Changes in Hematologic Parameters Induced by Thermal Treatment of Human Blood

Transcription

1 Annals of Clinical & Laboratory Science, vol. 32, no. 4, Changes in Hematologic Parameters Induced by Thermal Treatment of Human Blood Jong Weon Choi and Soo Hwan Pai Department of Clinical Pathology, College of Medicine, Inha University, Inchon, Korea Abstract. To investigate the effects of high temperature on hematologic parameters of human blood, we assessed complete blood cell counts, red cell indices, and platelet aggregability in 72 adult blood samples after exposure to varying degrees of temperature. There were no significant differences in erythrocyte counts, hemoglobin concentration, or mean corpuscular hemoglobin (MCH) between pre- and post-treated groups when blood samples were treated at 50 C for 5 min. Under the same conditions, however, mean corpuscular volume (MCV) and hematocrit became significantly higher in the post-treated group (p <0.01). A dramatic increase was observed in the numbers of platelets and eosinophils measured by electronic counter: the mean numbers of platelets and eosinophils in the post-treated group ( x 10 9 /L and 5.58 x 10 9 /L) were significantly higher than those for the pre-treated group (246.6 x 10 9 /L and 0.15 x 10 9 /L, p < 0.01). Platelet clumps of varying size were seen in EDTA-anticoagulated blood after exposure to 46 C for 90 sec; however, these clumps were not detected in citrate-, heparinized-, or washed EDTA-anticoagulated blood. Platelet aggregability to various agonists was profoundly decreased after treating the blood at 43 C for 5 min. In summary, this study shows that critical changes of hematologic parameters occur when blood is exposed to 50 C, and that the anticoagulating property of EDTA is altered upon exposure to high temperature. (received 9 April 2002; accepted 20 May 2002) Keywords: temperature, hematologic values, eosinophils, platelet aggregability. Introduction Echinocytosis is a morphologic change that is characteristic of red blood cells from thermally injured patients [1,2]. The temperature required for red cell fragmentation is an indicator of membrane abnormality, since the morphologic changes are readily detected and the critical temperature for normal red blood cells is constant [2]. Increased thermal sensitivity of red cell membrane has been demonstrated for pyropoikilocytes [3]. Pyropoikilocytosis are distinguished by their substantially lower critical temperature of fragmentation. The lowest temperature at which normal red blood cells undergo heat-induced Address correspondence to Soo Hwan Pai, M.D., Department of Clinical Pathology, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon, , Republic of Korea; tel ; fax ; shpaimd@inha.ac.kr. fragmentation is 49 C, whereas pyropoikilocytes undergo morphologic changes or fragmentation 45 C [4]. Fatality may occur at body temperature >42 C, at which temperature red blood cells undergo in vivo hemolysis. However, there are no significant changes of morphology when red blood cells are exposed to 42 C in vitro. Further, the stability of spectrin structure, the activity of ATPase, and the glycolysis of red blood cells are nearly optimal even at that temperature [5,6]. Changes of hematologic parameters induced by exposure of blood to varying degrees of thermal temperature have not been reported. The present study shows that a dramatic elevation occurs in the numbers of platelets and eosinophils after thermal treatment, when measured by an electronic counter. Our data reveal that platelet clumps are generated peculiarly in EDTA-anticoagulated blood after exposure to heat. This paper describes the striking changes that are observed in thermally treated /02/0400/0393 $ by the Association of Clinical Scientists, Inc.

