Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice

  • Katrien Deroost1,

    Affiliated with

    • Natacha Lays1,

      Affiliated with

      • Sam Noppen2, 3,

        Affiliated with

        • Erik Martens1,

          Affiliated with

          • Ghislain Opdenakker1 and

            Affiliated with

            • Philippe E Van den Steen1Email author

              Affiliated with

              Malaria Journal201211:166

              DOI: 10.1186/1475-2875-11-166

              Received: 17 November 2011

              Accepted: 7 March 2012

              Published: 14 May 2012



              Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.


              An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann–Whitney U test and Pearsons correlation analysis.


              Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.


              The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.


              Chemo-luminescence Densitometry Haemozoin quantification Malaria pigment Plasmodium


              With growing world populations in tropical countries, every year more people become exposed to malaria. As many areas of endemic malaria transmission overlap with regions of poverty, direct and indirect burdens of this infectious disease are important. In particular, a small percentage of the malaria-infected patients develop life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems, even if they have similar peripheral parasitaemia levels compared to patients with mild or asymptomatic malaria [1]. Efficient adjunct therapies against these immunopathological complications are still not available. Therefore, studying the mechanisms of disease development in severe malaria is paramount.

              Inside the red blood cell (RBC), almost 80% of the haemoglobin (Hb) is degraded, which means that high amounts of toxic haem (which is rapidly oxidized to haemin) are liberated in the food vacuole of the parasite capable of generating reactive oxygen species and damaging cell membranes and proteins [2, 3]. As a detoxification mechanism, the parasite biocrystallizes the haemin molecules into insoluble haemozoin (Hz) crystals or malaria pigment [4, 5]. When the schizont ruptures, Hz is released into the circulation and is rapidly removed by phagocytes inside several organs. Multiple in vitro and ex vivo pro-inflammatory and immunosuppressive effects of Hz have been described (reviewed in [47]). However, few in vivo data about the effects of Hz on the immune system exist. As large amounts of Hz are produced during infection and accumulate inside multiple organs, Hz may be important for the progress towards malaria-associated pathologies. This hypothesis is further strengthened by the fact that abundant Hz has been observed in brains [810] and placentas [1113] from malaria patients with cerebral and placental complications, respectively. In addition, Hz was detected in brains of mice with cerebral symptoms [14, 15] and in lungs of mice with malaria-associated acute respiratory distress syndrome (MA-ARDS) [16].

              Experimental mouse models offer useful tools to study malaria-related disease mechanisms. Depending on the mouse-parasite combination, different aspects of human malaria can be mimicked and investigated, even if these models are not exact replicas and should thus be extrapolated with caution. In this study, C57BL/6 J mice were infected with three different parasite strains with a varying degree of pathogenicity. The Plasmodium berghei ANKA (PbANKA) parasite induces typical symptoms of cerebral malaria (CM), such as paralysis or coma and mice succumb within seven to nine days. In this mouse model, the pathology critically depends on activation of leukocytes, including CD8+ T cells, and a local inflammatory reaction [1]. Although sequestration of Plasmodium falciparum in the brain is strongly associated with CM in patients, it is unclear whether specific cyto-adherence of PbANKA occurs in the brain [17]. However, local parasite accumulation in the brain is thought to be an important feature of this model [18]. Mice infected with Plasmodium berghei NK65 (PbNK65) do not develop such an encephalopathy but rather die from severe respiratory problems between nine and eleven days post-infection [16]. This respiratory pathology closely resembles human MA-ARDS, as in both mice and patients leukocytes (predominantly macrophages and lymphocytes) and infected RBCs (iRBCs) accumulate in the lungs, resulting in the disruption of endothelial barriers, severe edema and intra-alveolar hyaline membrane formation [16, 19, 20]. Plasmodium chabaudi AS (PcAS)-infections are self-limiting and the mice are able to recover. Moreover, C57BL/6 mice mount a protective immune response against PcAS-parasites mediated by phagocytes, CD4+ T cells and specific antibodies, which is very similar to the immune response generated against P. falciparum in humans [21].

              To investigate the organ-specific Hz deposition in these three mouse models, novel techniques are described in the present study to accurately quantify the Hz content in tissues. These methods were implemented to compare the amount of Hz between various organs and between similar organs from mice infected with parasites of different pathogenicity. Most Hz was found in livers and spleens. Far less Hz was detected in lungs and kidneys, whereas limited amounts of Hz were observed in hearts and brains, irrespectively of the parasite species. In addition, more Hz was found in mice infected with PbNK65 or PbANKA compared with PcAS-infected mice despite of similar peripheral parasitaemia levels.


              Chemical products

              All chemicals were purchased from Sigma-Aldrich (Bornem, Belgium), unless otherwise stated.

              Mice and parasites

              C57BL/6 J mice (seven to nine weeks old) were obtained from Janvier (Le Genest-Saint-Isle, France) and placed in a conventional animal house with food and water ad libitum. Parasite growth in mice was supported by supplementing the drinking water with 0.375 mg/mL 4-amino benzoic acid. Mice were intraperitoneally infected with 104 iRBCs by serial passage of tail vein blood obtained from a mouse that had been infected with one of the following parasite strains: PcAS, PbNK65 (kind gifts of the late Prof. D Walliker, University of Edinburgh, Scotland, UK) or PbANKA (Cl15CY1, a kind gift of Prof. C Jansse, Leiden University Medical Centre, The Netherlands). The percentage of infected erythrocytes in the peripheral blood was determined by microscopic analysis after Giemsa staining. Mice were sacrificed at the indicated time points after infection and blood was removed by heart puncture. Mice were perfused with Dulbecco’s phosphate-buffered saline (PBS) (Lonza, Verviers, Belgium) to remove circulating iRBCs from the organs. Livers, spleens, kidneys, lungs, hearts and brains were removed, weighed and stored at -80°C until further analysis. A part of the liver, spleen, lung and kidney was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and frozen in liquid nitrogen-cooled isopentane for histological analysis. All experiments were approved by the local ethical committee (License LA121251, Belgium).

