When using an automated biochemistry analyzer, we often encounter this situation: a test run on its own gives correct results, but when combined with certain other tests the results become abnormal. The reason is that the analyzer's washing system gradually loses effectiveness as service time accumulates, increasing the degree of reagent cross-contamination.

Cross-interference between reagents on automated biochemistry analyzers affects the accuracy and reliability of test results, introduces large deviations, and can sometimes mislead clinicians and delay diagnosis and treatment.

Avoiding interference between reagents is therefore very important and urgent in routine clinical biochemistry work.

Common Causes of Interference

The reagent contains the substrate to be measured in the next test, or one of its components reacts with the substrate of the next reaction, directly interfering with the next result.

Another cause is that the reaction triggered by the reagent indirectly disturbs the progress of the next test: when reagent contamination is present, the next measurement actually reflects the combined result of two consecutive reactions.

Typical Examples

① pH shift: carry-over between reaction buffers changes the pH of the next reaction, preventing it from reaching optimal conditions.

For example, in the biuret method for serum total protein, with other conditions equal, the reaction requires alkaline conditions (pH 8–9) for protein peptide bonds (—CONH—) to react with alkaline copper solution and form the purple complex, so that total serum protein is fully measured.

If pH < 8, total protein results are falsely low, directly affecting globulin and albumin/globulin results. Enzyme activity assays react fastest and are most sensitive at their optimal pH, but because buffering capacity is limited, carry-over contamination can invalidate enzymatic results.

Most enzyme reactions work at pH 6.0–7.5; ALP, γ-GT and LDH are best under alkaline conditions.

② TG, T-CH, UA and Mg reagents contain bile salts, which cause serious interference with cyclic enzyme assays of bile acids.

③ ALT (IFCC) and AST (IFCC) reagents contain high-activity LD and may interfere with LD measurement.

④ CK (NAC-activated) and CK-MB (NAC-activated) reagents contain Glu; their assay principle includes the hexokinase (HK) reaction of Glu, so they may interfere with Glu measurement — especially serious for Glu measured by the HK method.

⑤ Glu (HK), CK (NAC-activated), CK-MB (NAC-activated), TG, HDL-C and LDL-C (direct method) reagents use magnesium salts, which may interfere with subsequent Mg2+ measurement; the high Cu2+ concentration in TP reagent can also interfere with Mg2+ measurement.

⑥ ALT, AST, LDH, CK, CK-MB, UA, γ-GT and TP reagents contain K+, which shares the same or opposite reaction course as enzymatic K+ measurement, interfering with enzymatic K+ results.

⑦ ChE (butyrylthiocholine), TC (ChOD-PAP), Glu (GOD-PAP), UA (uricase) and α-HBDH (DGKC) reagents use phosphate buffers, which may interfere with inorganic phosphorus measurement.

⑧ The Mg2+ reagent (Calmagite) contains EGTA, a Ca2+ chelator, which may interfere with Ca2+ measurement.

⑨ CK testing should not precede ALP or Ca testing, because EDTA-Na in the CK reaction solution inhibits ALP activity and binds Ca, lowering results.

⑩ Enzymatic BUN measurement consumes NADH via glutamate dehydrogenase (GLDH), so it should not be placed immediately before tests whose substrate is NADH, such as ALT and AST.

Screening Tests

In fact, the package inserts provided by manufacturers often do not fully reflect reagent composition, and the factors influencing biochemical reactions are highly complex.

Discovering possible cross-contamination interference through experiments is therefore even more important.

The following tests can be used to screen for and identify possible interference between reagents:

Countermeasures

Based on the causes of common reagent interference and the screening tests above, targeted measures can be taken to eliminate interference. Common methods include:

Operators should also know the analyzer's working state thoroughly, and consider the possibility of cross-contamination when choosing the tests of one sample or the transition from the last test of one sample to the first of the next, so that results are more reliable.

Common biochemistry tests may be arranged in the following order:

P, Alb, DBIL, TBIL, K, Ca, CK, TBA, TG, AFU, ALT, CK-MB, APOA1, T-CH, APOB, Cl, HDL-C, LP(a), AST, GPDA, AMY, LDL-C, BUN, UA, CHE, ADA, γ-GT, Mg, Na, ALP, LDH, GPDA, TP, Cr.

Closing

With these methods most cross-contamination between reagents can be resolved. In practice, when an automated analyzer runs many tests continuously, the details of each reaction are hard to observe.

We must therefore understand the analyzer's working principle, methods, workflow and state thoroughly, and analyze reagent composition and reaction principles in depth — combining theory with practice to develop procedures suited to our own laboratory.

This article is reproduced from "MiaoBi Biochemistry" for exchange and learning only. Please contact us if any copyright issue arises, and we will handle it promptly.