Thematic Axis 1: Patient Preparation

Stonys and Vitkus mention that only a minority of patients tend to arrive well-prepared for blood sample collection, which is a problem in Lithuania. The causes of inadequate preparation include misinformation and variability in recommendations provided by laboratories, which affects the quality of the results ​. ¹⁷ Arredondo evaluated the importance of fasting in hematology and coagulation tests. Samples were taken while fasting, after the consumption of a standardized breakfast of carbohydrates, proteins, and lipids, and then at the first, second, and fourth hours post-consumption. The results showed no return to baseline values in the 4 hours following food intake, leading to the conclusion that, for the analytes included in the study, fasting time should be considered important. ¹⁸

This is corroborated by Lippi et al., who, in their publication on the importance of the pre-analytical phase in clinical studies, emphasized the relevance of patient preparation so that the blood sample effectively reflects the subject’s real conditions. Therefore, it is indicated that precise information should be collected on medication and supplement use, as well as the pathologies affecting the patient, reinforcing the importance of fasting time, the time of sample collection, abstinence from tobacco, avoiding coffee consumption, and refraining from strenuous physical activity for 48 hours. Moreover, patients should be seated for at least 10 minutes before extraction. ¹⁹

Continuing with Lippi, among the important steps he also recommends are the confirmation of the patient’s identity with at least two indicators, defining the sample matrix, specifying the sample volume, applying the tourniquet for less than one minute, defining the venipuncture site, performing venipuncture by phlebotomists, following the recommended order of extraction, and standardizing the mixing of samples. ¹⁹

Stonys and Vitkus, in their study on the attitude and lack of understanding of non-laboratory health professionals about the importance of patient preparation, applied an anonymous questionnaire that revealed that these professionals consider patient preparation in laboratory tests significant, such as fasting, alcohol, tobacco, or medication consumption (nurses more so than doctors). However, the attitude towards the impact of physical activity, the menstrual cycle day, and circadian rhythm showed significant differences depending on years of work experience (more or less than 20 years) and whether they had received relevant training. ²⁰⁾

Thematic Axis: Test Request

Kopcinovic and colleagues, in their study on body fluid analysis, made a recommendation to professionals involved in the collection and processing of these samples to standardize the procedure at a national level. For this, they conducted a survey and reviewed evidence through the literature, reinforcing the importance of the test request form and order of analysis, which should include: patient’s name and surname, gender, date of birth, unique identifier, date and time of collection, hospital unit, doctor’s identification and contact details, the identifier of the sample taker, collection procedure and site, and the anatomical origin of the sample. ²¹

In a study conducted by Fenta and colleagues to evaluate pre-analytical errors, they observed that 87.5 % of the test requests were incomplete regarding clinical diagnosis, 12.5 % did not include gender, and 15 % were missing age. ²² According to Kadic et al, errors in patient identification were associated with a higher occurrence of laboratory errors. In addition, 85 % of the requests did not have the time of sample collection. ²³⁾

A study conducted by Salek and colleagues, which consisted of a survey to investigate test request forms and result reports in the therapeutic drug monitoring service in laboratories, found that only 12 % implemented all the necessary elements for optimal therapeutic drug monitoring. These elements included age, body weight, sample timing, date of first administration, time of last administered dose, dose, dosing interval, route of administration, purpose of the test, and other co-administered medications. ²⁴

Thematic Axis 3: Sample Collection

Compton and colleagues, in the case of tissues for molecular tests, estimate that the ideal sample thickness is up to 5mm due to the penetration speed of formalin. For blood samples, factors such as the type of collection tube (EDTA tube or specialized tubes for cell stabilization), the tube fill level as per the manufacturer’s recommendation, and the tube order in multiple collections to avoid cross-contamination are emphasized. The recommended order is: tube without additive, coagulation tube with sodium citrate, tube with clot activator, tube with clot activator and serum separator, heparin tube (either sodium heparin or lithium heparin), EDTA tube, and tubes with other additives like citrate, dextrose-acid; oxalate/fluoride, and anti-glycolytic agent. Additionally, an adequate number of inversions should be performed for proper mixing of the analyte with the tube additive.²⁵⁾

