Exeley Inc.

Ahmed, Henni Sid

Caelen, Jean

The command of comfort in an intelligent building by speech classification and image classification for energy optimization

BILIAIEV, Mykola

PSHINKO, Oleksandr

RUSAKOVA, Tetiana

BILIAIEVA, Viktoriia

SŁADKOWSKI, Aleksander

APPLICATION OF LOCAL EXHAUST SYSTEMS TO REDUCE POLLUTION CONCENTRATION NEAR THE ROAD

PIETRZAK, Oliwia

PIETRZAK, Krystian

WAGNER, Natalia

MONTWIŁŁ, Andrzej

IMPROVING SEAPORT COMPETITIVENESS BY CREATING A CONNECTION TO THE NATIONAL RAIL NETWORK

Crawford, M.N.

T E C H  T I P  
Plastic sprayer: A 
device for washing 
microplates 
M. N.  CRAWFORD 
Inexpensive plastic pint or quart sprayers are 
satisfactory substitutes for “JET” (Cole-Parmer, 
Chicago, IL) or similar automatic pipets. The sprayers 
are available at Woolworth stores or garden supply 
shops. The cap should be removed from the nozzle 
and plastic tubing attached with an adapter such as 
a pipette tip scavenged from various sources. The 
plastic tubing should be of analyzer size (or from a 
plastic transfer pack). A big piece of rubber tubing will 
fit over the nozzle, and the large end of a big pipette 
tip can be fitted into the rubber tubing, allowing the 
tapered small end of the pipette tip to go into the 
plastic tubing. The eight-tip manifold (Robbins Scien- 
tific Co., Mountainview, CA) will also need an adapter 
for attachment to the tubing. All of the connections 
should be secured with tightened wires. 
The plastic bottle must be kept below the level of 
the microplate to avoid dripping. The “trigger” of the 
sprayer must be pulled once or twice, depending on 
the size of the wells and the volume of reagents 
therein, To simplify rinses of the equipment for storage 
or use, well-marked separate plastic bottles may be 
kept, one filled with saline and the other with distilled 
water. The nozzle and tubing may be transferred from 
one bottle to another. This $2 substitute saves almost 
$150 (Figure 1). 
Fig. 1. “Semi-automated” manifold for cell washings 
on microplates. 
V O L U M E  4, N U M B E R  2, 1 9 8 8  41 


Plastic sprayer: A device for washing microplates

DeLong, E. Nicole

Evaluation of a complement-dependent anti-Jka by various sensitization and detection methodologies: a case report

Henry, Stephen M.

