*Corresponding author: a [dot] albersen [at] lumc [dot] nl
Introduction: Commonly used free thyroxine (FT4) immunoassays can be falsely elevated due to interference causing misinterpreted thyroid function. We present two cases with high FT4 concentrations due to antibody interference. This study’s aim was to investigate the source of the FT4 immunoassay interference and possibility of its removal by two different techniques in order to correct the discrepancy between obtained FT4 values and the patient’s clinical status.
Materials and methods: Two patients presented at their general practitioners’ with elevated FT4 concentrations in combination with a normal and increased thyroid stimulating hormone (TSH) concentrations. Clinical symptoms differed between patients but did not correspond with the hyperthyroid status suggested by the laboratory results. FT4 concentrations from both patients were measured on four common commercial immunoassays and the dialysis method before and after treatment with heterophilic blocking tubes and protein A/G.
Results: Removal of interfering antibodies using protein A/G resulted in normal FT4 concentrations.
Conclusion: This report illustrates falsely elevated FT4 concentrations due to assay interference on the Immulite immunoassay analyser caused by heterophilic antibodies, which were eliminated by protein A/G treatment. We point out the importance of a close collaboration between doctors and the laboratory to avoid unnecessary clinical intervention.
Key words: FT4; thyroid function test; diagnostic errors; interference; immunoassay
In most patients euthyroidism, thyrotoxicosis or hypothyroidism is confirmed with the combination of free thyroxine (FT4) and thyroid stimulating hormone (TSH) measurements. Predominantly, thyroid function tests (TFT) are easily interpreted and correspond with the patient’s clinical status. However, in a limited number of cases the TFT and the clinical impression are discordant. A number of factors, including concurrent (non-thyroidal) illness, medication (e.g. amiodarone, thyroxine) and immunoassay interference, can cause these discrepancies (1-3). These factors should be excluded in order to prevent unnecessary diagnostic and therapeutic interventions (4).
Currently, the competitive immunoassay is a common method to determine FT4 concentrations in diagnostic laboratories. These assays measure the unbound thyroxine fraction whereby the vast excess is protein-bound (> 99.5%). Immunoassay interference can cause false (positive or negative) FT4 results that can lead to misdiagnosis and potentially harmful therapy (5).
Here we describe two patients with a discrepancy between the TFT and the patient’s clinical status. The aim of the study was to investigate the source and possible removal of the FT4 immunoassay interference in these two patients.
Material and methods
A 58-year-old woman presented with fatigue and weight gain at the general practitioner (GP). 14 years earlier, she was diagnosed with Hashimoto’s autoimmune thyroiditis and has been using levothyroxine since. She was evaluated and thyroid function test revealed a markedly elevated FT4 (28 pmol/L; reference range 11–21 pmol/L) with normal TSH (1.5 mU/L; reference range 0.16–4.6 mU/L). Two months later, upon return to the GP, levothyroxine dosage was decreased since she complained about palpations in combination with a still markedly increased FT4 (31 pmol/L) and normal TSH (1.1 mU/L). The GP clinically questioned her hyperthyroid status since she also complained about fatigue and weight gain, not in line with hyperthyroidism. Despite lowering her levothyroxine dosage, FT4 measurements kept increasing further now in combination with an increased TSH of 5.8 mU/L, after which she was referred to a medical specialist who in collaboration with the clinical chemist suspected an interference on the FT4 assay as the culprit (Table 1).
Table 1. Initial levels of TSH and FT4 obtained in patients by respectively Immulite, Cobas and DXi platforms
A 65-year-old male presented with abdominal pain, no change in defecation pattern or weight at the GP. He was using levothyroxine for 10 years after being diagnosed with hypothyroidism. Upon evaluation, his thyroid function tests revealed an increased FT4 (36 pmol/L) and TSH (7.9 mU/L). The GP contacted the endocrinologist and discussed the clinical symptoms in combination with the laboratory results. They concluded that either there was a problem with the patient’s medication compliance or a laboratory discrepancy with the TFT. Upon return to the GP two months later patient’s medication compliance was questioned but found adequate and laboratory was repeated with an increased FT4 (29 pmol/L) and TSH (11.5 mU/L). The GP contacted the clinical chemist since the clinical status of the patient did not match the TFT and interference on the FT4 assay was discovered by repeating the analysis on a different platform (Table 1).
Sera from the patients described were used in this study. Blood was collected in 10 mL serum vacuum tubes with silica clot activator and polymer gel separator: BD SST II Advance Tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Sera were obtained with informed consent from the patients following national and institutional guidelines.
In addition, commercial sera from two patients with a human anti-mouse antibodies (HAMA) concentration of 2464 ng/mL and a rheumatoid factor (RF) concentration of 440 IU/mL were included as known interfering factors for immunoassays (product number 3PH490, lot number SD3158-56 and product number 3PH330, lot number SD3142-31, respectively, from Scantibodies Laboratory, Santee, USA) (4).
