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When Abnormal is Normal: Recognizing Artifacts in Common
Diagnostics
Jean-Sébastien Palerme Small Animal Internal Medicine Service
Lloyd Veterinary Medical Center
Errors in Laboratory testing
➢Introduction ➢ Why do we care? ➢ Causes of laboratory errors
➢Patient-related factors ➢ Breed ➢ Pseudohyperkalemia ➢ Calcium measurements
➢Operator-related factors ➢ EDTA contamination ➢ Hemolysis
Why do we care?
Consequences • Lost money, lost time • Additional blood draws • Difficulty/stress for the patient • Unhappy clients
• Incorrect diagnosis or therapy • 12% of errors may impact patient care*
• Electrolyte supplementation • Initiation/discontinuation of medications • Dialysis
* Goldschmidt et al Klin Biochem Metab 1995
Stankovic and Smith, Am J Clin Pathol 2004
Fasting, Timing
Hemolysis
Contamination
Delayed separation,Freezing
Equipment calibration
“Typos”
Patient-related differencesDrugs
Where can things go wrong?
Patient-related factorsBreed idiosyncrasies Pseudohyperkalemia Corrected Calcium
Breed idiosyncrasies
Case Example
Anna o 10y FS Greyhound o Routine annual wellness o Health problems
• Ostearthritis o NSAIDs
Case Example
• CBC o Mild Thrombocytopenia
• 174 000/ul o Mild erythrocytosis
• PCV 59%
• Chemistry o Renal azotemia
• Creatinine 1.6 mg/dl with isothenuric urine o CKD? o Consequence of NSAIDs?
Case Example
Possible (wrong) courses of action? o Tick-borne Dz testing o Bone marrow o U/c o AUS o BP o Stop NSAIDs o Fluids for dehydration?
Greyhounds
Cam
pora
et a
l, JS
AP
2011
Greyhounds
Why do they have a low platelet count? o Multiple theories
• Sequestration in the spleen/lungs
• Infectious process o Tick-borne diseases (Babesia canis)
• Bipotential stem cell theory o Increased RBC production is favored over platelet production
Irwin VCNA 2010
Greyhounds
Biochemical abnormalities – Higher reference range creatinine values • Urea is similar to other breeds
Feeman et al Vet C
lin Path 2003
Greyhounds
Reasons for a higher creatinine – Decreased glomerular filtration rate in this
breed? • Greyhounds’ GFRs are in fact higher than other
breeds
–Meat based diet of racing greyhounds? • High creatinine is also seen in healthy retired dogs
eating normal diets
– Increased muscle mass?
Greyhounds
SDMA • New renal biomarker • Not influenced by muscle mass
Liffman et la Vet Path 2018
Other hematologic/biochemical differences?
• Many other abnormalities are commonly reported! o Increases
• Sodium • Albumin • ALP/ALT
o Decreases • WBC • Globulins • Phosphorus • Calcium
Steiss et al Vet Comp 2000
Other sight hounds?
Similar abnormalities have been found in related breeds
Case Example
Happy • 5 year old FS Cavalier
King Charles Spaniel
• Presents for pre-dental blood work
Case Example
Pede
rsen
et a
l, JV
IM 2
002
CBC o Thrombocytopenia
• 60 000 platelets/ul • Multiple
macroplatelets seen on smear
Case Example
Possible pathologies? • Immune-mediated disease • Infectious cause (tick-borne disease) • Bone marrow disease
Consequences • Delay dental until cause determined • Risk of bleeding?
• Initiate immunosuppression
Macrothrombocytopenia
Cavalier King Charles Spaniels • Reported in 40 – 50% of Cavaliers
Documented in other breeds • Norfolk and Cairn terriers • Chihuahua • Labradors • Poodles • Akitas • …
Hayakawa et al. Vet Clin Path 2016
Macrothrombocytopenia
Inheritance • Autosomal recessive
• Genetic testing available • β1- Tubulin mutations
• How low? • 30 000 – 90 000/ul • 56% of dogs have <100 000/ul
• No clinical bleeding associated • Normal BMBT
Pedersen et al, JVIM 2002
Macrothrombocytopenia
What to do? • Slide review!
