Pan American Health Organization
ADVISORY COMMITTEE ON MEDICAL RESEARCH
Fifth Meeting
Washington, D.C., 13-17 June 1966
Item 5 of the Agenda
COLLABORATIVE STUDIES IN NUTRITIONAL ANEMIAS
Ref: RES 5/14 1 June 1966 RES 5/14
Table of Contents
Paae
Collaborative Studies in Nutritional Anemias O. O O.. . O. O O . 1
Appendix I
Trip Report ..... o ..... O O 0 5 Appendix II Guide to the WHO/PAHO Study on the
Nutritional Anemias . . . . O O O 20 -1- RES 5/14
COLLABORATIVE STUDIES IN NUTRITIONAL ANEMIAS*
In the fall of 1963, a meeting sponsored by WHO/PAHO held in
Caracas, Venezuela, and attended by investigators from the Americas considered the questionaf anemia in Latin America and pointed out that anemias in its various forms are a significant problem there. While information collected for the meeting suggested that nutritional anemias represented the largest category, the available amount of precise and useful data was meager. A plan was evolved at this meeting for the study of nutritional anemias - based in part on prior deliberations by WHO advisory groups - and involving the establishment of a central laboratory affiliated with collaborating investigatora. The program as planned was financed for three years starting in January, 1965, by the Williams-
Waterman Fund, for the total amount of $41,280. The Reference Laboratory, under the direction of Dr. Miguel Layrisse, was located at the Instituto
Venezolano de Investigationes Cientificas (IVIC) in Caracas. A scientific advisory group was formed consisting of Drso Victor Herbert, William
Crosby, Maxwell Wintrobe and Clement Finch.
Within several months, the PAHO Reference Laboratory and Training
Center for Applied Research in Nutritional Anemias was functional and able to carry out the essential procedures for the study including plasma iron and iron binding capacity measurements, L. casei-folate assays, and
B12-Euglena assays of plasma. (At the present time, the B12 method is being changed to the isotopic method described by Herbert in Blood, 1965).
Experimental protocols were revised, translated into Spanish for use by the collaboratora (see Appendix II of this document).
* Prepared by Dr. Co A, Finch for the Fifth Meeting of the PAHO/ACMRo -2- RES 5/14
In January-February of 1965, a brief trip was made by Dr. CoAo Finch
to the laboratory of Dr, L. Sánchez-Medal in Mexico City, Dr, F, Viteri in
Guatemala City, Dr, Ho Vélez in Medellín, and Dr. C.Reynafarje in Lima.
Shortly thereafter, Drso Sánchez-Medal, Viteri and Vélez became official
collaborators in the study. Dr. J. G, Chopra had also been conducting a
study on anemias in Trinidad under U. S. Public Health Service funds. In
view of her need of the special laboratory assistance provided by the Center,
her program was also affiliated. The only additional funds received for
this study by any of the collaborators was approximately $7,000 received
from WHO by Dr. Sánchez-Medalo
The next step was to establish potential in the laboratories of the
collaborators for carrying out some of the special studies. Accordingly
professional personnel from the collaborating laboratories in Mexico,
Guatemala and Trinidad received orientation and training in methodology
at the Reference Laboratory. The Center has also conducted tests in
parallel with the reference center in London and South Africa to ensure
standardization of methods. Routine determinations for folate and B12 in sera from patients with anemia in Trinidad and in Caracas were con-
ducted, By January 1966, the Reference Laboratory was considered to be
:* functional and capable of both running samples which might be shipped
from collaborators and expanding its training program.
In March 1966, a visit was made to existing collaborators by Drs.
Herbert, Layrisse and Finch with the purpose of reviewing the status of
the study at each institution and coordination of future studies! It
seems that all participants should be able to proceed with the pregnancy
study as outlined in the protocol, and the data could be completed either
* See Annex I to this document. RES 5/14 in the local laboratory with appropriate cross-standardization with the
Reference Laboratory, or by shipments to that Laboratory for analysis by
January, 1967o It appeared also that tissue samples on medicolegal deaths could be shipped for iron analysis by the same deadlineo These data might well be the subject of a conference in the spring of 1967 to include both the collaborators who have contributed data and new collaborators who would be entering the program. Dr, Herbert continued his trip to explore the possibility of other collaborators. It would be hoped that Dr. Reyna- farje in Lima, Dr. MoAo Jamra in Sao Paulo, Dr. Ao Gutnisky in Corrientes,
Argentina; Drs. SO Kremenchuzky and Eo Rochna Viola in Buenos Aires,
AoPo Cristoffanini and P. Rubinstein in Santiago, Chile, might become collaborators and that all of them will be able to undertake the pregnancy and tissue iron studies in the near future. It would thus seem that the program might be well expanded to approximately 10 collaborating laboratories by the beginning of 1967o
It has been important to proceed with care in selecting collaborators, for the objective of this study is not only to obtain certain initial survey data, but to develop the potential to carry out definite studies as to the cause of the deficiency anemias as a secondary attack° All of the individuals mentioned are thought to have competence in hematology, investigative poten- tial, access to patients, the local backing in the program, and a high degree of personal motivation°
The next one or two years should give an inaication as to whether this group of outstanding investigators will be able to work profitably together in a general approach to nutritional anemia problems in Latin America° It does seem assurred that what information is collected will be adequately -4- RES 5/14
standardized and of a high degree of accuracy permitting comparison between different areas. It is also anticipated that the program will be of con- siderable educational value in introducing important hematologic methods into these countries. It would further seem that the common interests will provide a worthwhile opportunity for exchange of information at a scientific level between individuals in the different countries involved°
One problem that still faces this undertaking is adequate financingo To pay off, it would seem that the program should go a minimun of six years, and perhaps eight. Some investigatora will require small amounts of money in the neighborhood of $5,000 to $6,000 a year to carry out special studies relating to problems which they uncover. Therefore, some mechanism is needed to obtain between $20,000 and $50,000 a year for the running of this program. RES 5/14 Appendix 1 -5-
APPENDIX I
TRIP REPORT RES 5/14 - 6 - Appendix 1
REPORT OF CONSULTANT VISITS TO LATIN AMERICA MARCH 20 - APRIL 18, 1966*
The purpose of the visits to Mexico, Guatemala, Colombia, Trinidad
and Venezuela was to determine the current status of the laboratories col-
laborating in the nutritional anemias program, to discuss a coordinated
schedule of the research and training aspects of the study, and to provide
help in solving any problems which may have arisen. The purpose of the visits
to Brazil, Paraguay, Uruguay and Argentina was to identify possible collabor-
ators with the PAHO Program by visiting their laboratories, determining their
interest in helping PAHO acquire data on the incidence of nutritional anemia
in their countries, and determining the research potential of their labora-
tories, their particular needs, and their potential for helping define etiology
and practical public health control of nutritional anemias in their countries.