FT4 measurements were performed on four different immunoassay analyzers: Cobas Modular E (Roche Diagnostics, Basel, Switzerland), Immulite 2500 (Siemens, Munich, Germany), DXi (Beckman Coulter, Krefeld, Germany) and Vitros ECi (Ortho Clinical Diagnostics, Johnson & Johnson, Beerse, Belgium) and compared to the equilibrium dialysis as described by Docter et al. (7). The reference ranges for FT4 were as follows: for Cobas Modular 12–22 pmol/L, for Immulite 2500 12–23 pmol/L, for DXi 8–14 pmol/L and for Vitros 11–25 pmol/L. The FT4 reference range for the Dialysis method was determined in-house (14–29 pmol/L). FT4 measurements were normalized by dividing the results with the upper limit of normal (ULN) for each assay (FT4/ULN). Figure 1 schematically presents FT4 measurements on each immunoassay analyzer commonly used in routine clinical chemistry laboratories. FT4 measurements on Cobas Modular E, Immulite 2500, and DXi were performed in duplicate, whereas FT4 measurements on Vitros ECi and equilibrium dialysis was done in singlicate. For the duplicate FT4 measurements, intra-assay variation was determined by taking the 95% confidence interval of the standard error based on the difference between these measurements.
Treatment of serum with heterophilic blocking tubes (HBT) (Scantibodies Laboratory, Santee, USA) was performed according to manufacturer’s instructions. The recombinant fusion protein that combines immunoglobulin (Ig) G binding domains of both Protein A and Protein G (protein A/G) agarose beads; Pierce Protein A/G Plus Agarose (Thermo Scientific, Rockford, USA) were washed and incubated at 4 °C overnight with sera. The effect of HBT and protein A/G treatment on IgG and IgM concentrations of the sera was measured using Immulite 2500. Antibodies against thyroglobulin (TG) were determined by a sandwich immunoassay (ImmunoCap 250, Phadia Thermoscientific, Uppsala, Sweden). Presence of thyroid hormone auto-antibodies (THAA) were tested with agar gel electrophoresis (8).
The initial patient FT4 and TSH values were obtained using the Immulite analyzer; subsequently, FT4 and TSH were measured on DXi or DXi and Cobas for patient 1 and 2, respectively. Based on the obtained FT4 concentrations, as presented in Table 1, assay interference was suspected. As interference due to antibody presence was suspected and antibody removal was performed.
To establish a baseline before removal of the antibodies, we measured IgM and IgG in the patient sera in duplicate. IgM (patient 1 = 1.19 g/L and patient 2 = 0.95 g/L) and IgG (patient 1 = 12.8 g/L and patient 2 = 9.9 g/L) were within the reference range of 0.45–2.30 g/L for IgM and 7.0–15.5 g/L for IgG. After either Protein A/G or HBT treatment, IgM and IgG were measured again in duplicate to investigate the effectiveness of antibody removal. Analysis of all sera showed a 40% decrease of IgM concentrations upon HBT treatment whereas no significant effect on the IgG concentrations was detected (Figure 2). Only a quarter of the original IgM concentrations could be detected after protein A/G treatment whereas almost no IgG concentrations were detected (Figure 2).
Figure 2. Measurement of IgM and IgG in patients’ sera and commercial serum with HAMA or RF. Results obtained by untreated sera are shown with black bars, sera treated by HBT with grey bars and sera treated by protein A/G with white bars. Error bars represent standard deviation of the mean
Subsequent analysis of the patient sera showed elevated FT4 with the Immulite assay in comparison with the DXi, Cobas, Vitros and Dialysis method for patient 1 and 2 (Figure 3 A and B). No interference of HAMA (2464 ng/mL) or RF (440 IU/mL) was detected in the FT4 assay performed on 4 different analyzers or using the dialysis method (Figure 3 C and D). Removal of antibodies with protein A/G treatment resulted in lower FT4 concentrations for the Immulite and Cobas assays for sera 1 and 2, whereas no effect or a slight increase was seen in DxI, Vitros and Dialysis methods for these sera. FT4 results after treatment with HBT gave similar results as untreated sera (Figure 3).
Figure 3. Measurements of FT4 by four different analyzers and the dialysis method of patient 1 (A), patient 2 (B), commercial serum with HAMA (C) and commercial serum with RF (D). Results obtained by untreated sera are shown with black bars, sera treated by HBT with grey bars and sera treated by protein A/G with white bars. Error bars represent intra-assay variation.
TSH, total triiodothyronine and total thyroxine showed no aberrant results on the 3 different analyzers, indicating that interfering antibodies do not necessarily give aberrant results on the whole thyroid panel used in current hospital laboratories.
In order to determine the source of the interfering antibody, anti-thyreoglobuline (anti-TG) and THAA were measured in sera 1 and 2. Both samples contained high concentrations of anti-TG (388 U/mL and 66 U/mL for patient 1 and 2, respectively, with a reference value < 40 U/mL) but did not contain THAA. Therefore we speculated anti-TG might interfere with the Immulite assay in particular. However a random selection of 26 sera with high titers of anti-TG showed no discordance between Immulite and Roche FT4 results (data not shown).
After detection of FT4 assay interference in the first patient, levothyroxine therapy was increased to her regular dosage. Upon re-evaluation 3 months later she presented with normal TSH (0.81 mU/L), normal FT4 (13 pmol/L) and clinically euthyroid. In the second patient the interference was discovered in an earlier stage. Upon re-evaluation he presented with normal TSH (2.5 mU/L) and clinically euthyroid. For patients FT4 concentrations were no longer monitored using the Immulite method because of the interference.