Benny, Graeme

Woodfield, Graeme

Evaluation of monoclonal anti-A 
reagents by salivary inhibition 
J 
studies 
S.M. HENRY, A.G. BENNY, D.G. WOODFIELD 
Abstract: Ten commercially available monoclonal and 
polyclonal anti-A reagents were evaluated for use in the test for 
ABH secretor status after finding unexpected inhibition with a 
monoclonal anti-A (reagent Z). Two of the available monoclonal 
reagents were from different batches from the same clone as 
reagent Z. Those two reagents, as well as reagent Z, appeared to he 
partially neutralized by salivary substances from group A non- 
secretor individuals. This finding suggests that when a saliva inhihi- 
tion test is done, a nonsecretor control saliva of the same ABO 
group as the saliva to he tested should be used. 
Recently, monoclonal antibodies have become 
widely used in immunohematology Such reagents, in 
contrast to polyclonal human reagents, often have a 
narrower specificity and selectivity for their antigenic 
determinants. As a part of our evaluation before intro- 
ducing monoclonal antibodies for routine use, we 
investigated their accuracy in determining the pres- 
ence of salivary group A substance. 
The determination of ABH secretor status by testing 
saliva is generally performed using the lectin anti-H 
(Ulex europaeus) sup(1) for group O individuals and anti-A 
and antiB for non-group O individuals sup(2,3) It is well 
Table 1. 
Anti-A reagents used in test procedures 
known that the anti-A and antiS used for such testing 
should not come from donors who have been hyperim- 
munized to A and B antigens, since alloagglutinins in 
such sera are less susceptible to neutralization.’ 
Materials and Methods 
Four human and five monoclonal anti-A reagents 
were evaluated for use in the test for ABH secretor sta- 
tus (Table 1). 
Saliva and blood samples were collected from 25 
unrelated laboratory staff One sample was from an A, 
Le(a + b -) Indian and one from an AB, Le(a - b +) Chi- 
nese. The remaining samples were from Caucasians. 
All dilutions of saliva and blood grouping reagents 
were made in phosphate buffered saline (PBS), pH 7.3, 
using calibrated pipettes. Dilutions were prepared 
immediately prior to use. 
The presence of salivary group A substances was 
evaluated by inhibition titrations based on previously 
described methods.’ 
Brand 
Code Manufacturer Batch No. Source Names 
1. N Gamma, Houston, TX, USA 5A25-1 Human Omni Series 
2. P Cooper Biomedical Inc., Malvern, PA, USA 231-1 Human 
3. Q Biological Lab, Auckland. New Zealand 8668 Human 
4. R Commonwealth Serum Lab, Melbourne, Australia 082/3 Human 
Biotest, Frankfurt. West Germany 13 3046 Monoclonal Seraclone 
1. Biotest, Frankfurt. West Germany 11 1047 Monoclonal Seraclone 
2. V Bioscot, Edinburgh, UK NBF 701 Monoclonal (Sample) 
3. W Eurobio, Germain, Paris, France 028 Monoclonal 
4. X HD Supplies, Aylesbury, UK 534 Monoclonal 
5. Y Ortho, Raritan, NJ, USA BAAIOSC Monoclonal Bioclone 
Z sup(†) Biotest, Frankfurt, West Germany 12 1087 Monoclonal Seraclone 
*Different batches from the same clone as Z. 
‘Results using this reagent in a salivary inhibition test initiated this study. 
V O L U M E  4 ,  N U M B E R  4 ,  1 9 8 8  75 
S.M. HENRY ET AI.. 
Collection of saliva 
Seven to 10 mL of saliva were collected in a 10-mL 
siliconized vacutainer tube. Within two hours of col- 
lection, the samples were boiled for 10 minutes and 
then frozen at - 30°C. Just before use, samples were 
thawed, centrifuged, and the clear supernates (some 
slightly opalescent) removed for testing. Samples were 
repeatedly frozen and thawed between assays, as this 
procedure has been shown to have no effect on the sta- 
bility of the ABH salivary substances present. sup(4) 
Blood typing 
To determine individual red cell Lewis types, red 
cell samples were tested with three anti-Le sup(a) and three 
anti-Leb reagents of human, goat, and monoclonal ori- 
gin (Biotest [human] anti-Le” 111057 and anti-Leh 
111047, Ortho [goat] anti-Le” LA346A1 and anti-Leh 
LB544A2, and Biotest Seraclone [mouse monoclonal] 
anti-Le” 111037 and anti-Le” 112116). Manufacturers’ 
recommendations for the use of reagents were 
observed. 
Titration of anti-A 
The titers of the different anti-A reagents against 
group A sub(2) red cells using PVC V-bottom microtiter 
plates (No. 001-010-2601, Dynatech Laboratories, USA) 
were determined using PBS for the serial doubling 
dilutions. Three dilutions from each titer, representing 
the last three strong to moderate agglutination read- 
ings, were selected for use in the saliva inhibition test. 
Microtiter plate agglutination was graded using the fol- 
lowing symbols: 
+ = strong agglutination 
w = moderate agglutination 
v = weakagglutination 
- = no agglutination 
Salivary inhibition test method 
Saliva samples, serially diluted in PBS, were pre- 
pared in microtiter plates by placing 20 µL of appropri- 
ate saliva dilutions into the wells. The microtiter plates 
were frozen and stored at - 30°C and thawed immedi- 
ately prior to use. Twenty µL of the appropriate dilution 
of blood grouping reagent (anti-A) were added to wells 
of the microtiter plate containing the selected saliva 
dilutions, and the mixtures were allowed to react at 
room temperature. After 30 minutes, 20 µL of a 2 per. 
cent suspension of group A sub(2) red cells were added and 
the reactants allowed to incubate for 60 minutes at 
room temperature. The microtiter plates were 
inclined at an angle of 45 degrees on an illuminated 
view box and allowed to settle for three minutes; 
agglutination was graded within the following five 
minutes. 
Screening tests 
Initially, all saliva samples were serially diluted from 
1:1 to 1:8 and tested with monoclonal anti-A (reagent 
Z), which had previously been found to give unex- 
pected results with group A, Le(a + b -) samples. The 
saliva samples were then diluted 1:2 and tested against 
10 more anti-A reagents, including 2 (S, T) from the 
same clone as Z. Finally, the three salivas from group A 
nonsecretors and positive and negative inhibition con- 
trol salivas were each diluted from 1:2 to 1:16 and 
tested against the three dilutions selected from the 
titration results of the anti-A reagents listed in Table 2. 
Microplate technology control 
To confirm the accuracy of the microplate technol- 
ogy, positive and negative inhibition controls and 
three group A, Le(a + b -) saliva samples were retested 
by the tube technique against dilutions one, two, and 
three selected for reagents R, S, and T (Table 2). 
Results 
Red blood cell Lewis types of the 25 saliva 
donors 
The 25 Lewis types were as follows: Group 0: 3 
Le(a+ b -), 9 Le(a - b +), 1 Le(a - b -); group A: 3 
Le(a + b -), 6 Le(a - b +); group B: 1 Le(a - b +); group 
AB: 1 Le(a + b -), 1 Le(a - b +). 
Reagent anti-A titers 
Table 2 shows the results of titrations performed on 
the four human (polyclonal) and five monoclonal 
anti-A reagents. Titration endpoints ranged from 1:32 
to 1:512 with the highest titers seen with the 
monoclonal reagents. Table 2 also lists the three serial 
dilutions selected for each reagent used in salivary 
inhibition tests. 
Saliva inhibition screening tests 
The initial saliva inhibition screening test, per- 
formed with saliva samples diluted from 1:1 to 1:8 and 
dilution one (see Table 2) from the anti-A reagents, 
showed that the seven A and AB secretor salivas inhib- 
ited all the reagents; the 15 group O and B saliva sam- 
76 I M M U N O H E M A T O L O G Y  
Monoclonal anti-A and saliva inhibition tests 
Secretor Blood Saliva 
status groups dilution 
