Immunohematology Notes 2025 PDF

***Immunohematology Notes 2025!!*** Chapter..... RBC structure and Function Three areas of RBC biology are crucial for normal erythrocyte survival and function: 1. - - - **Integral membrane proteins**: - **Eg.** Glycophorin. **A-D** - **Peripheral proteins**: - **Eg.** S...

***Immunohematology Notes 2025!!*** Chapter..... RBC structure and Function Three areas of RBC biology are crucial for normal erythrocyte survival and function: 1. - - - **Integral membrane proteins**: - **Eg.** Glycophorin. **A-D** - **Peripheral proteins**: - **Eg.** Spectrin, Ankyrin, Actin, Protein 4.1 (**SA2P**) Throughout embryonic and fetal development, activation of globin genes progresses from : **Zeta → Alpha → Epsilon → Gamma → Delta → Beta** ***[Band 3 protein] - serves simultaneously as an anion transporter, of chloride and bicarbonate an anchor for the cytoskeleton, haemoglobin and glycolytic enzymes.*** **Deformability** - - **Permeability** - - - NB. **Consequences of Impairment:** - - - Rigid RBCs are sequestered by the spleen, resulting in reduced RBC survival. - **RBC Metabolism** 1. - - - 2. - - - 3. - - *[Hemoglobin's primary function is gas transport: oxygen delivery to the tissues and carbon dioxide (CO2) excretion. ]* **Effect of 2,3-DPG:** 1. 2. 3. ***Hemoglobin-Oxygen Dissociation Curve*** This curve illustrates the relationship between the partial pressure of oxygen and the percentage of hemoglobin saturation with oxygen. A left shift indicates increased oxygen affinity **(R)**while a right shift indicates decreased affinity **(T)** for O2 and is more likely to release it to tissues. **Transfusion of stored blood can cause a leftward shift.** When hemoglobin **releases oxygen**, it undergoes a structural change that creates more space between its chains, **allowing 2,3-DPG to bind**. This form is called the **tense (T)** state, which has a ***lower affinity for oxygen***. When hemoglobin **binds oxygen**, the structure changes again, ***breaking the salt bridges*** formed with 2,3-DPG and pulling the chains closer together. This new conformation is the **relaxed (R)** state, which has a ***higher affinity for oxygen.*** **Conditions Right Shift** **Conditions Left shift** ---------------------------- --------------------------------------- Anemia Multiple transfusions of stored blood Acidosis/(decrease pH) Alkalosis/ (increase pH) Increase to 2,3 DPG Decreased to 2,3 DPG Fever Hypothermia ***\''The normal resting state of oxygen is approximately 104 mmhg saturated at 98% in the lung upon entering tissues through arterial blood and now becoming venous blood oxygen is unloaded to tissues at approximately 40 mmhg which is 75% saturated. \''*** ***Factors Affecting RBC storage*** *[Stored blood often has low 2,3-DPG levels]*, *[which can cause a left shift in the oxygen dissociation curv]*e, making it harder for hemoglobin to release oxygen, leading to inadequate tissue oxygenation. ***In stored blood:*** \# of viable cells decrease -------------------- -------------- Glucose decrease ATP decrease PH decrease Lactic acid **increase** K+ **increase** **N.B** When collecting red blood cells for storage these changes will occur.Oxygen dissociation curve Shift to the left there is an increase in the need for hemoglobin and oxygen affinity thus less oxygen delivered to tissues) **Preservatives** - Adenine, incorporated into the CPD solution (CPDA-1) *increases ADP* levels, thereby driving glycolysis toward the synthesis of ATP. Adenine-supplemented blood can be **stored at 1°C to 6°C for 35 days**; the other anticoagulants are approved for 21 days. **Preservative** **Abbreviation** **Storage** -------------------------------------- ------------------ ------------- *Acid citrate dextrose* ***ACD*** 21 days *Acid Phosphate dextrose* ***CPD /CP2D*** 21 days *Citrate phosphate dextrose-adenine* ***CPDA*** 35 days The viability of transfused red blood cells (RBCs) can be affected by several factors, including the type of storage container used. It\'s important for the plastic material to be permeable to CO2 to help maintain higher pH levels during storage, blood is now stored in **polyvinyl chloride (PVC) plastic bags**. Another issue with PVC is its tendency to break at low temperatures; therefore, components frozen in PVC bags must be handled with care. **What is an additive solution?** - - - - - **[Benefits of Additives ]** Extends the shelf-life of RBCs to **42 days** by adding nutrients ***Allows for the harvesting of more plasma and platelets from the unit.*** Produces an RBC concentrate of lower viscosity that is easier to infuse especially in emergencies. **[RBC Freezing ]** - - 1. 2. **Freezing Procedure:** ***Cryoprotective Agent:** glycerol is added to RBCs that are **less than 6 days old**. **Glycerol protects the cells during freezing.**Glycerol is slowly mixed into the RBCs while shaking them vigorously, allowing it to penetrate the cells effectively.After mixing, the RBCs are quickly frozen and stored in a freezer at temperatures below **-65°C.** The exact temperature can vary based on the glycerol concentration used.* - - ### **Storage and Thawing of Frozen RBCs** - ### **Deglycerolization Process** 1. - - - +-----------------------------------+-----------------------------------+ | **Advantages** | **Disadvantages** | +===================================+===================================+ | Long-term storage **(10 years)** | A time-consuming process | +-----------------------------------+-----------------------------------+ | Maintenance of RBC viability | Expensive | | | | | and function | | +-----------------------------------+-----------------------------------+ | Low population of leukocytes and | Storage requirements (--65°C) | | platelets | | +-----------------------------------+-----------------------------------+ | | | +-----------------------------------+-----------------------------------+ ### **Impact of 2,3-DPG on Oxygen Delivery** ### Low 2,3-DPG levels can lead to: 1. ### **Increased** cardiac output. 