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A simple and rapid method of staining malarial parasites in thick blood smears
195 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. XXXIV. No. 2.
August, 1940.
A SIMPLE A N D RAPID M E T H O D OF S T A I N I N G M A L A R I A L PARASITES IN T H I C K BLOOD SMEARS. BY
J. W. FIELD, M.D*., From the Institute for Medical Research, Federated Malay States.
Dried smears of blood of a thickness not greater than about 50/~ can be made transparent enough for microscopic examination by lysing the red cells and removing the haemoglobin. Conventional methods of staining malarial parasites in thick blood smears depend on this fact. The ideal method of staining would be one in which the red cells were alone affected during lysis of the blood, but in practice other cells--leucocytes and blood protozoa--are also damaged and we have to be content with a compromise giving maximal * The assistance of Dr. M. H. WHYTEwho made many of the parasite counts to which reference is made, is gratefully acknowledged.
196
STAINING ,MALARIAL PARASITES.
destruction of red cells with a minimal effect on the other cellular elements. Ordinarily this compromise is attained by drying the blood for several hours before staining. Prolonged drying produces a slight fixation of all the cells of the blood which, while enough to protect the white cells from gross damage during lysis, is not enough to hinder the solution of the erythrocytes. If t h e period of drying is reduced m u c h below eight hours the white cells are liable to plasmolysis and may be so damaged during staining that they are scarcely recognizable. The difficulty of speeding up the standard Giemsa methods of staining thick blood smears is related mainly to these facts. Methods of preserving the outlines of the leucocytes in Giemsa-stained thick smears and of rapidly staining malarial parasites without undue delay for drying the blood have recently been described by PAMPANA and by SIMONS. PaMPANA (1938) draws attention to the fact that damage during staining to leucocytes and blood protozoa in thick blood smears may be reduced by diluting the stain with a phosphate buffer solution isotonic with blood serum --not, as is usual with distilled water or with a hypotonic buffer. Fresh and dried blood, he points out, do not behave in like manner towards isotonic solutions, for the red cells in dried blood are lysed almost as readily by isotonic solutions as by distilled water. For routine Giemsa staining he advocates the dilution of the stain with a buffer solution of phosphate equimolecular with blood serum and of p H 7"2.* SIMONS (1938) points out that leucocytes and blood protozoa are beautifully preserved in thick blood smears stained with a solution of methylene blue containing saponin and approximately isotonic with serum.+ Lysis of the red cells and penetration of the stain are exceptionally rapid. By this method the stain is allowed to act on the dried smear for two minutes, a cover-slip is applied and after the excess of the stain has been cautiously blotted away the preparation is ready for microscopic examination. Smears may be stained within a few minutes of preparation without fear that they will detach from the slide or that the leucocytes or blood protozoa will be broken up. The main drawbacks of SIMONS' method of staining are that the smears are examined through a considerable depth of aqueous stain, and hence the blood cells or protozoa must be picked up at different focal levels; that they must be examined before the stain is dry; and that the stain, acting as a blue light filter, obscures the yellow colour of malarial pigment. * P a m p a n a ' s i s o t o n i c Buffer Solution p H 7.2 : - NA~HPO~ 14.8 g r a m m e s . Distilled w a t e r KH~PO4 5"3 ,, t S i m o n s ' Stain : Distilled w a t e r 300 c.c. Methylene blue 0"6 g r a m m e s . S o d i u m chloride 1.8 ,,
1,000 c.c.
S o d i u m citrate 3'0 g r a m m e s . Saponin 2.0 ,, F o r m o l 15% 12 c.c.
J. w. FIELD.
197
These observations seem to suggest that speed of staining and freedom from distortion of leucocytes and protozoa in thick blood smears may perhaps best be sought by the use of basic stains in isotonic solution. Various experiments based on this assumption have been made in this laboratory and a method has been devised whereby thick blood smears may be stained within 1 second sufficiently well for the accurate identification of malarial parasites. The method is briefly described in this preliminary communication as it may interest other workers concerned with the rapid diagnosis of malaria. GENERAL PRINCIPLES OF THE METHOD.