2 394 Annals of Clinical & Laboratory Science human blood specimens. The results of this study may help to elucidate the changes in hematologic parameters that occur in blood of thermally-injured or hyperthermia-treated patients. Materials and Methods Seventy-two healthy volunteers (37 men, 35 women), the subjects of this study, were laboratory personnel who had taken no medications for 2 wk. Complete blood cell counts (CBC) were within the normal range. The age of the subjects averaged 32.4 yr (range 26 to 47 yr). Blood was collected in EDTA-, and citrate-, and heparin-containing Vacutainer tubes (Becton-Dickinson, Rutherford, NJ). Each blood sample was incubated in a temperature-controlled shaking water bath at temperatures ranging from 40 to 54 C. CBC analysis of pre- and post-treated blood specimens was performed using an electronic cell counter (SE 9000; Sysmex, Kobe, Japan). Peripheral blood smears of each specimen were reviewed for the confirmation of leukocyte differentials, morphologic changes of erythrocytes, and appearance of platelet clumps. Morphologic changes of red cells were examined with light and phase contrast microscopes. To evaluate the effect of EDTA on heat-induced platelet clumps, EDTA-anticoagulated blood specimens were washed 3 times with normal saline, and the washed blood samples were then incubated at varying temperatures in the water bath. To investigate changes in platelet aggregability induced by thermal treatment, blood was collected in Vacutainer tubes (Becton-Dickinson) containing M sodium citrate (1 vol anticoagulant to 9 vol whole blood). Platelet-rich plasma (PRP) was prepared from pre- and post-thermally treated samples by centrifugation at 200 x g for 20 min at room temperature. Platelet-poor plasma (PPP) was prepared by centrifugation of PRP at 3000 x g for 15 min. Platelet counts in PRP ranged from x 10 9 /L to x 10 9 /L. PRP was incubated at 37 C with stirring (1,100 rpm) for 3 min prior to the addition of any agents, and all experiments were completed within 4 hr of collection. Aggregating agonists (ie, epinephrine, 300 µm; ADP, 10 µm; collagen, 10 µg/ml; and ristocetin 1500 µg/ml; Helena Laboratories, Beaumont, TX), were used to induce platelet aggregation, which was monitored by continuous recording of light transmission in a PACKS-4 aggregometer (Platelet Aggregation Chromogenic Kinetic System-4; Helena Laboratories). The final reaction volume was 500 µl; the light transmission of PRP and PPP was adjusted to 0% and 100%, respectively, prior to recording the light transmission of each sample. Data analysis was performed using SAS statistical software (version 6.12; SAS Institute, Cary, NC). Differences of values between pre- and postthermally treated groups were evaluated by Student s paired-sample t-test (p <0.01 statistically significant). Results Hematologic data of 72 blood samples, which were treated at 50 C for 5 min, are listed in Table 1. There were no significant differences in erythrocyte counts, hemoglobin concentrations, or mean corpuscular hemoglobin (MCH) between pre- and post-treated groups. However, hematocrit and mean corpuscular Table 1. Hematologic parameters (mean ± SD) after incubation of blood samples (n = 72) at 50 C for 5 min. Parameters Pre-treatment Post-treatment RBC (x /L) ± ± 46.7 Hemoglobin (g/dl) 14.1 ± ± 1.7 Hematocrit (%) 41.4 ± ± 4.7* MCV (fl) 90.2 ± ± 5.2* MCH (pg) 30.6 ± ± 1.7 MCHC (%) 33.9 ± ± 1.6* Platelets (x10 9 /L) ± ± 987.3* RDW (%) 12.6 ± ± 3.0* PDW (%) 11.8 ± ± 3.2 MPV (fl) 10.0 ± ± 0.7 Eosinophils (x 10 9 /l) 0.15 ± ± 4.09* RBC = red blood cell, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, RDW = red cell distribution width, PDW = platelet distribution width, MPV = mean platelet volume. Heparin-anticoagulated blood samples were used for counting platelet numbers. * p <0.01 by Student s paired t-test.

3 Hematologic parameters and thermal treatment 395 Fig. 1. Erythrocyte morphology after in vitro exposure of human blood to 50 C for 30 sec. Thin filamentous projections of erythrocytes are evident (Wright-Giemsa stain, original magnification x 1000). Fig. 2. Changes in erythrocyte morphology after in vitro exposure of human blood to 50 C for 5 min. Many fragments of erythrocytes and microspherocytes are evident (Wright-Giemsa, original magnification x 1000). Fig. 3. Appearance of platelet clumps after thermal treatment of human blood at 46 C for 90 sec. A large clump of platelets (indicated with an arrow) is observed in EDTA-anticoagulated blood (Wright-Giemsa stain, original magnification x 1000). Fig. 4. No platelet clumps are seen in heparinized blood even after thermal treatment at 46 C for 90 sec. (individual platelets are indicated with arrows; Wright- Giemsa stain, original magnification x 1000).

4 396 Annals of Clinical & Laboratory Science volume (MCV) in the post-treated group averaged 47.6 ± 4.7% and ± 5.2 fl, which were significantly higher than in the pre-treated group (41.4 ± 4.2% and 90.2 ± 3.7 fl, respectively, p <0.01 in each case). Platelet and eosinophil counts, measured by electronic counter, were approximately 12- or 30-fold higher in the post-treated group than in the pre-treated group (p <0.01). On the other hand, when we examined the numbers of platelets and eosinophils in smear slides, no significant differences were observed between the two groups. No significant morphologic changes were observed in erythrocytes when the blood samples were incubated at 48 C for 5 min; however, erythrocytes began to show filamentous projections after exposure to 50 C for 30 sec (Fig. 1). These changes in erythrocytes were intensified as the exposure time was prolonged. In blood samples incubated at 50 C for 5 min, many microspherocytes and red cell fragments, which were broken from echinocyte spicules, were observed (Fig. 2). The reaction of platelets following exposure to elevated temperature is summarized in Table 2. No morphologic changes were observed in platelets of Table 2. Appearance of platelet clumps in peripheral blood smears after thermal treatment of blood specimens. Temp. Treatment Platelet clumps in blood smears* ( C) duration (sec) EDTA Citrate Heparin Washed EDTA * Platelet clumps are observed in blood smears from only EDTA-anticoagulated blood samples after exposure to high temperature. EDTA-anticoagulated blood samples were washed three times with normal saline. +++, frequent; ++, occasional; +, rare; -, not seen. peripheral blood smears until the blood was incubated at 44 C for 90 sec. As shown in Fig. 3, platelet clumps of varying size were observed in blood smears after blood samples were incubated at 46 C for 90 sec. The severity of platelet clumping was intensified as the temperature was elevated. The phenomenon of platelet clumping was present in EDTA-anticoagulated blood, but was not seen in citrate-anticoagulated or heparinized blood (Fig. 4). The platelet clumps were not observed when EDTA-anticoagulated blood was washed 3 times with normal saline, even when the blood samples were exposed to 54 C for 90 sec. Table 3 summarizes the changes in the maximal aggregability of platelets induced by various agonists after thermal treatment of citrate-anticoagulated blood. The mean values of platelet aggregability to agonists were markedly decreased after citratedanticoagulated blood specimens were incubated at 43 C for 5 min. Discussion In this study, we investigated the effects of in vitro exposures to high ambient temperatures on hematologic parameters of human blood and on platelet aggregation. We found that critical change in hematologic values and erythrocyte morphology occurs during the time that blood is exposed to 50 C. In particular, striking increase was noted in the numbers of platelets and eosinophils that were counted after exposure to high temperature. We also found that the anticoagulating property of EDTA is changed by exposure to temperature 46 C. Our results for morphologic changes in thermally-treated erythrocytes are in general agreement with the report Table 3. Platelet aggregability after thermal treatment of citrate-anticoagulated blood specimens at 43 C for 5 min. Agonists Maximal aggregation (%, mean ± SD) Pre-treated Post-treated ADP 96.2 ± ± 0.9 Collagen 92.3 ± ± 1.4 Epinephrine 86.4 ± ± 2.7 Ristocetin 94.1 ± ± 1.5