The following is a summary of the information obtained.
There appear to be six sources for funds which migh be available to
aid the program for investigations of nutritional anemias in Latin America.
These sources include PAHO/WHO itself, the Organization of American States, the
Alliance for Progress (which may be contacted locally through the Cultural
Attaché at the U. S. Embassy in each country), the AID (Agency for Inter-
~^ national Development), and the OIR#(Office of International Research) of the
NIH/USPHS. The Department of Defense funds come from ARPA (Advanced
Research Projects Agency), are committed three years in advance, and there-
*Prepared by Dr. Victor Herbert for the Fifth Meeting'of the PAHO/ACMR. Visits to Mexico, Guatemala, Colombia and Venezuela by Drso. Finch,Lay- riase and Herbert; to Trinidad by Drs. Finch and Herbert; to Brazil, Pa- raguay, Uruguay and Argentina by Dr. Herbert. **The Nutrition Section of OIR is the former ICNND; Dr. Arnold E. Schaefer is its Head. The OIR is administratively under the NIH, but its 'Nutrition Section (the former ICNND) is autonomous and funded by The Department of Defense, Atomic Energy Commission, and The Departmentsof Agriculture and of State. RES 5/14 Appendix 1 7-
fore are a relatively stable source of funds. There are also U. S. Public
Law 480 funds, which are monies paid to the U. S. A. but left in local
currency in local banks; their expenditure must be approved by the local
country. Finally, the W. K. Kellogg Foundation of Battle Creek, Michigan,
awards fellowships to Latin American universities for two years training of
their staff members in United States professional schools.
1. MexrQ-City, Mexico. Dr. Luis Sánchez-Medal (Dept. of Hematology, _Hos-
pital of Diseases-of Nutrition), received $ 7,000 from-'SHO for the period
September 1965 through August 1966 to begin his collaboration in the program.
He has already sent 200 medicolegal liver samples to Dr. Bothwell and to Dr.
Ramalingaswami for iron determinations. Working with him are Dr. Javier
Pizzuto and Dra. Delifa López. Dr. Pizzuto trained for two years in coagula-
tion with Dr. Wintrobe, and returned to Mexico City last October via short
visits to the laboratory of Dr. Finch to learn iron technics, and to the labora-
tory of Dr. Herbert to learn serum B12 and folate assays. Dr. López has been
trained at IVIC under the PAHO program to learn the reference laboratory
technics. They are now setting up serum B12 (coated charcoal method) and
serum folate (aseptic addition- L. casei method).
Of the 200 liver biopsies sent to Drs. Bothwell and Ramálingaswami by
Dr. Sánchez, more than 150 were males and all were medicolegal autopsy cases, which provide the best survey material.
They have carried out a survey study in the state of Tlaxcala, popu - lation 378,122 (69,788 adult males). In study of 586 samples from 586 males
over age 25 (in 21 villages), examinations were made of blood for hemoglobin, hematocrit, blood smear, iron, iron binding capacity, serology, cholesterol, total protein, albumin, and of stool for blood and parasites. Only one case of hookworm was found, but 16 per cent of subjects had E. histolytica in- fection. The 21 villages were divided into 4 regions,in which wide differ-
· , RES 5/14 - 8 - Appendix 1
ences in percentage of iron deficiency was found. Overall, there were 5 per
cent iron deficient males, manifested by serum iron below 60 and transferrin
saturation below 20%, with hematocrit below 44. Because of altitude of
7,500 feet, normal male hematocrit is 51.2, hemoglobin is 17.7 and MCV is
96 (Blood 3:660-681, 1948). In villages A and B, which were closely adjacent
tj each other, there were sharp differences in percentage iron deficiency
(16% in village A and 2% in village B), with approximately 50 males studies
in each village. Two of the males in village A had guaiac positive stools,
but none in village B. There were 6 E. histolytica positive stools in each
of the two villages. Because the villages are so close to each other, they
will be studied further in an attempt to determine if, with a larger series
of males,there is still the wide discrepancy in percentage iron deficiency between the two villages, and, if so, what the etiologic factor might be.