The method depends essentially on the fact that certain basic dyes in aqueous solution are able to penetrate a depth of approaching 50/J. of dried blood with extreme rapidity and are adsorbed by leucocytes and blood protozoa before lysis of the red cells is complete and before the red cell envelopes are stained so heavily that they obscure the microscopic field. The presence of free haemoglobin seems to retard the staining of the red cell residues which form the "background " of the stained thick smear and if the staining process is arrested at the moment when contrast is maximal, i.e., when ]eucocytes or protozoa have stained; but the red cell residues, protected by free haemoglobin, have not yet taken the stain--the leucocytes and protozoa are contrasted against a smooth pale yellow ground of residual haemoglobin. Contrast is optimal when the staining process is restricted to the pH range 6"6 to 7'0; and if, in addition, the stains are used in isotonic solution the outlines of leucocytes and blood protozoa are extraordinarily well preserved. The essential conditions appear to b e : - i. The use of certain basic stains in isotonic solution to conserve the outlines of the leucocytes. ii. Limitation of staining to about 1 second, at which time leucocytes and blood protozoa have taken the stain but the background is relatively unstained. iii. The buffering of the stain to pH 6"6 to 7"0--the pH range at which contrast is maximal. iv. The retention of some of the haemoglobin to provide a pale yellow background contrast to the stained parasites and to restrict the staining of the envelopes of the lysed red cells. Various stains have been tested, singly or in combination. Those which have given the best results are methylene-blue, brilliant cresyl blue, mixtures of methylene blue and eosin and an aqueous extract of combined Giemsa powder; of these brilliant cresyl blue has been the most generally satisfactory.* * T h e staining of the malarial parasites with brilliant cresyl blue is not new. SYDENSTmCKER and VRYONIS (1935) for example, have described and illustrated the appearances of P. falciparum and P. vivax in fresh blood stained with brilliant cresyl blue in physiological saline. T h e method, however, is a standard method of vital staining and differs radically from that described in this paper.
198
STAINING MALARIAL PARASITES. PREPARATION OF STAIN.
T h e composition of the stain recommended is as f o l l o w s : Brilliant cresyl blue ............ Disodium hydrogen phosphate (anhydrous) ... Potassium dihydrogen phosphate (anhydrous) Distilled waEer ...............
1"0 1"0 1"25 100
gramme gramme gramme c.c.
The phosphate salts are first dissolved. The solution is approximately isotonic with blood serum and is of p H 6"6. The stain is now added, solution being aided by grinding in a mortar. After filtration the stain is ready for use. Occasional filtration subsequently is desirable as a thin scum m a y form which is liable to deposit on the slides *
METHOD
OF USE.
The blood smears should not be more than 50/~ thick; if they are too thick there is too great a depth of residual haemoglobin. The thickness is about right if with the slide held near the face of a watch the hands can be dimly seen through the dried smear. The smears are rapidly dried by waving in the air or in a hot-air current and lightly heat-fixed by passing through a flame for about a second. T h e slides should not be made so hot that they cannot be held against the back of the hand. To stain the smears the slides are dipped for 1 second into a jar containing the stain, immediately removed and rinsed for 5 seconds by waving gently in a vessel of clean tap water. T h e y are now placed vertically on a rack to drain and dry and are then ready for microscopic examination. D u r i n g drying it will be noted that the haemoglobin continues to drain from the smear down the slide, leaving in the final dried smear four distinct zones depending on the a m o u n t of haemoglobin left behind. A. A zone at the upper thin edge of the smear from which most of the haemoglobin has drained away and in which on microscopic examination the background is seen to be stained a pale blue grey. In this zone minute blood parasites, such as young P. falciparurn rings, are best sought. * An alternative stain, giving rather less brilliant but otherwise similar staining, can be prepared from methylene blue and eosin in proportions as follows : Methylene blue 0.4 grammes, Eosin 0'1 gramme, Isotonic phosphate buffer 100 e.c. The stains are independently dissolved--the methylene blue in 80 c.c. of the phosphate solution and the eosin in 20 c.e.--and mixed by the addition of the eosin solution to that of the methylene blue. After filtration the stain is ready for use. Subsequent filtration is essential whenever a scum appears on the surface of the stain.
