ascp resident review course hemostasis and thrombosis ... · ascp resident review course hemostasis...

ASCP Resident Review Course

Hemostasis and ThrombosisSession # RES2-17

Karen A. Moser, M.D.Saint Louis University

Department of Pathology

September 8, 2017

Speaker Disclosure

•In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation.•This presentation will not include discussion of pharmaceuticals or devices that have not been approved by the FDA or unapproved or "off-label" uses of pharmaceuticals or devices.

From the ABP Website

Clinical Pathology examination•25% hematology (includes coagulation)•10-15% molecular (all topic areas)

Coagulation topics have the potential to fall under either of these categories

www.abpath.org

www.abpath.org

Clinical Pathology Exam Blueprint

WrittenPracticalw/ ImagesPractical

Approximate %

Goals For This Session

•Review basic hemostasis physiology (very basic!)•Answer boards-style multiple choice questions that cover a variety of common or highly testable topics•Not a comprehensive didactic on hemostasis and thrombosis

–Focused review of high-yield topics

Crash Course-Hemostasis Physiology

•High level overview–Vascular Injury–Vasoconstriction–Primary Hemostasis = platelet plug formation–Secondary Hemostasis = fibrin formation–Fibrin polymerization–Hemostatic control measures to prevent excess clotting

Platelet adhesion, activation, and aggregation are main tasks of primary hemostasis

Goal of Primary Hemostasis = formation of primary hemostatic plug

Collagen is most important extracellular matrix component for platelet adhesion

Platelet agonists such as ADP, TxA2 bind to platelet surface receptors to promote granule release

Fibrinogen binds platelet GPIIb/IIIa receptors, connecting adjacent platelets

Figure from: Robbins Pathologic Basis of Disease, 9th ed.

Which substance is contained within platelet alpha granules?

A.ATPB.β2 glycoprotein 1C.CalciumD.Platelet factor 4E.Serotonin

Which substance is contained within platelet alpha granules?

A.ATPB.β2 glycoprotein 1C.CalciumD.Platelet factor 4E.Serotonin

Platelet Granule ContentsAlpha granules contain larger proteins

•Platelet factor 4•B-thromboglobulin•von Willebrand factor•Thrombospondin•Fibrinogen•PDGF

Dense granules contain smaller molecules

•ADP•ATP•Serotonin•Calcium

Rule of Thumb

Very important for platelet aggregation

Very important cofactor for coagulation cascade

•The main point of secondary hemostasis is fibrin deposition

–Stabilizes platelet plug (secondary hemostatic plug)

Platelets = BricksFibrin = Mortar

Tissue factor exposed at injury site promotes local activation of secondary hemostasis

Phospholipids on platelet surfaces provide surface for cascade reactions

Figure from: Robbins Pathologic Basis of Disease, 9th ed.

How do we make fibrin?

FXI

FIX

FXII

FX

FVII

FII Thrombin

Fibrinogen Fibrin

FVIII

FV

Intrinsic pathway(Also includes

Prekallikrein and High Molecular Weight

Kininogen)

Extrinsic pathway

Coagulation Cascade(In Vitro coagulation)

Common Pathway

What happens at each step?

•Sequential enzymatic reactions leading to formation of fibrin

–Each step of the coagulation cascade includes the same basic components.

•Enzyme (activated coagulation factor)•Substrate (inactive coagulation factor- “pro-enzyme”)•Cofactor (helps reaction proceed)•Negatively charged phospholipid surface (provided by platelets)•Calcium (for steps involving factors II, VII, IX, X)

Study Break!

Here are a few (unscientific) mnemonic devices that might help you…

Reference: Ma A. ASH Clinical News. 2016;2(4):57-58.

Coagulation Simplified-The Extrinsic Pathway

7VX

The Extrinsic(PT) Pathway

Extrinsic Pathway = Short and Lucky• PT has two fewer

letters than APTT.• PT values are shorter

than APTT values. • Extrinsic (PT)

pathway is also shorter.

• How lucky that there are fewer steps to remember!

• The lucky PT pathway uses lucky Factor 7 to activate Factor X.

Coagulation Simplified-The Intrinsic Pathway

T

NE

T VX

E

The Intrinsic (APTT) Pathway

Remember that APTT is a basic TENET of hematology testing.

TENET stands for. . .

Coagulation Simplified-The Intrinsic Pathway

Twelve

NineEight

TenVX

Eleven

The Intrinsic (APTT) Pathway

Coagulation Simplified

X

The Intrinsic (APTT) Pathway

The Extrinsic (PT) Pathway

The PT and the PTT pathway meet at Factor X.

“X” marks the spot.

Coagulation Simplified

VX

The Intrinsic (APTT) Pathway

The Extrinsic (PT) Pathway

Factor V is a cofactor for Factor X, and you can remember this because V fits into the notch of the X.

