Improved method for the cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch.

Refinement of a method described previously (Cryobiology12, 110–118, April, 1975) made possible routine freezing of full units of packed erythrocytes after separation of platelet rich plasma, and buffy coats. The volume frozen was 405 ml which included packed red cells (190–220 ml), plasma (43–73 ml), and cryo-HES (142 ml, final concentration 14% $\text{w}\text{v}$). The units could be frozen with or without shaking by direct immersion in liquid nitrogen. Thawing was accomplished by transferring units quickly from liquid nitrogen storage to a shaking water bath at 54 °C. The average yield from units of red cells was 98.4%. The stability to a 50-fold dilution in 0.15 m NaCl was 87.8%. Thawing rate was the critical variable in producing the most stable thawed cells. Plasma expander HES was usable but the thawed units were more viscous and about 7% less stable. Red cells prewashed with 0.15 m NaCl and frozen without plasma showed no significant changes in cellular yield or stability. The optimum resuspension medium was 3% glucose. A morphologic study of cells fixed in 1% glutaraldehyde revealed that before freezing red cells were partially dehydrated in 14% HES. These were smooth, flat discs. Cells fixed on thawing were extensively dehydrated and seen as large, thin, smooth, flat discs with approximately 10% echinocytes. On dilution with 6% glucose (1:1) these swelled and reverted to biconcave discocytes except for approximately 5% echinocytes. Storage in liquid nitrogen measured in groups of three units of 15 units for 0, 3, 6, 9, and 12 weeks revealed normal postthawed oxygen delivery (P-50). The greatest measurable effect of freezing red cells in HES was a loss of cellular K⁺ compensated by a corresponding increase in Na⁺.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

NMR q-space analysis of canonical shapes of human erythrocytes: stomatocytes, discocytes, spherocytes and echinocytes

q-Space plots obtained experimentally using pulsed field-gradient stimulated echo (PGSTE) nuclear magnetic resonance (NMR) spectroscopy from water diffusing in red blood cells (RBCs) of different canonical (distinct variant) morphologies have “signature” features. The experimental q-space plots from suspensions of stomatocytes, echinocytes and spherocytes generated chemically had no diffraction features; in contrast a sample of blood from a patient with hereditary spherocytosis showed diffraction minima. To understand the forms of q-space plots, mathematical/geometrical models of discocytes, stomatocytes, echinocytes and spherocytes were used as restricting boundaries in simulations of water diffusion with Monte Carlo random walks. These simulations indicated that diffusion-diffraction minima are expected for each of the cell shapes considered. The absence of diffusion-diffraction minima in stomatocytes generated by dithiothreitol treatment was surmised to be due to non-alignment of the cells with the magnetic field of the NMR spectrometer. Differential interference contrast microscopy images of the chemically generated spherocyte and echinocyte suspensions showed them to be heterogeneous in cell shape. Therefore, we concluded that the shape heterogeneity caused the loss of the diffusion-diffraction features, which were observed in the more homogeneous sample from a patient with hereditary spherocytosis, and in the simulations of homogeneous cell suspensions. This understanding of factors that affect q-space plots from RBC suspensions will assist morphological studies of other cell and tissue types.     

Alkaline hemolysis fragility is dependent on cell shape: results from a morphology tracker.