199
j. w . FIELD,
B. An adjoining zone where there is sufficient residual haemoglobin to colour the background a smooth pale creamy yellow. In this zone colour contrast is maximal--vivid blue against pale yellow--and here most malarial parasites, excepting only small P. falciparum rings, are best definedi C. A central zone where there is too much residual haemoglobin for clear definition and where only the leucocytes and larger blood protozoa are well seen. D. A lower zone at the lower thin edge of the smear where leucocytes and the larger parasites are often beautifully contrasted against a smooth yellow ground. The technique of staining and examination m a y be smnmarized as follows : - 1. Rapidly dry the smear and lightly fix in a flame. 2. Dip the smear into a jar of stain for 1 second. 3. Differentiate in clean tap water for 5 seconds. 4. Drain and dry by placing vertically against a staining rack. 5. Examine Zone A for P. falciparum rings and either of Zones B or D for other malarial parasites. TABI.E.
Thin film diagnosis.
W r o n g species diagnosis in " rapid " t h i c k smear.
Species diagnosis u n c e r t a i n or parasites n o t recognized in " rapid " thick smear.
!
P. falciparum P. v i v a x ... P. malariae Mixed I
4 1 0 13 #
239 103 18 23
* O n e species recognized.
Using this technique and rapidly drying the stained smears in a current of hot air it has been found possible to diagnose malaria and to identify the species and phase of parasite with surprising accuracy within one and a half minutes of the first preparation of the smears.
Reliability. Thick blood smears stained by this rapid method have now been compared with Giemsa-stained thin films in 383 cases of acute malaria. The accuracy of diagnosis from the ;apidly stained fihns is indicated in the table above.
200
STAINING MALARIAL PARASITES.
Diagnostic Comparison of Rapidly Stained Thick Smears with Stained Thin Films in 383 Cases of Acute Malaria. Parasites were usually seen in the thick smears within the first few microscopic fields, sometimes when in the thin films they h a d been f o u n d only after a prolonged search. In 250 cases the parasites f o u n d in the first ten microscopic fields were counted. T h e relative thick s m e a r and thin film counts were as f o l l o w s : Average n u m b e r of parasites per 10 thin film fields Average n u m b e r of parasites per 10 thick s m e a r fields
6"2 99'2
T h e r e was thus a sixteen-times concentration of parasites in the thick smears, a figure which, when f a m i l i a r i t y with the appearances of the parasites in the thick smears has been acquired, is p r o b a b l y a fair indication of the relative speed of diagnosis. MORPHOLOGY OF MALARIAL PARASITES STAINED BY THIS
RAPID METHOD.
Malarial parasites in thick smears stained b y this rapid m e t h o d have a general resemblance to those seen in smears stained b y standard G i e m s a methods. As in Giemsa-stained smears the host cells have disappeared and the parasites are seen on a b a c k g r o u n d derived f r o m the lysed red cells. T h e m a i n departures f r o m the usual appearances in Giemsa-stained smears are that : - (a) T h e c y t o p l a s m tends to be m o r e vividly stained. (b) T h e c h r o m a t i n is poorly stained and not differentiated in colour. (c) Small " r i n g " f o r m s tend to be pale and s o m e w h a t ill-defined. (d) , N u c l e a r and reticular d6bris f r o m i m m a t u r e red cells is m o r e deeply stained. DESCRIPTION OF PLATE.