In this report we performed a side-by-side comparison of the efficacy of two interfering antibody removal techniques in FT4 assays across multiple immunoassay analyzers. We show that protein A/G treatment prior to analysis performed superiorly in comparison with the commonly used HBT treatment to remove interfering antibodies. Our data demonstrates its effectiveness for the first time by IgM and IgG measurement before and after protein A/G treatment.
This study showed that in the Immulite immunoassay discrepant FT4 results in two patients were due to interfering antibodies. Several interferences of the FT4 assay have been described including heterophilic antibodies (4), human anti mouse antibodies (HAMA) (4), thyroid hormone auto-antibodies (THAA), albumin variants (familial dysalbuminaemic hyperthyroxinaemia) and heparin use (9-14). Since patients did not use heparin it was postulated that an antibody caused the grossly elevated FT4 results on the Immulite 2500 in comparison with other analyzers and equilibrium dialysis. Several different immunoassay tests have been reported to give aberrant results when interfering antibodies are present (15-18). Antibody interference was eliminated by treatment with protein A/G sepharose but not with HBT. Furthermore, we have demonstrated that protein A/G treatment has little effect on FT4 immunoassay measurement.
When FT4 immunoassay interference is suspected, retesting with a different commercial assay is a useful strategy (18,19). However, comparison of FT4 results between different methods can be challenging (20,21). One should be aware of these differences between laboratory methods and use the appropriate reference intervals when interpreting results.
Different strategies have been reported for removal of interfering antibodies (1,4,10,22). Upon treatment of the sera we were able to partially remove IgM antibodies with HBT treatment whereas Protein A/G removes both IgM partially and IgG completely. The latter treatment led to normal FT4 concentrations on the Immulite assay comparable to concentrations found using DXi, Cobas, Vitros and dialysis methods. We chose to treat with protein A/G instead of polyethylene glycol precipitation (PEG) because of the specific ability of protein A/G to bind and remove only immunoglobulins. Known disadvantages for PEG treatment are the non-specific nature causing precipitation of proteins and ability to interfere with immunoassays (23). Since the protein A/G treatment corrected the FT4 results on the Immulite assay, we speculated THAA might be the culprit. THAA have been reported to be of the IgG class, which is almost completely removed by protein A/G treatment (24). However no THAA were detected in our sera. Since THAA coexists with antibodies to thyroglobulin, we screened an additional 26 sera on Immulite and Cobas but no discrepancies were found. We speculate that an antibody interferes with the Immulite assay since partial IgM and near complete IgG antibodies removal corrected the high results on the Immulite assay. Since none of the other assays are influenced by this antibody we postulate two possible hypotheses why the Immulite assay (one-step method) produced aberrant high FT4 results. Firstly, the interfering antibodies interacts with the capture antibodies and somehow prevents tracer, but not FT4 binding. Antibodies in the remaining assays differ and therefore do not interact with the interfering substance. The exact binding properties of the antibodies used are proprietary information and therefore could not be compared to possibly identify the source of interference. Secondly, the interfering antibody binds the tracer directly and prevents the tracer from binding to the capture antibody. Therefore, interfering antibody-tracer complex is washed away resulting in falsely elevated FT4 result (competitive immunoassay). This is only possible in a one-step assay since the interfering substance remains present during the entire procedure. The other one-step immunoassay, Cobas Modular E, has two consecutive incubations. Firstly, serum and capture antibody are incubated followed by a second incubation whereby tracer is added. The test is completed by a wash step. This assay is possibly also affected by the interfering antibody whereby protein A/G treatment reduces FT4 concentrations. The DXi method is a two-step assay with a separate washing step that potentially removes the interfering substance before analysis (Figure 1) and consequently avoiding direct contact with the tracer. The Vitros method uses a one-step principle like the Immulite assay but interference is seen only with the Immulite. Our hypothesis is that the antibody that interferes in the Immulite and Cobas assays is somehow blocked by the blocking agents used in the Vitros assay or the labeled anti-T4 antibody is modified in such a way that it is less likely to cause aberrant results by antibodies present in human serum. Since the dialysis method is independent of binding proteins and no antibodies are used the interfering immunoglobulin will not alter the results of the dialysis FT4 measurement.
HBT treatment might be an effective procedure in routine laboratories in case immunoassays results are suspected to be influenced by interfering antibodies (25,26). However, most commercial immunoassays already use blocking agents. This possibly explains the fact no differences were observed in HBT treated and untreated HAMA serum (Figure 2C).
Looking for the source of the interference we included sera that contained HAMA and RF antibodies. These have been reported to interfere with hormone assays (1) but in our study we did not find any effect on the FT4 measurements on the Immulite Modular, DXi, Vitros analyzers or the dialysis method.
In conclusion the interfering antibody in this study is only able to disturb the FT4 results in the Immulite assay. By comparing FT4 result of different commercial immunoassays interference was confirmed. HBT were not able to remove the interfering substance judging by the FT4 levels above the reference levels after treatment in the Immulite assay. After protein A/G treatment the FT4 results dropped to levels within reference, comparable to those measured with the other methods. Leading to the conclusion that protein A/G treatment effectively removes the interfering antibody. In addition this study shows the importance of proper collaboration between a doctor and clinical chemist when laboratory results are not in line with the clinical presentation.