Secretor A Le(a-b+)* 1:2 
Nonsecretor 0 Le(a+b-)* 1:2 
Nonsecretor AB Le(a+b-)* 1:2 
Table 2. 
Reagent titers and dilutions used for testing 
Dilution one Dilution two Dilution three 
Human Monoclonal Human Monoclonal Human Monoclonal Test 
N P Q R  S T V W X Y  N P Q R  S T V W X Y  N P Q R  S T V W X Y  status 
Positive control 
salivas 
-  -  -  -  - - - - - - - - - - -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  
+  + ‡ +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  
Negative control 
salivas 
+  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  
Anti-A Dilution Dilutions 
Code 0 1 2  1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 One Two Three 
Nonsecretor A Le(a+b-)† 1.2 
1:4 
1:8 
1:16 
- - - 1:8 1:16 1:32 1. N + t + + + W 
- - - 1:8 1:16 1 3 2  2. P + + + + + W 
- - - 1:8 1:16 1 3 2  3. Q + + + + + W 
- - - 1:8 1:16 1 3 2  4. R + + + + + W 
+ + + + + W W W V - - 1:16 1:32 1:64 
1. + + + + W W W V V - 1:16 1:32 1:64 
v - 1:16 1:32 1:64 2. v + + + + + W W 
1:16 1:32 3. w + + + + + W V V 
4. x + + + + + + W W V V - 1:32 1:64 1:128 
5. Y + + + + + W W W v v - 1:16 1:32 1:64 
- - 
- - 
- - 
- - 
- - 
1:8 - - - 
+  +  +  +  v v + + + + + + + + -  -  +  w  +  +  +  w  -  +  -  -  v  -  w  +  Testsalivas 
+  +  +  +  w  +  +  +  +  +  +  +  +  +  v  -  +  +  +  +  + + v + -  -  w  w  +  +  
+  +  +  +  +  +  +  +  +  +  +  +  +  +  w  w  +  +  +  + + v + -  -  +  +  +  +  
+  +  +  +  +  +  +  +  +  +  4 + + + + + + + + + + +  +  w  +  v  -  +  +  +  +  
*Different batches from the same clone. 
Note: Underlined dilutions refer to dilutions one, two, and three 
ples (secretors and nonsecretors) caused no inhibition 
but the three salivas from group A nonsecretors caused 
complete or partial neutralization of reagents S and T, 
representing different batches from the same clone as 
reagent 2. The remainder of the anti-A reagents were 
unaffected (results not shown). 
Confirmatory saliva inhibition test 
To evaluate further the inhibition of anti-A, selected 
control salivas and salivas from the three group A non- 
secretors were diluted from 1:2 to 1:16 and tested 
against the three dilutions of the 9 anti-A reagents 
listed in Table 2. Results are shown in Table 3. 
The group O control sample, as expected, showed 
Table 3. 
Inhibition test results 
no inhibition of anti-A. Control A, Le(a - b +) samples, 
at all dilutions, completely inhibited anti-A. 
Again, group A, Le(a + b -) saliva samples resulted in 
some degree of inhibition of all three dilutions of the 
Biotest monoclonal anti-A reagents (S and T). Using the 
weakest anti-A reagent dilution (No. 3), the group A, 
Le(a + b -) saliva samples were also able to inhibit two 
of the four human (polyclonal) reagents and three 
more monoclonal reagents. The group AB, Le(a + b -) 
sample inhibited none of the reagents. 
Microplate technique controls 
There were no significant differences in the results 
obtained from parallel tests done by the microplate 
V O L U M E  4 ,  N U M B E R  4 ,  1 9 8 8  77 
S.M. HENRY ET AL. 
method or the tube test method 
Discussion 
Analysis of these secretor and nonsecretor saliva 
samples indicated that some monoclonal anti-A 
reagents may be neutralized to a marked extent by 
group A nonsecretor salivas. The reagent that was con- 
sistently neutralized consisted of two batches (S, T) 
from the same clone as reagent Z, which provided the 
first clue on which this study was based. 
The ability of the group A, Le(a + b -) samples to 
inhibit the Biotest monoclonal reagents was not a 
result of excessive dilution of the reagents, as the non- 
group-A samples gave very strong reactions at the same 
dilutions. 
Most apparent nonsecretor individuals have been 
reported to secrete small quantities of ABH sub- 
stance~.*,~ Therefore, it is important that nonsecretor 
controls for a secretory assay must all be of the same 
ABO group. Dilution three in Table 3 clearly demon- 
strates the potential error when using non-group-A 
samples, or even group AB samples as controls for 
group A nonsecretors. Using dilution three, the group 
A, Le(a+ b -) samples caused inhibition with half of 
the polyclonal reagents as well as all but one of the rest 
of the monoclonal reagents. This additional inhibition 
is primarily a result of excessive dilution of the 
reagents; however, the group AB and O nonsecretor 
salivas caused no inhibition at that dilution. The dis- 
covery that some monoclonal reagents may be mark- 
edly affected by group A nonsecretor salivas within the 
dilution range recommended for use in an inhibition 
test demonstrates the need for appropriate controls 
and discretion when interpreting results. 
The group A salivary substances found in most 
g r o u p  A nonsecretors are of low molecular weight 
(1,500-13,000 daltons) having a similar antigenic con- 
figuration as the predominantly high molecular 
weight substances (200,000-5,000,000 daltons) of 
secretors. sup(5) It is conceivable that individual monoclonal 
antibodies are capable of specifically interacting with 
these low molecular weight substances and thus be 
neutralized. 
The failure of all monoclonal anti-A reagents to be 
significantly affected by group A salivary inhibition 
may be a consequence of the slightly different specific- 
ity of the different monoclonal reagents for mono- and 
difucosyl-A determinants." One demonstrable differ- 
ence between monoclonal reagents is the ability to 
detect the A, phenotype. There was no correlation in 
this study between the ability to detect this phenotype 
and inhibition of the reagents by group A, Le(a + b -) 
saliva. 
It is unclear why the group AB, Le(a + b -) sample 
failed to show inhibition of the anti-A reagents. It is 
possible that these individuals have lower levels of 
group A substance because of competition for precur- 
sor chains with the group B enzyme, or the salivary 
antigen may be fundamentally different in molecular 
configuration. 
These results emphasize the requirement for non- 
secretor controls in a secretory assay to be of the same 
ABO group as the samples tested. Moreover, the use of 
monoclonal reagents in place of polyclonal reagents 
must be viewed with caution and carefully evaluated. 
Acknowledgments 
The authors wish to thank the staff of the Auckland 
Regional Blood Centre who kindly donated saliva and 
red cell samples for this survey 
References 
1. Royd WC, Shapleigh E. Separation of individuals of any blood 
group into secretors and non-secretors by use of a plant agglu- 
tinin (lectin). Blood 1954;9:1195-7. 
2. Race RR, Sanger R. Blood groups in man, 6th ed. Oxford: Black- 
well Scientific Publications, 1975;323-49. 
3. McNeil C, Trentelman EF, Krentzer VO, Fullmer CD. Aberrant 
secretion of salivary A, B and H group substances in human 
beings. Am J Clin Pathol 195728:145-51. 
4. Sturgeon P, McQuiston D, Van Camp S. Quantitative studies on 
salivary blood group substances I. Methodology and physio- 
logical variables. Vox Sang 19773;24:97-113. 
5. Fiori A, Giusti GV, Panari G. Gel filtration of ABH blood group 
substances II. Individual gel chromatographic patterns of ABH 
substances in the saliva of secretors and non-secretors. J 
Chromatol 1971;55:351-63. 
6. Chen H, Kabat EA. lmmunochemical studies on blood groups. 
The combining site specificities of mouse monoclonal 
hybridoma anti-A and anti-B. J Bid Chem 1985;260:13208-17 
Stephen M. Henry, Dip MLT ANZIMLT, Graeme Benny 
MSc(Hons), Graeme Woodfield PhD FRCPE FRCPA, 
Auckland Regional Blood Centre, Auckland 
Hospital Auckland New Zealand. 
78 I M M U N O H E M A T O L O G Y  