FIG. l.--P, falciparum : " ring " forms in thick blood film stained for one second. The rings are pale and delicate; the chromatin is less well differentiated than in Giemsastained films. FIo. 2.--P. vivax: early schizonts in thick blood film stained for one second. The cytoplasm of the larger forms of P. vivax stains very intensely with brilliant cresyl blue, The field also contains one polymorph and two monocytes. Leucocytes are fairly well stained by this method ; their form tends to be better preserved than in films stained by standard Giemsa methods. FIG. 3.--P. falciparum: gametocytes in thick blood film stained for one second. The cytoplasm of gametocytes is vividly defined even with this brief period of staining as penetration of the stain is almost immediate. FIc. 4.--P. malariae: early schizonts in thick blood film stained for one second. The compact circular form usual in lysed and stained thick blood films is retained. The cytoplasm is well stained but the chromatin is poorly differentiated. The field also contains one leucocyte,
PHOTOMICROGRAPHS
OF MALARIAL
PARASITES
IN THICK
BLOOD
FILMS
STAINED
SECOND BY THE METHOD DESCRIBED IN THE TEXT. (Objective 1/12 inch oil immersion, ocular × 15, Wratten B filter.) To face page 200.
FOR
ONE
J. w . FIELD.
~)01
Trophozoites.--Young trophozoites may preserve the ring forms usual in fixed thin films but are often collapsed. The cytoplasm is fairly deeply stained except with very young forms of P. falciparum which may be so faint that they are seen only with critical microscopy. The cytoplasm of P. vivax trophozoites is apt to appear broken and identification may be difficult until one is familiar with their appearance. The chromatin dot appears smaller than in Giemsa-stained films, is more faintly stained and undifferentiated in colour. Schizonts.--Schizonts are deeply stained. Schizogony is less evident than in Giemsa-stained films but there is better preservation of outline. Generally speaking the larger the parasite the more intense the stain and schizonts are usually well contrasted against the creamy-yellow background. Gametocytes.--The differentiation of gametocytes from the larger trophozoites or schizonts is sometimes difficult except where, as with P. falciparurn, they have a distinctive shape. The chromatin is not well defined but the cytoplasm is deeply stained and the pigment is often seen well enough to afford assistance in identification. The differentiation of species is little more difficult than in Giemsa-stained films; ~the indications which aid diagnosis are the same--the size of the parasites, the numbers present, the uniformity or diversity of phase, the compactness or dispersion of the cytoplasm, the amount, appearance and arrangement of the pigment, the number of merozoites in the segmenting forms, etc. ADVANTAGES AND DRA~rBACKS.
The one advantage of this rapid staining method over conventional methods is the extremely rapid diagnosis which its use permits. The smears are stained for 1 second only. Provided there are facilities for rapidly drying the blood, stained dried smears with all the advantages of a sixteen-fold concentration ot parasites are available for examination in little over a minute after the blood is drawn.* The method has been adopted as a routine for immediate diagnosis in this laboratory and from tests in several hundreds of cases of acute malaria it has become clear that, for those who are familiar with the thick-smear morphology of malarial parasites, its use is consistent with fair accuracy. There are two drawbacks; the minuteness and pallor of very young " ring " forms of P. falciparum and the fact that the stain is liable to .emphasize the nuclear and reticular d~bris of immature red cells much more than is usual in Giemsa-stained smears. Young P. vivax trophozoites are sometimes found in immature cells with cytoplasm and reticulum so intermingled that the identification of the parasites is difficult, but a search for older forms will usually settle the diagnosis. Reticulocytes and the larger trophozoites of Crescents are stained by this rapid technique with remarkable clarity of outline when the blood smears are rapidly made on a hot slide, Immediate heat fixation is, however, unsuitable for ring forms.
202
STAINING MALARIAL PARASITES.
P. vivax have a superficial resemblance.
Nuclear d4bris from immature red cells may be confused with parasite chromatin. Granted good microscopy, however, and a knowledge of the appearance of reticulocytes and of the nuclear residues of young red cells in thick smears of anaemic blood, these difficulties should seldom leave the diagnosis in doubt. SUMMARY.
A method is described by which malarial parasites may be stained in thick blood smears in one second. Tests of this method in several hundreds of cases of acute malaria have shown that the great speed in malarial diagnosis which its use makes possible is consistent with fair accuracy. REFERENCES. PAMPANA, E.J. (1938). Riv..~Ialariol., 17, 300. SIMONS, H. (1938). Bull. Soc. Path. exot., 81, 100. SYDENSTRICKeR,V. P. & VRYONIS,G.P. (1935). ft. Lab. din. Med., 20, 1094.