The Common Pathway = Small Bills

V + X

II = prothrombin

I = fibrinogen

You can remember the factors in the common pathway by remembering the bills in your wallet smaller than a $20. Don’t forget the $2 bill!

Let’s put that coagulation cascade knowledge to the test!

A 4 day old male infant is brought to the emergency department by his mother. She notes that he has bleeding from both his circumcision site and the site of a heel stick. Laboratory values at presentation include-

Test Result (s) Reference Interval (s)

APTT 101.7 24.6-32.8APTT 1:1 mixing study

36.1 24.6-32.8

PT 12.2 9.0-13.0

This scenario is most likely caused by deficiency of which factor?A.Factor IIB.Factor VC.Factor VID.Factor VIIIE.Factor IX

A 4 day old male infant is brought to the emergency department by his mother. She notes that he has bleeding from both his circumcision site and the site of a heel stick. Laboratory values at presentation include-

Test Result (s) Reference Interval (s)

APTT 101.7 24.6-32.8APTT 1:1 mixing study

36.1 24.6-32.8

PT 12.2 9.0-13.0

This scenario is most likely caused by deficiency of which factor?A.Factor IIB.Factor VC.Factor VID.Factor VIIIE.Factor IX

FXI

FIX

FXII

FX

FVII

FII Thrombin

Fibrinogen Fibrin

FVIII

FV

APTTIntrinsic pathway

PTExtrinsic pathway

TT

Coagulation Cascade(In Vitro coagulation)

Prolonged APTT- DDx

•Intrinsic factor deficiency or specific factor inhibitor•Lupus anticoagulant•Anticoagulant medications

–Heparin–Direct thrombin inhibitors

•Argatroban•Dabigatran (variable prolongation with different reagents)

A 68 year old man has an APTT of 94.2 s identified in a pre-operative laboratory screening evaluation. He has no personal or family history of a bleeding disorder. What is the most likely cause of his APTT prolongation?

A.Factor II deficiencyB.Factor VIII deficiencyC.Factor IX deficiencyD.Factor XII deficiencyE.Factor XIII deficiency

A 68 year old man has an APTT of 94.2 s identified in a pre-operative laboratory screening evaluation. He has no personal or family history of a bleeding disorder. What is the most likely cause of his APTT prolongation?

A.Factor II deficiencyB.Factor VIII deficiencyC.Factor IX deficiencyD.Factor XII deficiencyE.Factor XIII deficiency

Note: Factor XIII deficiency should not prolong the APTT, as FXIII works to cross-link fibrin. APTT endpoint is fibrin clot formation.

Prekallikrein and high molecular weight kininogen are other contact factors that can cause APTT prolongation without bleeding diathesis.

Prolonged PT- DDx

•Extrinsic factor deficiency or specific factor inhibitor•Anticoagulant medications

–Warfarin–Direct Xa inhibitors (degree of prolongation varies by reagent and drug)

•Rivaroxaban•Apixaban •Edoxaban•Betrixaban

•Liver disease

What about lupus anticoagulant?Does not tend to prolong PT due to high amounts of phospholipid in PT reagent.

What about heparin?Most PT reagents can neutralize ~1-2 U/mL heparin.

Prolonged APTT and PT- DDx

•Liver disease•Vitamin K problem (deficiency, warfarin, superwarfarin)•Heparin•DIC•Common pathway factor deficiency or inhibitor (rare)•Lupus anticoagulant with hypoprothrombinemia (rare)•Dysfibrinogenemia (rare)

Common Factor InhibitorsClassic Association

•Acquired factor V inhibitors classically associated with previous use of fibrin glue containing bovine thrombin (substance used in cardiac and orthopedic procedures)

If the PT and APTT are normal, my patient doesn’t have a bleeding disorder.

True or False?

Bleeding Disorders with Normal APTT and PT

•von Willebrand Disease•Factor XIII deficiency•Qualitative platelet disorder

–Acquired- Uremia, myeloproliferative neoplasms, antiplatelet drugs, post-cardiopulmonary bypass–Inherited- Glanzmann thrombasthenia, Bernard-Soulier syndrome, others

A patient undergoing evaluation for a bleeding disorder has the following laboratory results-

Test Result Reference Interval

Factor VIII activity 15% 50-150%

VWF:Rco <10% 50-150%

VWF:Ag 27% 50-150%

vWF multimers Loss of high and intermediate weight multimers

Normal pattern and distribution

What is the most likely diagnosis?A.Normal patientB.Von Willebrand disease type 1C.Von Willebrand disease type 2AD.Von Willebrand disease type 2BE.Von Willebrand disease type 3

A patient undergoing evaluation for a bleeding disorder has the following laboratory results-