Evaluation of monoclonal anti-A reagents by salivary inhibition studies

Weaver-Dahm, Marty

Problem solving: a course for 
blood bankers 
M. WEAVER-DAHM 
Abstract: A structured program for the training of blood 
bank personnel and students entitled “Problem Solving” is pre- 
sented in its entirety The explanation includes the primary objec- 
tive for this program, course description, how to evaluate student 
results, a grading system used for errors, and preparation of speci- 
mens and practical examinations. An example of an actual problem 
is also presented. 
The training of blood bank personnel and students 
can pose a challenge to clinical instructors. A wide 
variety of specimens must be provided to simulate 
actual problems that a blood banker might encounter 
such as ABO discrepancies, auto- and alloantibody 
problems, hemolytic disease of the newborn, transfu- 
sion reactions, etc. Some of the samples may come 
from patient problems encountered during the routine 
workday Other samples can be “made up” using out- 
dated blood grouping reagents and reagent red cells. 
However, there is no guarantee that examples of all 
blood banking problems will be available when the 
trainee or student needs them. Rather than leaving 
blood bank instruction to chance, Kay Beattie and Jane 
Cullen of the American Red Cross, Southeastern Mich- 
igan Region, developed a structured program entitled 
“Problem Solving” as part of the Master’s/SBB curricu- 
lum at Wayne State University The intent of the pro- 
gram was to give students the opportunity to integrate 
and apply their didactic and practical training in 
resolving complex immunohematological problems. 
“Problem Solving” is offered in the later part of the 
SBB program so that the students, having completed 
most of their lectures and clinical rotations, are famil- 
iar with a wide variety of serological techniques and 
have the knowledge to resolve most blood banking 
problems. 
The Program 
Objective 
The primary objective for “Problem Solving” is, 
given a number of samples, each accompanied by a 
brief case history, the student will attain practical labo- 
ratory testing experience in solving serological prob- 
lems that may be encountered in a hospital blood bank 
or laboratory setting and therefore will be able to ful- 
fill the transfusion requirements of various kinds of 
patients. 
Couse description 
“Problem Solving” is an 11-week course consisting of 
nine “patient” samples, numerous paper problems cov- 
ering the areas of coagulation, hematology, and person- 
nel management, and a practical exam. Each student is 
provided with a specific patient sample and paper 
problems on a predetermined day of each week. Lim- 
ited amounts of sample are alloted so that students 
learn to be judicious in the use of specimens. A brief 
case history is provided and, in some cases, donor units 
are available for crossmatching. Textbooks, workshop 
manuals, and other written materials may be used as 
resource information; blood bank and laboratory per- 
sonnel may not be used as sources of information. The 
students may not discuss the specimens among them- 
selves or others, with the exception of the blood bank 
instructor. The use of rare test cells or reagent antibod- 
ies may be permitted only after consulting with the 
supervisor. On occasion and by request only, the stu- 
dent may be provided with additional information 
and/or samples by the blood bank instructor, such as 
transfusion history, results of other laboratory tests, 
family studies, saliva for secretor studies, or specimens 
on family members. The student must be prepared to 
defend the relevance of such requests to the resolution 
of the problem. At times, information will be intention- 
ally withheld to encourage the student to investigate 
all possible explanations of the problem at hand. At the 
end of each one-week period, the student must submit 
a written report to the instructor and give an oral pre- 
sentation. 
The criteria by which students must show their 
expertise are as follows: 
V O L U M E  4 ,  NUMBER 4 ,  1 9 8 8  79 
M. WEAVER-DAHM 
1. The students must complete all work on the origi- 
nal sample provided and not request additional 
patient sample. 
2. Any ABO discrepancies must be resolved using 
appropriate techniques (i.e., adsorption-elution, 
red cell separation, etc.). 
3. The correct Rh status of the patient and/or donor 
must be determined. 
4. Any unexpected antibodies must be identified 
within the confidence limits maintained by the 
AABB. 
5. Special techniques must be used when applicable 
(i.e., the use of enzymes, chloroquine diphosphate, 
adsorption and elution, hemagglutination- 
inhibition with soluble substances, homologous 
adsorption, Kleihauer-Betke stains, etc.). 
6. All mixed field reactions must be explained and 
investigated. 
7. Donor samples must be regarded as donor units 
received from another institution. 
8. Any discrepancies or problems encountered with 
the donor units must be investigated and the cor- 
rect disposition of the units determined. 
9. Selection of appropriate donor units for transfu- 
sion must be discussed when applicable. 
10. Proper controls must be used. 
Evaluation 
The student is evaluated on a written report of the 
problem that shows results of all serological testing, 
explains the problems encountered, discusses other 
possible explanations of the problems and how they 
were excluded, lists all references used in resolving 
the problems, and discusses selection of blood for 
transfusion including the logistics of finding compati- 
ble units in a random population. In addition, the stu- 
dent must give an oral presentation to the class and 
instructors in which the approach to solving the prob- 
lems, procedures used, and results obtained are dis- 
cussed, as well as how alternative presumptions were 
excluded, the conclusions, and selection of blood for 