We would like to thank Hans van Toor, Johan Tak, Heleen Angenent, Ton Kerdel, Rene Ysselstijn for technical assistance and Erwin Birnie for statistical advice.
Potential conflict of interest
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Goce Dimeski*, Kendra Bassett, Nigel Brown.Paraprotein interference with turbidimetric gentamicin assay. Biochemia Medica 2015;25(1):117-24 http://dx.doi.org/10.11613/BM.2015.014
Department of Chemical Pathology, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Australia
*Corresponding author: goce_dimeski [at] health [dot] qld [dot] gov [dot] au
Introduction: Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays.
Materials and methods: A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser.
Results: The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery.
Conclusion: IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol.
Key words: turbidimetry; IgM; paraprotein; interference
Gentamicin belongs to the aminoglycosides group of antibiotics which are among the oldest antibiotics available to treat serious infections caused primarily by gram-negative bacteria. When the use of aminoglycosides became more widespread, the toxic effects, ototoxicity and nephrotoxicity, became more apparent and there was a switch to other, safer, antimicrobial agents, and the use of aminoglycosides sharply declined. However, the development of multi-drug resistance among bacteria has now led to resurgent use of the aminoglycosides in the treatment of serious infections. Not only does gentamicin offer comparable low levels of resistance but it is rapid in its bactericidal activity. However due to toxicity, there is a need to monitor plasma concentrations to prevent the rare occurrence of sudden idiosyncratic deafness and nephrotoxicity with prolong therapy (1,2). Even though guidelines exist for monitoring plasma concentrations (3) a recent study reported 20% of collected samples were outside the required sampling window (6 -14 hours post dose), and 15% of doses were adjusted without monitoring and approximately half of all dose adjustments were based on inadequate information or inaccurate nomogram interpretation (4).
Serum protein abnormalities have been shown to interfere with turbidimetric assays such as vancomicin on the Beckman DxC800 general chemistry analyzer (5). Interferences have also been reported with other turbidimetric assays, C-reactive protein (6,7) phenytoin (8), and transferrin (9), as well as with nephelometric assays, IgA and IgG (10), and other non turbidimetric/nephelometric assays such as total bilirubin (11), thyroid-stimulating hormone (12), lactate dehydrogenase, uric acid and alkaline phosphatase (13), glucose and gamma-glutamyl transferase (14), HDL-cholesterol and as well as glucose interference being observed with glucose analyses on a hexokinase method but not on an oxidase method (15). From our searches no such interferences have been reported with gentamicin.
Materials and methods
A 93 year old female with severe dementia presented with leg cellulitis and sepsis and was administered 320 mg gentamicin (Pfizer, Perth, Australia) at one of our smaller hospitals. No further gentamicin was administered during her hospital stay. On presentation the patient’s sample was analysed on a Beckman DxC600 general chemistry analyser (Beckman Coulter, Brea, CA, USA) as per Beckman Coulter recommendations and the results were: creatinine 177 µmol/L (reference range (RR) 46-108), urea 12.0 mmol/L (RR 2.9-8.2), total protein 63 g/L (RR 60-83), albumin 22 g/L (RR 35-50), and globulins 41 g/L (RR 25-45). A blood sample was collected ~40 hours post gentamicin administration and the laboratory could not obtain a result due to persistent high blank absorbance errors on the Beckman DxC600 general chemistry analyzer (Beckman Coulter, Brea, CA, USA). The sample was diluted with normal saline with ratios starting from 1/3 and going as high as 1/20. The sample was then referred to our laboratory and dilutions were repeated on a Beckman DxC800 general chemistry analyzer using the exact same method. The absorbance error could not be eliminated to obtain a result as shown by the absorbance curves in Figure 1.
Figure 1. Reaction absorbance curves for 1. Gentamicin: A- normal sample, B -case study neat sample, C-case study 1/20 diluted sample; 2. Vancomicin: D- normal sample, E- case study neat sample; 3. Valproate: F- normal sample, G- case study neat sample.
A second sample was then collected at ~50 hours post gentamicin administration and this was dispatched for analysis on a Roche Cobas system (Roche Diagnostics, Mannheim, Germany) and the result was 3.3 mg/L. No further gentamicin was administered. Review of the absorbance curves suggested an interference related problem. Although the total protein level did not suggest presence of paraproteins the globulins level was high enough not to rule out the presence of paraproteins. The sample was then diluted off board with the reagent A (reaction buffer) in the ratio used in the method and no precipitation was observed. Unfortunately by this stage due to limited volume no further tests could be conducted.
A third sample was received at ~107 hours post gentamicin administration and the gentamicin result on the Roche Cobas method was 1.2 mg/L. The remaining portion of this sample was analysed for all the turbidimetric assays utilised on the Beckman DxC800: C-RP, carbamazepine, digoxin, haptoglobin, phenytoin, transferrin, tobramycin, theophylline, valproate, vancomicin, to see if any of these would exhibit any limitations. Like the gentamicin method all these methods are particle enhanced turbidimetric inhibition immunoassay methods. Only vancomicin and valproate showed high blank absorbance readings. Additionally the sample was analysed for immunoglobulins (rheumatoid factor, IgA, IgG and IgM) by nephelometry on a BNTM II System (Siemens Diagnostic, Deerfield, IL, USA) as per manufacturer recommendations.