              Haemozoin quantification in organ cryosections by densitometric analysis

              Cryosections with a thickness of 7 μm were prepared from frozen livers, spleens, lungs and kidneys and imaged by light microscopy. Transmitted light images were taken through a 20x/0.8 Plan-Apochromat objective of an Axiovert 200 M microscope equipped with an AxioCam MRm camera (Zeiss, Göttingen, Germany). For Hz quantification on liver sections, images were obtained from two rows of three consecutive fields. The densitometric analysis was performed with the AxioVision 4.6 software with a home-written script and the relative quantity of Hz/μm2 was calculated with the formula as shown in Figure 1G. The densitometric value (DV) of a pixel reflects the intensity of transmitted light at this position in the section. The densitometric background (DB) was determined in each picture by calculating the mean DV of all pixels with a DV above an empirically determined threshold. To test the linearity of the densitometric method to measure Hz on cryosections, gelatin blocks containing different concentrations of synthetic Hz (sHz) were analysed. sHz was prepared as described previously [22] and was homogenized and added in different concentrations to a 10% gelatin-PBS solution at 37°C in a 24-well plate. Upon solidification on ice (to prevent sedimentation of sHz), sHz-containing gelatin blocks were cut out of the wells, embedded in O.C.T and frozen at -80°C. For each concentration, five to six images were analysed from one section/sHz block and this was done for two blocks.

              Haem quantification by colorimetric analysis and haem-enhanced luminescence

              The Fe-ions in the haemin molecules that constitute the Hz crystal are in the oxidized state (Fe3+). Therefore, a dilution series of a haematin stock solution (10 μM – 1.2 nM), prepared by dissolving haemin in 100 mM NaOH, 2% SDS and 3 mM EDTA, was used as a standard to compare methods. In an alkaline environment, haematin produces a brown colour measurable spectrophotometrically (Biotech Powerwave XS) at 405 nm [23]. Background absorbance was evaluated from a blank sample and subtracted from the measurements. The method for quantification by luminescence was based on the method of Schwarzer et al.[24] and was optimized for working in a 96-well plate and modified according to Yuan et al.[25]. Different concentrations of haematin were added in 96-well plates suitable for luminescence (Perkin Elmer, Waltham, MA, USA) and diluted in a solution containing NaOH and Na2CO3 (four volumes of 100 mM NaOH, 2% SDS and 3 mM EDTA and one volume of 1 M Na2CO3, pH 10.4) (final volume 50 μL). After addition of 100 μL luminol (100 μg/mL 3-aminophtalhydrazide) and 100 μL of peroxide (7% tert-butyl hydroperoxide), both dissolved in the NaOH/Na2CO3-solution, light emitted in the presence of Fe3+ (present in the haematin core) was measured during one second using a Thermo Luminoskan Ascent apparatus. Peroxide catalysis into oxygen by Fe3+ is a fast reaction. Therefore, special care was taken to keep the time between the addition of the peroxide and the luminescence measurements minimal and as similar as possible between the different wells (maximum time deviation between individual wells was eight seconds). A sigmoidal relationship between the haematin concentration and the luminescence (events/sec) was obtained. Background luminescence was evaluated from a blank sample and subtracted from the measurements. The above method for haem-enhanced luminescence was used for all measurements unless differently stated. The time-dependence of the luminescence signal was measured with 125 nM haematin in duplicate every ten seconds for eight minutes during a kinetic reading without any other samples in the plate to allow fast repetitive reading of these two wells. A lag-time of twelve seconds existed between the addition of the peroxide and the start of the measurement, which was partly attributable to a shaking step.

              Haemozoin determination in tissues and trophozoites

              To extract haemozoin from perfused mouse tissues, approximately 30 – 60 mg (liver, spleen, kidney or lung), half brain or a full heart were homogenized with the Precellys Lysing Kit (VWR, Leuven, Belgium) in minimum five volumes of a solution containing 50 mM Tris/HCl pH 8.0, 5 mM CaCl2, 50 mM NaCl and 1% Triton X-100. The homogenate was supplemented with 1% Proteinase K and incubated overnight at 37°C. The next day the proteinase K digest was sonicated (VialTweeter, Hielscher Ultrasonics GmbH, Teltow, Germany) for 1 min (10 W, pulse 0.5 sec) and centrifuged at 11,000 x g for 45 min. The supernatant was discarded and the pellet was washed three times in 100 mM NaHCO3, pH 9.0 and 2% SDS with subsequent sonication and centrifugation for 30 min to remove degraded tissue, free haem and Hb. After the third wash, the pellet (Hz) was dissolved and sonicated in 100 mM NaOH, 2% SDS and 3 mM EDTA to form haematin and centrifuged to pellet any remaining insoluble material. To confirm that the isolated material was indeed Hz, it was examined for its birefringence character. For this purpose, isolated Hz that was washed three times as described above, was subsequently washed in distilled H2O to remove the salts, smeared on a glass slide and monitored by polarized light microscopy with a 40x/1.3 oil EC Plan-Neofluar objective of an Axiovert 200 M microscope.