transfusion. The students must be prepared to defend 
their results and conclusions. 
Grading system 
total of 100: 
Error in ABO grouping - 21 points 
Error in Rh typing - 21 points 
The following points would be subtracted from a 
Error in antibody identification 
-affecting compatibility testing - 21 points 
-not affecting compatibility testing - 10 points 
Antigen-typing errors - 5 points 
Calling compatible units incompatible - 5 points 
Failure to perform eluate when indicated - 10 points 
Failure to separate mixed cell population -10 
points 
Other errors or failures to proceed in the usual man- 
ner result in loss of points at the discretion of the 
instructor. Passing grade for this course is 80 percent 
overall. The breakdown of the grading system is as 
follows: 
Nine “wet” specimens 64% 
Practical problems 20% 
Paper problems 16% 
If a student fails to correctly resolve the serological 
problem provided, points may be recovered by provid- 
ing written documentation of further investigation 
into the problem, including an explanation of why the 
incorrect conclusion was reached (Le., a dosing anti-Jk sup(a) 
reactive only with polyspecific anti-human globulin). If 
deemed necessary by the education coordinator, the 
student may be provided with another specimen of 
equal complexity to prove expertise in a particular 
area of problem solving. The primary goal is to provide 
a learning experience for the student; this includes rec- 
ognizing pitfalls that may lead to incorrect interpreta- 
tions and developing tactics to avoid these pitfalls. 
Preparation of specimens 
In order to prepare for “Problem Solving,” several 
planning steps must be taken. First, the kinds of prob- 
lems that need to be covered must be identified: 
Antibodies 
-to multiple antigens 
-to high-frequency antigens 
-to low-frequency antigens 
Warm autoantibodies 
Cold autoantibodies 
Hemolytic disease of the newborn 
Transfusion reactions 
ABO typing problems 
Rh typing problems 
Miscellaneous—i.e., HTLA antibodies, antiSd sup(a), Bom- 
bay phenotype 
Second, specimens must be collected and frozen 
throughout the year. These samples can be obtained 
from donors, patients, student sample exchange pro- 
80 I M M U N O H E M A T O L O G Y  
Problem solving for blood bankers 
grams, or can be concocted using commercial re- 
agents. And finally, each student's sample must be thor- 
oughly tested by the instructor. A duplicate sample is 
always prepared in case the student challenges the se- 
rological results. A notebook is kept (under lock and 
key) with the worksheets and results on each of these 
samples as well as suggestions for further investigation. 
A coding system is used to label the specimens, in 
which each letter of the alphabet stands for a particular 
problem. For example: B-K-L-3-SP 
B = multiple antibodies 
K + weak subgroup in ABO system 
• L  =  problem with donor unit for crossmatch 
3 = the week scheduled for this problem 
SP = student's initials 
with similar codes. 
All samples, except the practical exam, are labeled 
Problem B-K-L+SP 
Mr. AN was admitted to the hospital one day prior to 
scheduled open heart surgery A blood bank sample 
was collected with an order for four units of blood for 
1. Synopsis of the problem: 
2. Specimens provided: 
surgery 
The patient's serum contains anti-E, -c, -Fy sup(a), and -K 
2 mLs packed red cells 
4 mLs serum 
4 donor units (2 mLs EDTA each) 
3. Direct antiglobulin test (DAT): not indicated on pa- 
tient's sample 
4. ABO: group O 
5. Rh phenotype: R sub(1)R sub(1), 
6. Antibody problem: 
Anti-E reacts 1 + at indirect antiglobulin test (IAT) 
Anti-c reacts 2 + at IAT 
Anti-Fy sup(a) reacts 2 + at IAT 
Anti-K reacts 3 + at IAT 
211-0 R sub(1)R sub(1), 3 + DAT, return to donor center 
212-0 R sub(1)R sub(1), Fy(a-), K- compatible 
213-0 R sub(o) Fy(a -), K- incompatible 
214-A sub(x) R sub(1)R sub(1), Fy(a+), K- incompatible 
7. Donor unit numbers: 
8. Further investigation: 
a. Request transfusion history on the patient—Mr. 
AN was transfused during the Vietnam conflict. 
b. Run selected cells to determine the specificities 
of the antibodies. Use of enzyme-treated cells 
may be helpful. 
c. Antigen type the red cells of Mr. AN to confirm 
antibody specificities and to help exclude other 
possibilities. 
d. If appropriate selected cells are not available, a 
homologous adsorption may be performed to re- 
move the anti-c, then identify/confirm the other 
antibodies. 
e. Determine causes of incompatible cross- 
matches. Identify ABO group of the A, donor 
unit. 
f. Calculate the number of units expected to be 
compatible with the patient's serum. 
9. Choice of units for transfusion: 
Group O R sub(1)R sub(1), Fy(a-), K- 
Practical examination 
At the end of the course, a practical exam is given 
that consists of seven patient samples and three donor 
specimens. No case histories are provided. A two-week 
time period is alloted for completion of all work. This 
practical exam is then presented both in written form 
and orally using the same criteria for evaluation as pre- 
viously discussed. 
Conclusion 
As an educator, it is difficult to ensure that the stu- 
dents have received a thorough education and have at- 
tained a certain level of expertise. Through structured 
programs such as the one presented here, learning is 
not left to chance. The students can incorporate all 
that they have learned and apply this knowledge to 
clinical problems and situations. A course like this is 
not only stimulating and challenging, but it serves as 
an excellent indicator for identifying the students' 
strengths and weaknesses. Students can then work to- 
ward improving proficiency in all areas of problem 
solving. 
Marty Weaver-Dahm MT(ASCP)SBB former Technical 
Education Coordinator; American Red Cross Blood 
Seruices Southeastern Michigan Region, P.O. Box 
33351, Detroit MI 48232-5351. 
V O L U M E  4 ,  N U M B E R  4 ,  1 9 8 8  81 