To determine if a suitable precipitation method could be implemented to provide more rapid results the Beckman calibrator sets for each of the three assays were precipitated to ensure accuracy is maintained post recovery from these known concentrations. We then precipitated 10 patient samples routinely requested independently for gentamicin, vancomicin and valproate were selected covering the broadest possible concentration range that is encountered for these analytes. The patient samples were precipitated by both 100% ethanol and polyethylene glycol (PEG), 24 g/100 mL (Fluka#812160, polyethylene glycol 6000, Sigma Aldrich) separately. Both of these reagents are commonly available in most laboratories. Based on the chemical structure of gentamicin (made up of amino groups attached to glycosides), and vancomicin (a tricyclic glycopeptide made up of glycans covalently attached to the side chains of the amino acid residues) it was possible PEG would not be suitable as it could precipitate these antibiotics, hence ethanol was considered as a milder alternative. The samples were processed in a single batch for each of the three analytes. Samples were mixed with equal volumes of the ethanol or PEG solution (0.25 mL : 0.25 mL), vortex mixed, than centrifuged (10 min, 3000 x g ), and the supernatant removed and analysed. For each sample the reaction curve was checked that it was normal to ensure there was no paraprotein interference.
Statistical analysis was performed using the Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and Analyse-It statistical add-on for Microsoft Excel (Analyse-It Software, Leeds, UK) for the Passing Bablok regression analysis. Recovery was calculated by subtracting the neat sample result from the post precipitation result, and the difference was then divided by the neat result and multiplied by 100% to provide the recovery percentage.
Besides the gentamicin only vancomicin and valproate were affected as shown from the absorbance plots, Figure 1. The patient medical record indicated the patient was not on vancomicin or valproate. Comparing the reaction curve with the normal sample reaction curve it was clear the precipitation in the gentamicin assay was occurring upon addition of the reactive reagents (reagent B and C). The vancomicin and valproate plots equally showed the precipitation was observed upon addition of the reactive reagents (vancomicin has reagent B and C and valproate only has reagent C) (Figure 1E and G).
The immunoglobulin analysis showed the following results: rheumatoid factor < 20 IU/mL (RR < 20); IgG 2.39 g/L (RR 7.0-16.0); IgA 2.56 g/L (RR 1.0-4.0); and IgM 18.9 g/L (RR 0.4-2.3). The results confirmed the Sia test findings that the absorbance error was induced by the paraprotein (IgM) precipitation (16).
The Passing Bablok regression correlation results, concentration ranges and recovery data is shown in Table 1. The PEG precipitation produced unsuitable recoveries (mean recovery of <30%) for the three analytes. Using the internal quality control performance data (two quality control levels per each assay) the mean CVs at two standard deviations for each of the assays were: a) gentamicin 15.2%, b) vancomicin 12.6% and c) valproate 17.8%. Hence, the ideal recovery for each assay should have been: a) gentamicin 84.8-115.2%, b) vancomicin 87.4-112.6% and c) valproate 82.3-117.8%.
Table 1a. The Passing Bablok regression analysis of concentration ranges and recovery results for the Beckman calibrators treated by 100% ethanol precipitation versus stated concentrations.
Table 1b. Comparison of drug concentrations (Passing Bablok regression analysis) measured in native patient samples and samples treated by 100% ethanol precipitation, N = 10.
From our searches, no interference has ever been reported with gentamicin or valproate turbidimetric assays by a paraprotein. Availability of absorbance curves is a powerful tool to highlighting potential assay problems. What the absorbance curves indicated was the precipitation/turbidity was occurring in the latter part of the reaction process, the non-blanking phase, upon addition of the reactive reagents hence for the high absorbance errors. As King et al. also highlight, the reverse would occur if the precipitation/turbidity is occurring in the blanking stage, and would lead to low absorbance errors, low results. This has been reported with vancomicin by previous publications (5,17,18).
Closer scrutiny of the proteins results, globulins specifically on presentation along with the age of the patient provided a trigger for further investigation, potential presence of a paraprotein. Although IgG is the most common paraprotein (~59-70%), followed by IgM (~17%) and IgA (~11-17%) (19,20). These paraprotein producing disorders increase with age rising from 3.2% in people < 50 years of age to 5.3% in people > 70 years of age (19), 4.5% in the population in the 45-75 years of age (20). Hence the reported paraprotein interference problems are in older patients as is the case here and from review of literature IgM is most frequently reported to be the cause of interference by turbidity or precipitation. This in turn was the reason we initiated testing of all the turbidimetric assays for potential interference.