Test Result Reference Interval

Factor VIII activity 15% 50-150%

VWF:Rco <10% 50-150%

VWF:Ag 27% 50-150%

vWF multimers Loss of high and intermediate weight multimers

Normal pattern and distribution

What is the most likely diagnosis?A.Normal patientB.Von Willebrand disease type 1C.Von Willebrand disease type 2AD.Von Willebrand disease type 2BE.Von Willebrand disease type 3

Types of vWD

•Type 1- partial quantitative deficiency•Type 2- qualitative deficiency

–2A- loss of intermediate and high MW multimers–2B- loss of high MW multimers

•Phenotypically similar to platelet-type vWD–2M, 2N- rare

•Type 3- severe quantitative deficiency

vWD screening profile

•Factor VIII activity- why?•vWF activity

–Most commonly ristocetin cofactor activity in US

•vWF antigen

•Activity and antigen concordantly decreased in types 1 and 3•Activity decreased > antigen in type 2A and 2B (abnormal activity:antigen ratio)

Table of vWD results by type

Am J Hematol. 84: 366-370, 2009.

vWF Multimer Gel

2B2A NormalType 3 (not pictured) = virtual absence of multimersNormal pattern seen in type 1, 2N, 2M, as well as unaffected pts.

A 25 year old woman presents with complaint of easy bruising and bleeding. She describes frequent nosebleeds, menorrhagia, and was told she had excessive bleeding after her wisdom teeth were removed 2 years ago. Platelet count and morphology are normal.

Her workup includes platelet function studies with the following pattern of results:

ADP Collagen Epi Ristocetin

What is the most likely defect leading to this patient’s clinical and laboratory findings?

A.Decreased thromboxane A2 synthesisB.GPIb/V/IX complex deficiency or defectC.GPIIb/IIIa receptor deficiency or defectD.Platelet alpha granule deficiencyE.Platelet dense granule deficiency

What is the most likely defect leading to this patient’s clinical and laboratory findings?

A.Decreased thromboxane A2 synthesisB.GPIb/V/IX complex deficiency or defectC.GPIIb/IIIa receptor deficiency or defectD.Platelet alpha granule deficiencyE.Platelet dense granule deficiency

Same question, another way…

What is the most likely diagnosis?

A.Aspirin effectB.Bernard-Soulier SyndromeC.Glanzmann thrombastheniaD.Gray platelet syndromeE.Dense granule storage pool defect

Aspirin Effect

Arachidonic Acid

Other Aggregation Patterns of Note

•Bernard-Soulier syndrome (defective GP1b receptor)

–Normal response with all agonists exceptristocetin (decreased)–von Willebrand disease can appear similar (defective vWF)

•Distinguish with–vW profile – normal in Bernard-Soulier–Giant platelets- present in Bernard-Soulier

A 55 year old woman with a history of Hashimoto thyroiditis (with secondary hypothyroidism) suddenly developed shortness of breath. She experienced a short syncopal episode, losing consciousness for approximately one minute immediately prior to presentation. Her medical history is significant for 4 normal deliveries without complications. Her cancer screening is up to date and has been negative (including mammogram, Pap smear, and colonoscopy). Initial laboratory evaluation included (reference interval in parentheses)- APTT 52 s (24-34 s), PT 12.1 s (10-13 s). A chest CT angiogram demonstrated large bilateral pulmonary emboli.

Which test, if abnormal on 2 or more occasions greater than 12 weeks apart, could contribute to a diagnosis of antiphospholipid syndrome in this patient?

A.Anti-β2-glycoprotein I IgAB.Anticardiolipin IgAC.CBC with manual differentialD.Dilute Russell Viper venom time (dRVVT)E.Prothrombin time (PT)

Which test, if abnormal on 2 or more occasions greater than 12 weeks apart, could contribute to a diagnosis of antiphospholipid syndrome in this patient?

A.Anti-β2-glycoprotein I IgAB.Anticardiolipin IgAC.CBC with manual differentialD.Dilute Russell Viper venom time (dRVVT)E.Prothrombin time (PT)

Classification Criteria•APS is present if 1+ clinical and 1+ lab criteria met

–Clinical Criteria•Vascular thrombosis

–1+ episodes of arterial, venous, small vessel thrombosis, any tissue or organ.

»Must be confirmed by imaging or histopathology•Pregnancy morbidity

–1+ unexplained deaths of morphologically normal fetus (documented by US or visual examination) at 10 weeks gestation or beyond–1+ premature births of morphologically normal neonate prior to 34 weeks gestation due to

»Eclampsia or severe pre-eclampsia»Placental insufficiency

–3+ unexplained consecutive spontaneous abortions prior to 10 weeks gestation

Classification Criteria

•APS is present if 1+ clinical and 1+ lab criteria met

–Laboratory Criteria•Lupus anticoagulant (LA) detected according to ISTH guidelines on 2+ occasions at least 12 weeks apart•Anticardiolipin (aCL) IgG or IgM detected on 2+ occasions at least 12 weeks apart

–“Medium or High Titer”»>40 GPL or MPL OR»>99th percentile (laboratory-defined)

•Anti-β2-glycoprotein-I (β2GPI) IgG or IgM detected on 2+ occasions at least 12 weeks apart

–>99th percentile (laboratory-defined)

What are the ISTH Guidelines for LA diagnosis?

