A Simple, Fast Correction Method of Triglyceride Interference in Blood Hemoglobin Automated Measurement

Abstract

Background

To establish a reliable correction method for automated hemoglobin (HGB) measurement by minimizing the interference from blood high triglyceride (TG).

Methods

Fifty whole blood samples and 50 plasma samples containing variable TG concentrations were used to determine the centrifugation speed and time. Complete blood cell counts (CBCs) were performed by an automated hematology analyzer for 102 blood samples, in which high‐level TG were artificially added. The same blood samples were centrifuged at low –speed to separate the plasma from blood cells. Then the plasma was analyzed by the same analyzer. By using the two CBC results, a correction formula was established to calculate the corrected HGB, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. Comparisons were also made of HGB, MCH, and MCHC values before and after correction of in‐patient individuals who received intralipid and developed lipemia.

Results

The percentage differences between the corrected and true values of HGB, MCH and MCHC were −0.28%, 0.06%, and −0.31%, respectively. The correlation coefficients of corrected values versus true values of HGB, MCH, and MCHC were 0.989, 0.935, and 0.717, respectively. This correction method was also effective for native lipemic samples.

Conclusion

High blood TG level can cause blood turbidity and erroneously high HGB results by hematology analyzers commonly used in clinical laboratories. Adding a simple step of low‐speed centrifugation and measurement of HGB in the plasma fraction allows a quick correction of HGB measurement in lipemic blood samples.

BACKGROUND

Hypertriglyceridemia is a well‐known factor that can interfere with the measurement of hemoglobin (HGB) using automated hematology analyzers. It can cause falsely high HGB values 1. Blood samples with high triglyceride (TG) contain chylomicrons (CMs) and very low density lipoprotein (VLDL) particles that scatter and absorb light, producing cloudiness, and turbidity in blood samples. TG in blood also replaces plasma volume. These two effects of high TG inevitably result in a false increase of HGB value.

Efforts to eliminate this lipid interference included a patent 2 that uses a special reagent to absorb TG and a correction method using three‐wavelength polychromatic analysis 3, 4. However, these correction methods failed to gain wide application in clinical practice due to their complexity and cost. Here, we report a simple but effective correction method to rectify the interference of high blood TG levels in measurement of blood HGB by automated hematology analyzers commonly used in clinical laboratories.

MATERIAL AND METHOD

Establishment of Optimal Centrifugal Force (g) and Time

Fifty fresh whole blood samples (2 ml each) with different concentration of HGB, without hemolysis, icterus, or lipemia (TG < 1.83 mmol/l) were drawn by atraumatic and sterile antecubital venipuncture into vacuum tubes containing the anticoagulant EDTA‐K₂. Another 50 plasma samples (2 ml each) containing variable TG concentrations (final TG concentration 6.50∼25.00 mmol/l) were prepared by adding intralipid mixtures (30% fat Emulsion injection (C_14–24), Sino‐Swed Pharmaceutical Corp. Ltd., Wuxi, China) to normal plasma with anticoagulant EDTA‐K₂. Complete blood cell counts (CBCs) of these samples were analyzed by XE‐2100 automated hematology analyzer (XE‐2100 analyzer, Sysmex, Kobe, Japan). Then, whole blood and plasma samples were both divided into five groups (10 samples in one group). The five groups of samples were centrifuged at 137 g for 10 min, 310 g for 5 min, 550 g for 3 min, 550 g for 5 min, 1,685 g for 5 min, respectively. Then same analyzer was used to detect the HGB concentration in the supernatant liquid fraction of the whole blood and plasma samples. An optimal centrifugal force and time were identified to separate plasma and blood cells and to exert the least influence on the CMs in the plasma.