5 Hematologic parameters and thermal treatment 397 of Zarkowsky [7], who found no significant changes in erythrocytes of healthy humans following heat treatment at 49 C. Utoh et al [8] reported that 97% of human erythrocytes underwent spherocytic and echinocytic transformation in vitro after incubation at 50 C for 1 hr; these transformations were observed in only 19% of calf erythrocytes under the same conditions. In contrast, in our study, echinocytes began to appear when blood was incubated at 50 C for 30 sec, and almost all erythrocytes underwent echinocytic transformation at 50 C for 90 sec. The apparent discrepancies among these studies in regard to in vitro exposures to high temperatures probably reflect differences in sample tubes, kinds of incubation, or sample volumes. In this study, echinocytes were not seen after incubation at 50 C for 5 min, whereas microspherocytes and erythrocyte fragments were characteristically observed. These results suggest that most echinocytes changed to microspherocytes after shedding their cytoplasmic projections into blood. In our study, dramatic elevations in the mean numbers of platelets and eosinophils were observed in thermally treated group, when measured by an electronic counter. However, there were no significant changes in the numbers of these cells, when counted on blood smears. These results suggest that microspherocytes or small fragments of erythrocytes are registered as platelets by electronic counting, which differentiates platelets from erythrocytes on the basis of size of a structure that passes through a small aperture. The electronic cell counter (SE-9000, Sysmex) that was used in this study measures the number of eosinophils after dissolving all blood cells, excepting eosinophils, using a lysis solution of Stromatolyser-EO II. To investigate the action of Stromatolyser-EO II on thermally-treated blood, we examined blood smears after incubating thermally-treated blood samples with the lysis solution. We observed that a considerable number of neutrophils remained persistently in the thermal-treated blood even after incubation with the lysis solution. These results suggest that Stromatolyzer-EO II-resistant neutrophils, which may be generated by thermal treatment, are measured in the electronic cell counter as eosinophils. It thus appears that careful attention should be paid to the interpretation of the numbers of platelet or eosinophils that are measured by electronic counter, especially in blood from severely burned patients or hyperthermia-treated patients. On the other hand, platelet clumps of varying size were observed in blood smears when blood samples were exposed to 46 C, and the degree of platelet clumping was intensified as the temperature was elevated. The platelet clumps were observed in EDTA-anticoagulated blood, but not in citrate- or heparin-anticoagulated blood. Interestingly, the platelet clumps were not seen in washed EDTAanticoagulated blood, even after the blood was exposed to 54 C. This suggests that the anticoagulating properties of EDTA may be altered by exposure to temperature. Under the experimental conditions, EDTA evidently induces platelet clumping and loses its anticoagulating property. Pasha et al [9] reported that platelet aggregability in response to ADP was decreased to 2.1% after platelets were exposed to 44 C. Rao et al [10] found that there were no significant changes in platelet aggregability or morphology when platelets were incubated at 43 C for 60 min; however, the aggregability in response to arachidonic acid was lost after platelets were exposed to 45 C for 90 min. In our study, there were no significant changes in platelet aggregabilty between pre- and post-treated blood specimens after exposure to 45 C for 90 sec. However, their aggregability was markedly decreased when platelets were incubated at 43 C for 5 min. These results indicate that the duration of exposure to elevated temperature is an important factor that influences platelet aggregability. In conclusion, a critical change in hematologic parameters occurs from the time that blood is exposed in vitro to 50 C. Platelet clumps are observed in EDTA-anticoagulated blood, but not in citrate-, heparin-, or washed EDTAanticoagulated blood, suggesting that the anticoagulating property of EDTA may be changed by exposure to high temperature. The present study emphasizes the need for care in interpreting the hematologic parameters for blood specimens from thermally injured patients that are produced by electronic cell counters, especially in regard to platelets and eosinophils.

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