•Current guidelines developed in 2009–Revision of previous guideline issued in 1995 by Brandt JT et al.

2009 ISTH Guideline- LA Diagnosis

•Choice of Test–Evaluation should include 2 tests based on different principles

•“Risk of false positives is increased to an unacceptable level if more than 2 screening tests are performed.”

–dRVVT should be first test–LA-sensitive APTT should be second test

•Phospholipid poor and silica activator in reagent•Example- hexagonal phospholipid neutralization assay

Other Laboratory Evaluation for Thrombophilia

•Inherited causes–Most common

•Factor V Leiden mutation•Prothrombin G20210A mutation

–Less common•Antithrombin deficiency•Protein C deficiency•Protein S deficiency•Use tests of protein function to screen for these disorders

–PC, AT activity; Free protein S antigen. PS activity also ok.

–Very Rare•Homozygous homocysteinuria•Defects of fibrinolysis

•Which vitamin K dependent protein is not a serine protease?

A.Factor IIB.Factor VIIC.Factor IXD.Protein CE.Protein S

•Which vitamin K dependent protein is not a serine protease?

A.Factor IIB.Factor VIIC.Factor IXD.Protein CE.Protein S Antithrombin (the third

major anticoagulant protein) is also a serine protease.

Endothelium

TMThrombin

APCPCPS

FVIIIa

FVa

Protein C and S Function In Vivo

Protein C and Protein S go together like…

A patient with recurrent episodes of venous and arterial thrombosis was given therapeutic doses of heparin but the heparin failed to exert an anticoagulant effect. The patient probably has a deficiency of:

A.Protein CB.Factor VIIIC.PlasminogenD.AntithrombinE.Platelets

Question Source: www.abpath.org, Clinical Pathology Written Exam sample questions

A patient with recurrent episodes of venous and arterial thrombosis was given therapeutic doses of heparin but the heparin failed to exert an anticoagulant effect. The patient probably has a deficiency of:

A.Protein CB.Factor VIIIC.PlasminogenD.AntithrombinE.Platelets

Question Source: www.abpath.org, Clinical Pathology Written Exam sample questions

How does heparin work?No Heparin

AT

FIIa FXa

Heparin (UFH)

FIIa FXa

ATAT

Heparin (LMWH)

FIIa

FXa

AT ATFondaparinux

FIIa FXa

ATAT

How do we monitor heparin?

•APTT or anti-Xa activity assay•CAP recommendation- APTT therapeutic range should be determined for each instrument/reagent combination

–Compare APTT results to anti-Xa activity in plasmas from patients on heparin–Correlation is not great–APTT therapeutic range corresponds to anti-Xa activity of 0.3-0.7 U/mL

Heparin- Important Complication

•Heparin induced thrombocytopenia (HIT)–Or, why we monitor platelet counts in patients on heparin

•Laboratory testing–Antigen assays

•Detect anti-heparin-PF4 antibodies•Predominantly ELISA-based assays

–Activation assays•Serotonin release assay- gold standard

What about warfarin monitoring?•Monitored with PT

–Significant variability between laboratories and between reagent lots

•INR = international normalized ratio–Attempt to standardize PT values

INR = (PT/MNPT)ISI

Where MNPT is geometric mean of normal PT (determined in a population of normal persons)Geo Mean = (PT1 x PT2 x… PTn)1/nISI is International Sensitivity Index of

thromboplastin (in PT reagent)

In a patient on dabigatran, which assay, if normal, best indicates that no drug is present?

A.APTTB.D-dimerC.dRVVTD.PTE.TT

In a patient on dabigatran, which assay, if normal, best indicates that no drug is present?

