Introduction
Hematological reporting is very important aspect in accurate diagnosis & investigations of various organic disease, parasitic manifestations & metabolic disorders as well.1 It includes complete blood count & peripheral smear which provides a definitive information to physicians or surgeons to diagnose, monitor & further management of patients.2 Complete hemogram is also necessary for patients who requires blood transfusion to estimate the quantity of blood to be transfused.3
The capability of sample to retain the initial value of a measured quantity for defined period within specific limits when stored under defined condition is termed as sample stability.4 This sample stability is essential to maintain accuracy & reliability of final results in case of delayed evaluation of blood samples.4
In routine hematological practice, EDTA is preferrable choice of anticoagulant for automated cell counts due to its general availability, ease of preparation, wide spread use & comparatively low cost.5, 6 However, the EDTA anticoagulated blood shows less stability of parameters & shows changes in morphology of cells in stored blood sample at room temperature in case of delayed analysis & finally result in wrong interpretation of data.2, 6 According to previous studies it has been verified that storing blood samples in EDTA for long time before analysis can affects morphology of blood cells, peculiarly red blood cells as they may causes altered osmotic fragility & shows their effects on cellular viability.6
Some factors such as lack of manpower & shortage of cell counters, weekends and high environmental temperature which can lead to longer storage of blood samples.4 In addition, many countries has been developed authorized central laboratories which receives blood samples from peripheral areas or other local laboratories which causes prolonged time of transportation & may lead to delayed evaluation of that particular hematological test. Hence, preanalytical phase needs a careful attention on procedures such as collection of blood samples, patient’s identification, preference of collection vials, labelling of specimens, accurate blood suction, clerical errors & storage.1
There is simple, modest method to maintain all hematological parameters & morphology of peripheral smear is storage of blood samples at 4°C refrigeration.7
Therefore, this study presents a quantitative data and peripheral blood smear changes for blood sample storage at fixed temperature 4°C refrigeration for 24 hrs. It is needful in routine clinical practice to increase accuracy & reliability of haematological investigations in case of delayed blood samples.
Aim
To assess the effect of room temperature & refrigerated storage on complete blood counts and on morphological features in peripheral blood smear on automated haematological parameters.
Objectives
To compare the effects of stored blood samples both at room temperature & 4°C refrigeration on automated haematology parameters & peripheral blood smear.
Materials and Methods
Subject
Total 150 blood samples from outpatient & inpatient section irrespective of any specific criteria, age & gender were collected randomly.
Study design
This prospective study was approved & accomplished at Bharati Vidyapeeth medical college & hospital, Sangli in April 2022 to June 2022.
Sampling collection
Approximately 2 ml of venous blood is collected in EDTA coated vacutainers under standard protocol of aseptic precautions.
Laboratory testing
The measurement of haematological parameters including WBC, RBC & Platelet count, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) was done on Sysmex XN 500 5 part differential which is on based on the principle of fluorescence flow cytometry method. Furthermore, their peripheral smears are made by proper mixing of blood samples & using Leishman stain.
In peripheral smear following morphological changes were studied -
Room temperature storage
Analysis of 75 blood samples is done at 0 hr for complete blood count as basis of measurement by using automated haematological analyzer 5 part differential & peripheral smear is prepared. Then subsequently these samples were stored at room temperature & re-analyzed after 24 hrs for all haematological parameters & peripheral smear.
Refrigerated storage
Analysis of another 75 blood samples is done at 0 hr for complete blood count as basis of measurement by using automated haematological analyzer 5 part differential & their peripheral smear is prepared. Subsequently samples were stored at 4°C refrigeration. After 24 hrs, samples were settled at room temperature for 30 mins, then re-analyzed for complete blood count & peripheral blood smear prepared.
Results
Table 1
Showing results of complete hemogram at room temperature storage of blood
Changes in stored blood samples at room temperature for 24 hrs
We observed that there is statistical significance (P values < 0.05) in WBC counts in blood samples stored at room temperature. Degranulation, increased granularity in neutrophils, vacuolization & bleb formation in cytoplasm & lobulation, pyknosis, smudging & vacuoles in nucleus are morphological changes seen WBC in stored blood samples at room temperature. Percentage of these artifacts plotted in Figure 1, Figure 2 & their images shown in Figure 3, Figure 4.
Graph 1
Showing percentage of nuclear changes in WBC in stored blood samples at room temperature for 24 hours
Graph 2
Showing percentage of cytoplasmic changes in WBC in stored blood samples at room temperature for 24 hours
Figure 1
Leishman stain (100 x showing nuclear degeneration & karyolysis) in stored blood samples for 24 hours at room temp
Figure 2
Leishman stain (100 x showing nuclear karyolysis & cytoplasmic degranulations) in stored blood samples at room temperature for 24 hours
The P values are not < 0.05 i.e no statistical significance in variation of haemoglobin values & RBC counts stored at room temperature for 24 hrs. However, some morphological artifacts in RBCs such as crenated RBCs & loss of central pallor revealed with stored sample at room temperature. These changes shown in Figure 6 & their percentage plotted in bar Figure 5.