BACKGROUND: The morphometric analysis of red blood cells (RBCs) is an important area of study and has been performed previously for fixed samples. We present a novel method for the analysis of morphologic changes of live erythrocytes as a function of time. We use this method to extract information on alkaline hemolysis fragility. Many other toxins lyse cells by membrane poration, which has been studied by averaging over cell populations. However, no quantitative data are available for changes in the morphology of individual cells during membrane poration-driven hemolysis or for the relation between cell shape and fragility. METHODS: Hydroxide, a porating agent, was generated in a microfluidic enclosure containing RBCs in suspension. Automatic cell recognition, tracking, and morphometric measurements were done by using a custom image analysis program. Cell area and circular shape factor (CSF) were measured over time for individual cells. Implementations were developed in MATLAB and on Kestrel, a parallel computer that affords higher speed that approaches real-time processing. RESULTS: The average CSF went through a first period of fast increase, corresponding to the conversion of discocytes to spherocytes under internal osmotic pressure, followed by another period of slow increase until the fast lysis event. For individual cells, the initial CSF was shown to be inversely correlated to cell lifetime (linear regression factor R=0.44), with discocytes surviving longer than spherocytes. The inflated cell surface area to volume ratio was also inversely correlated to lifetime (R=0.43) but not correlated to the CSF. Lifetime correlated best to the ratio of cell inflation volume (Vfinal-Vinitial) to surface area (R=0.65). CONCLUSIONS: RBCs inflate at a rate proportional to their surface area, in agreement with a constant flux model, and lyse after attaining a spherical morphology. Spherical RBCs display increased alkaline hemolysis fragility (shorter lifetimes), providing an explanation for the increased osmotic fragility of RBCs from patients who have spherocytosis.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Natural killer activity of kurloff cells: A direct demonstration on purified kurloff cell suspensions

In order to study their natural killer effect, guinea pig splenic Kurloff cells were fractionated by Percoll discontinuous density gradient centrifugation. Kurloff cells were collected and tested for cytotoxicity in a 24-hr chromium-release test. Comparison of different splenic cellular samples (of males or estrogenized females) with increasing percentage of Kurloff cells, revealed a highly significant positive correlation (r = 0.93, α < 0.01) with the cellular cytotoxicity developed against the K 562 target cells.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

In vivo circulation of mouse red blood cells frozen in the presence of dextran and glucose

Loading with monosaccharide can improve the quality of human red blood cells (hRBCs) frozen with polymer. But in vivo life span of hRBCs frozen with polymer and sugar is not determined. In this study, following incubation with glucose, mouse red blood cells (mRBCs) were frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. After thawing, hemolysis, exposure of PS, and osmotic fragility of frozen mRBCs were determined in vitro. After transfusion of fluorescein isothiocyanate (FITC)-labeled mRBCs, the 24 h recovery and half life span of frozen mRBCs were determined. The data indicated the postthaw hemolysis of mRBCs frozen with dextran and glucose were significantly less than that of cells frozen with dextran (17.23% ± 5.21% vs 25.96% ± 10.07%, P = 0.034). But freezing can also result in exposure of phosphatidylserine and increase of osmotic fragility of mRBCs. After transfusion, the 24 h recovery of mRBCs frozen in the absence or presence of glucose was similar to that of the control cells (P = 0.748 and 0.971). However, the half life span of mRBCs frozen in the absence or presence of glucose was significantly less than that of the control cells (P = 0.000). In addition, incubation with glucose can not increase the life span of frozen red blood cells (7.16 ± 0.93 d vs 7.15 ± 0.34 d, P = 0.982). In conclusion, incubation with monosaccharide could significantly increase the recovery of mRBCs frozen with polymer. Although freezing can significantly shorten the half life span of frozen cells, it can not influence the 24 h recovery of frozen mRBCs. In addition, incubation with monosaccharide before freezing can not increase the life span of frozen mRBCs. So according to the above data, to increase the life span of hRBCs frozen with polymer and monosaccharide, the osmotic fragility of the frozen RBCs must be decreased in the future.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.     