The proximity of the villages suggests that most faetors, such as diet, may be equal, and some other factor, perhaps a factor producing blood loss, operates to produce the greater degree of iron deficiency in village A.
Dr. Sánchez-Medal states that Dr. Hector Coll, Chief of Zone II of
PAHO,has been helpful in securing supplies from the United States. Dr.
Sánchez-Medal plans to follow the PAHO pregnancy protocol (Appendix II) using 100 third trimester pregnant women who will have determination of
B12, folate, iron, iron binding capacity, hemoglobin, etc.
He plans to study the etiology of iron deficiency in the various villages of Tlaxcala by 51Cr redcell survival and intestinal blood loss studies, as well as investigating iron content of the diet, intestinal ab- sorption of 59Fe from foods, total iron elimination using 55Fe, and exhaustion ef iron stores by bleeding normal subjects and then determining rate of hemoglobin recovery. In iron deficient subjects without anemia, a similar Res 5/14 Appendix 1 -9- O4-w procedure will be followed.
Dr. Sánchez-Medal is receiving PAHO funds to support his training
program for physicians at his institution, and expects completed, in the
near future, a new building financially supported by the Wenner-Gren
Foundation of Sweden. 'iC
Doctors Sánchez-Medal and Layrisse are interested in the formation of
a Pan American Society of Hematology which may be an instrument not only for
providing exchange of knowledge among Latin America countries, but also
an instrument for furthering study of nutritional anemias throughout Latin .4o1 America. A meeting on nutritional anemias in Latin America sponsored by -·z . PARO would be an excellent opportunity to found such a society.
2. Guatemala City, Guatemala. Dr. Fernando Viteri, of the Institue of Nutri- tion of Central America and Panama (INCAP), has set up determinations of serum e B12 (E. gracilis) and folate (L. casei) , iron and iron binding capacity
(both by Ramsey method), and is also engaged in a study of erythropoietin in children with kwashiorkor (this latter study in collaboration with Dr. Clement 4J Finch).
INCAP is supported in part by funds from the aix countries of Central America. It has funds for bringing medical students and graduate physicians from the United States for the summer or for longer periods, and can also r A~ pay their round trip transportation and a stipend while in Guatemala City. ,!i Working with Dr. Viteri are Dr. Jorge Alvarado and Dr. R. Wood, who
.ei-. k will be replaced in June by Dr. David Luthringer from Vanderbilt University.
They are primarily interested in the multiple nutritional deficiency syn- drome in children; their kwashiorkor consultant is Dr. Robert Hartman of : -- Vanderbilt University who is paid by the Nutrition Section of OIR. This
4 - 10 - RES 5/14 Appendix 1 section (formerly the ICNND) is now engaged in a nutrition survey of all the
Central American countries which is scheduled to end in March of 1967.
The ICNND survey includes studies of 20 families in each of 40 communi-
ties in each of the Central American countries who have a clinical examina-
tion, dental survey and blood pressure determination. Of each 20 families,
five will have serum vitamin E, A, carotene, C, transaminase, LDH; also urine
N-methyl nicotinamide, thiamine, nicotinamide; whole blood riboflavin; hemo-
globin, hematocrit, erythrocyte count, blood smear percentage eosinophils,
and serum iron and iron binding capacity. Folate and B12 will be determined
in the serum of 192 subjects in each country (48 at each of 4 different
altitudes), and in all study subjects who are pregnant women or children
under age 5.
In further pursuit of his participation in the PAHO survey, Dr. Viteri
plans to do serum B12 and folate determinations on each of the first two
mothers who deliver babies each day at Roosevelt Hospital in Guatemala City;
these women are from the medium-low economic group. He already has deter-
mined serum B12 and folate on 68 children below age 5; of these, five have
serum folate below 3ng/ml and 19 more have levels below 5; of 84 children,
one has a serum B12 level below 100 pg/ml and 21 more have levels below 200.
Of 14 pregnant women, one has a serum folate level below 3, 4 more have
levels below 5; of 19 pregnant women, 9 have serum B12 levels below 200 and
an additional one has a level below 100.
It appears to be difficult to secure specimens of liver for iron
determinations; Dr. Viteri plans to look further into this aspect of his
collaboration in the PAHO study. $ RES 5/14
Appendix 1 - 11 -
3. Colombia. Dr. Hernán Vélez, Chief of the Nutrition Section and Dr.
Alberto Restrepo, Chief of the Hématology Section, were Kellogg Fellows in the USA and are now in the Department of Internal Medicine of the Faculty of
Medicine at the University of Antioquia in Medellín. Dr. William Rojas,
Chief of the Department of Medicine, is interested in furthering their studies of nutritional anemia, which have been going on for several years, They have been expecially interested in children with kwashiorkor (protein deficiency; wet malnutrition) and marasmus (dry malnutrition; starvation). They have preliminary studies suggesting such children do not have a hematologic re- sponse to 1 gram but do to 5 grams of protein per day given as CARE non-fat dry milk powder. They are doing serum iron (Bothwell & Mállett method), IBC
(Ventura, J. Clin. Path. 2:271, 1952), aseptic addition method L. casei folates, but their B12 assay had not been working. 'The consultants may have helped to solve this problem. They plan to send one of their people to the
PAHO Reference Laboratory and Training Center to compare results of the above tests.