Establishment of Correction Method

XE‐2100 analyzer was used for CBC analysis to obtain HGB concentration of lipemic blood samples (HGB_LB). Lipemic blood samples were centrifuged at 550 g for 3 min. Then the plasma fraction was analyzed for hemoglobin concentration (HGB_LP). Then the following formula was used to calculate the corrected HGB levels:

$$\begin{array}{ccc}\hfill {\mathrm{HGB}}_{\mathrm{CORRECTED}}& =& {\mathrm{HGB}}_{\mathrm{LB}}-({\mathrm{HGB}}_{\mathrm{LP}}-{\mathrm{HGB}}_{\mathrm{LP}}\hfill \\ \times {\mathit{HCT}}_{\mathrm{LB}})\hfill \end{array}$$

$${\mathrm{MCH}}_{\mathrm{CORRECTED}}={\mathrm{HGB}}_{\mathrm{CORRECTED}}/{\mathrm{RBC}}_{\mathrm{LB}}$$

$${\mathrm{MCHC}}_{\mathrm{CORRECTED}}={\mathrm{HGB}}_{\mathrm{CORRECTED}}/{\mathrm{HCT}}_{\mathrm{LB}}$$

Anticipated results were as following:

$$\begin{array}{ccc}\hfill {\mathrm{HGB}}_{\mathrm{CORRECTED}}& =& {\mathrm{HGB}}_{\mathrm{TRUE}},{\mathrm{MCH}}_{\mathrm{CORRECTED}}\hfill \\ =& {\mathrm{MCH}}_{\mathrm{TRUE}},{\mathrm{MCH}}_{\mathrm{CCORRECTED}}\hfill \\ =& {\mathrm{MCHC}}_{\mathrm{TRUE}}.\hfill \end{array}$$

Validation of Correction Method

A total of 102 fresh whole blood samples (2 ml each) with no hemolysis, icterus, or lipemia (TG < 1.83 mmol/l) were collected in vacuum tubes containing EDTA‐K₂. A XE‐2100 analyzer was used to obtain CBC results, including the true concentration of HGB. The samples were centrifuged at 1,685 g for 5 min to separate blood cells from the plasma. Variable amount of the plasma in samples were replaced with intralipid to achieve different TG concentrations, raising the TG concentration to >4.0 mmol/l. Then the samples were mixed gently for 10 times to obtain artificial lipemic samples. CBCs were performed for these lipemic samples (HGB_LB). The lipemic blood samples were then centrifuged at 550 g for 3 min to separate the plasma. The concentrations of HGB in lipemic plasma (HGB_Lp) were measured. All three steps of HGB measurements were performed by the same mode in the same analyzer.

Comparison of Values Before and After Correction From Patients Received Intralipid

We applied this method on 10 randomly selected in‐patient individuals who had a clinical history data within 1 week. Patient's blood samples were nonlipemic before they received intralipid, and these results were defined as true values. Later, these patients received intralipid and developed lipemia. Sample from the patients were analyzed and corrected by this method.

Statistical Analysis

Test results were demonstrated as Mean ± standard deviation (SD). Paired samples t‐test was used for comparison HGB values of artificial lipemic blood before and after correction between the true values. The effect of interference and correction was showed by differences in percentage. Linear regression was used for correlation analysis. All data were processed by statistical software SPSS statistics 11.0. P < 0.05 was defined as statistical significance.

RESULTS

Establishment of Centrifugal Force and Time

Different centrifugal force and time were used to centrifuge 50 fresh whole blood samples with different HGB concentration and 50 mixtures of intralipid and plasma with different TG concentration. Changes in parameters before and after centrifugation were listed in Table 1.

Table 1.

Impact of Different Centrifugal Force and Time on Whole Blood and Intralipid Mixture

