TECHNIQUES FOR THE STUDY OF ANEMIAS IN MICE 561 



method in studies with mice lies not only in its speed and accuracy, but also in the fact 

 that it works very efficiently with blood samples of one lambda (0.001 ml.), making it 

 especially suitable for newborn and fetal counts. 



Hematocrit percentages can be determined with the use of Van-Allen centrifuge 

 tubes, 3 per cent oxalate diluent, and a standard clinical centrifuge. However, 

 recent improvement of microhematocrit methods 823 ' 1108, 1300 have been very useful 

 in murine as well as human hematology. Each determination uses a very small 

 volume of undiluted blood (+0.05 ml.), collected in an open-ended heparinized or 

 oxalated capillary tube (A. H. Thomas Co., Philadelphia). After heat fusion of one 

 end in a microburner flame, these tubes are spun for six minutes at 11,000 r.p.m. 

 in a special flat-angle hematocrit centrifuge (International Centrifuge Co., Boston). 

 The volume of packed red cells relative to total volume of blood is then read directly 

 in a convenient hematocrit-reader device, usually with excellent agreement between 

 samples from the same individual. The direct readings depend on the ratio of length 

 of column of packed cells to total blood column, so that the problem of filling a pipette 

 to a preset level is avoided. Special oxalated pipettes collecting approximately 

 0.01 ml. for each reading have proved useful for newborn and fetal hematocrit 

 determinations, but these are not read accurately in the hematocrit reader described 

 above. The author has had no personal experience with a special reader designed 

 to go with these tubes 1300 but has obtained fairly reliable results by reading column 

 heights in these tubes placed on a millimeter scale in the field of a dissecting microscope. 



The usual method for comparing size of erythrocytes in different animals is determi- 

 nation of their mean cell volumes from the ratio of hematocrit percentage to erythrocyte 

 count : 



packed cell volume erythrocyte number 

 total blood volume total blood volume 



Accuracy and repeatability of this determination depend on the variability 

 observed in each of the component values. 



A value proportional to the mean and distribution of erythrocyte volumes in a 

 single blood sample may also be calculated directly from the relative counts for different- 

 sized cells in the Coulter electronic counter. 477 



Determinations of hemaglobin content in blood of mice may be achieved with 

 considerable accuracy, especially if hemoglobin is converted to the stable cyanomethe- 

 moglobin. 230 Hemoglobin standards and reagents are available (Scientific Products, 

 Evanston, 111.) ; the latter may be made up locally, 1397 and concentrations are calculated 

 from optical density read in a photometer. 



It is frequently desirable to judge the rate at which new red cells are being added 

 to the circulation. For this, determination of the reticulocyte percentage is important. 

 An excellent stain for the murine reticulocyte is the new methylene blue method of 

 Brecher 124, 126 in which an approximately equal mixture of blood and dye is held in a 

 capillary pipette for ten minutes, smeared on a cover slip, air dried, and counted under 