They plan to start now to collect samples from 100 third-trimester pregnant women, and hope to complete this by the end of the year; 'they will also try, Within the year, to have 100 liver biopsies sent to India and
South Africa from medicolegal cases. Since their medical examiner will not weigh livers, they will record the height of each subject. (Weights are not recorded routinely, but heights are.)
The Kellogg Foundation of Battle Creek is about to start providing fellowships in Medellin; this is an extension of their program of providing fellowships for Latin Americans in the Uo So A.
e RES 5/14 Appendix 1 -12 -
Dr. Roberto Rueda-Williamson, Director of the National Institute of
Nutrition in Bogotá, Colombia states that a new nutrition unit has just been formed within his institute under Alfonso Villamil, who is particularly in- terested in studying nutritional anemia. It is suggested that Dr. Villamil be contacted.
4. Trinidad. Dr. Joginder Chopra, Nutrition Consultant to PAHO Zone I
(which includes most of the Caribbean Islands except Puerto Rico and the Virgin
Islands) has a research grant from the USPHS to study nutritional anemias in
Trinidad. This grant expires in July of 1966, Intimately involved with her. in this study are two men: Dr. Neville Byam, a citizen of Trinidad and a full-time internist paid by the government of Trinidad to work at Port-of-
Spain General Hospital running research in diabetes and nutritional anemia, and Dr. G. Lyn Brinkman, a Ph.D. biochemist who has set up, in Port-of-
Spain, assays for serum B12, folate, iron, and iron binding capacity.
Dr. Brinkman is supported by PAHO, but will probably leave at the expiration of his PAHO contract in September of 1966 unless the USPHS research grant is resumed. He feels that in the absence of this grant it will not be possible for him to study nutritional anemias effectively.
Dr. Byam has collected 86 sera from women in the last trimester of pregnancy for determination of B12, folate, iron and iron binding capacity.
Trinidad people do not like to give blood, and it is only possible to get blood from the hospital population and not from volunteers. There is no hematologist on Trinidad, and this lack makes more difficult the study of nutritional anemia on that island. Dr. Byam was trained in Edinburgh. He may go to England for six months of postgraduate study in the near future.
He has had difficulty in securing liver biopsies because of reluctance of RES 5/14 Appendix 1 13
the pathology department to provide such from accident victims. However,
there are sufficient violent deaths in Trinidad to make accumulation of 100
such liver biopsies possible if Dr. Byam or someone else can constantly
stimulate the pathologist to collect such biopsies. Hovever, Dr. Byam has
many other responsibilities in addition to the nutrition study, and there is
a serious shortage of physicians in Trinidad. ^
A preliminary survey of serum B12 and folate results done for Dr.
Brinkman by IVIC in Caracas suggests that the incidence of folate and of
B12 deficiency is 10% (for each deficiency) in randomly selected popula-
tion consisting primarily of women and children. In 1961, ICNND conducted
a survey of nutrition in the West Indies (Trinidad, Tobago, St. Lucia, St.
Christopher, Nevis, Anguilla). In Trinidad and Tobago, about 8% of all male
adults and almost 40% of the female adults had hemoglobin values below 12
grams per cent. Anemia was even greater among adults in the other islands.
No marked racial differences in prevalence of anemia were noted..
5. Venezuela. The PAHO Reference Laboratory and Training Center on
Nutritional Anemias continues fully operational under the direction of Dr.
Miguel Layrisse at the IVIC (Instituto Venezolano de Investigaciones Cienti-
ficas)o Functioning in part under a three year grant for the period 1964-1967
from the Williams-Waterman Fund, this laboratory has facilities to
determine serum iron and iron binding capacity, vitamin B1 2 and folate on duplicate samples from collaborating laboratories, in order to verify results.
It also offers a training program providing one to two weeks of orientation in study methods for responsible investigators and up to one month training for the laboratory technician of such an investigator to learn the methods used in the study. In addition to his studies of nutritional anemia per se, Dr.
Layrisse has continued his studies of hookworm anemia, and is engaged in RES 5/14 Appendix 1 -14 -
studies of absorption of iron from various foods, in collaboration with Dr.
Clement Finch.
6. Brazil. In the short time in Rio de Janeiro, it was not possible to find a potential collaborator for the program. Guanabara State Institute of Cardiology houses, on one of its floors, the hematology ward of the State
Institute of Hematology. This hematology ward of 22 beds plus eight bassinets is under the direction of Dr. Paulo da Costa Martins. They currently suffer from a shortage of funds and personnel. Because of low salaries, physicians in Rio generally must earn the bulk of their income from private practice.
Dr. Someswara Rao, nutrition consultant to PAHO Zone V, with home office in
Rio, may be able to help PAHO identify a collaborator from the Rio area, but the prospects do not look promising.
Possible collaborators in the city of Fortaleza include Dr. J. Murillo
Martins and Dr. Célio Brasil Girao of the Faculty of Medicine of the
University of Ceará in Fortaleza.
In Sao Paulo, Dr. Michel Abu Jamra, Chief of Hematology at the Hospital das Clinicas of the University of Sao Paulo School of Medicine believes that he can provide the PAHO study with sera from 100 women in the third trimester of pregnancy and specimens of liver from 1OO adult males. Dr. Jamra is one of the foremost hematologists in South America, is particularly interested in nutritional anemia, and has a very active group working with him. He has the space and personnel to set up coated charcoal serum B12 determinations, and lacks only a well-counter. It was suggested to him that the USPHS might look with favor upon his application for a well-counter, especially in view of the fact that he already has space, other equipment, and personnel. With such RES 5/14 Appendix 1 -.i5 -.
a counter he hopes to pursue studies of iron and vitamin B12 absorption,
transport, storage and excretion, as well as doing determinations of serum
vitamin B12 levels. .