A.APTTB.D-dimerC.dRVVTD.PTE.TT

Recently Approved Oral Anticoagulants

•Direct Thrombin Inhibitors –Dabigatran (Pradaxa)- 2010

•Direct Xa Inhibitors–Rivaroxaban (Xarelto)- 2011–Apixaban (Eliquis)- 2014–Edoxaban (Savaysa)- 2015– Betrixaban (Bevyxxa)- 2017

•Term for these drugs as a group- Direct oral anticoagulants (DOAC)

FXI

FIX

FXII

FX

FVII

FII Thrombin

Fibrinogen Fibrin

FVIII

FV

APTTIntrinsic pathway

PTExtrinsic pathway

TT

Coagulation Cascade(In Vitro coagulation)

Dxa•↑ APTT•↑ ↑ PT

DTI•↑ ↑ TT•↑ APTT

First Reversal Agent for Dabigatran FDA approved October 16, 2015• Trade name- Praxbind (idarucizumab)

• Humanized Fab• Binds dabigatran to neutralize effect

• Andexanet alfa (direct Xa inhibitor reversal agent) submitted but not approved by FDA in August, 2016• Clinical trials ongoing

Dabigatran and Rivaroxaban (Apixaban, Edoxaban) Effect on Coagulation Assays

•Drugs act as inhibitors in the laboratory:–Incomplete correction with 1:1 plasma mix–Non specific inhibitor effect in factor assays–Can cause a false positive Bethesda assay

•False positive Lupus Anticoagulant Assays

•Falsely elevated (normal) APCR, protein C clot-based activity, protein S activity, and possibly antithrombin activity (depending on drug and method)

•Dabigatran: Falsely low FXIII activity

•No effect on: D-dimer, VWF assays, free protein S antigen, chromogenic protein C activity, reptilase time, prothrombin G20210A and factor V Leiden mutations

Dabigatran and Rivaroxaban Effect on Coagulation Assays

0

25

50

75

100

125

150

175

200

FII FV FX FVIII FIX FXI AT PC PS APCR

Dabigatran

Rivaroxaban

PC and PS assays: clot-basedAT assays either IIa or Xa based

* Adcock DM, et al. AJCP. 2013;139:102-109; ECAT Proficiency Data 2012.

Laboratory assays

% c

hang

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mpa

red

tore

fere

nce

plas

ma

Questions?karen.moser@health.slu.edu

Good Luck!You can do it!

Additional Resources•The following books have information about hemostasis/thrombosis physiology and disorders with a focus on laboratory testing and issues.

–Bennett ST, Lehman CM, Rodgers GM, eds. Laboratory Hemostasis- A Practical Guide for Pathologists, 2nd ed. New York: Springer, 2015.–Kottke-Marchant K, ed. An Algorithmic Approach to Hemostasis Testing, 2nd ed. Chicago: CAP Press, 2016.–Kumar V, Abbas AK, Aster JC, eds. Robbins and Cotran Pathologic Basis of Disease, 9th ed. Philadelphia: Elsevier, 2015. (Chapters 4 and 14 have pertinent information)–Shaz BH, Hillyer CD, Roshal M, Abrams CS, eds. Transfusion Medicine and Hemostasis- Clinical and Laboratory Aspects, 2nd ed. New York: Elsevier, 2013.

Additional Resources•There are a couple of case study books available that you may also find helpful for self-study.

–Gulati G, Filicko-O’Hara J, Krause JR, eds. Case Studies in Hematology and Coagulation ASCP CaseSet. Chicago: ASCP Press, 2012.–Rodgers GM, Alday AE. Hemostasis Casebook- Lab Diagnosis and Management. Chicago: ASCP Press, 2013.

•Finally, this website provides both clear descriptions of laboratory tests in the hemostasis laboratory and has some helpful data interpretation exercises (basically unknown clinical cases) to test your knowledge. Best of all, access is free!

–practical-haemostasis.com

Image from Gateway Arch (www.gatewayarch.com)

Clinical Chemistry Board Review

T. Scott Isbell PhD, DABCCAssistant Professor of Pathology

Saint Louis University School of Medicine Medical Director of Clinical Chemistry and Point of Care Testing 

ASCP Annual Meeting, September 2017

Disclosure

• Grant/Contracts ‐ None• Scientific/Advisory Board Membership – None• Stocks ‐ None• Salary – None 

Clinical Chemistry

• Very broad subject within clinical pathology

• Area referred to as chemical pathology by ABP

• Represents 22% of the written portion and 37% of the practical portion of the CP exam

• 365 questions on CP exam (Written + Practical) 

• So ~70 chemical pathology questions in total  

Topics (Subsections) Within Chemical Pathology

• Specimen collection and safety• Technical methods• Analytes• Electrolytes, catecholamines, vitamins, porphyrins• Toxicology • Organ system• Endocrine• Reproduction, pregnancy, newborn• Immunology • Lab management

Source: American Board of Pathology website 

Immunology

• Hypersensitivity– Type I (Allergy) – Type II (Cytotoxic, Ab dependent) – Type III (Immune Complex) – Type IV (Delayed‐type, cell‐mediated)

• Autoimmunity • Immunodeficiency 

What makes a good test question in clinical chemistry? 

Questions that test your…1. ability to diagnose/interpret based on laboratory findings and clinical data

2. understanding of the analyte with respect to tissue expression, metabolism/clearance, sub‐cellular localization, etc. 

