Objectives

Blood Banking for • Discuss the structure and function of red components and their relationships to each other. Hematologists: a. Lipid Bi-layer b. Transmembrane (integral proteins) c. Cytoskeletal proteins (peripheral proteins) Or Is It The Other Way Around? • Describe the location of the red cell discussed.

Phyllis Kirchner, MT(ASCP)SH, SBB • Correlate the relationship of changes in red cell membrane proteins and their impact for the Bank and/or the lab

Blood Bank/Hematology Red cell vs Hepatocyte Connection: A Red Cell Perspective Membranes Hepatocyte RBC • Red cell membrane ER 51% • Structure Golgi 7% • Blood group antigens nuclear 2% • Functions mitochondria 37% The “somes” 1%+ plasma 2% 100 %

* % of total membrane

Lipid Bilayer Red Cell Membrane Structure CHO= glycolipid cholesterol • Membrane lipids "head" - polar region • Phospholipid anchored Hydrophilic proteins "tail" - non-polar region • Transmembrane (integral)proteins Hydrophobic

• Cytoskeleton (peripheral) proteins Cholesterol and phospholipids exchange w/plasma elements

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Phospholipids Schistocytes/Microspherocytes

PolarHeadGroup Choline Or Choline • Sign of damage to Ethanolamine RBCs Or Phosphate Serine Phosphate Or

Glycerol Inositol Sphyngosine • Double fatty acid tails Polar Head Group Head Polar can “seal” damaged

Non membrane; prevents

Fatty Acid tails -

polar polar Tails Hgb/Fe loss

polar Tails polar

-

Non

Intravascular Hematology Findings - Signs of Increased Destruction Complement lyses cell – • PB / BM response Nothing left to see • Increased PRODUCTION - polychromasia, increased retic count, may see some nRBCs

Immune vs Non-Immune Red Cell Antigens

• Spherocytes Sugar Chains Proteins • DAT + = immune • ABO • Rh • DAT - • Lewis • MNS • P1 • Kell • Schistocytes • Kx • Mechanical damage • Kidd • Duffy

• PI-Linked

2 GlycoPhosphotidyl Inositol Case #1 Anchor • 72 yo female has • Hematology follow-up visit 10 • H/H = 8g/dL / 24% days post hip • Spherocytes replacement. Says • she started to feel • DAT + good, then was • Anti-Jka eluted from getting a “bit weak”. red cells • Delayed Transfusion Reaction

www.hmds.org.uk/pnh_review.html

Phosphatidyl Inositol (PI)-linked Proteins CD 59 • PI links to membrane - have lots of lateral movement • Complement defense proteins • Decay accelerating factor - DAF (CD55) • Membrane inhibitor of reactive lysis - MIRL (CD59) • Acetylcholinesterase (Cartwright, JMH and Dombrock antigens) CD59 Membrane Inhibitor of Reactive Lysis (MIRL) CD 55 Decay Accelerating Factor (DAF)

Paroxysmal Nocturnal PNH - Lab Findings Hemoglobinurea (PNH)

• Defective stem cell with • Pancytopenia decreased/defective PI -linked proteins • HGBemia/HGBurea/hemosiderinuria with • Affects all blood cells + increased abnormal cells • Decreased complement defense proteins • Polychromasia,  retics, haptoglobin, • decay accelerating factor - DAF (CD55) bilirubin,  LD • membrane inhibitor of reactive lysis - MIRL (CD59)***

3 All cells positive FLAER PNH Assay - Neutrophils PNH Patient by FLAER Normal by FLAER

• CD55 & CD59 Normal

CD 55 CD 55 CD 59 CD59

Limited number of cells positive

• CD55 & CD59 PNH

CD 55 CD 55 CD 59 CD59

Membrane Proteins Transmembrane Protiens

• Transmembrane or integral - embedded in the – red cell antigens Multi-pass Single pass

• Peripheral proteins – cytoskeleton

• Some proteins structurally related to non- erythroid, others red cell specific Multi-pass

Transmembrane Proteins & RBC Antigens • Immunodominant groups • The part of antigenic determinant that binds most strongly • CHO • Usually the terminal non-reducing sugar residue of a glycoprotein/glycolipid • Protein • May be the terminal or exofacial a.a. sequence, a single a.a. difference or conformational structures along protein

4 Anion Exchanger-AE Cold Agglutinins (Band 3) • Multipass transmembrane protein • Lymphoid disorders, Mycoplasma - (14 transmembrane domains) pneumonia, infectious mono • Functions to transport chloride in, •  MCV,  MCHC bicarbonate out; CO2 transport • Autoanti-I or autoanti-i (many located on AE) • Dimers / Tetramers • (GPA) associated • ABH, I i antigens (CHOs) • Diego blood group • Malarial receptor • Complexed with Rh

Southeast Asian Ovalocytosis Rh Transmembrane Proteins

• Band 3 mutation – decreased #s on cell • Rh30 polypeptide(s) –RhD & RhCE • Double slit ovalocytes • RhAG (Rh50 • Malarial (Plasmodium falciparum) glycoprotein) resistance • Associated proteins • LW glycoprotein • CD 47 integrin- associated protein • Glycophorin B • Duffy glycoprotein Avent and Reid, Blood (2000)95:375 • Band 3 linkage N Engl J Med 2005;353

