Because blood cell analyzers have the advantages of speed, accuracy and convenience, they have been changing laboratory blood routine testing methods and testing procedures for more than 60 years. In recent years, due to the use of various new technologies, the detection performance of blood cell analyzers has become more and more powerful, and there are more and more detection parameters. In particular, the popularization and application of instruments with five-class leukocyte classification technology and fully automatic pipelined blood cell analysis systems The use of such equipment has made laboratories more and more dependent on such equipment to complete a large number of routine blood tests [1].
Under the specific environmental conditions of increased sample size and lack of personnel, the importance of peripheral blood smear examination has been gradually downplayed and ignored, resulting in some missed diagnoses and errors. This phenomenon has attracted the attention of the clinical testing community at home and abroad. Therefore, Professor P.W.Barnes and other professors advocated and formed the International Consensus Group for hematology Review, and in 2005 formulated the automatic blood cell counting and white blood cell classification Counting recheck standard [2], which was introduced by the International Society of Experimental Hematology (ISLH) as a recommended method for reference by professionals who use various automated blood analysis systems around the world.
The Testing Branch of the Chinese Medical Association also formed a national hematology retest expert group in 2006 to interpret the standard and publish it [3]. These are the 41 screening rules we are familiar with. Using different blood cell analysis systems, it has become an important task for user units of blood analyzers to establish retest rules that are suitable for their respective medical units and service groups. This is also an important inspection in the Ministry of Health's Laboratory Management Measures and ISO 15189 review. terms.
When applying various automated blood cell analysis equipment, review and re-examination are very important. We should conduct review or re-examination from the following points to find problems in time.
Pay attention to the screening of sample characteristics
When performing blood cell analysis and reexamination, it is very important to first pay attention to the characteristics of the specimen. Problems that significantly affect the test results include: whether the specimen collection volume meets the requirements, agglutination or clots in the blood specimen, chyloemia or lipemia, cold agglutination or cryoglobulin, hemolysis, hyperleukemia, and hyperbilirubinemia. wait.
1. Specimen collection volume: If a vacuum blood collection tube is used and the vacuum blood collection mode is used, there will generally not be a large difference in the collection volume of bleeding. However, if you encounter difficulty in drawing blood, there may be insufficient collection volume. At this time, you should communicate with the clinic to confirm whether the test results are affected. If not, use the concession test mode to issue a report, and make a note to explain the collection volume problem. If a syringe is used to collect and then inject into a blood collection tube, the collected amount may be inaccurate, too little or too much. Too much may cause an inappropriate ratio of anticoagulant to blood, leading to insufficient anticoagulation, thus affecting the test results of platelets and other indicators. In this case, unqualified specimens should be processed.
2. Coagulation specimens: The specimen can be found to have coagulated by visual inspection; shaking the test tube can reveal clots of different sizes in the test tube; open the test tube cap and see that there are clots in the cap; use a long bamboo skewer or construction tool to stir the specimen or absorb it. Used to detect clots or fibrin filaments not visible to the naked eye. Such specimens may be judged to be unqualified and shall be rejected and returned.
3, chyloemia or lipemia: can be found through natural precipitation or centrifugal sedimentation (Figure 1). Chylemia will lead to incorrect HGB measurement results detected by optical methods, which will affect the calculation results of MCH and MCHC. At this time, it is necessary to Use centrifugal plasma replacement mode to eliminate interference before measuring HGB, or use an instrument or function based on optical methods to measure HGB results, and re-evaluate and calculate MCH and MCHC. Hyperlipidemia cannot be detected by the naked eye. Biochemical test results can be queried through the LIS system, and then the WBC classification scatter plot can be viewed to evaluate whether the classification results are correct. If necessary, microscopic classification should be used to correct the white blood cell classification results.
4, cold agglutination and cryoglobulin: severe cold agglutination phenomenon can be seen with the naked eye, fine sand-like particles appear on the test tube wall (Figure 2). When there is weak cold agglutination, the specimen can be placed in a 4°C refrigerator environment for 3-5 minutes, that is, It can be detected with the naked eye, or a small amount of specimen can be dropped on a glass slide and viewed under a microscope.