              To isolate Hz from trophozoites, heparinized blood was obtained by heart puncture from PbNK65-infected mice and trophozoites were cultivated ex vivo overnight and harvested as described [26]. After determination of the total red cell number and the percentage of iRBCs, Hz was extracted from the cells as described above but without proteinase K treatment and subsequently dissolved as described.

              The extracted Hz was measured in different dilutions with the above-mentioned protocol for haem quantification by luminescence. A dilution series of haematin (10 μM – 1.2 nM) was used as a standard. The unknown Hz concentration was calculated from the calibration curve of the haematin concentration (nM) versus luminescence (events/sec). Background luminescence was evaluated from a blank sample and subtracted from the measurements. The amount of Hz (fmol or pmol haematin/mg tissue) was multiplied with the total weight of the concerning organ and expressed as pmol or nmol haematin/organ. An accuracy limit was estimated for each organ separately.

              Statistical analysis

              P-values for the differences between two groups were calculated with the Mann–Whitney U-test, using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). The same software was used for linear regression analyses and for calculating Spearman correlation coefficients. The slopes of the individual regression lines of the different groups were compared online [27]. A p-value less than 0.05 (p < 0.05) was taken as statistically significant.


              Haemozoin detection by densitometric analysis

              Pigment distribution was investigated on unstained cryosections from various organs of mice infected with PbNK65 or PcAS and monitored with light microscopy. Hz was observed on transmitted light images from infected mouse organs as brown pigments and was absent in organ sections from uninfected mice as is shown for the liver (Figure 1A–F). The pigment was found equally distributed throughout the liver (Figure 1B–C), whereas in the spleen (Figure 1D), the lungs (Figure 1E) or the kidneys (Figure 1F), it was located in the red pulp, the interstitial tissue or clustered in presumably the glomeruli, respectively. Brains and hearts contained such low amounts of Hz that it was almost unnoticeable on unstained sections. To estimate the amount of Hz on organ cryosections, semi-quantitative densitometry with the AxioVision 4.6 software using a home-written script was applied. The linearity of this densitometric method was investigated on cryosections from gelatin-blocks with different concentrations of sHz. A broad linear relationship was found between the sHz concentration and the obtained relative densitometric value (Figure 1G). The detected Hz signal was converted into the relative quantity of Hz/μm2 tissue as calculated with the formula described in Figure 1G. This technique was used to measure Hz in liver cryosections from non-infected, PcAS and PbNK65-infected mice. Despite similar mean peripheral parasitaemia levels in both groups of mice (PcAS 18%; PbNK65 12.7%; p = 0.4), significantly more Hz/μm2 tissue was detected in livers of PbNK65-infected mice compared with PcAS-infected mice (Figure 1H). This technique combines in-situ information with relative quantification and can be used to measure Hz in organs with a high and evenly distributed Hz content such as livers. However, this method is poorly applicable for organs in which the pigment is not equally distributed, e.g. spleen, lungs and kidneys. In brains and hearts, the amounts of Hz were too low to be quantified by densitometry.
              Figure 1

              Haemozoin detection by densitometric analysis. Transmitted light images (grey scale) were taken from unstained 7 μm thick cryosections from livers of uninfected mice (A) and from mice infected with PcAS (B) or PbNK65 (C), from PcAS-infected spleens (D), PbNK65-infected lungs (E) and kidneys (F). In panel G, the relative densitometric value obtained from cryosections of gelatin blocks with different concentrations of sHz were analyzed and the formula used to calculate the relative quantity of Hz/μm2 is shown. In panel H, the relative Hz content was measured in liver sections from uninfected (Con), PcAS and PbNK65-infected mice ten days post-infection. Each dot represents the result from an individual mouse. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001

              Comparison of haem quantification by different techniques

              Because the densitometric analysis is not suitable to quantify Hz in tissue sections from all organs, a more sensitive technique was designed to determine Hz in organ extracts. Several methods for quantifying Hz in blood are described in literature, including a colorimetric [23] and a chemo-luminescence assay [24]. These two techniques were adapted to a 96-well plate format and the sensitivity was compared by measuring different concentrations of haematin produced when haemin or Hz is dissolved in an alkaline environment. A sigmoidal relationship was obtained between the haematin concentration and the blank-subtracted absorbance at 405 nm (Figure 2A) or the blank-subtracted luminescence with the reagent concentrations used as described for a cuvette-based system by Schwarzer et al.[24] (Figure 2B). In the 96-well plate format adapted for measuring absorbance or luminescence with a plate reader, both techniques detected the presence of haematin starting from a minimal concentration of 1 μM. To improve the sensitivity, the chemo-luminescent assay was adjusted by stabilizing the pH around 10.4 and by raising the concentrations of luminol and peroxide 100-fold. In this way, a detection limit around 100 nM was obtained (Figure 2C). Furthermore, the time-dependence of the luminescence signal was examined by measuring the emitted light of a single concentration during a kinetic measurement. With the described protocol for haem-enhanced luminescence in a 96-well plate format, emitted light was measured when the luminescence signal was almost maximal (Figure 2D). The sensitivity of this 96-well plate-based assay is significantly lower than the sensitivity of the cuvette-based method described by Schwarzer et al.[24]. However, the current 96-well based method has the advantage of a higher throughput and the sensitivity appeared sufficient to measure Hz in organ extracts. To extract Hz from organs, the method described by Sullivan et al.[15] was optimized. The most important modification was the addition of an overnight proteinase K digestion step that eliminated high background signals e.g. in lung samples. After several wash steps to remove any free haemin, the Hz crystals were converted to free haematin by dissolving in a strong alkaline environment, so that the haematin concentration could be measured. Since the extraction procedure involved washing steps in the presence of SDS, possible quenching of the emitted light by SDS was investigated and had no effect on the luminescence catalyzed by haematin-Fe3+ (Figure 2E).
              Figure 2