• Consider genetic testing
• Hemostatic testing • BMBT
• Plateletcrit (PCT) • (MPV x plt count) / 1000 • Normal 0.129 – 0.403%
Pseudohyperkalemia
Case Example
Snowball o 16y MC Pomeranian
o Problems
• Stage II chronic kidney disease • Hyperadrenocorticism
• Chronic bronchitis
• Mitral valve disease o Recheck for CKD
Case Example
Snowball • CBC • Thrombocytosis (816 k/ul)
• Chemistry abnormalities • Increased ALP • Mild azotemia (Stable) • Hyperkalemia (6.4 mmol/L)
• Use of ACE inhibitor? • Iatrogenic Addison’s?
Pseudohyperkalemia
Pseudohyperkalemia • Release of intracellular potassium from cells • Measured serum potassium values not
representative of in vivo levels
• Absence of clinical abnormalities • ECG abnormalities
Courtesy of NCSU-Cardiology
Pseudohyperkalemia
Proposed mechanism
Release of intracellular potassium • Release of potassium secondary to RBC lysis • Passive leakage of intracellular potassium from
WBCs • Release of intracellular potassium from platelets
during clot formation
Pseudohyperkalemia
Red Blood Cell Lysis (hemolysis) • Most common cause in people
• Not a common cause in dogs and cats • Absence of Na/K pump in mature RBCs
• Responsible for keeping high intracellular [K+] • Breed exceptions
• Shiba Inu, Akita, Shar Pei • PFK deficiency (English Springer Spaniels)
Pseudohyperkalemia
White blood cells • Large stores of intracellular potassium • Well documented in cases of lymphoma and
leukemia in humans • Only one case report in veterinary medicine
• Chesapeake Bay Retriever with acute lymphoblastic leukemia*
*Henry et al, JAVMA 1996
Pseudohyperkalemia
Thrombocytosis (people)
o Release of intracellular K+ from platelets during clot formation
o Well documented in humans
Ong et al, Int J Lab Hematol 2008
Pseudohyperkalemia
Thrombocytosis (dogs)
o Positive correlation between platelet count and serum - plasma [K+]
o Positive correlation between processing time and serum [K+]
Reimann et al, JVIM 1989
Pseudohyperkalemia
Thrombocytosis (cats)
• Serum [K+] readings were consistently higher than plasma readings
oNo cats had documented thrombocytosis
• Delay in processing caused increases in [K+] (both serum and plasma)
Gunn-Moore et al, J Fel Med Surg 2006
Case Example
Back to Snowball o Serum [K+] : 6.4 mmol/L
o What about Addison’s???
• Na/K ratio?
o [Na+] = 146mEq/L
oNa/K = 23
Case Example
Back to Snowball o Serum [K+] : 6.4 mmol/L
• Platelet count 816k/ul oReal or spurious?
o Plasma [K+]: 4.1 mmol/L
• Normokalemic!
Pseudohyperkalemia
Tips • Minimize time before serum separation
• Keep Asian dog breeds in mind (hemolysis)
• If hyperkalemia is present • Verify platelet count • Run [K+] on heparinized (not EDTA)
plasma
Corrected Calcium
Abnormal serum calcium values are common!
• Increases o Hemoconcentration
• Decreases o Inflammatory disease
o Protein-losing nephropathies/enteropathies
o Liver failure
Calcium
Calcium
Protein boundComplexedIonized
Protein boundComplexedIonized
Physiologically active form
Calcium
Corrected calcium o Based on linear
relationship between total calcium and albumin
o If albumin goes up, so does total calcium
o If albumin goes down, so does total calcium
Meuten et al, JAVMA 1982
Calcium
Corrected calcium
= Measured tCa++ – albumin + 3.5
Meuten et al, JAVMA 1982
Calcium
Thelma 9y FS hound Parathyroidectomy post-op
tCa++ = 9.4 mg/dl (low) Albumin = 2.2g/dl (low)
Calcium
Thelma 9y FS hound Parathyroidectomy post-op
tCa++ = 9.4 mg/dl (low) Albumin = 2.2g/dl (low)
Corrected Ca= Measured tCa++ – albumin + 3.5 = 9.4 mg/dl – 2.2 g/dl + 3.5 = 10.7 mg/dl (normal!)
iCa++ (VBG) = 1.26 mmol/L (1.25 – 1.45 mmol/L !normal!)