Vol. XXXIV. No. 2.
August, 1940.
A SIMPLE A N D RAPID M E T H O D OF S T A I N I N G M A L A R I A L PARASITES IN T H I C K BLOOD SMEARS. BY
J. W. FIELD, M.D*., From the Institute for Medical Research, Federated Malay States.
Dried smears of blood of a thickness not greater than about 50/~ can be made transparent enough for microscopic examination by lysing the red cells and removing the haemoglobin. Conventional methods of staining malarial parasites in thick blood smears depend on this fact. The ideal method of staining would be one in which the red cells were alone affected during lysis of the blood, but in practice other cells--leucocytes and blood protozoa--are also damaged and we have to be content with a compromise giving maximal * The assistance of Dr. M. H. WHYTEwho made many of the parasite counts to which reference is made, is gratefully acknowledged.
196
STAINING ,MALARIAL PARASITES.
destruction of red cells with a minimal effect on the other cellular elements. Ordinarily this compromise is attained by drying the blood for several hours before staining. Prolonged drying produces a slight fixation of all the cells of the blood which, while enough to protect the white cells from gross damage during lysis, is not enough to hinder the solution of the erythrocytes. If t h e period of drying is reduced m u c h below eight hours the white cells are liable to plasmolysis and may be so damaged during staining that they are scarcely recognizable. The difficulty of speeding up the standard Giemsa methods of staining thick blood smears is related mainly to these facts. Methods of preserving the outlines of the leucocytes in Giemsa-stained thick smears and of rapidly staining malarial parasites without undue delay for drying the blood have recently been described by PAMPANA and by SIMONS. PaMPANA (1938) draws attention to the fact that damage during staining to leucocytes and blood protozoa in thick blood smears may be reduced by diluting the stain with a phosphate buffer solution isotonic with blood serum --not, as is usual with distilled water or with a hypotonic buffer. Fresh and dried blood, he points out, do not behave in like manner towards isotonic solutions, for the red cells in dried blood are lysed almost as readily by isotonic solutions as by distilled water. For routine Giemsa staining he advocates the dilution of the stain with a buffer solution of phosphate equimolecular with blood serum and of p H 7"2.* SIMONS (1938) points out that leucocytes and blood protozoa are beautifully preserved in thick blood smears stained with a solution of methylene blue containing saponin and approximately isotonic with serum.+ Lysis of the red cells and penetration of the stain are exceptionally rapid. By this method the stain is allowed to act on the dried smear for two minutes, a cover-slip is applied and after the excess of the stain has been cautiously blotted away the preparation is ready for microscopic examination. Smears may be stained within a few minutes of preparation without fear that they will detach from the slide or that the leucocytes or blood protozoa will be broken up. The main drawbacks of SIMONS' method of staining are that the smears are examined through a considerable depth of aqueous stain, and hence the blood cells or protozoa must be picked up at different focal levels; that they must be examined before the stain is dry; and that the stain, acting as a blue light filter, obscures the yellow colour of malarial pigment. * P a m p a n a ' s i s o t o n i c Buffer Solution p H 7.2 : - NA~HPO~ 14.8 g r a m m e s . Distilled w a t e r KH~PO4 5"3 ,, t S i m o n s ' Stain : Distilled w a t e r 300 c.c. Methylene blue 0"6 g r a m m e s . S o d i u m chloride 1.8 ,,
1,000 c.c.
S o d i u m citrate 3'0 g r a m m e s . Saponin 2.0 ,, F o r m o l 15% 12 c.c.
J. w. FIELD.
197
These observations seem to suggest that speed of staining and freedom from distortion of leucocytes and protozoa in thick blood smears may perhaps best be sought by the use of basic stains in isotonic solution. Various experiments based on this assumption have been made in this laboratory and a method has been devised whereby thick blood smears may be stained within 1 second sufficiently well for the accurate identification of malarial parasites. The method is briefly described in this preliminary communication as it may interest other workers concerned with the rapid diagnosis of malaria. GENERAL PRINCIPLES OF THE METHOD.