透析式洗涤冰冻红细胞去除白细胞和血小板的方法

目的：用低速离心的方法减少悬浮红细胞中自细胞和血小板的数量,使透析式洗涤机洗涤的冰冻红细胞中白细胞和血小板的残留量小于1％。方法：分别选用低速离心（700和500r／min）和常规离心（3800r／min）全血制备悬浮红细胞,计算白细胞和血小板的清除率;甘油低温冰冻保存,透析式冰冻红细胞洗涤机洗涤冰冻红细胞,对洗涤后的红细胞进行质量检测。结果：500r／min离心后白细胞和血小板的清除率（％）分别为53.82±6.83和85.23±4.21;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为0.843±0.058和0.903±0.035。700r／min离心后白细胞和血小板的清除率（％）分别为23.38±2.36和62.61±3.82;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为0.983±0.024和1.021±0.045。3800r／min离心后白细胞和血小板的清除率（％）分别为9.82±4.12和6.39±3.26;洗涤后的冰冻红细胞中白细胞和血小板残留量（％）分别为3.839±2.896和3.528±2.689。结论：用500r／min离心全血制备的悬浮红细胞,经甘油低温冻存,透析式洗涤机洗涤后,血小板和白细胞的残留量等各项指标均符合国家标准。     

Lysosomal enzyme secretion by macrophages during intracellular storage of particles

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase β-N-acetylglucosaminidase. Thus macrophage lysosomal enzyme secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.     

Mechanism of freeze-thaw damage in Pseudomonas F8

Pseudomonas F8 was tested for freeze-thaw sensitivity after being washed several times with water. Survival increased to 85% in the fourth wash. Addition of 1% sodium chloride decreased survival to the level obtained when cells were frozen in brain-heart infusion broth. No differences were observed between cells grown at 5 or 23 °C. The freeze-thaw sensitivity of Pseudomonas may be due to the cell's exposure to concentrated medium salts during cooling and warming.     

Hemoglobin Constant Spring: hemoglobin synthesis in heterozygous and homozygous states.

Thirteen adult and one newborn heterozygotes, and three homozygotes for hemoglobin Constant Spring were examined for globin chain synthesis. Reticulocytes from venous blood were incorporated with [³H]-leucine in an incubation mixture for 3 hours. Globin prepared from the radioactive, washed red cells was fractionated by CM-cellulose chromatography in 8 M urea and the total radioactivity of each globin chain was determined. The mean of $\text{α}\text{β}$ ratio in the heterozygotes was 1.34 ± SD 0.08, which is significantly different from that of 1.07 ± SD 0.03 in eleven normal controls. The $\text{α}\text{β+γ}$ ratio in the heterozygous neonate was also 1.39. The $\text{α}\text{β}$ ratios in the three homozygotes were around 1.6. The α-Constant Spring chain appears to be over produced, but it may be unstable or labile, not fully available for conjugation with the non alpha chains.     

The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity

The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=?0.94, P<0.0005}$) and a positive correlation for peritoneal lymphocytes ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=+0.97,P<0.0005}$) and spleen cells ($\text{r}{}_{\mathrm{<mtext>s</mtext>}}\text{=+0.76,P<0.005}$) existed.     

Physical-Chemical Basis of the Protection of Slowly Frozen Human Erythrocytes by Glycerol

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology, 13:9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J., 23:89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT₅₀) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT₅₀ for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT₅₀ corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.     

Na+/H+ exchange is increased in sickle cell anemia and young normal red cells

Summary Red cell volume regulation is important in sickle cell anemia because the rate and extent of HbS polymerization are strongly dependent on initial hemoglobin concentration. We have demonstrated that volume-sensitive K:Cl cotransport is highly active in SS whole blood and is capable of increasing MCHC. We now report that Na⁺/H⁺ exchange (Na/H EXC), which is capable of decreasing the MCHC of erythrocytes with pH_i<7.2, is also very active in the blood of patients homozygous for HbS. The activity of Na/H EXC (maximum rate) was determined by measuring net Na⁺ influx (mmol/liter cell·hr=FU) driven by an outward H⁺ gradient in oxygenated, acidloaded (pH_i 6.0), DIDS-treated SS cells. The Na/H EXC activity was 33±3 FU (mean±se) (n=19) in AA whites, 37±8 FU (n=8) in AA blacks, and 85±15 FU (n=14) in SS patients (P<0.005). Separation of SS cells into four density-defined fractions by density gradient revealed mean values of Na/H EXC four to five times higher in reticulocytes (SS1), discocytes (SS2) and dense discocytes (SS3), than in the fraction containing irreversibly sickled cells and dense discocytes (SS4). In contrast to K:Cl cotransport, which dramatically decreases after reticulocyte maturation, Na/H EXC persists well after reticulocyte maturation. In density-defined, normal AA red cells, Na/H EXC decreased monotonically as cell density increased. In SS and AA red cells, the magnitude of stimulation of Na/H EXC by cell shrinkage varied from individual to individual. We conclude that Na/H EXC is highly expressed in SS and AA young red cells and decays slowly after reticulocyte maturation.     