Rh Complex/ Band 3 Complex Rh Proteins • Function • RhAG - ammonium transporter • RhD/RhCE - structural CD47 Band 3 Band 3 Bi-Lipid • Rh null phenotype Layer • Diminished LW, GPB, CD47 • stomatocytosis, spherocytes

Ankyrin

Spectrin

Adapted from Bruce et al, Blood, May, 2003

5 Numerous Glycocalyx tetrasaccharides on each glycophorin

“Sugar –coated

Glycoprotein Cell”

Glycophorins - Red Cell Glycophorins Antigens • Single pass • Absence of normal GPC and GPD (Leach transmembrane phenotype – negative for Gerbich antigens) glycoprotein • Contains majority of RBC CHO "sugar • Some RBCs will become elliptical coated cells“ = glycocalyx - negatively charged • GPC/GPD tether to membrane cytoskeleton • GPA & GPB assoc with Rh/AE complex

Bloodsystemslaboratory.org Bloodsysyemslaboratory.org Altas.arabslab.com

Glycophorins - Red Cell Antigens Kell Proteins • T- expression (polyagglutination) most commonly seen when bacterial neuraminidase from • Single pass transmembrane protein linked cleave off neuraminic acid and expose to multipass protein cryptantigens New Antigen • Endopeptidase – RBC function unknown

Glycoprotein • K null morphologically normal

• Kx null (McLeod phenotype) cells form Neuraminidase acanthocytes

Kx null • Other cryptantigens exposed/created with other sugars removed or added either from enzymes, inherited or mutation

Adapted from L.Ernster, 2007

6 Duffy Proteins Kidd Proteins Kid(d) Fya, Fyb, Fy6 • Multipass (7) glycoproteins DARC • IL-8 receptor • Ags located on protein that forms a channel thru membrane that actively (DARC-Duffy Antigen Receptor for Chemokines) transports urea in and out of cell • Plasmodium vivax receptor (Fy6) • When exposed to urea, cells with Jka or a b • Fy -Fy - protected from P. vivax Jkb will swell, sphere and lyse • Jk(a-b-) cells will shrink and crenate; lyse very slowly

Jk(a+b-) or RBC Jk(a+b+) Jk(a-b-) Urea Lytic Curve Lysis Jk(a-b+) with Jk(a-b-) RBC’s

2M Normal Urea RBC’s

Natalie Pasyak, 2017: Used with permission

Case #2 Surface Summary

• CBC on 32 yo male • Patient sample sent from Philippines to Blood Bank – Protectors/ had count tested for Kidd “Sugar freaks” Receptors Repulsion/ > 5,000,000/L, but antigens – Signalers smear looked • Patient was Jk(a-b-) G normal – Could Receptors

correct with

extended lyse time. V M No hemoglobinopathy detected Transporters

7 Red Cell Membrane Cytoskeleton Hereditary Spherocytosis

• Matrix of proteins - spectrin, actin, ankyrin, • Spectrin lattice work affected band 3, band 4.1, band 4.2- creates the cytoskeleton of the - requires • Loss of surface area = spherocytes ATP to maintain

Color Atlas of Hematology, Glassy, CAP; 1998 Hematology, Hoffman, 2008

Case # 3 Hereditary Elliptocytosis (HE)

• 23 yo woman is • Sample in Blood • Deficiency/abnormal seen for her 1st Bank for Type and cytoskeletal proteins prenatal visit. CBC Screen. DAT is shows normal added. • 85% have no clinical symptoms results except smear has marked # • DAT is negative – • Others transfusion of spherocytes. family studies Patient shows no dependent (esp determine patient homozygotes/ double- evidence of acute has HS heterozygotes) hemolysis

Hematology and Blood Bank Acquired Morphologies • Clues that something is amiss Stored Blood • Signs of production • Dependant on anti-coagulant / time • Signs of destruction • Loss of cholesterol/plasma proteins • Increased ion concentration • Will this patient be transfused? •  ATP =  deformability • Have they already been transfused?

8 Objectives

• Discuss the structure and function of red cell membrane components and their relationships to each other. a. Lipid Bi-layer b. Transmembrane proteins (integral proteins) c. Cytoskeletal proteins (peripheral proteins)

• Describe the location of the red cell antigens discussed.

• Correlate the relationship of changes in red cell membrane proteins and their impact for the Blood Bank and/or the Hematology lab

References References

• Blood, Textbook of Hematology, 2nd Ed., Jandl • Scientific Basis of , 2nd Ed., Anderson • Williams' Hematology, 7th Ed., Lichtman • Immunobiology of Transfusion Medicine, Garratty • CAP-Color Atlas of Hematology, Glassy • Children’s Hospital of Oakland Research Institute, • in Clinical Medicine, 10th Ed., Dr. Frans Kuypers Mollison • Journal of Membrane Biology, C.Diaz, AJ.Schroit • Clinical Practices of Transfusion Medicine, 3rd Ed., • Annual Reviews of Physiology, K. Balasubramanian, AJ Petz Schroit

References

• Hematology; Basic Principles and Practice, 3rd Ed., Hoffman • Clinical Hematology and Fundamentals of Hemostasis, 4th Ed., Harmening • Applied Blood Group , 7th Ed, Issit

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