On the other hand, García del Pino and colleagues, in their study on pre-analytical conditions in glucose tests, sent a survey to different laboratories and observed that the conditions under which the tests were performed were not ideal. The largest percentage of samples was performed in serum tubes, followed by plasma tubes with lithium heparin and plasma tubes with glycolysis inhibitor such as sodium fluoride on a smaller scale (19 %). The importance of the selected serum tube does not meet the conditions of the fasting plasma glucose diagnostic criteria. ²⁶

De Laat-Kremers and colleagues observed significant diversity among laboratories in the pre-analytical phase for thrombin generation measurement. They conducted a study through a questionnaire to laboratories in different locations around the world to understand these differences. 68 % of laboratories use only platelet-poor plasma, 4 % use only platelet-rich plasma, 24 % combine both types, 1 % use whole blood, and 3 % combine all three types. 40 % draw blood using a butterfly needle, and 39 % with a straight needle. 73 % use plastic collection tubes and 14 % use glass. The preferred anticoagulant is trisodium citrate (83 %), and 71 % discard the first blood tube. ²⁷⁾

Simundic and colleagues observed that among the most important causes of hemolysis during phlebotomy were the use of inadequate equipment, such as syringes instead of evacuated tubes, needle diameter favoring turbulent flow, blood transfer from the syringe to the collection tube, and blood tubes with less volume than recommended. Phlebotomy from the antecubital fossa and gentle mixing of the tubes is recommended. ²⁸

The importance of documenting collection time is emphasized by Grankvist and colleagues in their study, as several factors can influence the integrity of biomarkers in serum and plasma samples. In the clinical biochemistry laboratory, samples are usually kept in their collection tubes until analysis, pending transport to the laboratory. Many analytes are stable for several hours without centrifugation; however, unstable ones require centrifugation and refrigerated transport or, in some cases, freezing before analysis. This is important to consider in peripheral sample collection centers, which should have supplies available to perform these pre-transport procedures. The stability of the analyte must be ensured by the laboratory. Laboratories requiring long-term storage should redouble their efforts to manage pre-analytical variations such as fasting, medication instructions, collection time restrictions, sample volume, maximum centrifugation time, and freezing. ²⁹

Thematic Axis 4: Sample Storage and Transportation

Niedrist et al. evaluated the impact of routine storage time and temperature conditions on anti-nucleocapsid (NC) antibodies, finding that their levels after approximately 3 months at less than -70°C or during 14 days at temperatures between 2-10°C did not decrease significantly from a statistical standpoint. However, for storage periods longer than 1.5 years, relevant deviations were observed, potentially increasing positivity rates in convalescent COVID-19 patients. ³⁰

Codish et al. reported that prolonged time between blood extraction and the separation of the cell mass can decrease glucose levels, influencing the diagnosis of hyperglycemia and hypoglycemia. Through the evaluation of fasting glucose tests in an adult population before and after an educational intervention, the establishment of five centrifugation centers at key city locations, and changes in transportation routes, samples were kept in insulated coolers with polystyrene at 20°C for a maximum of 2 hours until centrifugation. The transportation temperature was monitored using a barcode tracking system. After the implementation of these changes, glucose results over 100 mg/dL increased significantly from 9.83 % to 25.91 %, and hypoglycemia (below 50 mg/dL) decreased. ³¹

Another study on glucose variation was conducted by Potter et al. on pregnant women. In a protocol of delayed centrifugation versus early centrifugation (within 10 minutes of sample collection), higher glucose levels were obtained with early centrifugation, demonstrating variation in the rate of gestational diabetes diagnosis depending on the centrifugation time. ³²⁾

Ottestad et al. conducted a study to evaluate the pre-analytical handling of HMGB1 (a mediator of systemic inflammation in sepsis and trauma) by taking arterial and venous samples, with delayed centrifugation times of 15 minutes, 3, 6, 12, and 24 hours stored at room temperature. They found that the samples were stable up to 6 hours, and arterial samples presented 40 % lower concentrations than venous samples. ³³

A study by Grzych et al. also evaluated this type of transportation, suggesting that plasma potassium levels can be influenced, which could lead to erroneous diagnoses. ³⁴ Cool et al. explored whether pre-analytical quality was affected in blood samples taken at the homes of cancer patients undergoing treatment due to delayed centrifugation and transportation. They concluded that these interventions do not affect clinical decision-making in this case. ³⁵⁾