              Haem quantification by different techniques. Different concentrations of haematin (10 μM – 1.2 nM) were used to compare the sensitivity of previously described techniques to quantify haem in a 96-well based format. In panel A, haematin was measured with a spectrophotometer at 405 nm (n = 4 for each concentration). Panel B shows the measurements of haematin concentrations by haem-enhanced luminescence with the same reagent conditions as described by Schwarzer and colleagues (20) for a cuvette-based system. Background absorbance/luminescence from a blank sample was subtracted from all the measurements. The adjusted chemo-luminescence protocol (100-fold higher luminol and peroxide concentrations buffered around pH 10.4) was used to measure the same concentrations of haematin in panel C (n = 8 for each concentration in B and C). The horizontal dashed line denotes the accuracy limit of the assay. The time-dependence of the luminescence signal and the effect of 2% SDS are displayed in panel D and E, respectively. The arrow in panel D denotes the start of the kinetic reading after an initial delay of twelve seconds, and the vertical dashed line indicates the moment of luminescence detection during the experimental readings. In panel F, natural Hz was isolated from trophozoites (troph) and livers, its concentration was determined by absorption at 405 nm and the concentration-dependence of the haem-enhanced luminescence was measured. Inset of panel F is a picture demonstrating the birefringence of the isolated Hz. The amount of Hz/trophozoite was calculated and the corresponding number of trophozoites (# troph) is integrated in the figure as a second X-axis

              As a final check up for the appropriateness of the assay for quantifying natural Hz, the concentration dependence of the haem-enhanced luminescence measured with the plate reader was studied with natural Hz purified from trophozoites and from the liver. As shown in Figure 2F, haemin and Hz derived from trophozoites or livers behaved similarly in the assay confirming the suitability of using haemin as a standard for deducing the Hz concentration in the samples. In addition, as birefringence is a typical feature of Hz [28], Hz isolated from the liver was spread on a glass slide and monitored by polarized light microscopy. This confirmed that the isolated material consisted mainly of Hz (Figure 2F, inset).

              Quantitative analysis of haemozoin in various organs of mice infected with different parasite species

              The optimized haem-enhanced luminescence technique was used to study differences in the amount of Hz between various organs and between the same organs of mice infected with parasites of different pathogenicity (PbANKA, PbNK65 or PcAS). Mice were sacrificed at the indicated times post-infection and perfused systemically. Even though no difference was found in the amount of Hz before and after perfusion ( Additional file 1), it seemed more reasonable to apply perfusion on all samples tested. In this way, there could be no doubt that the detected Hz represented organ-trapped Hz and not Hz present in the circulation. Quantification of the total amount of Hz per organ revealed that most Hz was present in livers followed by spleens (Figure 3A–B). Far less Hz was detected in lungs and kidneys (Figure 3C–D), whereas subnanomolar levels of Hz were found in brains and hearts, irrespectively of the parasite species used (Figure 3E–F). Livers, lungs, kidneys and hearts from PbNK65-infected mice nine to ten days post-infection contained significantly more Hz compared with the same organs from PbANKA-infected mice seven to eight days post-infection or PcAS-infected mice ten days post-infection, even though livers of PcAS-infected mice were significantly larger (p < 0.0001 for liver weights between PcAS d10 and PbNK65 d9-10 and between PcAS d10 and PbANKA d7–8). In addition, lungs and hearts from PbANKA-infected mice seven to eight days post-infection had significantly more Hz compared with the same organs from PcAS-infected mice after ten days of infection. The total amount of Hz was similar in spleens of PcAS and PbNK65-infected mice ten days post-infection (Figure 3B), although the amount of Hz/mg spleen tissue was six-fold lower in mice infected with PcAS compared to PbNK65 (median value was 1164.4 pmol haematin/mg spleen for PbNK65 and 199.9 pmol haematin/mg spleen for PcAS; p < 0.0001). This was compensated by the three- to four-fold larger spleen size in PcAS-infected mice (p < 0.0001). Furthermore, similar amounts of Hz were observed in brains from PbNK65 and PbANKA-infected mice, whereas less Hz was found in brains of PcAS-infected mice.
              Figure 3

              Quantification of haemozoin in tissues. C57BL/6 J mice were infected with 104 PbANKA, PbNK65 or PcAS parasites or were left uninfected (Con). At the indicated time intervals after infection, mice were dissected after heart puncture and perfusion. Extracted Hz from 30 – 60 mg tissue of livers (A), spleens (B), lungs (C) and kidneys (D), from half brains (E) and from whole hearts (F) were quantified by haem-enhanced luminescence and expressed as nmol haematin/organ (liver, spleen, lungs and kidneys) or pmol haematin/organ (brain and heart). Each group consisted of 15 to 20 mice, with each dot indicating individual data points. Horizontal dashed lines were used to denote the accuracy limit of the assay for each organ separately. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001

              Different total haemozoin levels in PcAS-infected mice and Plasmodium berghei-infected mice

