Monoclonal Protein Interferences in Clinical Chemistry

Dr Sutirtha Chakraborty,

MD (Biochemistry), FACB (USA)

Chief Consultant , Dept. of Biochemistry

Peerless Hospital, Kolkata 1

CASE STUDY

• 58 year old male , known hypertensive on treatment.

• Presents to hospital with progressive fatigue & weakness for the past 2 months.

• Patient also complained of shortness of breath for the past 2 weeks.

• Hb 9.5 gm/dL( RI: 12 – 15)

• Normocytic, Normochromic type

• WBC count(total & differential) –Normal2

• Creatinine 2.05 mg/dl (RI 0.6 – 1.2 mg/dl)

• Urine Albumin(dipstick): Trace

• Serum Sodium 137 mmol/L (RI: 135-145)

• Serum Potassium 3.9 mmol/L (3.5 – 5.3)

Nephrologist Consult:Serum Calcium, Phosphate and Uric Acid

Case Study 1…….Contd.

3

• Serum Calcium : 10 mg/dL (RI: 8.5 – 10.5)

• Serum Uric Acid: 9.0 mg/dL (RI: 3.5 – 7.0)

• Serum Phosphate: 28.4 mg/dl (RI: 2.5 – 4.5)

• Plasma PTH: 22 pg/mL (RI: 15 – 65)

•What could be the cause of extreme hyperphosphatemia in this patient?

4

Spurious Lab Results

• Significant number of laboratory results andreports are factitious or misinterpreted.

• Such laboratory results often lead tounnecessary testing or treatment.

• An observant Laboratory Physician shouldidentify, intervene & investigate the error.

• Spurious/ Factitious results most commonlyoccour due to assay Interference in the“Analytical Phase”

• Major cause of reputational risk for labs.5

Monoclonal Gammopathy

• Expansion of a single Ig-secreting plasma cell population.

• Most cases involve IgG or IgA monoclonal cell populations. About 15-20% are composed of IgMmonoclonal cells.

• Disorders associated with Monoclonal Protein:

MGUS, Multiple Myeloma (MM), Waldenströmmacroglobulinemia (WM), amyloidosis (AL) or other lymphoproliferative disorders.

6

Ig G-Kappa mediated Spurious Hyperphosphatemia

• Loss of linearity

• ? Monoclonal Protein

• Serum Total Protein: 8.1 g/dL, Albumin 3.5 g/dL• Serum Protein Electrophoresis : Sharp Band of restricted mobility in Gamma region

Immunofixation:Positive for Ig G - Kappa

7

8

Interference as a result of Ig G – Kappa monoclonal protein

1 2 3

Image: from Dr Graham Jones, Australia9

Pseudohyperphosphatemia

• The frequency of immunoglobulin-inducedlaboratory errors is variable and probablyunderreported.

• up to 1 in 4 patients with monoclonalgammopathy show interference in PO4 assay.

• Pseudohypophosphatemia can also be seenas a result of such interference.

• Various “wet chemistry” phosphomolybdateassay is vulnerable to this interference.

10

Spurious Hyperbilirubinemia

• Monoclonal protein interferences occur withtotal bilirubin measurements leading to falsehigh results.

• Results typically show T Bil > 15 mg/dL withcorresponding increased Indirect Bilirubin asresult of normal direct Bil.

• Apparently results mimic Hemolytic anemia.

• Common Culprit: Ig G-Lambda , Ig M (in WM)

11

Pantanwitz et al. Arch of Pathology 200412

Ig M interference with HDL Cholesterol Assay

Female, 58 years

Diagnosed with WM

Total C : 202 mg/dL

HDL C: 11 mg/dL

LDL C : 139 mg/dL

TG: 165 mg/dL

VLDL : 52 mg/dL

13

Spuriously Elevated CRP

• CRP > 300 mg/L

• Procalcitonin : 0.07 ng/mL

• Full Blood Counts: Normal

• Blood Cultures: - Ve

• Final Diagnosis:

Light chain myeloma

14

Hypercalcemia & Myeloma

• Ig A Myeloma

• Extensive bone lytic lesions.

• BM Plasma Cells 30 %

• SPE : M band ( 4.8 g/dL)

• Serum Calcium 19.3 mg/dL

(Arsenazo Method)

To treat or not to treat this hypercalcemia ?????????

15

Monoclonal Protein InterferencesDalal et al. AJCP 2009

16

Strategies: What to do?

• Check the serum colour to see if it is truly icteric.

• Spectrophotometric measurement using an Iindex, if available on the autoanalyzer. (LIHindex)

• Rerun the assay to demonstrate any imprecisionbeyond what is typically observed.

• Check for loss of linearity (Serial dilutions)

• Measure the analyte using a different method.

• Always Correlate result with the clinicalinformation.

17

Multi-layer Thin Film Dry Slide Technology

18

Simple techniques for removing Monoclonal Protein Interferences

1) Salting Out Methods2) Precipitation with TCA3) Ultrafiltration4) Dialysis5) Polyethylene Glycol (PEG)

Caution: Make sure that the analyte of interest is insensitive to this procedure. (Works well with Phosphate, Uric Acid, Bilirubin but not with CRP, HDL-C)

19

20

Pseudohyponatremia

• Caused by displacementof serum water byelevated concentrationsof serum lipids orproteins.

• Indirect ISE involvessample dilution and willproduce spuriously lowsodium.

• Occurs when TP > 12g/dL

Fortgens P, Archives of Path & Lab Med ,201121

Monoclonal protein interference in the Preanalytical Phase

Chakraborty et al. CCLM 2014 22

23

Which reagent do you choose ?

• Total Protein Regent 1

• Method: Biuret

• CV% - 1.8 %

• EQAS Outliers - None

• Pack Size – Same

• CPT: Rs 5/test

• Total Protein Regent 2

• Method: Biuret

• CV% - 1.9%

• EQAS Outliers - None

• Pack Size – Same

• CPT: Rs 1.25/test

24

Case Study

• A 50-year-old woman was being treated for sudden-onset sensorineural deafness.

• LFT showed Total protein 10.1 g/dL and albumin was 3.5 g/dL.

• Results Rechecked and released with a comment “Marked A:G ratio reversal noted advised Serum Protein Electrophoresis”

• Subsequent tests requested : SPE, IF, Sr. Calcium

25Chakraborty , Clin Chem 2015

• Normal Electrophoreticpattern with IF negative.

• Dextran is used in the management of sudden hearing loss.

• Dextran Interference on Biuret Assay?????

• So which reagent do we choose?

26

JCLA, DECEMBER 2014

Conclusion:

“The results show that it is possible to use analytical interference for diagnostic purposes, and most importantly, almost all cases were identified at an early stage of the disease, when associated clinical manifestations were not yet observed”.

27

Key Learning Points• Since systemic deproteinisation of serum is no longer applied

in modern assays, interferences caused by monoclonal proteins will continue to occur.

• All chemistry assays in patients with known monoclonal gammopathies should be reviewed.

• Always check Manufacturer Kit Insert for possible interferences.

Awareness is the key!

28

Thank You

29