Problem solving: a course for blood bankers

Kumar, Rajeev

Kumar, Dilip

Kumar, Dinesh

Performance enhancement of in-network probabilistic aggregation (PEPA) for vehicular ad-hoc networks

Beattie, Kathryn M.

the Rh-positive, D sup(u)-variant mother. Transfusion 1982;22:76-7. 
2. lacey PA, Caskey CR, Werner DJ, Moulds JJ. Fetal hemolytic dis- 
ease of a newborn due to anti-D in an Rh-positive D sup(u)-variant 
mother. Transfusion 1983;23:91-4. 
3. Brornan 8. Swedish thoughts on D sup(u) nomenclature. Transfusion 
1984;24:85. 
4. Moore BPL. D sup(u) terminology revisited. Transfusion 1983;23401. 
5. Issitt PD. Applied blood group serology. 3rd ed. Miami: Mont- 
6. Widmann FK. Questions and answers. AABB News Briefs 
7. Zuck TF. D terminology revisited. Transfusion 1983;23:90. 
8. lssitt PD. Applied blood group serology 3rd ed. Miami: Mont- 
9. Widmann FK, ed. Technical manual. 9th ed. Arlington,VA: Amer- 
gomery Scientific Publications, 1985:647. 
1987;10(No. 3):13-4. 
gomery Scientific Publications, 1985:234-5. 
ican Association of Blood Banks, 1985:319-20. 
Ohio Association of Blood Banks; Scientific and Education Com- 
mittee. Results compiled by Judy Duckett, MT(ASCP)SBB, The 
Christ Hospital 2139 Auburn Avenue, Cincinnati, OH 45219. and 
Mary Leaman Morrison, MS MT(ASCP)SBB, Hoxworth Blood 
Center, 3231 Burnet Avenue, Cincinnati, OH 4526-4055. 
THE IMMUNOHEMATOLOGY 
CONSULTATION REPORT 
WHAT, WHEN, HOW MUCH? 
Kathryn M. Beattie 
Introduction 
There is no doubt about the mandate to maintain 
blood bank records and reports. The Code of Federal 
Regulations (CFR 606.160) states: 
Records shall be maintained that include, but 
(b) (4) Compatibility test records: 
are not limited to the following: 
(i) Results of all compatibility tests, 
including crossmatching, testing of 
patient samples, antibody screening 
and identification. 
(ii) Results of confirmatory testing. 
(6)  Transfusion reaction reports and com- 
plaints, including records of investiga- 
tions and follow-up. 
The American Red Cross (ARC) Blood Service Direc- 
tive 6.5 explains in detail how ARC reference labora- 
tories must comply with the Federal Regulations for 
records. It states: 
Each patient specimen studied must have the 
A. Antibody identification including special 
following records: 
cell typing and a report to the submitting 
hospital. 
B. Units screened and found compatible. 
C. All special typings or procedures 
performed. 
It also says these records must be kept indefinitely. 
Immunohematology Reference Laboratory 
Reports 
What information should be included in a reference 
laboratory report? The closest direction about content 
can be found in the ARC Good Manufacturing Prac- 
tices (GMP) Inspection Checklist which asks: "Do 
patient consultation report forms include name and 
address of Red Cross Center, patient name and identifi- 
cation numbers, date, serologic data, transfusion rec- 
ommendations or clinical significance of antibody, 
referral laboratory, and name of person sending 
report?" 
Identification 
What is the need for patient identification? There is 
no doubt that the name and address of the submitting 
and reference laboratories and the name of the patient 
must appear in the report. The submitting hospital's 
identification number for the patient should appear so 
that the report will be placed in the correct chart. As 
patients are now rapidly discharged, the reference lab- 
oratory report may not arrive at the submitting hos- 
pital until after the patient is discharged, which means 
that the correct chart will have to be found in the med- 
ical records files. 
Many patients have similar or identical first and last 
names and hospital admission codes can change with 
each admission. Therefore, social security numbers 
and birthdates would allow better identification of the 
patient by the referral reference laboratory when the 
patient is subsequently admitted, perhaps at another 
hospital, and further work is requested. 
Turnaround time 
Three dates-the date of specimen collection, the 
date of receipt by the referral laboratory, and the date 
the report is sent-should appear in the report. The 
first two dates aid in establishing the chronology of 
events if a series of specimens is sent and an evolving 
picture develops, such as the detection of new anti- 
bodies, interim transfusions, transfusion reaction 
investigations, etc. 
The third date—the date the report is sent- 
provides a means of documenting the turnaround 
time. Turnaround time can be affected by a number of 
factors, including volume and complexity of cases, 
number of reference personnel available, overtime 
availability, etc. Knowledge of turnaround time allows 
laboratory personnel to examine the system and to 
pinpoint areas that need attention. 
Many reference laboratories telephone the report to 
the submitting laboratory as soon as  the work is com- 
pleted. If blood is needed, information concerning 
availability of compatible units and crossmatching 
techniques need not await a written report. If speci- 
ficity of the antibody or antibodies has not been deter- 
mined by the end of a normal work day, the urgency of 
the patient's need for blood can be ascertained, and 
from this telephone consultation a decision can be 
made concerning the need for overtime. 
If there is no urgent need for blood and the case re- 
quires more extensive or complex testing, a prelimi- 
nary written report can be sent, indicating that a final 
report will follow later. 
What should be considered a normal turnaround 
time? This will depend on the urgency of the situation. 
Apriority policy for antibody identification or requests 
for antigen-negative blood may be based upon the fol- 
lowing prioritized list: 
1. Patients who are actively bleeding, need emer- 
gency surgery, or have hemoglobin values <= 7 
g/dL and for whom blood has been requested. 
2. Patients for whom no blood has been requested 
but- 
a. have a history of GI bleeding. 
b. are scheduled for elective surgery. 
3. Specimens for antibody identification only. 
Turnaround time for work on samples from bleeding 
patients should be as short as possible. For the second 
group, the service is usually performed in less than 24 
hours. Telephone reports should be given on both of 
these patient problems. The written report should go 
out within two to three work days. Weekends, particu- 
larly long holiday weekends, can delay the report for 
another one to two days. Specimens in the third group 
are completed within a reasonable time, which is dic- 
tated by the constraints of the first two sample groups. 
Body of the report 
The personnel at the submitting laboratory want to 
know a number of things depending upon the type of 
case: 
How did the serum and/or eluate react? 
What was done to resolve the problem? 
What antibodies were found? 
Were clinically significant antibodies excluded? 
Is there any special information about the anti- 
Can references be cited? 
Is there evidence of a transfusion reaction or 
hemolytic disease of the newborn (HDN)? 
What type of blood should be given? 
How should compatibility testing be done? 
Is there a need to contact siblings or a rare donor 
Are there contributing factors to account for 
Why does the submitting laboratory need to know 
how the patient’s specimen reacted in the reference 
laboratory? If the findings were different, there may be 
ways to explain these differences. For example, the 
submitting transfusion service laboratory’s work may 
show reactions at room temperature that became 
weaker or nonreactive in the indirect antiglobulin test. 
The referred specimen arrived and was refrigerated 
overnight before being worked on in the reference lab- 
oratory. Agglutination was not demonstrable in any 
testing phase. This information, transmitted along with 
an explanation that the referring laboratory’s results 
were probably caused by auto-anti-I and that auto- 
adsorption at 4°C can resolve that problem, could 
bodies that should be offered? 
organization for rare blood? 
serological discrepancies? 
be useful in further testing. 
Another actual situation involved a discrepant re- 