3. ability to recognize and explain spurious laboratory data 

4. ability to interpret laboratory data, e.g. Hbelectrophoresis, amino acid patterns, SPEP, SFIX

5. ability to perform biochemical calculations6. understanding of methodology/analytical principles 7. ability to detect and understand pre‐analytical errors 8. knowledge about which tests is better diagnostically9. ability to understand current diagnostic guidelines

1. Serum protein electrophoresis demonstrates a restricted band of electrophoretic mobility migrating in the gamma region possibly representing a monoclonal protein. What additional test is required to confirm the presence and type of monoclonoal protein? 

A. ImmunodiffusionB. ImmunofixationC. Serum free light chain quantitation D. Urine protein electrophoresis E. Immunoglobulin quantitation 

monoclonal protein in the gamma region

A B C D E

A = normal serum

B = hypergammaglobulinemia

C = patient

D = patient 

E = patient 

Serum Immunofixation

Good for identification of monoclonal protein, not quantitative, greater analytical sensitivity than protein electrophoresis 

Monoclonal Protein

• Immunoglobulins with a single defined amino acid sequence produced from a single clone of plasma cells 

• Detectable in the laboratory as a discreet band or peak on electrophoresis– Usually appears in the gamma regions (especially IgG)– Sometimes in the beta region (especially IgA) – Rarely in the alpha‐2 region  

• Discreet band is referred to as an M‐spike or M‐protein

Monoclonal Protein

• The M protein can be IgG, IgA, IgM, IgD (rare), or IgE (rare) paired with either kappa or lambda light chains– Most common M‐protein is IgG (55% of patients) followed by IgA (25% of patients) 

– IgM is seen primarily in Waldenström’smacroglobulinemia

• Excessive production and accumulation of IgM monoclonal protein leads to hyperviscosity syndrome

• Sometimes the M protein consist of free light chains only (kappa or lambda). Seen in about 20% of patients. 

2. Urinalysis demonstrates a +2 reaction for blood. Which of the following can be distinguished from hemoglobinuria on a urine dipstick via urine microscopy?

A.MyoglobinuriaB.HematuriaC.Proteinuria D.BilirubinemiaE.Cholesterol 

Question adopted from Quick Compendium Companion for Clinical Pathology. ASCP Press. 2014

Urinalysis • Components 

– Macroscopic – color, turbidity,  

– Microscopic – cells, casts, microorganisms, crystals 

– Biochemical • Glucose • Protein • Bilirubin • pH• Blood• Ketones • Nitrite• Leukocytes

Blood: CHP + TMBZ  H2O + Cumene + Oxidation Dye (Cyan Color)  Reaction catalyzed by peroxidase activity of heme proteins: hemoglobin, myoglobin

Reaction occurs in presence of cell free hemoglobin and in the presence of intact RBCs. Therefore microscopic analysis is required to differentiate between hematuria and hemoglobinuria

Ketones: “ketones” + sodium nitroprusside ketone complex (purple color)Does not detect beta‐hydroxybutyrate! 

3. Which of the following analyte is useful to differentiate between exogenous administration of insulin and endogenous production of insulin?

A. Pro‐insulinB. Pre‐proinsulinC. C‐peptide D. Somatostatin

NT‐Pro‐BNP Has Longer Half‐life than BNP

Proteins exist in different forms with varying utility 

4.Which of the following techniques is most suitable for the detection and quantification of ferric (Fe3+) hemoglobin? A. Arterial blood gas analysisB. Pulse oximetryC. Co‐oximetryD. Peripheral blood smearE. All are equivalent 

Pulse oximetry measures  a patient’s oxygen saturation

Other hemoglobins encountered clinically include HbCO (smoking and exposure) and metHb (acquired vs. congenital) 

Co‐oximetry uses multiple wavelengths of light to detect HbO2, Hb, HbCO, and metHb; performed on blood gas analyzers, handheld pulse co‐ox also available

Methemoglobinemia

Acquired• Drug, toxin, chemical 

mediated oxidation of ferrous iron to ferric iron

• Sulfonamides, lidocaine, aniline derivatives (sulfanilamide), nitrates (nitroglycerin), and nitrites (e.g. poppers: amyl nitrite, isobutyl nitrite)  

Congenital • Mutations leading to 

dysfunctional methemoglobin reductase

• Hemoglobins M– Mutations at the proximal or 

distal histidyl residues in the heme pocket   

5. Carryover is a major concern for most automated clinical chemistry analyzers. Which analyte has the highest likelihood to contribute to carryover? 