Cold agglutination can lead to low red blood cell measurement results, which can lead to incorrect calculations of MCH and MCHC, inaccurate MCV measurement, and inaccurate WBC counts. It should be solved by first using a 37°C water bath for 30~60 minutes and then measuring again. Cryoglobulins are extremely difficult to detect, often leading to falsely elevated PLT results. They can be initially discovered through abnormalities in blood smears and scatter plots, and ultimately need to be confirmed by immunofixation electrophoresis or capillary immunotyping results. At this time, the PLT results cannot be released easily. It may be necessary to wait in a 37°C water bath for 30 to 60 minutes before measuring again to eliminate the interference of cryoglobulin.
5. Hemolysis: Centrifuge the specimen to find out whether there is hemolysis. If there is hemolysis, it can be rejected as an unqualified specimen.
6. Hyperleukemia: The white blood cells of patients with leukemia often rise to more than 100×109/L. It is generally believed that when the number of white blood cells is >200×109/L, the colorimetric HGB measurement results will be incorrect. Patients with severe anemia may even It will also affect the accuracy of RBC detection results, and thus the accuracy of MCH and MCHC calculation results.
After the specimen has been sedimented for a period of time, an obvious white blood cell sedimentation layer appears between the red blood cell layer and the plasma (Figure 3). Comprehensive analysis of the relationship between HGB, RBC, and HCT and the results of MCH and MCHC can be used to confirm whether the blood routine results comply with the rules. Once affected, the impact of the number of white blood cells on red blood cells and the turbidity interference caused by HGB should be subtracted. If there is an error, please correct it and send the report again. Hyperleukemia (especially neutrophils) can also affect blood glucose test results, leading to a decrease [10]. You should communicate with colleagues in charge of blood glucose testing in the department in a timely manner.
7. High bilirubin: High bilirubin can be initially judged by observing the plasma color through precipitation or centrifugation. It can also be judged after checking the serum Bil measurement results through the LIS system. High bilirubin will cause the HGB results of the colorimetric method to be higher, which will lead to inaccurate calculation results of MCH and MCHC. Abnormalities in these two calculation parameters can also be used to evaluate whether the specimen is affected by high bilirubin and leads to higher results. . If you encounter this problem, you can use the plasma replacement method to re-measure the HGB results and correct the HCH and MCHC calculation results.
8. Others: includes specimen collection errors, no barcodes or labels, labels falling off, test tube rupture, test tube caps not tightly capped causing specimen leakage, etc., which should be rejected.
Microscope smear review
This is very important! There are two methods for re-examination and review of blood smears, blood smear browsing (review) and blood smear classification (differential). In many cases, we only need to review the blood film to confirm the conclusions drawn by the blood cell analyzer, such as the size of the red blood cells (the abnormal shape, staining, and inclusions of the red blood cells found should be described and reported at the same time), the size does not etc., the increase or decrease of certain types of cells (excluding morphology report content). For problems suggested by leukocyte morphology, incorrect classification, or problems that cannot be classified, blood films should be classified under a microscope and reclassified in the report.
So what issues should be paid attention to and solved in the re-inspection rules? The following suggestions are for reference:
1. First browse the blood smear. At this time, you can use a low-power (or high-power) microscope to browse and observe the blood smear production effect, cell distribution, staining effect, and whether there is any The appearance of large cells with pathological significance, etc. If it is EDTA anticoagulant, the platelets should be evenly distributed and there should be no aggregation. If there is obvious platelet aggregation, especially when large agglutination occurs at both ends and tail of the slide (Figure 4), it will cause the instrument to malfunction. The important reason for the false low of platelet count using the method is also the first thing to pay attention to during re-examination, and it is also the content that the laboratory department should not miss the most.
When observing blood films, it is also necessary to observe the distribution and arrangement of red blood cells, and discover the coin-like arrangement phenomenon and red blood cell agglutination phenomenon, so as to initially determine whether there are multiple myeloma, macroglobulinemia, high blood viscosity or cold agglutination. Syndrome, etc., and even consider whether it will affect the accuracy of red blood cell count.
2. By browsing and observing the blood smear, the cell count results measured by the blood cell analyzer can be reviewed and the reliability and consistency of the instrument count results can be initially evaluated. For example, white blood cells can be reviewed by referring to the relationship in Table 1 [4]; or the estimation method can be used for review, and a uniformly distributed area in the blood film can be selected for evaluation. White blood cell count (×109/L) = the average number of white blood cells per high-power field of view. Number×2×109/L[5].
can also preliminarily evaluate the consistency and review of the platelet count results of the instrument based on the platelet distribution on the blood smear. If there is an average of 1 platelet per oil lens field of view, it is equivalent to 10×109/L of the PLT count result, and so on. Or infer the number of platelets through the blood smear, select an area at the body-tail junction of the blood smear that is evenly distributed and without abnormal aggregation or fibrin filaments, and browse the blood smear to roughly estimate the number of platelets. The method is: platelet number (×109/L) = The average number of platelets in each oil field of view is ×15 × 109/L [5].
3. A very important issue during re-examination is not to miss blood system diseases (Figure 5, Figure 6), especially blood system tumors. Although many brands of blood cell analyzers can give prompts such as immature granulocytes (IG), atypical lymphocytes (ATYP), blast cells (BLAST), nuclear left shift (LEFT SHIFT), large unstained cells (LUC), etc. Alarm messages, and even quantitative result messages, but they still have limitations. According to the current technical level, the blood cell analyzer based on the five-classification principle cannot yet give a clear definition of a variety of abnormal cells, let alone give a correct white blood cell classification report for blood system diseases. Therefore, experienced laboratory physicians or technicians are required to conduct detailed microscopic classification reexamination.
When we perform microscopic smear examination, in addition to paying attention to and discovering primitive or immature cells of various lines, we should also pay attention to the granular changes in white blood cells, such as multigranular neutrophils and few (poor) granular neutrophils; Neutrophil toxic changes, such as toxic granules (also called multigranular neutrophils), vacuolar degeneration, Döhle bodies and unequal leukocyte sizes; nuclear left and right shifts; Pelger-Huet malformation (Figure 7 ), May-Hegglin malformation, Chediak-Higashi malformation, whether phagocytosis of pathogenic microorganisms, etc. The emergence of these problems is of great significance for the diagnosis of infectious diseases, certain serious infections, poisoning, malignant tumors, scarlet fever, diphtheria, MDS, etc., and is also helpful for the treatment and prognosis of diseases. Cases with abnormal increases in eosinophils, basophils and monocytes also need to be reviewed and confirmed under a microscope to prevent false increases.
In addition, it is necessary to observe whether there are other abnormal cells, such as atypical lymphocytes, plasma cells, myeloma cells, hairy cells, Sezary cells (Figure 8), lymphoma cells, etc., in order to initially screen for certain Blood system diseases. When abnormal white blood cell parameters appear and an alarm prompts, it is generally necessary to consider classifying white blood cells under a microscope, giving an appropriate percentage report for immature or abnormal white blood cells, and correcting or reclassifying the results of incorrect classification by the instrument. If some alarm information is too sensitive, microscopic examination confirms that no cells indicated by the abnormal alarm information or no abnormal cells are found, and this must also be recorded and explained in the report.
4. Pay attention to the morphological information of red blood cells to provide preliminary identification information for the diagnosis of anemia. General blood cell analyzers will provide a lot of red blood cell alarm information, such as large cells (Macro), small cells (Micro), different sizes of red blood cells (Aniso), uneven hemoglobin content (HC VAR), hypochromicity (Hypo), Morphological information such as hyperpigmentation (Hyper).
The author believes that this red blood cell morphology information is more accurate in informing the size or size of red blood cells in a sample. It is more objective, more sensitive, and more accurate and quantitative in understanding the size and pigment changes of red blood cells. It is a comparative Credible parameters, and the accuracy of these parameters should be based on qualified instrument calibration, in-control instrument quality control, and complete control of the three floating average indicators of MCV\MCH\MCHC.Manual observation and description of these red blood cell information may be affected by personal experience factors. The author believes that its sensitivity and consistency are not as accurate and stable as the results of instrumental analysis. The clinical significance of red blood cell size and morphological changes is shown in Table 2.
Red blood cell analysis can also provide prompt alarms for some phenomena. For example, red blood cell aggregation (RBC Agglutination) indicates that the specimen may have cold agglutination; an increase in HGB turbidity (HGB Interf) may be due to the influence of chylous blood factors; an increase in nucleated red blood cells. (NRBC), red blood cell bimodal distribution (Dimorphic Pobulation), red blood cell fragment (RBC Fragment) interference, etc. These tips can make preliminary judgments on some red blood cell and hemoglobin abnormalities, but NRBC, red blood cell aggregation, red blood cell fragmentation, etc. still need to be examined under a microscope Confirm below. If these phenomena occur, it will affect the accuracy of white blood cell and red blood cell counting, and then affect the accuracy of red blood cell-related calculation parameters. Therefore, blood films need to be rechecked; an increase in red blood cell fragments will interfere with the platelet count results of the impedance method, and the instrument histogram must also be passed. Initial screening of graphs and scatter plots, confirmed by reexamination of blood films, and affected count results need to be corrected.
What we need to pay attention to when re-examining blood films is that when there are abnormal changes in the morphology, staining and internal structure of many red blood cells, the relevant parameters and alarm information of the blood cell analyzer cannot be fully covered or cannot be correctly prompted, such as disorders of globin production. Target-shaped red blood cells that appear in anemia and HbC; hereditary elliptocytosis; increased spherocytes in autoimmune hemolytic anemia (Figure 9); caused by liver disease, hemolytic anemia, and pediatric digestive system diseases Increased stomatocytes (Figure 10); increased acanthocytes in chorea and severe liver injury (11), tear drops in myelofibrosis, megaloblastic anemia, and ellipsoids Increased elliptocytes in polycythemia (Fig. 12); as well as sickle cell anemia, basophilic stippling red cells, Cabot rings, and haemoglobin The occurrence and increase of Howell-Jolly bodies and other conditions require observation of red blood cell morphology to identify and participate in clinical auxiliary diagnosis.
In 2015, the International Council for Standardization in Haematology (ICSH) launched the standardization of red blood cell morphological terminology and the grading standards for peripheral blood cell morphological characteristics (ICSH recommendations for the standardization) based on the research and recommendations of more than a dozen experts. of nomenclature and grading of peripheral blood cell morphological features), see Table 3. This recommended standard provides valuable reference guidance for the standardization of cell morphology terminology in the future, especially in terms of criteria for judging abnormal cell morphology [6]. There is a certain gap between the terminology and classification principles of red blood cell morphology and the original nomenclature and classification of red blood cell morphology in professional books or textbooks in my country. This aspect should attract the attention of our Chinese colleagues. At the same time, the author also recommends that the domestic testing society and hematology experts discuss as soon as possible to formulate or establish standardized terms and translations in Chinese and English, and standardize the definitions of various cell types. This is of positive significance to the development of the profession.
5. Pay special attention to platelets. Platelets are small in size and prone to aggregation and inaccurate counting. Therefore, they are one of the re-examination contents that are very prone to problems. When performing blood smear examination or re-examination, first pay attention to whether there is a platelet aggregation (PLT Clump) alarm message or an abnormal platelet distribution (PLT Abn Distribution) prompt. Especially when using EDTA anticoagulated blood specimens, pay attention to prevent platelets. False reduction occurs. The pseudothrombocytopenia caused by EDTA anticoagulants can lead to clinical misdiagnosis or delayed treatment.
In addition, you need to pay attention to the alarm (Large PLT) whether there is a significant increase in large platelets. It will cause a false decrease in platelets counted by the impedance method. At this time, you need to consider whether to use equipment with other detection principles or use a microscope counting method. If corrected, excessive presence of giant and large platelets may even cause the instrument to "miss counts". These giant and large platelets that exceed the size of red blood cells can only be discovered through microscopic examination (Figure 13). The presence of more platelets with missing or reduced granules, giant platelets, abnormally morphological platelets, and significantly different platelet sizes in peripheral blood is suggestive of diseases related to bone marrow pathology, hematopoiesis and coagulation disorders, such as myelodysplastic syndrome. , chronic myeloproliferative diseases, etc. In the past two years, the author of
has also discovered cases of patients with cryoglobulinemia, resulting in a false increase in platelet count. If the false increase reaches an extremely high level (such as >1000×109/L), through our The re-examination rules can also find that if the platelets are low and falsely increase to normal levels, or even the instrument does not display any alarm information, it is very difficult to detect and re-examine. The blood smear can be indirectly detected through microscopic examination. Take a slice and pay attention to the coagulation traces formed by irregular protein aggregation between red blood cells. At the same time, evaluate whether the number of platelets is consistent with the counting results of the instrument. If they are not consistent, especially if the counting results of the instrument are significantly higher, cryoglobulin can be suspected. interference. We found two such patients in 2013 and 2015. Therefore, when cryoglobulinemia is discovered, the platelet count results must be confirmed by manual re-examination. This is an issue that has not been mentioned in the previous re-examination rules [7].