              As brains and hearts contained only subnanomolar amounts of Hz, individual total Hz levels were calculated by making the sum of organ-specific Hz levels from liver, spleen, lungs and kidneys. Mice infected with parasites of the P. berghei strains contained significantly more Hz than mice infected with PcAS-parasites (Figure 4A). Moreover, the total amounts of Hz correlated with the peripheral parasitaemia levels for all parasite strains (Figure 4B and C). PbANKA and PbNK65-parasites had a similar Hz-production pattern, i.e. comparable amounts of Hz at similar peripheral parasitaemia levels. In contrast, PcAS-infected mice seemed to have significantly less Hz in relation with their parasitaemia compared with PbANKA or PbNK65 as indicated by the lower slope on the regression curve. The difference between the slopes of the regression lines was significantly different between PbNK65 and PcAS-infected mice (p < 0.0001).
              Figure 4

              Pc AS-infected mice contain less total Hz than Plasmodium berghei -infected mice. The total amount of Hz (nmol haematin)/mouse was estimated from the sum of the Hz content in the individual organs. Panel A shows the average amount of Hz found in C57BL/6 J mice infected with PbANKA, PbNK65 or PcAS on different time points post-infection, with the contribution of each organ to the total amount of Hz ± SEM. Asterisks on top of data sets indicate the statistical significances compared with the uninfected control group. Horizontal lines with asterisks on top indicate statistical differences between infected groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. In panel B, the total amount of Hz (nmol haematin) was correlated with the level of parasitaemia (%) for each parasite strain separately and the individual regression lines are shown. The regression line of PbANKA was prolonged under the form of a hatched line to better distinguish it from the PbNK65 line. The equation of the regression lines and the R2-values, together with Spearman r-values and p-values are shown in panel C


              The first observations of black pigment in necroptic spleens and brains go back to the 18th Century (reviewed in [5]). About 130 years later, a publication mentioned brown-grey colourations of brain, spleen and liver, which turned out to arise from pigment deposition. At first believed to be melatonin, it was later linked to a parasitic disease. Presently, many in vitro and ex vivo immunomodulating effects have been ascribed to Hz [47]. However, data about the fate and properties of Hz in the in vivo situation are still scarce. Hz is released in the circulation in considerable amounts after schizont rupture where it may interact with a whole range of different cell types. The majority of the liberated Hz is presumably captured and phagocytosed by circulating and tissue resident monocytes/macrophages in which it can persist for a long time. In this way, Hz may be capable of causing considerable inflammation that might progress to tissue injury. In this study, techniques for sensitively quantifying the amount of Hz in tissues were examined and the organ-specific Hz content was compared between parasite species with a varying degree of pathogenicity.

              As Hz crystals were observed on unstained cryosections from livers, spleens, lungs and kidneys, a technique for estimating the amount of Hz in these sections by densitometric analysis was developed. As Hz is proportionally distributed throughout the liver, the estimation of the amount of Hz by densitometry was quite reliable. In other organs, however, Hz was found in specific structures such as the red pulp in the spleen, the interstitial tissue in the lungs or presumably the glomeruli in the kidneys. This may in part be attributed to the differential localization of tissue-resident phagocytes. This implies that Hz distribution is a confounding factor for the accuracy of the Hz measurements on organ cryosections by densitometry. In addition, this technique is time-consuming, labour-intensive, semi-quantitative and not suitable for organs with a low Hz content and was thus not further explored. Therefore, a more sensitive, analytical and quantitative method for determining the Hz content in tissues was investigated. To isolate Hz from organs, a protocol described by Sullivan and colleagues [15] was modified. The main adaptation was the digestion of the homogenates with proteinase K. This digestion eliminated high background signals, which were presumably due to the binding of Hb to otherwise insoluble extracellular matrix components. Upon conversion of the isolated Hz into soluble haematin, a chemo-luminescence assay was used for quantification. This assay was based on the method of Schwarzer et al.[24] and adapted to microtiter plate format. The obtained sensitivity with the optimized protocol was lower compared with the haem-enhanced luminescence assay described by Schwarzer et al. This was not due to quenching of the luminescence signal by SDS nor was it caused by the altered time frame during which the emitted light was measured (two seconds/sample versus approximately ninety six seconds/plate), but probably originated from the use of different luminescence detector systems (cuvette system versus microplate reader). Nevertheless, the microplate-adjusted approach offers the advantage of measuring several samples in varying concentrations simultaneously with a sensitivity that is optimal for the quantification of Hz in malaria-infected organs.

              As an application, the distribution of Hz throughout the body of infected mice was studied and compared between diverse parasite strains with varying pathogenicity. Sullivan and colleagues already quantified the Hz content in brains, livers and spleens of mice [15, 29] and in human placentas [11], but no detailed comparison between organs and between parasite species was described. Almost 95% of the total pool of Hz was found in livers and spleens. This was expected as large volumes of blood are filtered through these organs and both contain a vast population of tissue-resident monocytes/macrophages capable of rapidly removing the crystalline material from the circulation by means of phagocytosis. It was also important to consider the liver and spleen sizes when determining the total Hz amounts, as these sizes evolve in a different way during infection with different parasites (i.e. induction of hepatosplenomegaly by PcAS). As the absolute Hz concentration in the organs could be determined by the luminescence assay, this was easily taken into account by multiplication with the organ weights.

              Furthermore, substantial amounts of Hz were detected in lungs of malaria-infected mice. In a new mouse model of MA-ARDS [16], considerable amounts of Hz were observed on histological sections of the lungs. By quantifying the Hz content in the lungs, significantly higher Hz levels were validated in lungs from P. berghei-infected mice (lung pathology) compared to PcAS-infected mice (no lung pathology), indicating that Hz may have a role in the development of malaria-associated lung disease.