verse grouping that was not reproducible in the refer- 
ence laboratory until the sample was tested with the 
hospital’s commercially prepared reagent group A and 
B cells.* The patient had an antibody to EDTA, and the 
diluent of the reagent red cells contained EDTA to pre- 
vent lysis of the cells by hemolytic anti-A and/or antiB 
in a patient’s serum. The inhouse prepared reverse 
grouping cells, used first, did not contain EDTA. Tests 
of the patient’s serum and group O red cells suspended 
in EDTA also showed agglutination. When the hospi- 
tal’s commercial reverse grouping cells were washed, 
they gave the proper reverse grouping for this patient. 
If the investigation had not been thorough and had 
been reported as “no ABO discrepancy,” the reference 
laboratory would have lost credibility and an opportu- 
nity to study an interesting finding. 
Discrepancies between referring and reference lab- 
oratories can be due to the use of different technical 
procedures. A single elution method may not yield an 
expected antibody. For example, a recent serum 
sample containing anti-At sup(a) and anti-D required separa- 
tion by adsorption-elution. The anti-At sup(a) was adsorbed 
onto D negative At(a + ) RBC; it was recovered using a 
concentrated ether eluate but was not recovered using 
an acid elution procedure. 
In a case with a positive direct antiglobulin test 
(DAT), it may be necessary to remove IgG from the red 
cells with chloroquine or glycine-EDTA to type them. 
This should be noted on the report so the submitting 
laboratory will know that typing results were not in- 
validated by the positive DAT. In cases where the pa- 
tient has been transfused, it should be noted that red 
cell typings were performed using the patient’s 
neocytes. 
What antibodies were found? There may he dif- 
ferences between what the reference laboratory and 
what the submitting hospital find. One reason may he 
faulty interpretation. Examination of the worksheets 
from the hospital, which should accompany the 
sample, may be enlightening. Another explanation is 
that antibodies previously identified and no longer de- 
monstrable were recorded on the accompanying 
paperwork. 
Were the detected antibodies clinically significant? 
What clinically significant antibodies have been 
Is there any special information about the 
Can references be cited to support the special 
Is there evidence of a transfusion reaction? Here the 
report should be worded very carefully, especially in 
the case of an immediate reaction. Personnel should be 
prepared to testify about the legal implications of 
findings. 
excluded? 
detected antibodies that should be offered? 
information? 
“This case was published in Immunohematology, Vol. 3, NO 3 (1987) 
53 
Is there evidence of HDN? Detecting an antibody in 
an infant's serum or on the red cells does not imply 
clinical HDN. The serological findings should be re- 
ported and a possible association with HDN discussed. 
What type of blood should be transfused? How 
should compatibility testing be performed? For clini- 
cally significant antibodies, there is no doubt that 
antigen-negative units are recommended. For anti- 
bodies serologically nonreactive at 30°C or below, the 
report may suggest doing crossmatches using a pre- 
warming technique. 
When clinically significant antibodies have been 
excluded in a specimen containing an HTLA antibody 
of undetermined specificity, it is acceptable to recom- 
mend that blood be selected on the basis of current se- 
rological compatibility but, when there is need for 
transfusion in the future, efforts must be made to first 
detect clinically significant antibodies. 
It is wise to let the transfusion service staff know the 
degree of difficulty in obtaining compatible units for 
future transfusion. While it may not appear critical to 
report that 91 percent of random donor units should be 
compatible with a patient's anti-K, it can be important 
to the clinician reading the report. 
When recommending antigen-negative blood for pa- 
tients with antibody to a high-incidence antigen, labo- 
ratory personnel have an opportunity to do several 
things. The patient's siblings can be recruited as a 
source of compatible units, or the patient can be en- 
couraged to store autologous units if able to do so. The 
patient's physician can be informed of the extent of dif- 
ficulty in obtaining rare blood, both for present and fu- 
ture transfusions. Is the blood available in the frozen 
inventory, or must it be recruited locally or nationally? 
Generating the report 
Some labs use a checklist of responses to be selected 
relating to the patient's case. However, a personalized 
report has more depth. Fill-in spaces for recording the 
test phases at which reactions were observed can be 
coupled with remarks about interpretations and con- 
clusions. The applicable remarks to be added can be se- 
lected from a list of comments generated for this 
purpose.* 
The final report 
The medical director, director of technical services, 
or the reference lab supervisor may review the report. 
The final written report should not depend on individ- 
uals who may not be readily available, as this will ap- 
preciably delay the mailing. 
A service offered by some reference laboratories is 
to furnish antibody identification cards (ID) to be 
carried by the patient. The card is only prepared for pa- 
tients with clinically significant antibodies. It is mailed 
along with the reference laboratory report to the hos- 
pital blood bank supervisor or transfusion service di- 
rector. Two letters should accompany the card: One of 
these letters informs the patient's physician of the clin- 
ically significant antibody found and the need to for- 
ward the ID card and other letter to the patient; the 
second letter to the patient instructs him or her to 
carry the card and present it when admitted to a hos- 
pital for surgery or pregnancy or whenever blood bank 
services are requested. 
Complete, clear, and helpful reference laboratory re- 
ports can be invaluable to the patient and to submitting 
hospital blood bank personnel. They can also gain de- 
served recognition for the reference laboratory. 
Kathryn M. Beattie, MT(ASCP)SBB, Director, Reference Laboratory 
and Technical Education, American Red Cross Blood Services, 
Southeastern Michigan Region, PO Box 33351, Detroit, MI 
48232-5351. 
A CASE REPORT PATERNITY CASE 
WITH THREE ALLEGED FATHERS 
AND A SET OF TRIPLETS 
Mary Lou Guizzo, Nancy Lang 
Introduction 
Parentage testing using combinations of genetic 
markers (red blood cell [RBC] antigens, serum pro- 
teins, RBC enzymes, and histocompatibility locus-A 
human antigens [HLA]) is capable of establishing non- 
paternity in 99 percent of the cases in which an alleged 
father is falsely accused.' If exclusion is not obtained, 
the likelihood of paternity can be determined. 
In this paternity case, three alleged fathers, a 
mother, and a set of triplets (two girls and a boy) were 
evaluated. Antigen typings of the HLA and RBC sys- 
tems and evaluation of red cell enzymes and serum 
proteins were performed. 
Materials and Methods 
Testing for red cell antigens was performed with 
commercially obtained antisera according to the man- 
ufacturer's directions. Red cell typing included testing 
with the following antisera: anti-A, -B, -AB, -A sub(1), -D, -C, 
-c, -E, -e, -C sup(w), -M, -N, -S, -s, -K, -k, -Fy sup(a), -Fy sup(b), -Jk sup(a), and -Jk sup(b). 
HLA-A and -B lymphocyte antigens were determined 
using standard lymphocytotoxicity tests sup(2) with antisera 
obtained from commercial sources. Testing was done 
in duplicate by two technologists working 
independently. 
Samples submitted for electrophoresis (serum pro- 
teins and red cell enzymes) were tested by an indepen- 
dent laboratory specializing in both conventional elec- 
trophoresis and iso-electric focusing. 
Calculations were determined using a sperm proba- 
bility test sup(3) to obtain a Paternity Index (PI) and a Rela- 
tive Chance of Paternity (RCP). The PI was defined as 
the probability that the alleged father could donate 
one sperm carrying all the obligatory genes (X), di- 
54 