2. ### **Decreased** mixed *[venous oxygen tension.]* **HOW?** *[When 2,3-DPG levels decrease, hemoglobin has a higher affinity for oxygen, meaning oxygen remains bound and is less readily released to tissues. This leads to:Increased Cardiac Output:Thus tissues receive less oxygen due to reduced oxygen unloading, they experience hypoxia.The body compensates by increasing cardiac output (heart rate and stroke volume) to deliver more oxygenated blood to tissues.Decreased Mixed Venous Oxygen Tension (PvO₂):represents the oxygen content in venous blood returning to the heart after tissue perfusion will increased hemoglobin oxygen affinity, less oxygen is released to the tissues.]* ### **Factors Affecting RBC Viability** **Storage Container Material** - - - **Temperature and Handling** - ### ### **Laboratory Procedures** ### **ABO Blood Typing** #### **Principle** - #### **Procedure** 1. 2. 3. 4. 5. 6. **NB. Forward Grouping (Cell Typing):\ ** - **Reverse Grouping (Serum Typing):\ ** - - NB. *Red top = serum (Reverse)= Serum + A/B cells* *Purple top= RBC (Forward) = Antisera + RBC* ### **Reagent Preparation** #### **10% Bleach Solution** - To prepare 100 mL of 10% bleach: - - i. #### **0.85% Saline Solution** - i. - - **Eg. 1 liter (L) is equal to 1000 milliliters (mL)** **NB.** *0.85% saline is used for RBC washing because it is isotonic to red blood cells. This means that the concentration of solutes in the saline solution is the same as the concentration of solutes inside the red blood cells. As a result, there is no net movement of water across the cell membrane, which helps to prevent the cells from swelling or shrinking and rupturing* **Cell Washing (Suspension):** - - - - - - - - **Make suspension:** 1. 2. ***\"If you get a red top and a purple top, have the serum from the red top and the cell from EDTA whole blood (purple). If you get only a purple top, take off the serum first. "*** ***COOMBS Check Cells (CCC)*** ***procedure:*** **Topic :Antigens** ***Bulletin board:*** - - ***Why do we grade our reactions ???*** - - - ***4+**= one solid agglutinate* ***3+**= medium to large agglutinates* ***2+**= small to medium **clear background*** ***1+**= small agglutinates with a **turbulent background*** ***[Include the key of only the reactions you got !!!!!]*** **Todays lecture** Topic :Antigen/antibody reaction Stage 1 agglutination ptt Antibody excess = prozone effect antibodies will bind and sensitize but it will not be enough to cause agglutination = false negative reaction When the cells are wash/diluted it is to get them in there right proportions to increase the likelihood of agglutination ***Platelet Structure\ ***Platelets contain several important components: - - - - ***In the U.S., platelets can only be stored for 5 days due to concerns about bacterial contamination.*** **Effects of Storage\ **During storage: - - **Limitation:** Platelet transfusions are essential for patients with low platelet counts (**thrombocytopenia**) to prevent bleeding. The success of these transfusions is often evaluated using a corrected count increment **(CCI**)---this measures how much the platelet level rises after transfusion but does not assess the platelets\' function. Stage 2 lattice formation: IGG is a monomer take two to activate C IGM Why is the ABO reactions generally 3+/4+ in relation to antigen antibody binding ANS= The larger the polymer the more the binding site thus the larger the agglutinate. Under centrifuge = false negative Over centrifuge= false positive due to the cells being packed together by force which is why there is a time for centrifugation and why the cell button should be dislodged prior to grading. Rouleaux is due to increase protein in circulation resulting in pseudoAgg pathologies that carry this similar trait = Mix Field Agglutination results after recent transfusion eg. patient is A receive O cells when testing A cells will AGG but O cells will return to suspension= \#+ with a turbid background. **Potentiator used to enhance AGG** LISS ALbumin PEG Salien **Blood Group System** **Warm/Cold** ------------------------ --------------- ---------------------------- Kidd Weaker reactions strengths Duffy Kell When can an antibody be used to find another antibody/(protein)? IgG - is an incomplete antibody while it may facilitate sensitization it cannot achieve lattice formation because of this it may not be detected at the immediate spin stage but the antihuman globulin phase may pick it up. (AHG) DAT- used poly specific reagent if the spin is positive then proceed to the mono specific IaT/ACT Principle To detect antibodies that has been sensitize Differenciate between Polychlonal and monoclonal antibodies How are polyclonal antibodies made?? Monoclonal antibodies is targeted at a single antigen unlike poly chlonal poly chlonal- multiple antibody are produced (multiple clones) to target different antigens What are clinically significant antibodies ? ANS=Slide 17 they must have IGg IGG based antibodies are warm antibodies Hydroma b cells are fuse with the tumor cells to make them immortal Direct indirect test CCC is used to validate a negative AHG test CCC must be a postzone phenomenon **Principle:** **CCC Procedure:** 1. 2. 3. 4. 5. 6. **AHG validation;** **1** drop of AHG to **2** drops of CCC centrifuge for 15-30 sec (screen) then observe for **2+** Agg anything else = under sensitize/ oversensitize cells which will cause interference such as: **Autoimmune Hemolytic Anemia** **NB. Invert the tube before taking cells to make your CCC if you take from the pack cell it will be too postzone.** 1.