			Fresh complete blood				Intralipid and plasma mixture
Centrifugal time	Centrifugal force	No.	RBC (×1012/l)		HGB (g/l)		RBC (×1012/l)		HGB (g/l)
(min)	(g)	(n)	Before	After	Before	After	Before	After	Before	After
10	137	10	3.94 ± 0.81	0.00 ± 0.00	116 ± 21.78	0.30 ± 0.48	0.00 ± 0.00	0.00 ± 0.00	30.4 ± 25.07	31.2 ± 25.17*
5	310	10	4.17 ± 0.90	0.00 ± 0.00	121 ± 22.82	0.40 ± 0.52	0.00 ± 0.00	0.00 ± 0.00	29.5 ± 23.20	30.0 ± 23.05*
3	550	10	4.40 ± 0.77	0.00 ± 0.00	132 ± 28.72	0.70 ± 0.48	0.00 ± 0.00	0.00 ± 0.00	30.5 ± 21.59	29.9 ± 22.24*
5	550	10	4.35 ± 0.55	0.01 ± 0.01	128 ± 15.83	0.80 ± 0.63	0.00 ± 0.00	0.00 ± 0.00	31.4 ± 24.28	30.7 ± 22.92*
5	1,685	10	4.82 ± 0.73	0.00 ± 0.00	136 ± 20.31	0.30 ± 0.48	0.00 ± 0.00	0.00 ± 0.00	32.3 ± 26.31	32.1 ± 25.68*

*P > 0.05.

Verification of the Correction Method

The deviation percentages and correlation coefficient of HGB, MCH, and MCHC of 102 lipemic blood samples before and after correction, in comparison with the true values, were shown in Tables 2 and 3, while the linear regression analysis was shown in Figure 1.

Table 2.

Comparison of True Values of HGB, MCH, MCHC, and Values Before Correction (n = 102)

Parameters	HGB (g/l)	MCH (pg)	MCHC (g/l)
True value	110.5 ± 17.21	29.3 ± 2.06	313.5 ± 7.20
Lipemic value	132.8 ± 24.65	35.4 ± 4.75	379.5 ± 42.63
Difference percentage (%)	20.18	21.03	21.07
Correlation coefficient (r)	0.791	0.463	0.207
P	<0.001	<0.001	<0.001

Table 3.

Comparison of HGB, MCH, MCHC in 102 Fresh Complete Blood Samples After Correction

Parameters	HGB (g/l)	MCH (pg)	MCHC (g/l)
True value	110.5 ± 17.21	29.3 ± 2.06	313.5 ± 7.20
Lipemic value	110.2 ± 17.57	29.3 ± 2.22	312.5 ± 11.17
Difference percentage (%)	−0.28	0.06	−0.31
Correlation coefficient (r)	0.989	0.935	0.717
P	0.157	0.802	0.204

Figure 1.

Correlation of true and lipemic values of HGB, MCH, MCHC before (A, C, E) and after (B, D, F) correction.

Comparison of Values Before and After Correction From Patients Received Intralipid

Values of HGB, MCH, and MCHC before and after correction of 10 randomly selected in‐patient individuals who received intralipid and developed lipemia were listed in Table 4.

Table 4.

Comparison of True HGB Values and HGB Values Before and After Correction in 10 Patient Samples

			HGB (g/l)		MCH (pg)		MCHC (g/l)
Sample	Plasma	TG
no.	turbidity	(mmol/l)	Lipemic	Corrected	Lipemic	Corrected	Lipemic	Corrected
1	+	20.60	167	156	40.4	28.2	365	340
2	+	10.72	78	72	40.6	37.3	373	343
3	+	26.32	110	70	51.6	32.8	556	353
4	+	10.61	84	65	38	29.2	438	337
5	+	12.12	121	115	27.9	26.4	344	325
6	+	18.20	142	131	30.9	28.5	347	320
7	+	10.89	95	89	21.5	20.1	301	281
8	+	26.10	89	59	31.3	20.7	320	338
9	+	9.83	123	109	34.2	30.2	348	308
10	+	17.46	118	102	29.9	25.9	313	272

DISCUSSION

Patients with hyperlipidemia are on the rise in practice and there is an increase in clinical use of intralipid for parenteral nutrition. Therefore, blood samples with high TG levels present a real challenge to clinical laboratories. High TG concentration in blood significantly interferes with HGB measurement by automated hematology analyzers. Thus, a simple and effective laboratory correction technique is needed.