A. SodiumB. DigoxinC. Human choriogonadotropinD. Phosphorus E. Phencyclidine

Glycoprotein Hormone Family 

Fig from David Grenache PhD,University of Utah/ARUP 

Tietz Textbook of Clinical Chemistry, 3rd ed, 1998

• Extends functional life of corpus leuteum• Maintains high progesterone concentrations in 

early pregnancy • Measured by immunoassay quantitatively • Measured by immunochromatography qualitatively 

(Point of Care or OTC) 

Look for the analyte that has the highest biological range

A. Pituitary B. AdrenalsC. Hypothalamus D. Gonads 

6. Given the following laboratory data which of the following glands is most likely to be dysfunctional? 

Based on these findings what is the most likely dx? 

Addison’s Disease 

• Aka adrenal insufficiency, aka adrenocortical insufficiency 

• Damage or dysfunction of adrenals  Failure of the adrenal glands to produce steroid hormones– Aldosterone – Cortisol 

• Hyperpigmentation 2/2 increased MSH stimulation of melanocytes (MSH and ACTH derive from a common precursor – POMC)  

Endocrinopathies make good questions…

• What is the best time to collect samples of salivary cortisol to be used as a screening tool for Cushing syndrome? 

• What is the most common cause of congenital adrenal hyperplasia? 

• What two tests are useful for the screening of thyroid dysfunction?

• When is it appropriate to order PTH related peptide? 

Late night, midnight

21‐hydroxylase deficiency 

TSH + Free T4

Elevated iCa2+ with low to normal PTH

7. Measurement of circulating thyroglobulin is a useful biomarker for the follow‐up of differentiated follicular cell derived thyroid carcinoma. What additional tests are required for proper interpretation of circulating thyroglobulin concentrations? 

A.TSH and free T4B.Anti‐thyroglobulin antibodies C.Total T4D.Reverse T3  

Thyroglobulin (Tg) as a Tumor Marker

• Current clinical guidelines consider a serum Tg of >1 ng/mL in an athyrotic individual as suspicious of possible residual or recurrent disease.

• The presence of anti‐thyroglobulin autoantibodies (TgAb), which occur in 15% to 30% of thyroid cancer patients, could lead to misleading Tg results. In immunometric assays, the presence of TgAb can lead to false‐low results; whereas it might lead to false‐high results in competitive assays.

Reference: Mayo Medical Laboratories 

8.Which of the following is diagnostic for diabetes mellitus according to the American Association of Diabetes (ADA)?  

A. HbA1c = 6.0%B. Fasting plasma glucose = 123 mg/dL (6.8 

mmol/L) C. 2 hr plasma glucose =215 mg/dL (11.9 

mmol/L) following a an Oral Glucose Tolerance Test (OGTT)

D. Random plasma glucose = 180 mg/dLE. Post‐prandial serum insulin = 2.6 mcIU/mL

Know your diagnostic criteria 

9. Medium‐chain acyl‐CoA dehydrogenase deficiency (MCADD) is characterized by an increased concentration of which metabolite in newborn screening dried blood spot specimens?

A. C18 acylcarnitineB. C10 acylcarnitineC. C8 acylcarnitineD. C5 dicarboxylic acylcartinineE. C4 acylcarnitine

Beta Oxidation of Fatty Acids

Reference: Nature Education

LC‐MS/MS Acylcarnitineprofile of human plasma 

(C8)

Inborn Errors of Metabolism 

Defects in Carbohydrate Metabolism 

Defects in Amino Acid Metabolism 

Defects in Lipid Metabolism 

Defects in Urea Metabolism 

Defects in Lysosomal Storage 

GlycogenStorage Disease

PKU, MSUD 

MCADD, LCADD

OTC Deficiency 

Gaucher’s, Neimanm‐Picks

10. A 67 year old bipolar female is brought into the emergency room non‐responsive. Labs reveal a sodium of 142 mmol/L, potassium of 3.8 mmol/L, chloride of 115 mmol/L, anion gap  of 1 mmol/L, bicarbonate of 26 mmol/L, creatinine of 1.0 mg/dL, BUN of 18.4 mg/dL, and glucose of 88.2 mg/dL. Measured osmolality was 324 mOsm/kg. What is her osmol gap?

A. 20 mOsm/LB. 25 mOsm/LC. 29 mOsm/LD. 30 mOsm/LE. 32 mOsm/L

Osmolality = a measure of the number of dissolved particles in a solution, expressed as mOsmol/kg. Useful for assessment of water and electrolyte balance (hyponatremia work up). Useful for evaluation of alcohol (ethanol, methanol, ethylene glycol) intoxication 

Calculated Osmolality =  (2*Na+)  + Glucose + BUN18 2.8(mOsmol/kg)

Osmolality is measured by either freezing point depression or by vapor pressure depression 

Osmolal Gap = Measured Osmol – Calculated OsmolGap > 10 mOsmol/kg indicates presence of osmoticallyactive substance in blood   