The Beckman method inserts state gentamicin, vancomicin and valproate were tested with IgM concentration up to 5 g/L without effect. In our experience the IgM concentration level is not the sole determinant for precipitation and subsequent interference. Precipitation occurs as a result of physicochemical conditions (pH, ionic strength, presence of surfactants and other chemicals in the reagents) being in the right balance, where the pH and the isoelectric point being the same and the protein charges being neutralised. This balance or uniqueness to achieve precipitation can be due to the IgM type (lambda or kappa), assay reagents or they can be influenced by other compounds like heparin (14). Ideally manufacturers should test for IgM interference with much higher concentration levels e.g. > 15 g/L, specifically with turbidimetric assays in order to better challenge the method.
Review of the Beckman method inserts of the tested turbidimetric assays does not provide ability to try and extrapolate as to why only three of the assays exhibited interference. The inserts do not contain data on the reaction buffer used (reagent A), its type or the pH and only minimal data, antibody type only on the reactive reagent(s) (reagent B or C, or B and C) being used. It is assumed the pH of the reactive reagents in these assays was sufficient to achieve the appropriate pI and cause the precipitation. The Roche gentamicin method showed no interference and this was most likely due to the analytical method difference, fluorescence polarization type rather than turbidimetric as is the Beckman method.
The available option for laboratories in obtaining an accurately representative result is predominantly to analyse samples on an alternative system/method which is not always easily accessible. Precipitation of proteins along with the interfering protein while retaining the analyte of interest in the supernatant is an alternative option. Precipitation can depend on the analyte chemical composition, and choice of precipitant and its availability. In general precipitation is most successful with inorganic compounds e.g. digoxin (21). With gentamicin and vancomicin containing amino acids ethanol was shown to be a suitable precipitant to obtain desirable recoveries with the three analytes affected in this case. The use of filtration methods is another option but they are only readily available in a few large laboratories.
Incidences or encounters like this can be a trigger to identifying the presence of unidentified pathological abnormalities and there is a need to immediately communicate to clinical staff for best patient care as was done in this case. A limitation of this study was that due to insufficient patient samples the type of IgM was not determined and neither was precipitation performed for the gentamicin to compare results to the Roche system. A second limitation was the small number (10 patient samples) tested without paraprotein interference, and the larger number of samples could also lead to improvement in the recovery.
Not just gentamicin but vancomicin and valproate are also associated with potential toxicity and the plasma levels need to be monitored. Being able to measure analytes’ concentrations and provide results both timely and accurately is the key to the existence of pathology. Having options or tools to overcome adversity, namely interferences, in house goes a long way to ensuring laboratory services are prompt and subsequent patient care decisions are optimised. When interference is detected with one analyte it is always valuable to run such sample on assays using same analytical techniques as the minimum, specifically for immunoassay based techniques.
In summary, the findings from this case showed for the first time IgM interference with gentamicin, valproate and vancomicin where the interference led to high absorbance which has never been reported with these analytes. Equally, it was shown ethanol can be used to precipitate proteins and produce acceptable recovery results. This allows all laboratories to use this technique to overcome such interferences.
Potential conflict of interest
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Şerif Ercan*1, Mustafa Çalışkan2, Erhan Koptur3. 70-year old female patient with mismatch between hematocrit and hemoglobin values: the effects of cold agglutinin on complete blood count. Biochemia Medica 2014;24(3):391-5. http://dx.doi.org/10.11613/BM.2014.042
1Department of Clinical Biochemistry, Lüleburgaz State Hospital, Kırklareli, Turkey
2Departments of Clinical Microbiology, Lüleburgaz State Hospital, Kırklareli, Turkey
3Home Health Services, Lüleburgaz State Hospital, Kırklareli, Turkey
*Corresponding author: serifercan [at] yahoo [dot] com [dot] tr
Introduction: There are a number of pre-analytical and analytical factors, which cause false results in the complete blood count. The present case identifies cold agglutinins as the cause for the mismatch between hematocrit and hemoglobin values.
Materials and methods: 70-year old female patient had a history of cerebrovascular diseases and rheumatoid arthritis. During routine laboratory examination, the patient had normal leukocyte and platelet counts; however, the hemoglobin (Hb: 105 g/L) and hematocrit (HCT: 0.214 L/L) results were discordant. Hemolysis, lipemia and cold agglutinin were evaluated as possible reasons for the mismatch between hematocrit and hemoglobin values.
Results: First blood sample was slightly hemolysed. Redrawn sample without hemolysis or lipemia was analyzed but the mismatch became even more distinct (Hb: 104 g/L and HCT: 0.08 L/L). In this sample, the titration of the cold agglutinin was determined and found to be positive at 1:64 dilution ratios. After an incubation of the sample at 37°C for 2 hours, reversibility of agglutination was observed.
Conclusion: We conclude that cold agglutinins may interfere with the analysis of erythrocyte and erythrocyte-related parameters (HCT, MCV, MCH and MCHC); however, Hb, leukocyte and platelet counts are not affected.
Key words: complete blood count; cold agglutinin; hematocrit; hemoglobin; interference
The use of automated hematology analyzers to examine complete blood count (CBC) is very common. The CBC is applied to diagnose anemia, to identify acute and chronic illness, bleeding tendencies and white blood cell disorders by both general practitioners and other medical specialties. There are a number of pre-analytical and analytical factors that cause false CBC results. Red blood cell (RBC) counts, hemoglobin (Hb), mean cell volume (MCV), white blood cell (WBC) count and platelet (PLT) count are parameters measured by hematology analyzers that are affected by spurious values in several situations. These situations commonly include the agglutination in the presence of ethylenediamine tetra-acetic acid (EDTA), insufficiently lysed RBC, erythroblast, platelet aggregates, cryoglobulins, agglutinins, lipids, hemolysis, and elevated WBC counts (1).