Currently, two major methods were applied to correct the interference of lipemia. Nevertheless, there were some obvious shortcomings. In Lin's patent, the first step of correction was high‐speed centrifugation. But the high‐speed centrifugation was limited not only by its poor popularity, but also by its complex and time‐consuming operation. In addition, it was found that fat particles aggregate to form floating mass on the surface of plasma after high‐speed centrifugation, which was difficult to clear and accurately measure its volume. The second step was to replace the plasma of lipemic blood sample with saline or another diluent. Since lipemic plasma can hardly be fully replaced, this method had a low accuracy. Kalache 5 reported an indirect measurement of HGB concentration. MCV was measured by standard electronic counting, and then the MCV/MCH ratio was calculated. A special formula was applied to correct HGB 6. Unfortunately, most blood cell analyzers were unable to perform standard electronic counting of MCV, this method did not gain wide application.

Our previous research data demonstrated that the interference of HGB from TG was less than 2% (data not shown) when TG concentration was <4.0 mmol/l (about 355 mg/dl). While, the Clinical Laboratory Improvement Amendments’ 88 (CLIA’88) provided the acceptable performance limit for HGB test was ± 7%. So, high blood TG was defined as a blood TG concentration >4.0 mmol/l in this study.

As shown in Table 1, a variety of centrifugation conditions (137 g for 10 min, 310 g for 5 min, 550 g for 3 min, 550 g for 5 min, or 1,685 g for 5 min) were tested. RBCs in all five groups of fresh whole blood samples settled at the bottom completely at all tested centrifugation conditions. This indicated that low‐speed centrifugation is sufficient to separate RBCs from the plasma in fresh blood samples. The RBC remained 0 before and after centrifugation in intralipid and plasma mixture. While the mean values of HGB in intralipid and plasma mixture before and after centrifugation in five centrifugation conditions were 30.4 and 31.2 g/l, 29.5 and 30.0 g/l, 30.5 and 29.9 g/l, 31.4 and 30.7 g/l, 32.3 and 32.1 g/l, respectively. This provided evidence that the HGB concentration of intralipid and plasma mixture was not affected by different centrifugal conditions. Therefore, the least time‐consuming centrifugation parameter was adopted.

Results in Table 2 demonstrated that adding intralipid in blood caused a change of HGB, MCH, and MCHC in 102 fresh whole blood samples (20.18%, 21.03%, and 21.07%, respectively, P < 0.05). After correction, the deviation percentages dropped to below ±2% (–0.28%, 0.06%, and −0.31% respectively, P > 0.05). Figure 1 also showed perfect correlation between true and corrected HGB, MCH, and MCHC, indicating that this correction method worked well on blood samples with intralipid added in vitro. Though Bornhost's report 7 had shown that samples with added intralipid could not fully simulate native lipemia samples, as showed in Table 4, this correction method worked well in clinical practice, especially for those patients who received intralipid injection.

In a clinical hematology laboratory, we could find a lipemic sample by the following methods: (1) TG > 4 mmol/l; or (2) difference between HGB, MCH, and MCHC values and historical data exceeded 20%; or (3) MCHC ≥ 365 g/l; or (4) HGB value and RBC count were not coincidence; or (5); a lot of nonstaining fat globules with different sizes were seen in the blood smear; or (6) hematology analyzer alarmed “Turbidity/HGB Interference”; or (7) plasma visible turbidity. When any of them appeared in blood sample, values of HGB, MCH, and MCHC should be corrected. As we all known, these rules could not valid everywhere. TG values and historical data were not available for every patient, and not all the lipemic samples had a MCHC ≥ 365 g/l (Table. 4). Rule No. 4 needed calculation and meanwhile rule No. 5 needed a smear preparation. And some samples did not have alarm “Turbidity/HGB Interference” such as sample No.8 in Table 4. So we recommended a way to find the plasma turbidity that was proved completely effective, that the CBC samples should be observed by naked eyes of medical technologists after RBCs natural sedimented for a few minutes before the results were reported (Table 4).

By adding a simple step of low‐speed centrifugation, our method was able to provide reliable results of HGB, MCH, and MCHC in highly lipemic blood samples without special equipments.