The method depends essentially on the fact that certain basic dyes in aqueous solution are able to penetrate a depth of approaching 50/J. of dried blood with extreme rapidity and are adsorbed by leucocytes and blood protozoa before lysis of the red cells is complete and before the red cell envelopes are stained so heavily that they obscure the microscopic field. The presence of free haemoglobin seems to retard the staining of the red cell residues which form the "background " of the stained thick smear and if the staining process is arrested at the moment when contrast is maximal, i.e., when ]eucocytes or protozoa have stained; but the red cell residues, protected by free haemoglobin, have not yet taken the stain--the leucocytes and protozoa are contrasted against a smooth pale yellow ground of residual haemoglobin. Contrast is optimal when the staining process is restricted to the pH range 6"6 to 7'0; and if, in addition, the stains are used in isotonic solution the outlines of leucocytes and blood protozoa are extraordinarily well preserved. The essential conditions appear to b e : - i. The use of certain basic stains in isotonic solution to conserve the outlines of the leucocytes. ii. Limitation of staining to about 1 second, at which time leucocytes and blood protozoa have taken the stain but the background is relatively unstained. iii. The buffering of the stain to pH 6"6 to 7"0--the pH range at which contrast is maximal. iv. The retention of some of the haemoglobin to provide a pale yellow background contrast to the stained parasites and to restrict the staining of the envelopes of the lysed red cells. Various stains have been tested, singly or in combination. Those which have given the best results are methylene-blue, brilliant cresyl blue, mixtures of methylene blue and eosin and an aqueous extract of combined Giemsa powder; of these brilliant cresyl blue has been the most generally satisfactory.* * T h e staining of the malarial parasites with brilliant cresyl blue is not new. SYDENSTmCKER and VRYONIS (1935) for example, have described and illustrated the appearances of P. falciparum and P. vivax in fresh blood stained with brilliant cresyl blue in physiological saline. T h e method, however, is a standard method of vital staining and differs radically from that described in this paper.
198
STAINING MALARIAL PARASITES. PREPARATION OF STAIN.
T h e composition of the stain recommended is as f o l l o w s : Brilliant cresyl blue ............ Disodium hydrogen phosphate (anhydrous) ... Potassium dihydrogen phosphate (anhydrous) Distilled waEer ...............
1"0 1"0 1"25 100
gramme gramme gramme c.c.
The phosphate salts are first dissolved. The solution is approximately isotonic with blood serum and is of p H 6"6. The stain is now added, solution being aided by grinding in a mortar. After filtration the stain is ready for use. Occasional filtration subsequently is desirable as a thin scum m a y form which is liable to deposit on the slides *
METHOD
OF USE.
The blood smears should not be more than 50/~ thick; if they are too thick there is too great a depth of residual haemoglobin. The thickness is about right if with the slide held near the face of a watch the hands can be dimly seen through the dried smear. The smears are rapidly dried by waving in the air or in a hot-air current and lightly heat-fixed by passing through a flame for about a second. T h e slides should not be made so hot that they cannot be held against the back of the hand. To stain the smears the slides are dipped for 1 second into a jar containing the stain, immediately removed and rinsed for 5 seconds by waving gently in a vessel of clean tap water. T h e y are now placed vertically on a rack to drain and dry and are then ready for microscopic examination. D u r i n g drying it will be noted that the haemoglobin continues to drain from the smear down the slide, leaving in the final dried smear four distinct zones depending on the a m o u n t of haemoglobin left behind. A. A zone at the upper thin edge of the smear from which most of the haemoglobin has drained away and in which on microscopic examination the background is seen to be stained a pale blue grey. In this zone minute blood parasites, such as young P. falciparurn rings, are best sought. * An alternative stain, giving rather less brilliant but otherwise similar staining, can be prepared from methylene blue and eosin in proportions as follows : Methylene blue 0.4 grammes, Eosin 0'1 gramme, Isotonic phosphate buffer 100 e.c. The stains are independently dissolved--the methylene blue in 80 c.c. of the phosphate solution and the eosin in 20 c.e.--and mixed by the addition of the eosin solution to that of the methylene blue. After filtration the stain is ready for use. Subsequent filtration is essential whenever a scum appears on the surface of the stain.
