WAYS TO CHALLENGE A BLOOD TEST IN WYOMINGE R I N G E R S T E N Z A N G - E H G L AW F I R M - F E B R U A R Y 6 , 2 0 1 5

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THE PROCESSD R AW T R A N S P O R T,

S T O R A G E , P R E P T E S T I N G

CHALLENGE!

C H A L L E N G E T H E S W A B U S E D D U R I N G T H E P R O C E D U R E

• “Alcohol solutions should not be used as a skin antiseptic.” Rules and

Regulations for Chemical Analysis for Alcohol Testing Chapter 3, section 3(b)

• Ethanol-based hand sanitizers (EBHSs) are used in most health care facilities in the United States

C H A L L E N G E T H E A N T I S E P T I C P R O T O C O L B E F O R E T H E D R A W

• Swabbed in a circular motion away from the site of the draw (don’t just move the dirt around!)

FA N C Y S C I E N C E W O R D

Candida Albicans = yeast that lives on human skin and creates alcohol as a waste product

CHALLENGE!

AUTO-BREWERY SYNDROME

C H A L L E N G E T H E N E E D L E U S E D T O D R A W B L O O D

• Phlebotomists switch out the blood kit needle for an elongated tube butterfly needle or a swap out for a smaller gauge needle (easier/causes less pain)

• “Collected using standard medical procedures and equipment for obtaining blood samples.” Rules and

Regulations for Chemical Analysis for Alcohol Testing Chapter 3, section 3(a)

CHALLENGE!

R E D B L O O D C E L L S A R E B R E A K A B L E

• A needle that is too small can cause the blood cells to rupture

• Rigorous shaking from mixing preservatives or shipping by mail

FA N C Y S C I E N C E W O R D

Hemolysis = the breaking of blood cells, most of which are red blood cells.

C H A L L E N G E T H E T U B E S U S E D T O C O L L E C T T H E S A M P L E

• 2 gray tubes

• The tube must “contain an appropriate anticoagulant and a preservative, such as potassium oxalate or EDTA (ethylenediaminetetraacetic acid) and a minimum of 0.1% sodium fluoride.” Rules and Regulations for Chemical Analysis

for Alcohol Testing Chapter 3, section 3(c).

CHALLENGE!

W H Y P R E S E R VAT I V E S ? R E M E M B E R O U R F R I E N D C A N D I D A A L B I C A N S

J. Chang & S.E. Kollman, The Effect of Temperature on the Formation of Ethanol by Candida Albicans in Blood, Journal of Forensic Sciences, Vol. 34, 105-109 (1989)

• The ability of yeast to convert sugar into ethanol has been harnessed by the biotechnology industry to produce ethanol fuel

• A diabetic will have large amounts of glucose present in their blood making it very hospitable to yeast — leading to larger amounts of fermented ethanol

S O W H AT I F T U B E I S E X P I R E D ?

• Yeast is everywhere

• You can contaminate a blood sample by simply touching the area where the needle enters the blood tube, prior to inserting the needle into the tube

T U B E I N V E R S I O N

According to BD, “inversion should be slow enough to allow the air bubble in the tube to completely traverse the length of the tube.” 

• The tube must be inverted in accordance with the manufacturer’s recommendations(5-8 times) following the collection of blood, so that the powder is homogeneously dispersed throughout the collection tube

THE PROCESSD R AW T R A N S P O R T,

S T O R A G E , P R E P T E S T I N G

OKAY, LET’S DIG IN!G A S C H R O M AT O G R A P H Y

C O L U M N F - I - D

C O L U M N

T E S T S A M P L E

F - I - D

TELEGRAPH TELEGRAM

CHROMATOGRAPH CHROMATOGRAM

Note: Thanks to Justin McShane for this analogy!

YOUR BLOOD ISN'T JUST THIS...

45% FORMED ELEMENTS 55% PLASMA

PLATELETS (4.8%)

RED BLOOD CELLS (95.1%)

WHITE BLOOD CELLS

(0.1%)

NEUTROPHILS (54-62%)

EOSINOPHILS (1-3%)

BASOPHILS (< 1%)

MONOCYTES (3-9%)

LYMPHOCYTES (25-33%)

ELECTRO- LYTES

WATER (92%)

PROTEINS (7%)

WASTES (95.1%)

NUTRIENTS VITAMINS

HORMONES

GASES

N2 O2 CO2ALBUMINS GLOBULINS FIBRINOGEN

THE SCIENCE OF SEPARATION

A METAPHOR: THE CHROMATOGRAPH IS JUST LIKE A RACE

LOTS OF DIFFERENT MOLECULES DISSOLVE INTO THE PLASMA

N-PROPANOL

ETHANOL

METHANOL

ACETONE

ACETALDEHYDE

ISOPROPANOL

LOTS OF DIFFERENT MOLECULES DISSOLVE INTO THE PLASMA

THE TRACK SURFACE IS “STICKY” WHICH SLOWS DOWN THE MOLECULES

Photo credit: Anthony Scott Photography

Chromatography — The separation of a mixture of "stuff" through a medium (liquid or gas) through which the molecules travel at different speeds.

R E A L LY FA N C Y S C I E N C E W O R D

CHROMATOGRAPHY

Good$chromatogram$•  Tall,$skinny$peaks$•  Resolu8on$of$ethanol$and$$$$$$$$internal$standard$

CLEAN RACE RESULTS

Thanks to Dr. Jimmie Valentine for these chromatograms

PHOTO FINISH!

CO-ELUTION (PHOTO FINISH)

Ethanol(not(separated(from(acetone(–(a(diabe4c(has(acetone(in(their(blood(

Slide&courtesy&of&Dr.&Jimmie&L.&Valen7ne&

C H A L L E N G E C O -E L U T I O N O F S A M P L E S O N C H R O M AT O G R A M

FA N C Y S C I E N C E W O R D

Co-elution = Samples leaving the column and entering the flame ionization detector at the same time.

• Two samples leaving at the same time makes it impossible to tell how much of each sample was in the blood.

CHALLENGE!

Thanks to Dr. Jimmie Valentine

Peak%tailing%Poor%chromatography%showing%peak%tailing%producing%addi6onal%area%to%be%included%in%quan6ta6on%

C H A L L E N G E P E A K S T H AT A R E N O N -S TA N D A R D

Co-elution = Samples leaving the column and entering the flame ionization detector at the same time.

• Peaks that are asymmetrical, either “tailing” (as on left) or “fronting” peaks.

• Peaks that “wiggle” or that are too fat

• Peaks that go below the baseline

• “Sudden issues with tailing indicates column contamination, possibly caused by a recent dirty sample”

CHALLENGE!

Thanks to Dr. Jimmie Valentine