HAABB Scholarship Presentation

Transcription

1 HAABB Scholarship Presentation y Roert T. Fischer MLT, ASCP Saint Louis Cord Blood Bank SSM Cardinal Glennon Children s Medical Center Saint Louis, MO and 2012 graduate of the Clinical Laoratory Technology Program Saint Louis Community College at Forest Park Saint Louis, MO

2 Potential Applications of MALDI-TOF MS for the Immunohematology Laoratory: Could the advantages of rapid identification of antiodies and antigens transform transfusion medicine?

3 Why consider alternatives to traditional testing in immunohematology?

4 Procedures for Antigen and Antiody Testing for Assessing Compatiility DAT Forward ABO Typing Reverse Typing Direct Antigloulin Testing Atypical Antiody Screening Atypical Antiody Identification Antigen Typing of Donor RBCs Crossmatching

5 Recipient Antigen Profile Can this Pathologic Scenario in Transfusion Medicine e Avoided Donor X Unit Antigen Profile Donor Y Unit Antigen Profile Donor Z Unit Antigen Profile B D C e k K p J s J k F y Le Lu M s B D c e K K p J s J k F y Le Lu M s Anti - c Anti - K B D C e k K p J s J k F y Le Lu N S Anti - c Anti - K Anti - N Anti - S B D C e k K p J s J k a F y Le Lu M s Anti - c Anti K Anti - J a k Anti - N Anti - S

6 RBC Antigen and Plasma Atypical Antiody Detection y Hemagglutination y Tue y Micro Typing Systems Gel Cards

7 Red Cell Antigen Reagent Antisera Cost Providing a Comprehensive Antigen Phenotypic Profile is not Resource or Cost Effective.

8 What lesson might e learned from the Clinical Microiology La?

9 Complexity versus Efficiency Complexity

10 Complexity versus Efficiency Bruker MALDI Biotyper system Efficiency ~ 1 minute to analyze < $ 1 consumale cost per analysis Rapidly acquires multiple analyte signals Requires no purification User friendly operation MALDI-TOF MS generates a parent ion signature of all small and macromolecules in the sample, including proteins, glycans, glycoproteins and nucleic acids. Parameters can e preset for simultaneously acquiring an array of analytes of interest in a crude sample. It has a road dynamic range, ale to acquire mass ranges of molecules up to 300,000 Daltons.

11 Species Specific Polymorphisms of the 16S rrna Complex Proteins Serve as Markers for Microial Identification S4 S7 S8 S15 S17 S20 S5 S6 S9 S12 S13 S16 S18 S19 S2 S3 S10 S11 S14 S21

12 Clinical Infectious Diseases (Infectious Diseases Society of America) Volume 49, no. 4 (2009): pp

13 What is MALDI-TOF MS? What is its niche in mass spectrometry?

14 Noel laureate Sir J. J. Thomson with the first mass spectrometer used to prove the existence of stale atomic isotopes y resolving two isotopes of neon, 20 Ne from 22 Ne, in Mass spectrometers are durale and versatile tools having uiquitous applications. They are often employed on NASA space proes such as on the Phoenix Lander mission to Mars in 2008.

15 Strong versus Soft Ionization in Mass Spectrometry Electron Impact MS of Lanosterol M + or [M + H] + M M + with 13 C Chemical Ionization MS of Lanosterol [M + H] Molecular Parent Ions M + with 18 O

16 M + M + As illustrated y this parent ion profile of an intact protein, with larger molecules there is an increased statistical odds of incorporating stale atomic isotopes of 2 H, 13 C, 15 N, 17 O, 18 O, 33 S or 34 S into the structure, as is reflected in its respective parent ion pattern, known as its isotopologue profile.

17 Various Mass Spectrometry (MS) Technologies Molecular m/z m/z Ionization Resolution Detection Strong Ionization (for fragmenting molecules to derive structural characterizations) Electron Impact (EI) Capacitive Field Photographic Plate Soft Ionization (for generating intact molecular parent ions) Chemical Ionization (CI) Quadrupole (QP) Dynode Electron Multiplier Detector Fast Atom Bomardment (FAB) Quadrupole Trap (QPT) Faraday Cups Thermospray (TS) Magnetic Sector (MS) Oritrap Matrix Assisted Laser Time of Flight (TOF) Microchannel Desorption Ionization (MALDI) Plate Detectors

18 How might MALDI-TOF MS e applied to traditional testing in immunohematology?

19 If microial identification of over 1,600 species y detecting variances in 20 riosomal proteins from a single analysis is a reality, then the potential for identifying clinically significant RBC antigens and corresponding atypical antiodies is a real possiility. Microial 16S riosomal proteins A 1 BH D C E K K p a J s a J k a F y a Le a M S Lu a A 3 B acq H o D u c e k K p J s J k F y Le N s Lu

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23 Detection of Atypical Antiodies y Mass Shift Pre-Equilirate Reagent Antigen in Deuterated Buffer After Antiody Binding Re-Equilirate in Aqueous Buffer If Atypical Antiody Present, Laile Reagent Antigen Amides Retain Deuterium

24 Detection of Atypical Antiodies y Mass Shift

25 Recipient s Plasma Atypical Antiody ID Panel Recipient RBCs Comprehensive Antigen Panel Donor Unit RBCs Comprehensive Antigen Panel Acquisition of multiple analyte m/z signals may afford 3 Assays, using ~30 ml, in 3 Minutes, for < $

26 Reduced Risk of Alloimmunization from Multiple Transfusions Recipient Antigen Profile Donor X Unit Antigen Profile Donor Y Unit Antigen Profile Donor Z Unit Antigen Profile B D C e k K p J s J k F y Le Lu M s B D C e k K p J s J k F y Le Lu M s B D C e k K p J s J k F y Le Lu M S B D C e k K p J s J k F y Le Lu M s

27 MALDI-TOF MS is Applicale to Noninvasive Fetal Genotyping Noninvasive genotyping fetal Kell lood group (KEL1) using cell-free fetal DNA in maternal plasma y MALDI-TOF mass spectrometry Ying Li, Kirstin Finning, Geoff Daniels, Sinuhe Hahn, Xiaoyan Zhong, Wolfgang Holzgreve Prenatal Diagnosis, Volume 28, Issue 3, pages , March 2008

28 Single Donor Platelet Screening for Microial Contamination, Affording Earlier Product Release for Transfusion

29 What is Needed is a Long-Term Perspective to the Development of MALDI-TOF MS for Immunohematology and Transfusion Medicine. Identification of the proper antigen and antiody paratopic signal assignments. Development of dataase liraries and corresponding computational analysis for the accurate extraction of pertinent data. Ascertain if any sample modifications are necessary, or if RBCs and plasma can e analyzed directly. Clinical validation of newly developed MALDI-TOF processing with clinical and reference methods.

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34 Angela Njoku MS, MT ASCP. Professor, Director of Clinical Laoratory Technology program, Saint Louis Community College at Forest Park, Saint Louis MO.