199
j. w . FIELD,
B. An adjoining zone where there is sufficient residual haemoglobin to colour the background a smooth pale creamy yellow. In this zone colour contrast is maximal--vivid blue against pale yellow--and here most malarial parasites, excepting only small P. falciparum rings, are best definedi C. A central zone where there is too much residual haemoglobin for clear definition and where only the leucocytes and larger blood protozoa are well seen. D. A lower zone at the lower thin edge of the smear where leucocytes and the larger parasites are often beautifully contrasted against a smooth yellow ground. The technique of staining and examination m a y be smnmarized as follows : - 1. Rapidly dry the smear and lightly fix in a flame. 2. Dip the smear into a jar of stain for 1 second. 3. Differentiate in clean tap water for 5 seconds. 4. Drain and dry by placing vertically against a staining rack. 5. Examine Zone A for P. falciparum rings and either of Zones B or D for other malarial parasites. TABI.E.
Thin film diagnosis.
W r o n g species diagnosis in " rapid " t h i c k smear.
Species diagnosis u n c e r t a i n or parasites n o t recognized in " rapid " thick smear.
!
P. falciparum P. v i v a x ... P. malariae Mixed I
4 1 0 13 #
239 103 18 23
* O n e species recognized.
Using this technique and rapidly drying the stained smears in a current of hot air it has been found possible to diagnose malaria and to identify the species and phase of parasite with surprising accuracy within one and a half minutes of the first preparation of the smears.
Reliability. Thick blood smears stained by this rapid method have now been compared with Giemsa-stained thin films in 383 cases of acute malaria. The accuracy of diagnosis from the ;apidly stained fihns is indicated in the table above.
200
STAINING MALARIAL PARASITES.
Diagnostic Comparison of Rapidly Stained Thick Smears with Stained Thin Films in 383 Cases of Acute Malaria. Parasites were usually seen in the thick smears within the first few microscopic fields, sometimes when in the thin films they h a d been f o u n d only after a prolonged search. In 250 cases the parasites f o u n d in the first ten microscopic fields were counted. T h e relative thick s m e a r and thin film counts were as f o l l o w s : Average n u m b e r of parasites per 10 thin film fields Average n u m b e r of parasites per 10 thick s m e a r fields
6"2 99'2
T h e r e was thus a sixteen-times concentration of parasites in the thick smears, a figure which, when f a m i l i a r i t y with the appearances of the parasites in the thick smears has been acquired, is p r o b a b l y a fair indication of the relative speed of diagnosis. MORPHOLOGY OF MALARIAL PARASITES STAINED BY THIS
RAPID METHOD.
Malarial parasites in thick smears stained b y this rapid m e t h o d have a general resemblance to those seen in smears stained b y standard G i e m s a methods. As in Giemsa-stained smears the host cells have disappeared and the parasites are seen on a b a c k g r o u n d derived f r o m the lysed red cells. T h e m a i n departures f r o m the usual appearances in Giemsa-stained smears are that : - (a) T h e c y t o p l a s m tends to be m o r e vividly stained. (b) T h e c h r o m a t i n is poorly stained and not differentiated in colour. (c) Small " r i n g " f o r m s tend to be pale and s o m e w h a t ill-defined. (d) , N u c l e a r and reticular d6bris f r o m i m m a t u r e red cells is m o r e deeply stained. DESCRIPTION OF PLATE.