              Low but detectable amounts of Hz were found in kidneys, hearts and brains of malaria-infected mice. Most Hz was detected in kidneys and hearts from PbNK65-infected mice ten days post-infection compared with PbANKA and PcAS-infected mice seven to eight and ten days post-infection, respectively. However, a different pattern was observed in the brains, i.e. Hz was undetectable in brains from PcAS-infected mice whereas similar amounts of Hz were detected in brains of PbNK65 and PbANKA-infected mice. A possible explanation for this difference is their diverse parasite synchronicity. At the moment of sacrificing the mice and organ removal, the PcAS-parasites in the circulation were all in the ring and young trophozoite stage. As these developmental stages do not yet contain abundant Hz [3, 10], it seemed reasonable that Hz was not detected in brains from mice infected with this parasite species. It is also possible that no Hz was detected because PcAS-parasites may not sequester in the brains as is the case for Plasmodium vivax-infected erythrocytes [10]. On the contrary, several developmental stages of P. berghei parasites are found in the circulation simultaneously and accumulation of P. berghei in the brain is still a debated issue. The observation of similar brain Hz contents in PbANKA and PbNK65-infected mice cannot be explained by their parasitaemia levels as significantly higher parasitaemias were found in mice that were infected with PbNK65 than in mice infected with PbANKA. The data however do suggest that Hz as such is not sufficient for the development of this immunopathology as PbNK65-infected C57BL/6 J mice do not develop cerebral complications [16]. These data are in contrast with data from Coban et al.[14] and Sullivan et al.[15] who found that brains from mice with cerebral pathology contained more Hz than healthy brains from infected mice. However, this may be explained by differences in the timing of analysis after infection and in the mouse or parasite strains used in the studies.

              Organ-trapped Hz may originate from two sources. As free Hz is rapidly removed from the circulation, it is found either inside phagocytes or inside cyto-adhering iRBCs along the endothelial lining of the organs’ microvasculature. Systemic perfusion removes circulating iRBCs but not sequestering iRBCs or Hz inside resident phagocytes, although inadequate perfusion can result from obstruction due to organ-specific cyto-adherence and haemorrhages. Furthermore, it is still not completely clarified if sequestration by murine malaria parasites occurs and which organs are the main targets. Local parasite accumulation has been demonstrated in brains and lungs of PbANKA-infected mice suffering from cerebral symptoms [18, 30], but no reports exist on PbNK65-parasite sequestration.

              After calculating the total amount of Hz in the mice, it was found that PbANKA and PbNK65-infected mice contained similar amounts of Hz at comparable parasitaemia levels. This suggests that both parasites produced similar amounts of Hz, or that their schizonts presumably consumed comparable amounts of Hb. On the contrary, lower amounts of Hz were retrieved in PcAS-infected mice despite of similar peripheral parasitaemia. Several explanations can be given for this finding. PcAS-parasites may produce less Hz, e.g. by digesting less Hb or by using other haem detoxification mechanisms (transport of haem out of the food vacuole or anti-oxidative defense mechanisms of the parasite) or PcAS Hz could be more easily degraded. Interestingly, Noland et al.[31] demonstrated that Hz crystals from different Plasmodium species have different shapes and dimensions, supporting the notion that Hz from different species may have different properties. In addition, Hz contents are variable in RBC infected with different Plasmodium falciparum strains [32].

              Another possibility is that peripheral parasitaemia, estimated by counting the percentage of iRBCs by microscopic analysis of Giemsa-stained blood smears, are not a true reflection of the total parasite biomass as they do not take sequestered parasites into account. Consequently, it is possible that PcAS-infected mice contain less Hz because of lower total parasite burdens. These observations may well translate to the situation in human malaria, where various parasite species have different degrees of virulence. Total parasite biomass in P. falciparum infections is higher than peripheral parasitaemia levels and the difference between these two parameters increases with disease severity [33]. Similarly, Hz-containing peripheral leukocytes are a marker for disease severity [3436], and accumulation of Hz in brain micro-vessels is associated with a subtype of cerebral malaria [37]. No data are available yet about total parasite burdens in P. vivax-infections and it is still questionable if P. vivax-iRBCs can adhere to the endothelial micro-vascular lining. However, cytoadhesion of P. vivax-infected erythrocytes was demonstrated in vitro[38] and, despite of the absence of sequestration in the brain [10], it was hypothesized that parasitized RBCs might sequester in lungs from patients with P. vivax malaria [39]. Similarly, very little knowledge exists on the role of Hz in P. vivax infections.

              Besides differences in pathogenicity, another interesting difference between P. berghei and PcAS is that PcAS can be cleared from the circulation in several mouse strains, including C57BL/6 mice, whereas PbANKA and PbNK65 cannot. The amounts of Hz produced by these parasites may also contribute to these differences, as Hz is known to suppress macrophage activity in vitro[40] and in vivo[41]. Interestingly, Spaccapelo et al. found that plasmepsin 4-deficient PbANKA-parasites, which produce less Hz, cause less immunopathology and are more easily cleared by some mouse strains [42].


              This paper describes newly developed and improved methods for sensitive Hz quantification in mouse organs. Different amounts of Hz were detected in the analysed organs and total Hz contents were highest in mice that were infected with lethal parasite strains. Therefore, it is clear that these techniques will be valuable in the investigation of a possible relationship between Hz and organ-specific malaria pathologies.