In the present case, we evaluated cold agglutinins as a reason for the mismatch between hematocrit and hemoglobin values. There are several case studies associated with cold agglutinins; however, these studies have been generally presented in a clinical perspective. Laboratory findings were also not sufficiently available in these studies. Only few cases have recently drawn attention to the importance of pre-analytical affect of cold agglutinins on CBC results (2,3). To our knowledge, the present study is also a unique case report of spurious CBC results associated with cold agglutinins using the ABX Pentra 80 (Horiba Medical, Montpellier, France) hematology analyzer.
Materials and methods
An anticoagulated blood sample (K3EDTA, 2 mL, Golden Vac-Tube, GongDong Medical Technology Co., Zhejiang, China) drawn from a 70-year old female patient was analyzed during routine laboratory examination. Patient had WBC 7.1 x 109/L and PLT count 327 x 109/L; however, hemoglobin (Hb: 105 g/L) and hematocrit (HCT: 0.214 L/L) results were discordant [if Hb value (mg/dL) is multiplied by three, it gives ± 3 HCT value (%)], as shown in Table 1 (Sample 1). Blood samples have been transferred from patient’s home to our laboratory after routine examination by a family physician. Patient had a history of cerebrovascular diseases and rheumatoid arthritis.
We first suspected hemolysis. When the tube with K3EDTA was centrifuged at 500 x g for 3 minutes, slight hemolysis appeared. Thus, to rule out problems with blood sample drawing, the physician and phlebotomist were informed to redraw the tube with K3EDTA. One tube with K3EDTA was drawn by home health services in non-fasting state and a CBC was run, but the results were not corrected; in fact, the mismatch became even more distinct (Hb: 104 g/L and HCT: 0.08 L/L) (Sample 2 in Table 1). Moreover, the sample had not hemolysis or lipemia. We then suspected cold agglutinin. In this sample, the titer of cold agglutinin was determined and a peripheral blood smear was also prepared.
The determination of cold agglutinin titer
The titer of cold agglutinin was determined according to a previously described method (4). Ten tubes were prepared to determine the titration of cold agglutinins. 1.5 mL of physiologic saline was placed in tube 1 and 1.0 mL of physiologic saline in tubes 2-10. 0.5 mL of patient’s serum was added to tube 1, mixed and 1 mL of the mix was transferred to tube 2, from which 1.0 mL was then transferred to tube 3; the process was reiterated until tube 9 (tube 10 served as a cell control). By this method, dilutions from 1:4 to 1:1024 were obtained. 0.1 mL of patient’s own erythrocyte suspension (2-5%) was added to each tube, and the contents were mixed by vigorously shaking and then placed in racks at 4 °C overnight. Tubes were removed from the refrigerator and read immediately. Readings were made by shaking the tube 3-5 times, firmly enough to make a silk-like suspension in negative tests. The agglutination degree was recorded as 1+ to 5+, 1+ representing just visible agglutination and 5+, a solid clump of cells. After reading, tubes were placed at 37 °C for 2 hours and re-visualized.
The titration of the cold agglutinin was found to be 4+ at 1:4 dilution ratio and 1+ at 1:64 dilution ratios. After incubation at 37 °C for 2 hours, reversibility of agglutination was observed. Thus, agglutination was confirmed as cold agglutinin.
As shown in Figure 1A, when the blood smear was examined under the microscope, clusters of erythrocytes were observed in each field.
After obtaining these findings, an expert phlebotomist and specialist of medical biochemistry visited the patient to draw blood at her home. The drawn specimen in non-fasting state was immediately transferred to the laboratory. During transfer, the specimen had not been exposed to the cold. The flat plastic container (500 mL) had been filled with water warmed to approximately 40 °C. The specimen had been placed in this hot plastic container and transferred inside the Styrofoam box. The K3EDTA tube was immediately run on the hematology analyzer. It seemed that all the CBC results were valid (Sample 3 in Table 1). Clusters of erythrocyte were not observed in the blood smear prepared from this sample (Figure 1B). Subsequently, this specimen was placed at 4 °C for 30 min, and re-analyzed. Although leukocyte and platelet values were not changed, a mismatch between HCT and Hb was again observed (Sample 3a in Table 1). The specimen was then stored at room temperature for 1-hour and re-analyzed, but the results remained the same (Sample 3b in Table 1).
Table 1. The results of CBC analysis in different samples.
Figure 1. Peripheral blood smear, May-Grunwald-Giemsa stain, 1000x. A: Redrawn first sample with clusters of erythrocyte, B: Sample transferred without exposition to cold.ples.