FIG. l.--P, falciparum : " ring " forms in thick blood film stained for one second. The rings are pale and delicate; the chromatin is less well differentiated than in Giemsastained films. FIo. 2.--P. vivax: early schizonts in thick blood film stained for one second. The cytoplasm of the larger forms of P. vivax stains very intensely with brilliant cresyl blue, The field also contains one polymorph and two monocytes. Leucocytes are fairly well stained by this method ; their form tends to be better preserved than in films stained by standard Giemsa methods. FIG. 3.--P. falciparum: gametocytes in thick blood film stained for one second. The cytoplasm of gametocytes is vividly defined even with this brief period of staining as penetration of the stain is almost immediate. FIc. 4.--P. malariae: early schizonts in thick blood film stained for one second. The compact circular form usual in lysed and stained thick blood films is retained. The cytoplasm is well stained but the chromatin is poorly differentiated. The field also contains one leucocyte,
PHOTOMICROGRAPHS
OF MALARIAL
PARASITES
IN THICK
BLOOD
FILMS
STAINED
SECOND BY THE METHOD DESCRIBED IN THE TEXT. (Objective 1/12 inch oil immersion, ocular × 15, Wratten B filter.) To face page 200.
FOR
ONE
J. w . FIELD.
~)01
Trophozoites.--Young trophozoites may preserve the ring forms usual in fixed thin films but are often collapsed. The cytoplasm is fairly deeply stained except with very young forms of P. falciparum which may be so faint that they are seen only with critical microscopy. The cytoplasm of P. vivax trophozoites is apt to appear broken and identification may be difficult until one is familiar with their appearance. The chromatin dot appears smaller than in Giemsa-stained films, is more faintly stained and undifferentiated in colour. Schizonts.--Schizonts are deeply stained. Schizogony is less evident than in Giemsa-stained films but there is better preservation of outline. Generally speaking the larger the parasite the more intense the stain and schizonts are usually well contrasted against the creamy-yellow background. Gametocytes.--The differentiation of gametocytes from the larger trophozoites or schizonts is sometimes difficult except where, as with P. falciparurn, they have a distinctive shape. The chromatin is not well defined but the cytoplasm is deeply stained and the pigment is often seen well enough to afford assistance in identification. The differentiation of species is little more difficult than in Giemsa-stained films; ~the indications which aid diagnosis are the same--the size of the parasites, the numbers present, the uniformity or diversity of phase, the compactness or dispersion of the cytoplasm, the amount, appearance and arrangement of the pigment, the number of merozoites in the segmenting forms, etc. ADVANTAGES AND DRA~rBACKS.
The one advantage of this rapid staining method over conventional methods is the extremely rapid diagnosis which its use permits. The smears are stained for 1 second only. Provided there are facilities for rapidly drying the blood, stained dried smears with all the advantages of a sixteen-fold concentration ot parasites are available for examination in little over a minute after the blood is drawn.* The method has been adopted as a routine for immediate diagnosis in this laboratory and from tests in several hundreds of cases of acute malaria it has become clear that, for those who are familiar with the thick-smear morphology of malarial parasites, its use is consistent with fair accuracy. There are two drawbacks; the minuteness and pallor of very young " ring " forms of P. falciparum and the fact that the stain is liable to .emphasize the nuclear and reticular d~bris of immature red cells much more than is usual in Giemsa-stained smears. Young P. vivax trophozoites are sometimes found in immature cells with cytoplasm and reticulum so intermingled that the identification of the parasites is difficult, but a search for older forms will usually settle the diagnosis. Reticulocytes and the larger trophozoites of Crescents are stained by this rapid technique with remarkable clarity of outline when the blood smears are rapidly made on a hot slide, Immediate heat fixation is, however, unsuitable for ring forms.
202
STAINING MALARIAL PARASITES.
P. vivax have a superficial resemblance.
Nuclear d4bris from immature red cells may be confused with parasite chromatin. Granted good microscopy, however, and a knowledge of the appearance of reticulocytes and of the nuclear residues of young red cells in thick smears of anaemic blood, these difficulties should seldom leave the diagnosis in doubt. SUMMARY.
A method is described by which malarial parasites may be stained in thick blood smears in one second. Tests of this method in several hundreds of cases of acute malaria have shown that the great speed in malarial diagnosis which its use makes possible is consistent with fair accuracy. REFERENCES. PAMPANA, E.J. (1938). Riv..~Ialariol., 17, 300. SIMONS, H. (1938). Bull. Soc. Path. exot., 81, 100. SYDENSTRICKeR,V. P. & VRYONIS,G.P. (1935). ft. Lab. din. Med., 20, 1094.