              Cerebral malaria


              Densitometric background


              Densitometric value






              Malaria-associated acute respiratory distress syndrome


              Plasmodium berghei ANKA


              Plasmodium berghei NK65


              Plasmodium chabaudi AS


              Infected red blood cell


              Synthetic haemozoin (beta-haematin).



              We want to thank Prof. Jo Van Damme and Prof. Sofie Struyf for the usage of the Axiovert 200 M microscope and Prof. Johan Neyts for the usage of the Precellys homogenizer and the luminometer. Furthermore, we are grateful to Prof. E Schwarzer and Prof. P Arese (University of Turin, Italy) for helpful discussions.

              KD is a research assistant of the “Instituut voor Wetenschap en Technologie (IWT)”, Belgium. This study was supported by the IWT, the “Fonds voor Wetenschappelijk Onderzoek in Vlaanderen (FWO-Vlaanderen)”, the “Geconcerteerde OnderzoeksActies (GOA 2007–2011)”, the Rega Centre of Excellence (EF/05/015) and the Research Fund KULeuven (CREA/09/023).

              Authors’ Affiliations

              Laboratory of Immunobiology, Rega Institute, University of Leuven
              Laboratory of Molecular Immunology, Rega Institute, University of Leuven
              Currently at the laboratory of Virology and Chemotherapy, Rega Institute, University of Leuven