7. Paragua.y. Three men, working together, may be the nutritional
group for Paraguay. They are: Dr. Felipe Giménez Velazco, Hematologist to
the Cátedra de Semiología Médica.of the Hospital de Clínicas of the University
Medical School in Asunci6n;. his associate in private practice, Professor
Doctor Alejandro Arce Queirolo; and Mro Martin López. Dr. GiménezVelazco
spends his mornings working at the Hospital de Clínicas, and his afternoons and
evinings in private practice with Dr. Arce. The address of their hematology
clinic is Eligio Ayala 1037, telephone number 4122 and 4123. Mr. Martin
López works mornings in the Central Laboratory of Public Heálth and after-
noons in the Institute of Sciences. -He trained for a Ph.D. degree in the
United States, speaks fluent English, in collaboration with Dr. Giménez
Velazco is already doing serum iron determinations and is interested in
learning the techniques for assay of serum vitamin B12 and folate. Drs.
Giménez Velazco and Arce are particularly interested in coagulation prob-
lems, and are involved in the study of thrombosis associated with hookworm
infection and anemia. Their experience has been that hookworm iron defi-
ciency anemia is present in almost the entire population of Paraguay.- Last
year an ICNND study was conducted in Paraguay; the report of that survey
is currently in preparation.
A suggestion for therapy of iron deficiency arose in a discussion with
Professor Arce in Asunción. Nearly everyone in Paraguay daily eats chancaca,
a molasses-like candy made from the sugar cane grown in Paraguay. If the government were to require that ferrous sulfate be added to chancaca during its preparation, this might well sharply reduce iron deficiency in Paraguay. RES 5/14 - 16 - Appendix 1
8. Uruguay. Dr. Roberto Caldeyro-Barcia, Servicio de Fisiologia
Obstétrica of the Hospital de Clinicas,put me in touch with Dr. Nelly Temesio,
Research Associate in Rematology at the Institute of Pediatrics (Hospital
Pereira Rossell) of the School of Medicine in Montevideo. Working with her in the area of pediatric hematology are Dr. Alberto Perez Scremini, Dr. Julio
Lorenzo, and Dr. Washington Giguens, all Clinical Assistant Professors of
Pediatrics in the Medical School. The chief of adult hematology is Dr. Pedro
Paseyro at the Hospital de Clinicas. All agree that nutritional anemias is uncommon in Uruguay , due partly to the almost complete absence of severe poverty because of the extensive government welfare program, and partly to the high average daily per capita meat consumption (one pound). They also find hookworm not common, and Echinococcus more of a problem in their country. Rickets is a greater problem than anemia in children due to the fact that children are kept indoors because of strong winds for much of eight months out of each year.
Dr. Temesio provided the following data: In the 15 years from
January 1951 to December 1965, in the Institute of Pediatrics in Montevideo, only 134 cases of hypochromic anemia were collected. Of these, 63% were males; 43% were below age 1, 50% between ages 1 and 3, and 7% greater than age 3. Sixty-two per cent came from the capital and 38% from the interior.
The most frequently associated conditions were: repeated infections, 58% prematurity, 34%; rickets, 20%; social problems, 13.4%; nultiple prior preg- nancies of mother, 1l%. Only 2.8% had parasitism, and of these, 2 were
Giardia and 0.8% Ascaris. Of these cases, bone marrow was examined in 37, and only one marrow was megaloblastic. Of 34 cases studied, achlorhydria was present in 18 (40%). RES 5/14 Appendix 1 -~ 17 ~
As a general recommendation, PARO might suggest to a collaborator in -_ each country that his own participation in the PAHO program will constitute a separate publication, by him, in the literature of his own country. This will facilitate dispersion within each country of knowledge of the nature and extent of nutritional anemia in that country. Thus, 100 serum samples collected from women in the third trimester of pregnancy in Uruguay, which have had determination of B12, folate, iron and iron binding capacity at the Reference Center in Caracas could then be published by the collaborat- or in Uruguay with IVIC as "Nutritional Anemia in Uruguay: A Preliminary
...... - Study in Collaboration with the Pan American Health Organization." This study would be published'in a medical journal of Uruguay, for example, "El
Día Médico Uruguayo." Such publication would not preclude overall publica- tion of the entire PAHO study at a subsequent date.
9. Argentina. In Corrientes, Abraham Gutnisky, Director of the
Institute of Physiology of the Faculty of Medicine of the National University of the Northeast in' Corrientes, is primarily interested in hematology and has turned his department into an impressive group of laboratories for hema- tologic studies, especially related to erythropoietin. He is in a new building, with good ward facilities next door, consisting of beds for children and women with severe anemia, which is common throughout the area due to hookworm. His group has been concentrating erythropoietin from the urine of these patients in collaboration with Dro Van Dyke and others in the United Státes. He has three well-counters, all being used, a probe counter for studies of cell sequestration, a Mettler balance, a refrigerated centrifuge, pH meter, and many other pieces of equipment in eight separate laboratory rooms. He also has a large, well equipped animal quarters for the rats and mice of his e studies with erythropoietin. Dr. Florencio Blanco, Minister of Education RES 5/14 Appendix 1 - 18 - and Public Health for the Province of Corrientes, is a young man who is a former student of Gutnisky, who studied hematology in Barcelona for a year and who wants to be a hematologist. In his position, he is of great aid to
Dr. Gutnisky. He and Dr. Gutnisky, and the Dean of the University, are anxious to participate in the PAHO program, and have the enthusiasm, facili- ties, patient material, and ability to do so. Dr. Gutnisky has just written to Dr. Layrisse directly, requesting two weeks at IVIC to learn the serum
B12 and folate assays (preferably the second and third week in June of this year).