Quantitative analysis of pyridine nucleotides in red blood cells: a single-step extraction procedure.

Quantitative analysis of red cell pyridine nucleotides has been unreliable in the past because of technical problems in extracting them in the presence of hemoglobin. A simple alcoholic extraction procedure for analysis of pyridine nucleotides in red blood cells is described in this paper. Pyridine nucleotides extracted in the presence of hemoglobin in solution show recoveries of NADH, NAD, and NADP averaging over 70%, while recoveries of NADPH were about 60%. In order to show that these techniques could detect actual intracellular differences in nucleotides inside red cells, two experiments were performed in which the ratios of the nucleotides would be predictably altered. Intact cells incubated in the presence of methylene blue show a decrease in the $\text{NADPH}\text{NADP}$ ratio, and intact cells incubated in the presence of hydrazine and lactate show an increase in the $\text{NADH}\text{NAD}$ ratio. The changes in pyridine nucleotide ratios in these experiments are in the expected direction and were easily detected. Levels of pyridine nucleotides in red blood cells of normal human adults are also presented.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

A New Lectin from Meadow Saffron (Colchicum automnale)

A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M_r 100,000) composed of 4A- and 4B-subunits of M_r 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes.     

Are fingernail lead levels a reliable biomarker of lead internal dose?

BackgroundPotentially toxic elements, such as lead, can bioaccumulate and alter human physiology. Human biomonitoring is an essential tool to evaluate chemical exposures in different biological matrices (blood, urine, saliva, nails, and hair). Of these biological matrices, nails are favorable for their ease of sampling, transport and storage. The aim of this study was to investigate possible correlations between blood lead levels (BLL) and washed and non-washed fingernail lead levels (FLL) in 55 adults living in a lead-contaminated area.MethodVenous blood and fingernail (thumbs and forefingers) samples were collected. Nails from the left hand were washed with Triton X-100 (0.5 % m/v) and HNO₃ solution, while nails from the right hand were not submitted to the pre-analytical procedures. Samples were analyzed by graphite furnace atomic absorption spectrometry, and pairwise correlations were used to correlate lead concentrations between BLL and FLL; nails from fingers of the same hand and between washed and unwashed fingernails. Principal component analysis was performed and scatter diagrams were plotted to investigate correlations.ResultsA non-significant positive correlation was found between BLL and washed forefinger nails lead (r = 0.219, p = 0.112) and between BLL and thumbnail lead levels (r = 0.182, p = 0.191). Comparison of fingernails from the same hand (thumb and forefinger), showed that lead concentrations of non-washed nails varied widely, even on analyses of transversal fragments from the same nail. Lead levels in non-washed forefinger nails were not correlated with non-washed thumbnails (r = 0.169, p = 0.219). Conversely, washed thumb and forefinger nails were found to be correlated (r = 0.39, p = 0.003). Washed and non-washed nails were also found to be correlated (p < 0.0001).ConclusionIn conclusion, the results showed that non-washed nails are not a reliable biomarker for lead exposure. Although washing nails before analysis may reduce external contamination, the correlation of lead concentrations between fingers is poor for fingernail lead levels to serve as an internal dose biomarker to lead exposure. In addition, levels in washed nails were not significantly correlated with blood lead levels. Fingernail lead levels seem to serve as an indicator of lead exposure sources in contact with the individual, but not as a reliable biomarker of internal dose.