Graph 3
Showing percentage of changes in RBC in stored blood samples at room temperature for 24 hours
While, P value < 0.05 i.e statistical significant changes seen in platelet counts in room temperature storage of blood & also shows morphological changes like aggregation of platelets & large platelets. Changes shown in Figure 8 & their percentage in bar Figure 7.
Graph 4
Bar graph showing percentage of changes in Platelets in stored blood samples at room temperature for 24 hours
In addition, room temperature storage also showed increase in mean value of MCV & decrease in mean value of MCHC with statistical significance (P value < 0.05).(Table 2)
Table 2
Showing results of complete hemogram at 4°C refrigerated temperature storage of blood
Changes in stored blood samples at refrigeration at 4°C for 24 hrs
This study has revealed that there is no statistical significance seen in values of haemoglobin, RBC count, WBC count, platelet count, values of MCV & MCHC.
Changes in counts of haematological parameters & morphology of RBC, WBC & platelets in blood smear which was noted in room temperature storage has shown stabilized with refrigeration at 4°C.
Discussion
Complete hemogram is basic laboratory investigation which assess the number, size, morphology of hematological parameters like white blood cells, red blood cells, platelets & related indices of the blood such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC).8 According to guidelines of International Society for Laboratory Hematology, morphological artifacts in blood cells begin within 30 minutes of collection of samples.5, 9 while, other previous studies also demonstrated that prolongation of storage of blood samples may lead to significant changes in haematological parameters & their cellular morphology.10, 9
In regular haematological workup, more than 70% of all errors commonly occurs during preanalytical phase which includes preparation of patient, collection, transportation, storage of collected blood samples & preparation for analysis.11 It is also necessary to consider the environmental temperature at different places, particularly in hot climate areas e.g. southern countries which affects stability of hematological parameters & final results.5
In recent years, a large central authorized laboratories encounters the common problems like proper transfer, storage duration of collected blood samples, long distance dispatchment of specimens which causes the delayed analysis of tests.12 Therefore, delayed sampling is one of the common problems in routine clinical practice, if quick analysis is not possible or if sample is required for retesting.11
According to Imeri et al., RBC counts & haemoglobin values are stable at both room temperature & 4°C refrigeration.13
Our study has demonstrated that haemoglobin values & RBC counts are not affected in room temperature storage for 24 hours, but there is significant increase in MCV because of crenated RBC in blood samples stored at room temperature. This change is stabilized by storage of blood samples at 4°C refrigeration for 24 hours. These similar findings were observed in prior studies.7
Other studies indicated that the degenerative changes in stored blood samples at room temperature allows the influx of water into cellular membrane result into increase in red cell size with time & eventually lead to increased mean corpuscular volume. & other morphological artifacts such as spherocytosis, echinocytosis & sphero-echinocytosis may found after 48 hr of storage at room temperature.6
While, MCHC values shows significant reduction in room temperature stored blood samples. MCHC is calculated by ratio of haemoglobin to haematocrit multiplied by 100. Thus, haematocrit increases while haemoglobin remains stable which leads to decrease in MCHC value.12
Room temperature storage also shows significant decrease in WBC count which was prohibited by refrigerated storage. Hence, our observance is identical to study Brent L. wood et al.7 Causes for reduced WBC count can be cytoplasmic changes such as degranulation, vacuolization, degeneration, sometimes blebs formation & karyolitic changes & lobulations in their nucleus.14
In relation with prior studies, it is stated that the statistically significant decrease in platelet count at all temperature begins after 6 hrs of sample collection.2, 15 While, in this present study, we have observed that platelet count also has reduced in stored blood samples at room temperature because of aggregation & swelling of platelets. These changes are corresponds with other previous studies.4 This change can be misinterpreted as pseudo-thrombocytopenia which can be prevented by refrigeration of blood samples at 4°C.
Overall, these morphological artifacts usually starts within 6 hrs of collection of samples & further increases with time & temperature.5 In our study we have observed the morphological changes like nuclear lobulations, nuclear karyolysis or pyknosis, nuclear vacuolization & then cytoplasmic changes like degranulation. Platelet aggregates & swelling are also seen. This observance is similar to other prior studies.14
Hence, in order to get standard haematological results it is necessary to maintain blood samples at fixed 4°C in conditions of extended period of evaluation, because previous studies proved that blood storage at 2°C may lead to freezing injury to RBCs results in hemolysis. However, storage at > 6°C lead to overgrowth of unspecified bacteria during sampling of blood.14, 1
Conclusion
The result of our study suggests that there are significant changes in haematological parameters from automated cell analyzer & their morphology of cells seen in peripheral blood smear, if blood samples stored at room temperature for extended period of 24 hrs after sample collection. Hence, storage of blood samples at room temperature should be avoided during transportation, within laboratory or in case of delayed evaluation. Finally, we have concluded that refrigerated storage at 4°C is the recommended method of preservation of blood samples for an accurate & standard test results in case evaluation of delayed samples.
Article DOI