DDx of Elevated Osmolar Gap 

• Isoproponol• N‐proponol• Propylene glycol • Ethylene glycol • Methanol • Formaldehyde• Mannitol • Diethyl ether ingestion  • Lithium overdose

Propylene glycol

Mannitol 

methanol

isopropanoln‐propanol

ethylene glycol

Laboratory calculations make good questions…

• Creatinine clearance• eGFR• Corrected calcium • Anion Gap (AG)• Estimated Average Glucose (eAG) • Fractional Excretion• Friedewald equation (calculated LDL) • Mean and Coefficient of Variation for replicate measurements

• Henderson‐Hasselbalch Equation 

A 25 year old female with a known history of poly‐substance abuse presents to the ED unconscious, with depressed respirations, and pinpoint pupils. Urine drug screen (UDS) is positive only for THC. Ethanol is not detected. Naloxone is administered IV and the patient responds appropriately. 

11. What is the best explanation for the apparent discordance between the clinical presentation of opioid intoxication and the negative opioid screening by UDS?  

A. Dilute urine B. Interfering substance leading to falsely low signal C. Specimen mix up D. Opioid(s) present in patient’s urine does not react with antibody in UDS

What opioid is most likely responsible for this patient’s intoxication? 

Fentanyl 

M. M

ilone. J M

ed Tox. 2014

12. Which of the following effects on laboratory results occurs during pregnancy?

A. Increases in serum ironB. Transient hypoglycemiaC. Decreases in thyroid hormones T3 and 

T4D. Decrease in cholesterol and 

triglyceridesE. Increase in creatinine clearance

Increased plasma volume 

HemodilutionDecreased proteinconcentration

Increased thyroid hormones, TSH usually suppressed 

Iron and ferritin decreased 

Increased lipids 

Increased CrCl due to enhanced GFR 

Pregnancy, age, sex, exercise, smoking, menstruation, time of day, position (supine, sitting, standing) are all pre‐analytical variables that can impact interpretation of the measurement

13. CA125 is a tumor marker for ovarian cancer. Given that CA125 has a sensitivity of 80% and a specificity of 99% in unselected women, and the prevalence of ovarian cancer is 25 per 100,000 women, calculate the positive predictive value (PPV) and the negative predictive value (NPV) for a CA125 result. Identify the correct statement below.  

A. PPV and NPV are 2% and 99.99%, respectively. This test is useful for screening unselected women. 

B. PPV and NPV are 80% and 99%, respectively. This test is useful for screening unselected women. 

C. PPV and NPV are 80% and 99%, respectively. This test is not useful for screening unselected women. 

D. PPV and NPV cannot be calculated from this dataE. PPV and NPV are 2% and 99.99%, respectively. This test is 

not useful for screening unselected women

How to calculate the PPV and NPV with the information provided 

Ovarian Cancer

No Ovarian Cancer 

Total 

+ Test 

‐ Test 

Total 

Sensitivity = 80% (0.80)Specificity = 99% (0.99)Prevalence = 25/100,000PPV = ? NPV = ? 

PPV = TP/[TP+FP] * 100NPV = TN/[TN+FN]*100 

Low Prevalence of 0.00025

400,000*0.00025 = 100 100 399,900 400,000

(TP) (FP)

(FN) (TN)

Sensitivity (0.80) * 100 = 80Specificity (0.99) * 399,900 = 395,901

80

20 395,901

3,999 4,079

395,921

PPV = 80/[80+3,999] = 0.0196 or 2%NPV = 395,901/[395,901+20] = 0.9999 or 99.99%

13. CA125 is a tumor marker for ovarian cancer. Given that CA125 has a sensitivity of 80% and a specificity of 99% in unselected women, and the prevalence of ovarian cancer is 25 per 100,000 women, calculate the positive predictive value (PPV) and the negative predictive value (NPV) for a CA125 result. Identify the correct statement below.  

A. PPV and NPV are 2% and 99.99%, respectively. This test is useful for screening unselected women. 

B. PPV and NPV are 80% and 99%, respectively. This test is useful for screening unselected women. 

C. PPV and NPV are 80% and 99%, respectively. This test is not useful for screening unselected women. 

D. PPV and NPV cannot be calculated from this dataE. PPV and NPV are 2% and 99.99%, respectively. This test is 

not useful for screening unselected women

Resources 

• From the website– AACC Learning Lab for Laboratory Medicine on NEJM Knowledge+ is an adaptive learning platform that combines gold standard content with the most efficient, engaging, and effective way to help laboratory medicine professionals increase proficiency, expand knowledge, and enhance careers.

– The material covers all disciplines of Laboratory Medicine in 5 program areas:

• Clinical Chemistry• Hematology and Coagulation• Transfusion Medicine• Microbiology• Molecular Diagnostics

scott.isbell@health.slu.edu