The Immunohematology Consultation Report: What, When, How Much?

Waider, Kayla L.

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 91
Rouleaux and saline replacement
K.L. Waider
Rouleaux is a phenomenon that commonly occurs in patients who 
have an increased number of circulating protein macromolecules. 
It is a benign, in vitro reaction that appears microscopically as red 
blood cells (RBCs) line up against each other; many liken the RBC 
aggregation to “stacked coins.” This unexpected reactivity may 
cause confusion in direct agglutination testing such as reverse 
blood typing and crossmatching. Saline replacement is the 
established method to resolve rouleaux. True agglutination will 
remain when plasma is replaced with saline for resuspension of 
the RBC button. Rouleaux will no longer be seen when the plasma 
proteins are removed. Immunohematology 2018;34:91–92.
Key Words: rouleaux, stacked coins, saline replacement, 
macromolecule
Principle
Rouleaux is a type of red blood cell (RBC) aggregation 
with a distinct “stacked coins” appearance when observed 
microscopically. These aggregates frequently have a more 
refractive appearance than classic RBC agglutination. There 
are typically many “stacks” in the sample, and their size can 
vary greatly. The rouleaux aggregates disperse differently 
from the main RBC button during resuspension, such that 
experienced technologists can sometimes recognize this 
phenomenon even before confirming it microscopically. 
It is most typically seen in patients with disease states that 
cause an increase in plasma protein macromolecules, such as 
multiple myeloma, diabetes mellitus, and many infections.1 
The macromolecules are thought to cause rouleaux by two 
unrelated mechanisms: the bridging model or the depletion 
model. One or both may occur in an individual. In the bridging 
model, the macromolecules partially adsorb into the RBC and 
bridge to the RBC closest to it. In the depletion model, the 
macromolecules move away from the RBCs and change the 
osmotic pressure between RBCs causing them to aggregate 
(Fig. 1).2 Once recognized, rouleaux can be resolved using the 
saline replacement method. By replacing the patient’s plasma/
serum with saline, the macromolecules are removed, and the 
RBCs dissociate.
Indications
Rouleaux may be present in any direct agglutination 
test containing plasma or serum. Examples are reverse ABO 
typing, immediate spin crossmatch, and immediate spin and/
or 37°C reading of antibody detection and/or identification 
testing. If unexpected positive results present after routine 
incubation and centrifugation, rouleaux should be considered. 
Once confirmed, the saline replacement method3 should 
be performed using the tube method. It is important to 
remember that rouleaux can only be found in the presence of 
patient plasma or serum, since this is where the interfering 
macromolecules are found. Rouleaux is not seen in the 
antiglobulin phase of testing because the wash step rids the 
test system of the patient’s plasma/serum.
Procedure
Examine suspected reactivity microscopically. Rouleaux 
will appear as stacked coins, whereas true agglutinates are 
Reagents/Supplies
Reagents Supplies
• 0.9% saline or PBS (pH 
6.5–7.5)
• Transfer pipettes
• 10 × 75 or 12 × 75 mm tubes
• Calibrated serologic centrifuge
• Microscope
PBS = phosphate-buffered saline.
Procedural Steps
• Confirm the presence of rouleaux microscopically following initial 
RBC resuspension.
• Re-centrifuge the test tube.
• Remove patient’s plasma/serum using transfer pipettes.
• Replace with saline.
• Resuspend. Confirm.
RBC = red blood cell.
S
E
R
O
L
O
G
IC
 M
E
T
H
O
D
 R
E
V
IE
W
92 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018
K.L. Waider
amorphous clumps. After confirming the presence of rouleaux, 
re-centrifuge the plasma (or serum) and cell mixture. Carefully 
remove the plasma using transfer pipettes, leaving the RBC 
button undisturbed. This effect can be achieved by tilting the 
tube so that the plasma is away from the cell button, allowing 
the pipette to aspirate it without touching the RBCs. Gently 
replace the plasma with an equal volume of saline without 
disturbing the RBC button. Resuspend the RBC button, 
and observe for agglutination. Rouleaux will disperse when 
suspended in saline, but true agglutination is stable in the 
presence of saline and will remain.
Once saline replacement has been performed, only direct 
agglutination testing may be interpreted. Serum/plasma 
may not be reintroduced to the test system. If test reading at 
additional phases are desired, the test must be repeated using 
a separate tube for each phase where a reading will occur.
Limitations
Rouleaux does not typically cause interference in column 
testing, although high protein levels can lead to false positives 
or hazy reactions in this method.4 Confirmation of rouleaux 
and resolution must be performed with the tube method.
Acknowledgments
The author would like to thank Jan Hamilton for her 
guidance while working on this manuscript.
References
 1. Samsel RW, Perelson AS. Kinetics of rouleau formation. I. A 
mass action approach with geometric features. Biophys J 
1982;37:493–514.
 2. Bäumler H, Neu B, Donath E, et al. Basic phenomena of red 
blood cell rouleaux formation. Biorheology 1999;36:439–42.
 3. Fung MK, Ed. Method 3-7: Detecting antibodies in the 
presence of rouleaux-saline replacement. In: Technical manual. 
19th ed. Bethesda, MD: AABB, 2017.
 4. Blood grouping reagent MTS A/B/D monoclonal and reverse 
grouping card [package insert]. Pompano Beach, FL: Micro 
Typing Systems Inc., 2008.
Kayla L. Waider, BS, MLS(ASCP)CM, Immunohematology Reference 
Laboratory Technologist III, American Red Cross Biomedical 
Services, Southeastern Michigan Region, 100 Mack Avenue, Detroit, 
MI 48201, Kayla.Waider@redcross.org.
Fig. 1 (A) Schematic drawing of the bridging model. The 
adsorption of dextran into single cells and the formation of bridges 
during the absence of shear flow lead to rouleaux formation of 
cells. (B) Schematic drawing of the depletion model. Depletion 
of macromolecules from the interface (with or without a weak 
adsorption) leads to rouleaux formation if the cells come into close 
proximity due to the osmotic pressure difference between the 
pressure in the bulk phase and in the gap. (Reprinted from Bäumler 
et al.2 with permission from Biorheology.)
(A)
(B)


Rouleaux and saline replacement

Not a member yet