Study of Interference Produced by Haemolysis In 73 Analytical Tests

One of the main objectives of a clinical laboratory is to inform
exact and accurate values, since the results of the tests requested to
the laboratory influence between 60-70% of medical decisions..


Introduction
One of the main objectives of a clinical laboratory is to inform exact and accurate values, since the results of the tests requested to the laboratory influence between 60-70% of medical decisions [1].
The presence of interferences, whether endogenous or exogenous, may produce erroneous results in a laboratory report that lead to a wrong diagnosis and even harmful clinical decisions. Among the possible endogenous interferences that may exist, hemolysis is one of the most important and in the main cause of preanalytical rejection of samples. The prevalence in our hospital (Getafe University Hospital, Madrid, Spain) in 2018 was 2.63% considering as hemolyzed serum those with hemolytic index ≥30mg/dL [2][3][4].
Hemolysis is a phenomenon that can take place both in vitro and in vivo [5]. In vitro hemolysis occurs when the red blood cells breaks in the blood sample, releasing its content to the blood. This process is called interference, and it could appear in every stage of the preanalytical process (from a wrong extraction to a wrong The interference produced by hemolysis could be because of different mechanisms [2,5]:

1.
Overlap in the measurement of the absorption spectrum (the released hemoglobin has high absorbance between 400-600nm).

4.
By dilution of the sample components (albumin) [6]. report, the distinction between analytically significant interference and clinically significant interference must always be established.
"Clinically significant interference" is defined as those interferences that generally lead to significant errors in the interpretation of laboratory results [9,10]. The objective of this study is to establish the limits in which hemolysis produces a clinically significant interference and can lead to confusion in the interpretation of results as well as fails in patient safety.

Material and Methods
The analytical procedure was performed based on the protocol published by the Spanish Society of Laboratory Medicine (SEQCml): "Procedure for the study of interference by hemolysis, bilirubin and turbidity and for the verification of hemolysis, jaundice and lipemia indices" [11]. To carry out the interfering hemolysate, blood The techniques analyzed were: 1.
In the ISE modules the ions are analyzed: chlorine, sodium and potassium (K).

2.
Through photometric techniques were analyzed: To determine how hemolysis affects each analytical test, we followed a serial of steps. First, the results were expressed as a percentage, according to the Glick et al method [13]. Then the percentage of interference was plotted against the hemolytic index for each analyte studied (interferogram) [10].
The percentage of interference was calculated using the formula: Cd: average of the values of each test for each dilution of the interference.
C1: average of the concentration obtained for dilution without interference.
Next, and taking into account the coefficient of analytical  [2]. This study allowed us to compare the results obtained with the information provided by the inserts, founding differences in several of the techniques analyzed (Table 2).
Although it is true that in some tests there is controversy between the different studies consulted, this may be due to the fact that the interference produced by hemolysis in the different determinations is influenced both by the analyzer and by the technique used.
For example, for GGT our study determines that there is no interference by hemolysis as reported by two other studies [5,7].
However, in three other studies [4,10,16], they conclude that interference to an HI 130-260 does occur. In the case of ALT, it can be observed that although the same analytical technique is used as in other studies [5,7,16], the same results regarding interference are not obtained. Therefore, it is very important to emphasize that each laboratory has to carry out its own study to determine what type of interference and at what concentration of hemolysis occurs, since the results of each laboratory cannot be generalized with others instruments or reagents [5,7,10,16]. As we have said at the beginning, there are different mechanisms that produce hemolysis.
For example, for K, LDH and AST, the increase in their value as a result of hemolysis is due to the release of these constituents from the inside of the red blood cell [4,6]. Another example would be the decrease in the direct bilirubin value due to analytical interference in the technique [6], which is consistent with our study.
Magnesium is another test that also increases due to analytical interference [6]. In our study it increases from 500 mg/dL, while in other studies this increase occurs from a hemolysis index of 130 mg/dL [10]. Despite of other authors determine that there is no interference for magnesium [17]. For immunochemistry techniques there is not enough available literature of interference by hemolysis. Therefore, our results cannot be compared in that sense. However, we consider highly recommended to perform an interference study, especially for certain immunochemical techniques on which our study concludes that there is interference with low hemolysis rates. For example, it is observed that there is interference for the determination of Tn-This, insulin, and folic acid too. As seen in Figure 1, there is a positive interference for folic acid due to the outflow of the red blood cell contents, while the decrease observed in insulin is caused by proteolysis [6]. Hemoglobin can produce chemical or spectrophotometric interference in many determinations, such as in the case of Tn-Ths which generates an undervaluation of its value with the consequent danger of underestimation of acute myocardial infarction in the case of not considering this possible interference [6].
Having performed the hemolysis study in the same equipment where it will be applied, puts at our disposal the information of the It is also important to use these indexes in terms of quality management. There must be a quality indicator for the hemolytic index that tells us how many samples arrive hemolyzed at the laboratory and what is their origin. In that way, we can keep an adequate record throughout the year and establish a monthly quality limit that must not be exceeded. In case of being overcome, we can establish quality meetings with the phlebotomists, raising awareness about the importance of good laboratory practices to avoid hemolyzed samples as much as possible. In our study, we have introduced several levels of interference for all tests (hemoglobin concentration from 0 to 1000 mg/dL), but it would also be interesting to carry out the study on two levels of concentration, one of them close to the clinical decision value due to some times the interference may depend on the concentration of the analyte [15,18].
It is very necessary the existence of a standardization between clinical laboratories that homogenize the degree of hemolysis from which it is considered clinically significant, and what is the degree of hemolysis considered for the rejection of the samples if appropriate, due to the level of clinical significance may vary depending on the article you consult. In our case, thanks to the realization of this work, important differences were discovered with the inserts provided by the commercial house, as well as differences respecting the indexes established for the previously used equipment, probably due to the change of techniques of some analyzers to others. In addition, improvements were made to the laboratory report adding whether hemolysis causes an underestimation or overvaluation, information that is necessary for clinicians.

Conclusion
In conclusion, when a laboratory introduces new equipment for the majority of biochemical and immunochemical tests in its catalog, different studies are needed to adapt to the possible differences that may arise. Among these studies one of the most important, due to its high prevalence, is the study of interference by hemolysis.