Calcium
Percy 12y MC Labrador Retriever Previous diagnosis of multiple myeloma (Undergoing treatment)
tCa++ = 12.0 mg/dl (high) Albumin = 3.0 g/dl (normal)
Calcium
Percy 12y MC Labrador Retriever Previous diagnosis of multiple myeloma (Undergoing treatment)
tCa++ = 12.0 mg/dl (high) Albumin = 3.0 g/dl (normal)
Corrected Ca++= Measured tCa++ – albumin + 3.5 Corrected Ca++= 12.0 mg/dl – 3.0g/dl + 3.5 = 12.5 mg/dl (9.7 – 11.3 mg/dl !high) iCa++ (VBG) = 1.32 mmol/L (1.25 – 1.45 mmol/L !normal)
Calcium
Caveats: 1. Formula is an estimate for total calcium • Not necessarily reflective of active form of
calcium (iCa) 2. Formula was generated using a specific
assay and values would likely be different if another assay were used
3. Formula only accounts for albumin –Globulins?
Calcium
Dogs Healthy • 38% disagreement between corrected and ionized
calcium Chronic kidney disease • 53% disagreement between corrected and ionized
calcium Critically ill • 68% disagreement between corrected and ionized
calcium
Schenck and Chew, Am J Vet Res 2005Sharp et al. JVECC 2009
Calcium
Cats – Poor correlation between tCa++ and either
albumin or total protein1, 2
– Retrospective comparison of ionized/corrected calcium in cats2 • 434 feline samples
– 40% were incorrectly identified as hypo, hyper or normocalcemic
Flanders et al, JAVMA 1989Schenck and Chew, Can J Vet Res 2010
Calcium
Tips • Corrected calcium is an estimate (at best)
• Strongly consider ionized calcium be performed • Critical patients • CKD patients
Operator-related factorsEDTA Contamination
Hemolysis
EDTA contamination
CASE EXAMPLE
Bodhi • 3y MC Jack Russell Terrier • Acute onset seizures • Chemistry • Increased liver enzymes values • Ca++ : 5.9 mg/dl " • Mg++ : 1.1 mg/dl " • K+ : 8.0 mmol/L #
• Classic for….
CASE EXAMPLE
Bodhi • 3y MC Jack Russell Terrier • Acute onset seizures • Chemistry • Increased liver enzymes values • Ca++ : 5.9 mg/dl " • Mg++ : 1.1 mg/dl " • K+ : 8.0 mmol/L #
• Classic for….EDTA contamination
EDTA (Ethylenediaminetetraacetic acid)
Used as an anticoagulant for collection of blood for CBCs
• Used in various salt forms • Di/tripotassium • Di/trisodium
Strongly binds Calcium • Blocks intrinsic and common
coagulation pathway
Good conservation of cell morphology
EDTA Contamination
• Consequences of EDTA contamination
• Spurious results • Increased serum [potassium] • Decreased serum [calcium] and [magnesium]
EDTA Contamination
Common problem? • Dependent on training of phlebotomists • Students?