Cold agglutinins are antibodies that are usually specific for I antigen, an erythrocyte surface carbohydrate macromolecule. Cold agglutinins bind to the erythrocyte surface antigen at a temperature optimum of 0-4 °C, which causes agglutination of erythrocytes and, thereby, impaired microcirculation ranging from moderate acrocyanosis to severe Raynaud’s phenomenon (5). The antigen-antibody complex also induces the classical complement pathway, resulting in extravascular hemolysis. The physiologic cooling of blood in peripheral vessels is usually sufficient to cause hemolysis and circulatory symptoms in patients with high thermal amplitude of cold agglutinins (6). The clinical effects of cold agglutinins are dependent on both the titer and the thermal amplitude of antibody (7).
Cold agglutinins are rare and only low titers can be found in the serum of healthy individuals. Cold agglutinins may be monoclonal or polyclonal. Monoclonal antibodies are usually found in patients with idiopathic forms of cold agglutinin disease or lymphoproliferative disorders. Polyclonal antibodies usually appear after infection, most often with Mycoplasma pneumonia, Ebstein-Barr virus or cytomegalovirus (7).
Cold agglutinins can cause hemolysis in patients undergoing cardiac surgery during a hypothermic cardiopulmonary bypass. Screening for cold agglutinin in all patients is not recommended; however, it can be useful in specific patient groups (8).
In the presence of cold agglutinins, we found decreased HCT and elevated MCV values. Hemoglobin concentration was unaffected by cold agglutinins, thereby, calculated MCH and MCHC values were prominently elevated. Leukocyte and platelet counts were also found to be unaffected by cold agglutinins. In the present case, the analysis of CBC was performed on ABX Pentra 80 hematology analyzer. RBC is measured by an electronic impedance variation principle on ABX Pentra 80. MCV and HCT are calculated directly from the RBC histogram. The impedance method of counting and sizing cells is based on measurable changes in electrical resistance produced by non-conductive cells suspended in conductive isotonic electrolyte solution. This electrical resistance is manifested as a pulse that has a height directly proportional to the cell size. Red blood cells, platelets and leukocytes are counted and sized according to the total number and heights of produced pulses (9). Cells between 30 to 300 fL are considered and counted as red blood cells on Horiba ABX Pentra. The microaggregates of erythrocyte are counted as single cells and large clumps of cells are excluded from the count, which lead to decreased RBC count (9). These abnormal RBC values will lead to abnormal MCV and HCT results.
We met with the present case in winter. Therefore, the cold agglutinin forming might be the result of the fact that blood samples were exposed to cold weather during transfer from patient’s home to our laboratory. When the patient’s previous results were reviewed, CBC results were found to be valid. This is most likely due to drawing the blood samples at the hospital, without exposure to cold. In addition, we found that CBC results were valid if the blood drawn from the patient was immediately transferred to the laboratory without being exposed to cold.
The interference of cold agglutinin on RBC and HCT results has previously been reported using an older automated hematology analyzer (10,11). It is seems that cold agglutinins can still cause artifactual changes on RBC and related parameters. This might be due to the counting of RBC by the impedance method, which is widespread among hematology analyzers produced different manufactures. In accordance with our case report, Kakkar (2) revealed spurious CBC results due to cold agglutinins on Advia 60 (Bayer diagnostics) hematology analyzer. In his report of two cases, he reported HCT to be spuriously low and discordant with Hb. MCV, MCH and MCHC values have also been found to be elevated.
Similarly, Breuer et al. (3) reported four patients with cold agglutinins whose RBC parameters described were incompatible and spurious. They showed that cold agglutinins caused artificially decreased red blood cell counts and increased MCV and MCHC in the automated Coulter STKS hematology analyzer. It was also noted that leukocyte and platelet counts remained unchanged. In a more recent case, Reddy et al. (12) reported elevated MCHC and MCV in a 12-year-old girl with mixed autoimmune hemolytic anemia.
The in vitro phenomenon of cold agglutination results in a spurious increase in MCV and MCHC and a decrease in the RBC count given by automated hematology analyzer; however, different findings have been reported in some clinical case reports.
In a 55-year old woman with a history of rheumatoid arthritis, Bizzaro et al. (13) presented in vitro platelet clumping due to EDTA-dependent autoantibodies and RBC agglutination due to cold agglutinins. Although they observed elevated MCHC in laboratory examination, the MCV value was reported as normal. In another study, Lee et al. (14) identified cold agglutinin in a case of 75-year old woman patient with non-Hodgkin’s lymphoma. They identified RBC agglutination related to cold agglutinins; however, a normal MCHC value was reported. Laboratory methodology and findings were, unfortunately, not obtained from these studies. Therefore, a satisfactory comparison could not be done.
In conclusion, cold agglutinins interfere with the analysis of RBC and RBC-related parameters (HCT, MCV, MCH and MCHC); however, Hb, WBC and PLT counts are not affected. In addition, the presence of cold agglutinins is easily displayed by the cold agglutinin titer test. It is also visible in a blood smear. Also, the effect of cold agglutinin on CBC results may be prevented if the blood drawn from the patient is immediately transferred to the laboratory without exposure to cold. Lastly, clinicians should be aware of the fact that anemia diagnosis based on low HCT alone may be misleading.
We thank Ercan Güneş, Adem Taka, and Pervin Köksal for their technical assistance. We thank Deniz İnci for helping to take the images of blood smears.
Potential conflict of interest
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Blood collection device history
Blood collection tubes
Clot activators and water-soluble agents
Order of draw
Original scientific paper: