Blood Draw Can Vacuum Collapse a Vein

ABSTRACT

Objective

To determine a method to reduce specimen hemolysis rates in pediatric blood specimens.

Methods

A total of 290 blood specimens from pediatric patients were classified into the capped group or uncapped group. The hemolysis index and levels of lactate dehydrogenase (LDH) were measured using an automated biochemical analyzer. Also, we performed a paired test to measure the concentration of free hemoglobin in specimens from 25 randomly selected healthy adult volunteers, using a direct spectrophotometric technique.

Results

The hemolytic rate of capped specimens was 2-fold higher than that of uncapped specimens. We found significant differences for LDH. Also, there was a significant difference in the concentration of free hemoglobin in the random-volunteers test.

Conclusions

Eliminating the residual negative pressure of vacuum blood-collection tubes was effective at reducing the macrohemolysis and/or microhemolysis rate.

Specimen rejection for laboratory diagnosis due to hemolysis is a serious problem in pediatrics. The prevalence of hemolysis is approximately 3.3%, which accounts for 40% to 70% of all unsuitable specimens. ¹ Hemolysis occurs in vivo or in vitro. However, in vitro hemolysis, particularly during blood drawing, is the major reason for specimen rejection. ²

Various factors leading to specimen hemolysis have been identified during blood drawing, including high negative pressure in collection tubes, ^3,4 residual alcohol in blood specimens, small or vulnerable veins, excessive shaking of specimens, prolonged tourniquet time, underfilled tubes, improper blood-to-additive ratios, and improper size of the needle gauge. ¹

Hemolysis affects the results of most tests, including coagulation, iron, serum potassium, aspartate aminotransferase (AST), ⁵ alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). Therefore, it is important to develop alternative approaches to reduce hemolysis during blood drawing.

Statistically, hemolysis rates in children are particularly high compared to those in adults. ⁶ In our hospital, during the first half of 2016, an average of 21.4% of specimens from children were hemolyzed, whereas during the same time period, an average of less than 5% of adult specimens were hemolyzed.

We postulated that the low blood volume of pediatric specimens may contribute to the high hemolysis rate. Almost all pediatric specimens are less than 2 mL within a 5 mL collection tube. By contrast, only 5.9% of adult specimens are less than 2 mL. When the blood volume is low, high residual vacuum pressure is likely maintained, which may contribute to a high hemolysis rate, particularly in pediatrics.

Because of the unique situation in pediatrics, it is hard to reduce the remnant vacuum pressure by increasing the volume of drawn blood. By contrast, if the negative pressure is reduced in vacuum tubes before blood drawing, there is unlikely to be enough driving force. ⁷ The problem of low blood volume in pediatric specimens can be solved by using commercially available low-draw-volume blood-collection tubes, which have already been used in a few hospitals; however, in most other hospitals, blood collection tubes are irreplaceable because the size needs to specifically match the automatic instrumentation. Also, the economic cost of tubes is a serious impediment to using low-draw-volume blood-collection tubes.

In this study, we tested whether opening the caps of blood-collection tubes can reduce the incidence of hemolysis in pediatric specimens. The objective of the present study was to explore whether an effective method of reducing hemolysis rates would be to eliminate the residual negative pressure of vacuum blood-collection tubes.

Materials and Methods

Selection of Participants

This study contained 2 parts. The first part involved blood specimens from participants (n = 290) from the Pediatric Department of the First Affiliated Hospital of Shantou University, China. All specimens from patients with liver disease, prolonged clotting time, autoimmune diseases, sickle-cell disease, other conditions affecting erythrocytes or hemoglobin, and other blood disorders were excluded. Specimens were collected from 113 girls and 177 boys, all of whom were aged 14 years or younger.

Capped specimens served as the control group. Uncapped specimens were the experimental group; those specimens underwent the additional step, immediately after drawing blood, of having the tube cap opened for release of negative pressure, after which the cap was placed back on immediately. Also, the collected blood specimens were transported within an hour by the worker who collected them. The blood flowed slowly down the walls of the collection tubes during blood collection. Before analysis, serum was separated by centrifugation (3000 g for 8 minutes) at room temperature.

The second part involved blood specimens from 25 healthy adult volunteers, with ages ranging from 18 to 50 years, including 11 men and 14 women. Participant selection was completely random. All specimens were labeled with the participant name and a unique number.

Study Design and Data Collection

Retrospectively, we recorded the monthly incidence of hemolysis during the first half of 2016, which was available from the laboratory and hospital information systems. We discovered a correlation between a high incidence of hemolysis and the low volume of blood drawn in pediatric specimens. Therefore, we proposed a method, which we would implemented starting in July 2016, to remove residual negative pressure by cap opening of the tubes immediately after drawing blood.

Of the 290 blood specimens from the Department of Pediatrics, 145 specimens were capped, whereas 145 were uncapped. All specimens were collected in 5-mL serum gel tubes; then, we analyzed the sera analyzed using an AU5800 biochemical analyzer (Beckman Coulter, Inc.). Values for routine clinical biochemical examination, including hemolysis index (HI) and LDH, were obtained automatically.

HI was categorized as −, +, ++, +++, or ++++, to represent serum hemoglobin concentrations in the range of less than 500 mg per L, 500 to 900 mg per L, 1000 to 1990 mg per L, 2000 to 2990 mg per L, and 3000 to 4990 mg per L, respectively. Specimens categorized with 1 or more "+" symbols were regarded as being hemolyzed. In a previous study, ⁴ HI was assessed with a biochemical analyzer that had a detection limit of 500 mg per L, which might have underestimated the true rate of hemolysis. Therefore, we designed a paired test, involving 25 healthy adult volunteers. Free hemoglobin was determined manually using a spectrophotometer (Jinghua). Specifically, 2 blood specimens were drawn from each volunteer, by the same experienced phlebotomist, into 2 different collection tubes, as follows:

• A total of 2 mL of blood was drawn into a 5-mL negative-pressure serum gel tube, creating a 3-mL volume of air at negative pressure.

• A total of 2 mL of blood was drawn into a 5-mL serum gel tube with 2-mL negative air pressure, leaving no residual negative pressure.

After standing at room temperature for 120 minutes, blood specimens were measured for concentration of free hemoglobin, using a free hemoglobin assay kit according to the procedure provided by the company. To avoid differences in hemolysis due to differences in usage, all collection steps for each specimen were standardized based on the guidelines of the Clinical & Laboratory Standards Institute (CLSI). ⁸

Statistical Analysis

We analyzed data using SPSS Statistics software, version 20.0 (IBM, Inc.). Because means were normally distributed and had equality of variances, the statistical analysis included the independent-samples t test for random specimens, and the Wilcoxon test for paired samples (for continuous variables). The results were considered statistically significant for P values of less than .05.

Results

Hemolysis rates of pediatric specimens during the first half of 2016 are shown in Table 1. The incidence of hemolysis per month was greater than 19.00% (19.21%–24.50%); most of the hemolyzed specimens were from the HI with + and ++.

Table 1.

Hemolysis Incidence during the First Half of the Year 2016

Month	Patients, No.	Hemolyzed Specimens. No.	Hemolysis Rate, %	Hemolysis Index
				+	++	+++	++++	+++++
January	328	63	19.21%	42	15	3	3	0
February	347	85	24.50%	52	24	6	3	0
March	352	75	21.31%	49	24	1	0	1
April	377	78	20.69%	59	18	0	1	0
May	400	77	19.25%	54	20	2	1	0
June	370	87	23.51%	59	25	3	0	0

Month	Patients, No.	Hemolyzed Specimens. No.	Hemolysis Rate, %	Hemolysis Index
				+	++	+++	++++	+++++
January	328	63	19.21%	42	15	3	3	0
February	347	85	24.50%	52	24	6	3	0
March	352	75	21.31%	49	24	1	0	1
April	377	78	20.69%	59	18	0	1	0
May	400	77	19.25%	54	20	2	1	0
June	370	87	23.51%	59	25	3	0	0

Table 1.

Hemolysis Incidence during the First Half of the Year 2016

Month	Patients, No.	Hemolyzed Specimens. No.	Hemolysis Rate, %	Hemolysis Index
				+	++	+++	++++	+++++
January	328	63	19.21%	42	15	3	3	0
February	347	85	24.50%	52	24	6	3	0
March	352	75	21.31%	49	24	1	0	1
April	377	78	20.69%	59	18	0	1	0
May	400	77	19.25%	54	20	2	1	0
June	370	87	23.51%	59	25	3	0	0

Month	Patients, No.	Hemolyzed Specimens. No.	Hemolysis Rate, %	Hemolysis Index
				+	++	+++	++++	+++++
January	328	63	19.21%	42	15	3	3	0
February	347	85	24.50%	52	24	6	3	0
March	352	75	21.31%	49	24	1	0	1
April	377	78	20.69%	59	18	0	1	0
May	400	77	19.25%	54	20	2	1	0
June	370	87	23.51%	59	25	3	0	0

Of the 145 capped and 145 uncapped specimens, the number of hemolyzed specimens was 38 and 11, respectively. Consequently, the hemolysis rate of the capped specimens (26.2%) was more than 3-fold that of the uncapped specimens (7.6%). There was a significant difference between these 2 groups (χ² test, P <.001) (Table 2).

Table 2.

Hemolysis of Capped and Uncapped Specimens

Variable	No. (%)a
	Capped	Uncapped
Hemolyzed	38 (26.2%)	11 (7.6%)
Nonhemolyzed	107 (73.8%)	134 (92.4%)
Total	145	145

Variable	No. (%)a
	Capped	Uncapped
Hemolyzed	38 (26.2%)	11 (7.6%)
Nonhemolyzed	107 (73.8%)	134 (92.4%)
Total	145	145

^a P <.001, estimated with χ² test.

Table 2.

Hemolysis of Capped and Uncapped Specimens

Variable	No. (%)a
	Capped	Uncapped
Hemolyzed	38 (26.2%)	11 (7.6%)
Nonhemolyzed	107 (73.8%)	134 (92.4%)
Total	145	145

Variable	No. (%)a
	Capped	Uncapped
Hemolyzed	38 (26.2%)	11 (7.6%)
Nonhemolyzed	107 (73.8%)	134 (92.4%)
Total	145	145

^a P <.001, estimated with χ² test.

A total of 145 capped and 145 uncapped specimens were selected for comparison of values for LDH. As shown in Table 3, the capped specimens (mean [SD], 337.52 [103.56]) yield higher values than uncapped specimens (296.29 [89.24]), with a statistically significant difference (P <.001). This result was also derived from a box-plot analysis of LDH in Figure 1.

Table 3.

Values of Lactate Dehydrogenase between Capped and Uncapped Specimens

Group	No.	Mean	SD	SE	F value	P Value (2-tailed)a
Capped	145	337.52	103.56	8.60	2.249	<.001
Uncapped	145	296.29	89.24	7.41

Group	No.	Mean	SD	SE	F value	P Value (2-tailed)a
Capped	145	337.52	103.56	8.60	2.249	<.001
Uncapped	145	296.29	89.24	7.41

^aEstimated using the independent-samples t test.

Table 3.

Values of Lactate Dehydrogenase between Capped and Uncapped Specimens

Group	No.	Mean	SD	SE	F value	P Value (2-tailed)a
Capped	145	337.52	103.56	8.60	2.249	<.001
Uncapped	145	296.29	89.24	7.41

Group	No.	Mean	SD	SE	F value	P Value (2-tailed)a
Capped	145	337.52	103.56	8.60	2.249	<.001
Uncapped	145	296.29	89.24	7.41

^aEstimated using the independent-samples t test.

Figure 1

Comparison of lactate dehydrogenase (LDH) values between capped and uncapped specimens via box-plot analysis.

Figure 1

Comparison of lactate dehydrogenase (LDH) values between capped and uncapped specimens via box-plot analysis.

Self-paired test data from 25 healthy volunteers are shown in Table 4. Two blood specimens were drawn from each volunteer, using 2 different vacuum-collection tubes. The concentration of free hemoglobin with 5-mL serum gel tubes, which contained 2 mL of negative pressure, was significantly reduced, compared with 5-mL serum gel tubes that contained 5 mL of negative pressure (Wilcoxon test; P = .001). Also, a cutoff value for hemolysis is free hemoglobin level higher than 50 mg per L. We found no significant difference for hemolyzed specimens (concentration of free hemoglobin >50 mg/L) (Fisher exact test, 2-tailed; P = .23).

Table 4.

Concentration of Free Hemoglobin and Number of Hemolyzed Specimens in 2-mL Vacuum Tubes vs 5-mL Serum Gel Tubes

Variable	2 mL Vacuum Tubes	5 mL Serum Gel Tubes	P Value
Free hemoglobin (mg/L), median (IQR)	18.63 (14.79–24.96)	43.61 (29.15–53.28)	.001a
Free hemoglobin >50 mg/L, no (%)c	1 (4.0%)	8 (32.0%)	.23b

Variable	2 mL Vacuum Tubes	5 mL Serum Gel Tubes	P Value
Free hemoglobin (mg/L), median (IQR)	18.63 (14.79–24.96)	43.61 (29.15–53.28)	.001a
Free hemoglobin >50 mg/L, no (%)c	1 (4.0%)	8 (32.0%)	.23b

IQR, interquartile range.

^a P value estimated with Wilcoxon test.

^b P value estimated using the Fisher exact test.

^c n = 25.

Table 4.

Concentration of Free Hemoglobin and Number of Hemolyzed Specimens in 2-mL Vacuum Tubes vs 5-mL Serum Gel Tubes

Variable	2 mL Vacuum Tubes	5 mL Serum Gel Tubes	P Value
Free hemoglobin (mg/L), median (IQR)	18.63 (14.79–24.96)	43.61 (29.15–53.28)	.001a
Free hemoglobin >50 mg/L, no (%)c	1 (4.0%)	8 (32.0%)	.23b

Variable	2 mL Vacuum Tubes	5 mL Serum Gel Tubes	P Value
Free hemoglobin (mg/L), median (IQR)	18.63 (14.79–24.96)	43.61 (29.15–53.28)	.001a
Free hemoglobin >50 mg/L, no (%)c	1 (4.0%)	8 (32.0%)	.23b

IQR, interquartile range.

^a P value estimated with Wilcoxon test.

^b P value estimated using the Fisher exact test.

^c n = 25.

The hemolysis incidence from January to September 2016 in pediatric specimens is shown in Figure 2. The method for eliminating residual negative pressure in the vacuum tube was initiated in July of that year. The incidence of hemolysis was greater than 19.0% before July, and decreased after July 2016 (<9.0%), indicating that elimination of residual negative pressure decreases hemolysis.

Figure 2

Hemolysis ratio from January to September 2016 in specimens from the Department of Pediatrics (concentration of free hemoglobin >500 mg/L; a cutoff was used to define hemolysis).

Figure 2

Hemolysis ratio from January to September 2016 in specimens from the Department of Pediatrics (concentration of free hemoglobin >500 mg/L; a cutoff was used to define hemolysis).

Discussion

We showed that the hemolysis rate of capped specimens (26.21%) was more than 3-fold greater than that of the uncapped specimens (7.6%). These results indicate that cap opening is effective at reducing macrohemolysis and/or microhemolysis resulting from blood collection. Thus, release of negative pressure has a significant effect on hemolysis resulting from blood collection. Therefore, we recommend that vacuum blood-collection tubes should be opened immediately, to eliminate residual negative pressure in specimens from pediatric patients, particularly when the volume of the collected blood is low.

The results of a previous study by most of us ⁹ showed that the concentration of free hemoglobin in a 3-mL negative-pressure tube is significantly higher than that in a negative pressure–eliminating tube, and specimens that are allowed to stand for 4 hours show higher free hemoglobin concentrations than those that are allowed to stand for 1, 2, or 3 hours. In the capped specimens, higher values were obtained for LDH; for those specimens, we found a statically significant difference (P <.001). Hence, the method used to reduce hemolysis rates can make test results credible.

Also, the results from 25 healthy adult volunteers showed that free hemoglobin is increased in capped specimens, compared with uncapped specimens. For hemolysis, the upper reference limit for free hemoglobin is 50 mg per L for serum. ^1,2 The reasons for selecting adult volunteers rather than children are that it is difficult to enlist child volunteers and it is difficult to obtain 2 tubes of blood from children. Adult red blood cells (RBCs) are more mature and stable than RBCs from children; also, adult RBCs are not easily destroyed. So, our method to reduce hemolysis rates is principally useful in pediatrics but also in any department in which blood draws involve low volumes.

Vacuum blood-collection tubes are used routinely for blood specimen collection in laboratory diagnostics worldwide. Compared with blood drawing with a syringe, the use of a closed vacuum tube guarantees clean specimens and phlebotomist safety due to less touching and transferring of blood. Vacuum tubes also have other advantages, such as shortened time of blood draws, ¹⁰ increased efficiency of diagnostic tests, and reduced risk of blood contamination and needlestick injuries. However, because of the negative-pressure vacuum, there is a strict standard for the volume of blood drawing. According to the International Standard Organization (ISO), the tightness of a vacuum tube cap must keep the level of vacuum stable at 101.3 kPa for 24 months. Also, the CLSI standards and guidelines suggest that the blood specimen should fully fill the tube and use the entire negative pressure. ⁸ Based on our practice in pediatrics, it is difficult to fulfill that standard for low volumes of blood.

Many difficulties are encountered when drawing blood from children. For example, venipuncture may cause detrimental physical and psychological effects. Further, children have lower total blood volume than adults and are more likely to develop anemia after blood draws, particularly critically ill children. ^11-13 Also, many parents and children consider venipuncture to be exceedingly distressing. ¹³ These factors lead to low blood volumes and high hemolysis rates in pediatric blood specimens.

The results of a previous study ¹⁴ showed that the mean volume of hemolyzed specimens is lower than that of nonhemolyzed specimens. A previous study by most of us ⁹ also found that residual negative pressure could damage RBCs and increase the concentration of free hemoglobin. Specimens of poor quality may contribute to unreliable diagnostic reports and repeated specimen collections. Redrawing of blood may have several negative effects, such as increasing discomfort and stress for the patient, wasting the time of medical workers, and affecting the efficiency of medical treatment.

The findings of this study demonstrate that the concentration of free hemoglobin increases in specimens in tubes with 3 mL of remnant negative pressure, after standing at room temperature for 120 minutes. Ideally, blood specimens should be processed quickly—within 30 to 60 minutes of collection—to protect specimen integrity. ⁶ However, it is extremely difficult to apply this standard in clinical practice. By contrast, we can eliminate remnant negative pressure to reduce hemolysis rates in pediatric specimens. Cap opening is a feasible and advantageous method to reduce hemolysis, particularly when the volume of collected blood is low. Diminishing blood-specimen volumes for diagnostic testing is inevitable in several critical circumstances, particularly for patients with small, difficult, obscure, or atypical veins.

To date, in vitro hemolysis remains the major cause of specimen rejection in laboratories. Hemolysis is the most unacceptable occurrence that affects the reliability and precision of laboratory testing and negatively affects diagnostic reasoning. The main problems occur because of incorrect specimen-collection methods, such as inappropriate addition of additives, egregious shaking of the blood, ¹⁵ wet alcohol admixed in the blood, needles being too small, ¹⁶ small veins, use of excessive negative pressure, excessive squeezing at the site of puncture, retardation of separation of blood specimens (separation gel tubes), and the use of pneumatic tube systems. Among these factors, negative pressure is a serious but controllable factor. Although different hospitals worldwide may have different detection instruments and use different specimen-collection tubes, reducing negative pressure may be a universal and useful method to reduce hemolysis. Therefore, we recommend immediate opening of the vacuum tube to discharge excess negative pressure.

Of note, the study has several drawbacks and limitations. Uncapping the specimens has the potential to increase the risk of contamination or occupational exposure for the phlebotomist; therefore, we can reduce the risk by raising vigilance and performing proper precautionary measures. The uncapping of blood specimens might change the carbon dioxide content and pH. To our knowledge, there are no relevant data indicating the level of hemolysis required for interference of measurement of individual analytes, such as potassium, AST, ALT, and LDH; this issue deserves further exploration in the future.

Conclusion

Eliminating the residual negative pressure in vacuum blood-collection tubes is effective at reducing macrohemolysis and/or microhemolysis that sometimes occurs in collection of pediatric blood specimens. We suggest opening the cap of the vacuum tube immediately after blood collection, when the volume of the blood specimen is low. This method can reduce the destruction of RBCs and ensure the quality of diagnosis, which can decrease the rates of specimen rejection and improve the efficiency of clinical treatment.

The preanalysis phase remains a fallible link in laboratory diagnosis and may lead to unqualified specimens and unreliable results. The main findings in this study describe a method to reduce hemolysis in blood specimens; this intervention does not guarantee (but does improve) quality at the preanalytical stage. Reducing hemolysis in blood specimens, particularly for pediatric patients, can decrease stress experienced by phlebotomists, mistakes in diagnosis, and harm to patient health. This method of eliminating remnant negative pressure is helpful in resolving a difficult problem regarding low blood volumes and high hemolysis rates.

Abbreviations

Abbreviations

• AST

  aspartate aminotransferase

• ALT

• LDH

• HI

• CLSI

  Clinical & Laboratory Standards Institute

• RBCs

• ISO

  International Standard Organization

• IQR

Acknowledgments

This study was approved by the First Affiliated Hospital of Shantou University Medical College Ethics Committee, China.

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Author notes

Contributed equally to the manuscript as first coauthors

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Blood Draw Can Vacuum Collapse a Vein

Source: https://academic.oup.com/labmed/article/51/1/41/5513921

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