In Buenos Aires, two young doctors seem ideal for incorporation into the PAHO studies. They are Dr. Silvio Kremenchuzky and Dra. Erna Margot
Rochna Viola. Dr. Kremenchuzky recently returned from two years with
Dr. David Mollin in London and Dra. Rochna from two years with Dr. Mollison in London. They are both full-time workers in the Division of Hematology of the Center of Nuclear Medicine in the brand new Hospital-School José de
San Martin. Their financial support and that of their laboratory comes from the Atomic Energy Commission of Argentina. They have already set up serum vitamin B12 assays using the coated charcoal method and are in procesa of setting up serum folate levels. They are bright, energetic, and much inter- ested in nutritional anemias and in full-time careers in academic medicine.
The laboratory is well equipped. Their hospital, which is brand new, will be receiving the entire patient load from the old main teaching hospital of the medical school, and thus will become the main teaching hospital of the medical school of the University of Buenos Aires. RES 5/14 Appendix 1 - 19-
10. Chile.* Dr. Alberto P. Cristoffanini of the Faculty of Medicine
of the University of Chile at the J. J. Aguirre Hospital has been doing research in nutritional anemia and has indicated his willingness to participate in the collaborative study.
As of September of 1966, Dr. Pablo Rubinstein will be at the Blood Bank of this same hospital. Dr. Rubinstein is currently in New York in the Dept. of
Hematology at The Mount Sinai Hospital. He is learning in Dr. Herbert's laboratory there the techniques for performing vitamin B12 and folate assays, and will bring those techniques back with him to Chile, where he will teach them to the individual designated by Dr. Cristoffanini to carry them out on a permanent basis. Should any problems arise in establishing these assays, which Dr. Rubinstein may not be able to solve, then either Dr. Cristoffanini or Dr. Rubinstein, or a designated associate may go to the Reference Laboratory in Caracas.
Dr. Rubinstein will be:returning to Santiago via Holland, where he will be spending July and part of August. He has agreed to stop at the Reference
Laboratory in Caracas early in September on his way back to Santiago.
-: - . . ,. , ,. .e : . . . .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4 *Because of a rescheduling of itinerary, the Consultant did not visit this country as planned. RES 5/14 - 20 - Appendix II
APPENDIX II
GUIDE TO THE WHO/PAHO STUDY ON THE NUTRITIONAL ANEMIAS GUIDE TO THE
WHO/PAHO STUDY ON THE NUTRITIONAL ANEMIAS:
1. IN PREGNANCY
2. TISSUE IRON STORES
Adapted from the Protocol for the
WHO Collaborative Study on Nutritional Anemias
PA 247.64
Ref: RES 65.1 1 December 1965
PAN AMERICAN HEALTH ORGANIZATION Pan American Sanitary Bureau, Regional Office of the WORLD HEALTH ORGANIZATION RES 65.1
GUIDE TO THE WHO/PAHO STUDY ON THE NUTRITIONAL ANEMIAS
Table of Contents
Page
1. Guide to the Protocol for the Study of Nutritional Anemias in Pregnancy ...... o. 1
2. Guide to the Protocol for the Study of Tissue Iron Stores ...... 11
3o Literature Citations...... 14
4. Appendices
A. Pregnancy Protocol
B. Protocol for Non-pregnant Women
C. Protocol for Control Males
D. Suggested Method of Packing Serum in Dry Ice for Sending by Air Freight RES 6501
GUIDE TO THE WIQ/PAHO STUDY ON THE NUTRITIONAL ANEMIAS
1. GUIDE TO THE PROTOCOL FOR THE STUDY OF NUTRITIONAL ANEMIA IN PREGNANCY
1.1 Purpose of the Study
To compare the incidence of anemia and of iron, vitamin B12 and folic acid deficiencies in different countries by studying their appearance in the following population groups:
a) a representative group of pregnant women of low
socio-economic status,
b) a comparable group of non-pregnant, non-lactating women
drawn as far as possible from the community from-which
the pregnant women are derived and matched as far as
possible for age, parity and socio-economic status, and
if possible in
c) a group of males from the same community.
1.2 Population to be Studied
The population to be studied should be clinically normal pregnant
women who come to antenatal clinics or hospitals during the third trimester
and who have not been treated with iron, vitamin B12 or folic acid during
this pregnancyo To avoid bias, consecutive cases seen by the attending
physician should be investigated. The number of patients needed for the.
study is not exactly known but may be indicated by the data on the first
100 patients after analysis by statisticians of PAHO/WHO. RES 65.1 - 2- .
The non-pregnant women and the group of male controls should, if possible, be members of the families of the pregnant women studied. These families should be randomly selected. Volunteers should not be sought since this may weigh.the series, e.g. anemic subjects might volunteer more readily than healthy subjects.
The observations considered desirable are listed in the summarized protocols (see Appendices A, B and C attached). In some instances it may not be possible to obtain all the data included in these forms. Under these circumstances, the protocol should be completed as far as possible, for any information, however incomplete, supplied at this stage of the study is valuable.