• Variable incidence • 28/117 (24%) hyperkalemic samples1
• 9/289 (3%) hyperkalemic samples2
1. Cornes et al, Ann Clin Biochem 20082. Sharratt et al. Int J Clin Path 2009
EDTA Contamination
Routes of contamination 1. Backflow of EDTA when using Vacutainer®
systems
1. Decanting blood from EDTA tubes into other tubes
Sharratt et al. Int J Clin Path 2009
EDTA Contamination
Routes of contamination 3. Contamination of syringe needles when blood is
delivered into EDTA tubes prior to other tubes
Fitzpatrick et al, J Clin Pathol 1987
EDTA Contamination
Back to Bodhi Classic electrolyte abnormalities Concurrent CBC was submitted
Recheck chemistry • Ca++ : 5.9 mg/dl ! 10.2 mg/dl • Mg++ : 1.1 mg/dl ! 1.8 mg/dl • K+ : 8.0 mmol/L ! 4.1 mmol/l
EDTA Contamination
Solutions • Enforce appropriate blood collection techniques
• Don’t dump EDTA samples into serum tubes!!! • Dispense sample into serum tube first • Use a different needle between tubes
• Note if CBC sample was taken at same time as serum • Repeat measurements • EDTA measurement
Hemolysis
Chuggy • 8y MC Pug X • Immune-mediated
thrombocytopenia • Treated with prednisone
and azathioprine • Chemistry
o Increased liver enzymes and bilirubin
Case Example
Concern for liver disease?
o Drug toxicity o Neoplasia o Cholangiohepatitis
Difficult blood draw o Multiple veins tried o Small sample
Case Example
Hemolysis
Free hemoglobin in serum of patient o >0.3g/L (>18.8mmol/L) o Pink to red hue
Ozcan et al, Turk J Biochem 2012
Consequences of hemolysis
H2ODECREASE in relative concentration of some analytes (dilution)
Electrolytes/metabolites
INCREASE in concentration of some analytes
HemoglobinCellular proteins
INCREASE/DECREASE due to chemical or optical Interference
AST/ALT/ALPCobalamine
Hormones (cortisol, ACTH, PTH)Coagulation test (PT/PTT/D-dimers)
FolatePotassium*
Albumin
Clinical consequences of hemolysis
• Common occurrence o 5 – 30% of submitted samples o Most common reason for sample rejection
• 60% of rejected samples
• Clinical consequences o Incorrect diagnosis/therapy o Delay in reporting results o Necessity for repeated phlebotomy
• Income loss • > $200k / year*
*Eng Hock Ong et al, Am J Med 2009
When can hemolysis occur?
• In vivo causes o Intrinsic to the patient o Underlying diseases
• IMHA
• Hypophosphatemia
• RBC membrane defects
• Mechanical injury • Toxicity (Heinz body anemia)
• In vitro causes o Operator dependent
Factors associated with hemolysis
Location of venipuncture o Humans
• Antecubital venipuncture ! 12.6% hemolysis
• Non-antecubital venipuncture (hand, foot...) ! 33.7% hemolysis
• Larger vessel, easily accessible ! less trauma
o Veterinary medicine
• Prioritize jugular sampling when possible
Factors associated with hemolysis
Technique of sampling o Catheter
o Syringe
o Vacutainer®
www.BD.com
Factors associated with hemolysis
Technique of sampling o Catheter
• 3 to 20-fold increase in hemolysis compared to needle
o Pressure causing catheter collapse
o Partial obstruction by existing clot
o Needle
• Gold standard in veterinary medicine
• Allows control of negative pressure
o Vacutainer®
• Gold standard in human medicine
• Lower incidence of hemolysis than needle?
*Tanable et al, Acad Emerg 2003
Factors associated with hemolysis
Other factors o Needle size
• Needle sizes smaller than 22 gauge
o Excessive negative pressure
• Use of needle + syringe can allow better control
o Prolonged tourniquet time
• Maintained tourniquet for > 2 minutes
*Tanable et al, Acad Emerg 2003
Hemolysis – What to do?
Three possibilities o Hemolysis “correction”
• Likely inconsistent and prone to over- or under-corrections
o Hemolysis alert with report
• Alert staff to possible artifact affecting specific analytes
o Repeat sample collection
Hemolysis – Summary
Tips 1. Sedate difficult patients prior to phlebotomy
2. Use larger blood vessels whenever possible using larger bore needles (≥22Ga)
3. Avoid IV catheter sampling
4. Control negative pressure
5. Limit delays in sample processing
• Time with lab pick up
• Separate from serum prior to dispatch
Know your patient! o Keep breed idiosyncrasies in mind
o Does the bloodwork match the patient?
Careful sampling o Correct sampling techniques
o Eliminate delays in sample processing
o Beware of sample cross contamination (EDTA)
Take home points