6. Another issue that requires special attention during blood film examination is blood parasites. Some patients may not pay attention to parasitic infections due to atypical clinical symptoms in some patients. We can find Plasmodium [8], microfilariae, babesiosis, trypanosoma, Toxoplasma and other infections in the blood. You must know that the discovery of these pathogens is actually a diagnostic report issued to the clinic, which is a blood test. It is very important that a diagnosis report can be issued. If a clinical patient has relevant symptoms, or the clinician prompts to check for blood parasites, the blood film should be carefully browsed, or several more blood films, including thick blood films, should be taken for detailed examination. Currently, in the South, especially those returning from epidemic-affected countries, the malaria parasite infection rate is very high, which deserves vigilance and careful inspection.
Re-examination sometimes requires the cooperation of traditional microscopy methods. Although the accuracy, precision and detection speed of blood cell analyzers are much higher than traditional manual measurement methods, there is no doubt. However, in some specific cases, manual methods may be needed for verification and auxiliary analysis. If you encounter a case where both EDTA and sodium citrate anticoagulants can cause platelet aggregation, you need to return to microscopic counting. In cases of severe cold agglutination, cryoglobulinemia, or increased red blood cell fragments, it may lead to errors in red blood cell count and platelet count, and it may be necessary to return to microscopic counting to solve the problem. Excessively high reticulocytes in patients with hemolytic anemia may also lead to low or incorrect counting results due to instrument reagents or linearity problems, and the 41 rules do not involve retesting rules for such problems, so it is also necessary to use It is corrected by traditional brilliant tar blue staining and microscopic counting methods [9].
Repeat and recheck
Repeat and recheck are also mentioned many times in the 41 retest rules, and retest specimens, retest after diluting specimens, count by alternative methods, and proceed according to laboratory SOP, etc. This means that after a certain rule is triggered, the laboratory needs to use re-examination, diluted sample re-examination or other methods for verification and confirmation.
Retest the sample: When you encounter related problems, you can confirm by retesting the sample. It is best to use another instrument, or even another brand or an instrument with a different principle to repeat, which will help to find the problem (prerequisite All instruments in the laboratory should be compared indoors and meet the comparison requirements).Re-measure after diluting the sample: When the count result exceeds the linear range set by the laboratory, the method of dilution and measurement should be used to obtain more accurate results. It is recommended to use the sheath fluid or diluent supplied with the instrument for quantitative dilution, and remember the dilution ratio to calculate the final result.
Alternative method counting: Laboratories should confirm the use of traditional counting methods to verify and count certain specimens that cannot be accurately measured by the instrument. The laboratory should develop appropriate SOPs and prepare appropriate equipment and reagents. The author suggests that platelet diluent, leukocyte diluent, bright tar blue stain, etc. should be the first and necessary reagents in the laboratory (Figure 17).
Auxiliary re-examination of image method equipment
Currently, there are blood cell inspection systems with digital images. These digital image devices can even be configured on the blood cell analyzer assembly line and run fully automatically. The system will store the data in the system according to the retest rules formulated by the laboratory. When the sample triggers the retest rules, the equipment can automatically transport the blood to the smear staining equipment to produce a standard blood smear. The blood smear is then placed under an automatic microscope to capture digital images, and then digital recognition software classifies white blood cells. It completely simulates microscopic classification technology for morphological blood cell examination, which is a useful advancement. However, there may still be some bottlenecks that have not yet been resolved. Perhaps due to the ever-changing morphology of blood cells, primitive and immature cells, abnormal cell morphology, etc., and under the interference of certain components, the correct recognition rate still needs to be further improved.
At present, for the application of such instruments, the author's suggestion is to first conduct a screen review, correct misidentified cells, eliminate interfering components, and re-observe and identify samples under the microscope for difficult-to-identify samples.
Currently, in the context of the widespread use of automated blood cell analyzers of various brands to test a large number of routine blood samples, and under the realistic conditions of increased workload, insufficient testing staff, and limited testing report time, we have developed a system suitable for our own laboratories. , It is very important to have re-inspection rules that can ensure the quality of experiments and minimize missed inspections and experimental errors. What is more important is that with the re-examination rules, these rules should be implemented and implemented in detail, and should not be a formality, so as to ensure that the reports issued by the laboratory are credible and of high quality, and to maximize the satisfaction of clinicians' diagnosis and treatment work for clinical testing. needs.
Source: Inspection Vision Network
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Editor: Da Wenwu Reviewer: Chen Xueli