              1. Schofield L, Grau GE: Immunological processes in malaria pathogenesis. Nat Rev Immunol 2005, 5:722–735.PubMedView Article
              2. Goldberg DE: Hemoglobin degradation. Curr Top Microbiol Immunol 2005, 295:275–291.PubMedView Article
              3. Rosenthal PJ, Meshnick SR: Hemoglobin catabolism and iron utilization by malaria parasites. Mol Biochem Parasitol 1996, 83:131–139.PubMedView Article
              4. Shio MT, Kassa FA, Bellemare MJ, Olivier M: Innate inflammatory response to the malarial pigment hemozoin. Microbes Infect 2010, 12:889–899.PubMedView Article
              5. Hanscheid T, Egan TJ, Grobusch MP: Haemozoin: from melatonin pigment to drug target, diagnostic tool, and immune modulator. Lancet Infect Dis 2007, 7:675–685.PubMedView Article
              6. Schwarzer E, Skorokhod OA, Barrera V, Arese P: Hemozoin and the human monocyte–a brief review of their interactions. Parassitologia 2008, 50:143–145.PubMed
              7. Arese P, Schwarzer E: Malarial pigment (haemozoin): a very active ‘inert’ substance. Ann Trop Med Parasitol 1997, 91:501–516.PubMedView Article
              8. Grau GE, Mackenzie CD, Carr RA, Redard M, Pizzolato G, Allasia C, Cataldo C, Taylor TE, Molyneux ME: Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria. J Infect Dis 2003, 187:461–466.PubMedView Article
              9. Dorovini-Zis K, Schmidt K, Huynh H, Fu W, Whitten RO, Milner D, Kamiza S, Molyneux M, Taylor TE: The neuropathology of fatal cerebral malaria in malawian children. Am J Pathol 2011, 178:2146–2158.PubMedView Article
              10. Silamut K, Phu NH, Whitty C, Turner GD, Louwrier K, Mai NT, Simpson JA, Hien TT, White NJ: A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain. Am J Pathol 1999, 155:395–410.PubMedView Article
              11. Sullivan AD, Nyirenda T, Cullinan T, Taylor T, Lau A, Meshnick SR: Placental haemozoin and malaria in pregnancy. Placenta 2000, 21:417–421.PubMedView Article
              12. Ismail MR, Ordi J, Menendez C, Ventura PJ, Aponte JJ, Kahigwa E, Hirt R, Cardesa A, Alonso PL: Placental pathology in malaria: a histological, immunohistochemical, and quantitative study. Hum Pathol 2000, 31:85–93.PubMedView Article
              13. Muehlenbachs A, Fried M, McGready R, Harrington WE, Mutabingwa TK, Nosten F, Duffy PE: A novel histological grading scheme for placental malaria applied in areas of high and low malaria transmission. J Infect Dis 2010, 202:1608–1616.PubMedView Article
              14. Coban C, Ishii KJ, Uematsu S, Arisue N, Sato S, Yamamoto M, Kawai T, Takeuchi O, Hisaeda H, Horii T, Akira S: Pathological role of Toll-like receptor signaling in cerebral malaria. Int Immunol 2007, 19:67–79.PubMedView Article
              15. Sullivan AD, Ittarat I, Meshnick SR: Patterns of haemozoin accumulation in tissue. Parasitology 1996,112(Pt 3):285–294.PubMedView Article
              16. Van den Steen PE, Geurts N, Deroost K, Van AI, Verhenne S, Heremans H, Van Damme J, Opdenakker G: Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome. Am J Respir Crit Care Med 2010, 181:957–968.PubMedView Article
              17. Craig AG, Grau GE, Janse C, Kazura JW, Milner D, Barnwell JW, Turner G, Langhorne J: The role of animal models for research on severe malaria. PLoS Pathog 2012, 8:e1002401.PubMedView Article
              18. Haque A, Best SE, Unosson K, Amante FH, de Labastida F, Anstey NM, Karupiah G, Smyth MJ, Heath WR, Engwerda CR: Granzyme B expression by CD8+ T cells is required for the development of experimental cerebral malaria. J Immunol 2011, 186:6148–6156.PubMedView Article
              19. Mohan A, Sharma SK, Bollineni S: Acute lung injury and acute respiratory distress syndrome in malaria. J Vector Borne Dis 2008, 45:179–193.PubMed
              20. Valecha N, Pinto RG, Turner GD, Kumar A, Rodrigues S, Dubhashi NG, Rodrigues E, Banaulikar SS, Singh R, Dash AP, Baird JK: Histopathology of fatal respiratory distress caused by Plasmodium vivax malaria. Am J Trop Med Hyg 2009, 81:758–762.PubMedView Article
              21. Stephens R, Culleton RL, Lamb TJ: The contribution of Plasmodium chabaudi to our understanding of malaria. Trends Parasitol 2012, 28:73–82.PubMedView Article
              22. Geurts N, Martens E, Van Aelst I, Proost P, Opdenakker G, Van den Steen PE: Beta-hematin interaction with the hemopexin domain of gelatinase B/MMP-9 provokes autocatalytic processing of the propeptide, thereby priming activation by MMP-3. Biochemistry 2008, 47:2689–2699.PubMedView Article
              23. Ncokazi KK, Egan TJ: A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds. Anal Biochem 2005, 338:306–319.PubMedView Article
              24. Schwarzer E, Turrini F, Arese P: A luminescence method for the quantitative determination of phagocytosis of erythrocytes, of malaria-parasitized erythrocytes and of malarial pigment. Br J Haematol 1994, 88:740–745.PubMedView Article
              25. Yuan J, Shiller AM: Determination of subnanomolar levels of hydrogen peroxide in seawater by reagent-injection chemiluminescence detection. Anal Chem 1999, 71:1975–1980.View Article
              26. Janse CJ, Ramesar J, Waters AP: High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc 2006, 1:346–356.PubMedView Article
              27. http://​www.​stattools.​net/​ http://​www.​stattools.​net/​
              28. Lawrence C, Olson JA: Birefringent hemozoin identifies malaria. Am J Clin Pathol 1986, 86:360–363.PubMed
              29. Levesque MA, Sullivan AD, Meshnick SR: Splenic and hepatic hemozoin in mice after malaria parasite clearance. J Parasitol 1999, 85:570–573.PubMedView Article
              30. Fonager J, Pasini EM, Braks JA, Klop O, Ramesar J, Remarque EJ, Vroegrijk IO, van Duinen SG, Thomas AW, Khan SM, Mann M, Kocken CH, Janse CJ, Franke-Fayard BM: Reduced CD36-dependent tissue sequestration of Plasmodium-infected erythrocytes is detrimental to malaria parasite growth in vivo. J Exp Med 2012, 209:93–107.PubMedView Article
              31. Noland GS, Briones N, Sullivan DJ Jr: The shape and size of hemozoin crystals distinguishes diverse Plasmodium species. Mol Biochem Parasitol 2003, 130:91–99.PubMedView Article
              32. Orjih AU, Fitch CD: Hemozoin production by Plasmodium falciparum: variation with strain and exposure to chloroquine. Biochim Biophys Acta 1993, 1157:270–274.PubMedView Article
              33. Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NP: Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med 2005, 2:e204.PubMedView Article
              34. Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ: Intraleucocytic malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med Hyg 1995, 89:200–204.PubMedView Article
              35. Amodu OK, Adeyemo AA, Olumese PE, Gbadegesin RA: Intraleucocytic malaria pigment and clinical severity of malaria in children. Trans R Soc Trop Med Hyg 1998, 92:54–56.PubMedView Article
              36. Hanscheid T, Langin M, Lell B, Potschke M, Oyakhirome S, Kremsner PG, Grobusch MP: Full blood count and haemozoin-containing leukocytes in children with malaria: diagnostic value and association with disease severity. Malar J 2008, 7:109.PubMedView Article
              37. Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, Lewallen S, Liomba NG, Molyneux ME: Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med 2004, 10:143–145.PubMedView Article
              38. Carvalho BO, Lopes SC, Nogueira PA, Orlandi PP, Bargieri DY, Blanco YC, Mamoni R, Leite JA, Rodrigues MM, Soares IS, Oliveira TR, Wunderlich G, Lacerda MV, del Portillo HA, Araujo MO, Russell B, Suwanarusk R, Snounou G, Renia L, Costa FT: On the cytoadhesion of Plasmodium vivax-infected erythrocytes. J Infect Dis 2010, 202:638–647.PubMedView Article
              39. Anstey NM, Handojo T, Pain MC, Kenangalem E, Tjitra E, Price RN, Maguire GP: Lung injury in vivax malaria: pathophysiological evidence for pulmonary vascular sequestration and posttreatment alveolar-capillary inflammation. J Infect Dis 2007, 195:589–596.PubMedView Article
              40. Schwarzer E, Turrini F, Ulliers D, Giribaldi G, Ginsburg H, Arese P: Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment. J Exp Med 1992, 176:1033–1041.PubMedView Article
              41. Scorza T, Magez S, Brys L, De Baetselier P: Hemozoin is a key factor in the induction of malaria-associated immunosuppression. Parasite Immunol 1999, 21:545–554.PubMedView Article
              42. Spaccapelo R, Janse CJ, Caterbi S, Franke-Fayard B, Bonilla JA, Syphard LM, Di Cristina M, Dottorini T, Savarino A, Cassone A, Bistoni F, Waters AP, Dame JB, Crisanti A: Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria. Am J Pathol 2010, 176:205–217.PubMedView Article


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