1.3 History and Physical Examination
Details of history and physical examination are not necessary, but a history of blood loss, blood donation, stool character and habits, fever, geophagia or any associated conditions likely to lead to anemia should be noted under "Remarks" on the Protocol. An attempt should also be made to assess the quality of the patient's diet, and a note about this should be included separately under "Remarks"o Where possible detailed dietary histories should be taken on randomly selected patients. If such detailed histories are taken they should be attached to the Protocol. The blood< pressure reading, the appearance of the optic fundus and the results of urinary analysis should be recorded in pregnant women where possible. If pregnancy is multiple this should also be recorded. In outpatient depart- ments where the stools of pregnant women are routinely examined for ova and worms the results of this investigation should be included on the
Protocol under "Remarks." - 3 - R-E 65.1
1.4 Blood Samples
These should be obtained using plain vacutainers without anti- coagulant (Becton, Dickison & Co., Rutherford, New Jersey, U.SoA.). These will be supplied by the Nutrition Unit, WHO, Geneva, on request.
The amount of blood required will depend on the methods used by the various laboratories but in general it is always good policy to take as much blood as possible. An attempt should be made to obtain 20 - 25 ml of blood. The requirements for the different tests can be calculated roughly
as follows:
Hematological tests (Hb, PCV, WBC, peripheral blood film and tests for fetal and abnormal hemoglobins 2 ml of sequestrenised blood).
Vitamin B12 assay*
Euglena assay, or 2.5 ml. of serum
L. leichmannii assay, or 4.0 ml of serum
E. coli assay 3.0 ml of serum
Folate assay* 2.5 ml of serum
Serum iron and binding capacity 3°0 ml of serum
Total protein and A/G ratio 0.5 ml of serum
Peripheral blood films. It is essential that well
spread, properly fixed and stained peripheral blood films should be
obtained from each subject. At least four films should be made from drops
* The amounts of serum suggested for the microbiological assays are calculated on the assumptions (1) that serum will be assayed in 2 assay batches and (2) that in each batch, each serum will be assayed in duplicate at two' dilutions or alternatively, in triplicate at one dilutiono The amount suggested is greater than that which is absolutely essential but in practice it is convenient to have some leeway to allow for mishaps and occasional bad assay batches. RES 65.1 - 4- obtained from the tip of the needle of the vacutainer after withdrawal from the vein.' Two films should be stained'as soon as possible after spreading; two should be fixed in methanolo All films should be filed and kept for future reference.
Blood for hematological investirations should be transferred immediately to a suitable sequestrenised container.
Blood for assay should be allowed to clot at room temperature. After 30 minutes.the blood should be centrifuged. and the serum removed with the usual sterile precautions. This procedure is preferable to the usual one of allowing the blood to stand at room temperature until retraction is complete, as there is always danger that serum folate may deteriorate at high room temperatures. If this procedure is impractical,then blood should be allowed to stand at 40C until re- $ traction is complete, the clot can be ringed, the tube centrifuged and the serum removed in the usual wayo
Storage. The serum should be stored at -20°C until assayed. The folate assay should be carried'out within three weeks of collection to avoid significant deterioration. If this is not possible then serum for folate assay should be stored with added ascorbic acid
(5 mg per ml), and this should always be done if ·there is any possibility of the serum remaining at room temperature for more than an hour or two.
If ascorbic acid is to be added to serum, the serum for the vitamin B12 and folate assays must be stored separately since ascorbic acid will destroy hydroxocobalamin in serum.
e -5- RES 65.1
1.5 Hematological methods (Cartwright, 1963; Dacie & Lewis, 1964)
Hemoglobin should be measured by a cyanmethemoglobin method. Cyanmethemoglobin standarda (100% = 14.8 gm/ 100 ml) will be distributed by the PAHO Reference Laboratory on Nutritional Anemias in
Caracas, Venezuela, together with instructions for their use.
Packed cell volume. Either micro- or macro-hematocrit methods may be used to measure the PCV but when micromethods are used they should be calibrated at suitable invervals against the usual method described by Wintrobeo
White blood counts.should be carried out from the sequestrenised blood using a Coulter counter if one is available.
Reticulocyte counts are desirable wherever possible.
Examination of blood filmsa Detailed reports should be made on the morphology of the red and white cells on the lines suggested in Appendix A,
The distribution of lobes in 100 white cells should also be determinedo In carrying out the lobe count it is probably advisable to take the same precautions to avoid causing errors due to abnormal distribution of cells as are taken for the ordinary differential white count, Results should be expressed (a) as the average lobe count (Herbert, 1964) and (b) as the percentage of polymorphs having more than 5 lobes in the nucleus.
The classification and differentiation.of lobes should be that described by Arnetho This is illustrated in Wintrobe (1961)o RES 65.1 - 6 -
., The wide differences in the results obtained by different workers counting the same film makes it essential that each worker establish his own normal range by carrying'out lobe count.s on subjects known to have normal serum B12 and folate levels (Herbert, 1964). The average figures should be given in'the protocol and it should be indicated whether the result is normal or abnormal for that laboratory (see Appendices A, B and
It should be emphasized that a lobe count should not replace the detailed and careful study of the morphology of the red and white cells in the peripheral blood smear.
If possible a differential white cell count should be done at the time of the average lobe count and the.result entered under "Remarks" on the protocol.
