• The most common infection from transfusions is sepsis, caused by bacteria in platelets.

Culture-based FDA-approved platelet bacterial screening tests::

• BacT/ALERT (bioMerieux) checks for rising CO₂, which means bacteria are growing.

• eBDS (Pall Corp.): looks for dropping oxygen levels, which also signals bacterial growth.

• Scansystem (Hemosystem)

Labs wait 24 hours before testing so bacteria can grow enough to be detect, but this delays platelet use for 48 hours.

The first bit of blood (about 20 to 30 mL) are collected into a separate but connected diversion pouch.

• This procedure minimizes the placement of skin plugs, the most common source of bacterial contamination, into the WBD platelet products.

Two fast tests—Pan Genera Detection (PGD) test (Verax Biomedical) & BacTx (Immunetics)—can quickly check platelets for bacteria.

Both of these tests are Immunoassays (Lateral Flow Immunochromatography) that detect lipoteichoic acids on gram-positive bacteria and lipopolysaccharides on gram-negative bacteria.

• Both aerobes (needing oxygen) and anaerobes (without oxygen) are detected.

• A sample of only 500 µL is required
  

Pathogen inactivation (PI) means treating blood to kill or reduce germs before it’s used.

INTERCEPT Blood System is the only FDA-approved method in the U.S. to make safer platelets and plasma by reducing germs

In the U.S., platelets can be stored for 7 days—but only if collected in special FDA-approved containers.

These 7-day containers must follow strict rules:

1. Every platelet unit must be tested with an FDA-approved bacteria test.

2. Platelets are cultured after 24 hours, then retested right before transfusion.
   

Vitalant Blood Center in Sacramento uses 7-day containers and does the first 24 hours bacteria test.

• Nearby hospitals do the second test before giving the platelets

Current Trends in Platelet Preservation Research

Autologous therapy uses the patient's own cells,

• while allogeneic therapy uses cells from a donor

Develop processes to Cryo-preserve (freeze) platelets

Frozen platelets are sometimes used in the U.S. for patients who don’t respond to allogenic (donated) platelets and need their own (auto-logous)

Platelets are collected using apheresis, mixed with a chemical (DMSO which is the cryo-preservative) to protect them, and frozen at minus 80°C.

Apheresis is a blood collection method where:

• Blood is drawn from a donor

• A machine separates and collects only platelets

• The rest of the blood (red cells, plasma, etc.) is returned to the donor

These frozen platelets can be stored for 2 years.

1. How much blood and preservative solution is typically in one unit of whole blood?

2. How often can a donor give whole blood?

3. What percentage of ATP in red blood cells is produced by glycolysis?

4. What other pathway produces ATP? And how much?

5. What percentage of RBCs must survive after transfusion for it to be considered successful?

6. Which preservative solutions allow RBC storage at 1°–6°C for 21 days?

7. Which preservative solution allows RBC storage for 35 days?

8. Which additive solutions allow RBC storage for 42 days in the U.S.?

9. Why are additive solution RBCs considered suitable for neonates and pediatric patients?
   
   
   
   

1. Each unit of whole blood collected contains approximately 450 mL of blood with 63 mL of anticoagulant preservative solution or approximately 500 mL of blood with 70 mL of anticoagulant preservative solution.

2. A donor can give blood every 8 weeks.

3. Glycolysis generates approximately 90% of the ATP needed by RBCs,

4. 10% of ATP is provided by the pentose phosphate pathway (PPP)

5. 75% post-transfusion survival of RBCs is necessary for a successful transfusion.

6. ACD, CPD, and CP2D are approved preservative solutions for storage of RBCs at 1°- 6°C for 21 days, and

7. CPDA-1 is approved for 35 days at 1°- 6°C

8. Additive solutions (Adsol, Nutricel, Optisol, SOLX) are approved in the US for RBC storage for 42 days.

9. Additive solution RBCs have been shown to be appropriate for neonates (new born) and pediatric (children) patients.

   • Because they maintain quality and safety during extended storage.

1. How long can RBCs be frozen if glycerolized and frozen within 6 days of collection in CPD or CPDA-1?

2. What is the only FDA-approved solution used to rejuvenate RBCs before freezing?

3. What types of RBC units are typically rejuvenated using Rejuvesol?

4. What are two types of RBC substitutes currently being researched?

5. What is the goal of current research on additive solutions for RBCs?

6. What FDA-approved system was introduced in 2016 for pathogen reduction in platelets stored in 100% plasma?

7. What are the two FDA-approved platelet additive solutions used in the U.S.?

8. Under what condition has the FDA approved the use of 7-day platelets?
   

1. RBCs can be frozen for 10 years from the date of freezing if they are glycerolized and frozen within 6 days of whole blood collection in CPD or CPDA-1.

2. Rejuvesol is the only FDA-approved rejuvenation solution used in some blood centers to regenerate ATP and 2,3-DPG levels before RBC freezing.

3. Rejuvenation is used primarily to salvage O type and rare RBC units that are outdated or used with specific anticoagulant preservative solution up to 3 days outdated (expired).

4. RBC substitutes under investigation include hemoglobin-based oxygen carriers and perfluorocarbons.

5. Research is being conducted to improve on the effectiveness of the current additive solutions.

6. In 2016, INTERCEPT was approved by the FDA for pathogen reduction of platelets in 100% plasma.

7. Two platelet additive solutions, InterSol and Isoplate, have been approved for use in the United States.

8. The FDA has approved the use of 7-day platelets as long as specific criteria are met.