Another study conducted by Chang et al. used a questionnaire to evaluate the functioning of primary care clinics in relation to pre-analytical phase management. Since most clinics do not have their own laboratories and send samples to reference laboratories, inadequate practices were found: 29.1 % of respondents reported a lack of centrifuges at the clinic, almost half had instructions on sample storage, and the samples were transported once a day during workdays. ³⁶⁾

Thematic axis 5: Pre-Analytical Errors

Nordin et al. ³⁷ observed that 82.6 % of pre-analytical errors are caused by human error, while technical errors account for 4.3 %, which is also consistent with Van Moll et al.,³⁸⁾ who found that human factors were more frequent (58.7 %) while technical causes accounted for 12.5 %. Additionally, they agree with the literature that pre-analytical errors are more frequent than analytical and post-analytical errors, with rates of 77.1 %, 13.5 %, and 8 %, respectively.

Mukhopadhyay et al. conducted a study comparing pre-analytical errors before and after the COVID-19 pandemic, finding that the rejection rate for blood samples was significantly higher during the pandemic (3 % vs. 1.1 %). Coagulated samples were the most common indicator of pre-analytical error in both stages, along with a significant increase in the rate of mislabeled samples. Hemolysis was the second most common error before the pandemic and the fourth most common during the pandemic. ³⁹

A similar study conducted by Eren et al. showed that samples not received were significantly more common during the pandemic, concluding that the pre-analytical phase was the most affected during the pandemic. ⁴⁰⁾

Tashkandi et al., in their study on a pilot model of Advanced Care Organization, mentioned that rejected samples corresponded to 68.3 % for requests without a sample, 6.95 % for incorrect tube usage, and 2.85 % for hemolysis. ⁴¹

In the study by Alshadhdali et al., of the total samples analyzed, 9.3 % presented pre-analytical errors, with the most common being coagulated samples (3.6 %) and samples not received (3.5 %). ⁴² The former is repeated in the study conducted by Kadic et al., where the error rate was 1.7 % of samples, with the causes being coagulated samples (39.87 %) and hemolysis (48.5 %). ⁴³

Hemolysis is a widespread error worldwide, as found in the study conducted by Zorbozan and Zorbozan, with a pre-analytical error rate of 0.22 %, highlighting samples with excessive transport time and samples collected in incorrect containers. ⁴⁴ Mesganaw et al. also observed hemolysis as a primary error in pre-analytical rejection, along with insufficient volume. ⁴⁵

Other studies have shown that errors can occur at different stages of the pre-analytical phase. Keppens et al. noted missing or incorrect information on the test request form, ⁴⁶ and Troiano et al. studied samples not received (3.7 %), which mostly occurred in the emergency department, following up with interviews with the involved staff to evaluate the cause. ⁴⁷

Studies have also mentioned the issue of fasting in laboratory tests. Kadic et al. found that only 37.5 % of patients arrived adequately prepared at the laboratory. ⁽⁴⁸

Laboratory errors are not exclusive to blood samples. There are also pre-analytical phase problems in the collection of culture swabs, where incorrect labeling has been evidenced in a study by Leonard et al. ⁴⁹ and in tissue samples, as seen in the study by Shinde and Dhanve. ⁵⁰

According to Reddy et al., the pre-analytical phase is mainly responsible for 97.4 % of the rejections of HIV serology samples, with the need for a separate sample being the most common cause (57.44 %). These errors are attributed to deficiencies in sample collection and handling, leading to increased costs and representing 82.6 % of total rejections. Additionally, the need to improve healthcare staff training is highlighted to minimize these errors, improve laboratory efficiency, reduce costs, and ensure accurate and timely diagnostic results. ⁵¹

Šálek et al. observed that only 67 % of laboratories specified the type of sampling tube on their request forms, which is critical to avoiding pre-analytical errors, such as drug absorption by serum separator gels. Furthermore, it was observed that the manual entry of data from paper forms into electronic systems is prone to transcription errors, which can compromise result quality. These deficiencies in the pre-analytical phase underscore the need to improve harmonization and therapeutic drug monitoring practices in laboratories. ²⁴