A comment on the"relative numbers of platelets in the peripheral blood film should be made.
It is suggested that bone-marrow aspiration should be done wherever possible. In pregnancy the iliac crest is perhaps the best site for bone-marrow aspiration. Here again a number of well-fixed, well-stained permanently mounted films should be kept for record' and review purposes. The-method of preparation of bone-marrow films varies in different laboratories. Ideally, and to avoid controversy, films should be made by both the "squash" and the "spread" technique. Unstained films should be examined for hemosiderin granules and where possible at least two marrow films should be stained for iron using Perl's reaction as described in Dacie & Lewis (1964), .If'this is done, iron-positive control bone- marrows should be included with every batch of iron stains. - 7 - RES 65.1
1.6 Assays
By far the greatest variation in the results of microbiological
assays is due to "cross-batch" differences. It is therefore essential
that serum should be assayed in at least two successive batches. Re- plicate assays within the same assay batch are also advisable. Ideally
each serum should be assayed in duplicate at two dilutions in two
consecutive assay batcheso However, if one knows from one's own experience
with an assay that there is no difference in the results at different
dilutions then serum may be assayed in triplicate at one dilution.
It is always advisable to assay two standard control sera in
each batch. They should be assayed in triplicate at two or more dilutions
and, in the case of the B12 assays, the recovery of B12 added to each
control serum should also be determined. Recovery experiments are
impractical in the folate assay.
Standard sera are best collected in bulk at one time, one batch
of serum from a deficient subject and one from a normal subject. The
bulk sera can then be divided into small samples, each containing enough
serum for one assay batch. Ascorbic acid (5 mg/ml) should be added to
the standard serum for the folate assay. The same sera can then be
assayed in consecutive batches over a long period and serve as a guide to
the overall behavior of the assay. Serious inaccuracy in B12 and folate
assays is usually due to contamination of samples either with traces of
the vitamin or more commonly with bacteria (Mollin, Anderson & Waters,
1963)o It is therefore essential that sera should be stetile and. for this
reason a closed blood collection system (vacutainer syringes) is recommended.
In cases of doubt, a serum sample should be centrifuged before assay and RES 65.1 - 8 -
the serum transferred to another sterile tube for the assay procedureo
The deposit from the initial tubes shoyld be spread, stained and sub-
sequently examined for bacteria. Relatively small numbers of bacteria
.can cause significant alteration in serum B12 and folate, but the serum B12 and folate levels of samples found to be 'sterile" by this relatively
crude method can be acceptedo
1.6.1 Serum B1 assay.0 The Euglena assay described by Anderson (1964) is accurate and sensitive and is ideal for the assay of large numbers of samples. The Lactobacillus leichmannii assay.(Spray,
1955) .and Eo coli (Grossowicz et al. 1954) are, however, also satisfactory assays and may. be used if desired. A copy of a suitable Lactobacillus leichmannii method will.. be circulated to participants if required°
106o2 Serum folate assay° The method of lHerbert (1961) is the recommended one. The 'whole serum' method has certain advantages but extraction methods may be used providing it can be shown .that comparable results are obtained with reference standards0
106.3 Serum.iron estimation° A modification of the l method of.Bothwell and Mallett (1955). is recommended, but other methods may be used if desired providing they yield comparable results with reference standards.
1 6o4. Reference .samles. As everyone isaware, it is ' extremely difficult to get agreement between different laboratories carrying out the same. procedures. This is bound to be .particularly difficult if microbiological assays-are used° Nevertheless, some attempt has to be made. to standardize our techniques and the following procedures are suggestedo . . -9- RES 65o1
Serum B. and folate reference sampleo It is suggested that a designated reference center (Dro Do Lo Mollin, Rematology
Department, British Post-Graduate Medical School, London) circulate a freeze-dried standard reference sample of serum at 3-monthly intervals to the collaborating laboratories and the reconstituted sample serves as the standard control serum° A large batch sample of a standard serum is being prepared for regular distribution. Each sample will be the equivalent of
5 ml. of serum when reconstituted.
Test samples should also be sent back periodically from collaborating laboratories to the reference center for checking. In the early stages it is suggested that serum from 6 different individuals should be sent to the reference center at intervals of 3 months. If possible, serum from normal and vitamin B12 and folate deficient subjects should be included°
It would be desirable to send 7 - 8 ml of serum from each patient and it would be preferable if these samples were freeze-dried. At the same time, liquid samples of the same sera might also be sent by air in sealed
'polyethylene envelopes surrounded by dry ice as is illustrated ·in Appendix
D. A comparison of the results of these two methods of dispatch might yield information which might be useful when studies are extended to co-
operating laboratories which do not have freeze-drying facilities. If no
facilities exist for freeze-drying then liquid samples only need be sent°
If liquid samples are to be sent in dry ice, then it is advisable
that an agreement be made with airlines so that the receiving reference
center is notified as soon as the material arrives at the airporto If
dried serum is sent in ampules it is well to remember that air services
are often brutal in their handling of packageso Samples should be labelled RES 65.1 - 10 -
4,
'Fragile'.and should be carefully packed.. Standard serum for folate
assay should-·not be- kept at room temperature or ordinary refrigerator 4 temperature for-longer than is absolutely essential.
Serum iron and TIBC Standards -Freeze-dried
serum can also be used for the standard reference sample for the serum .- , iron and total iron binding capacity measurements. A standard Sample-
will be circulated by Dr. Bothwell at 3-monthly intervals.
The assay of serum B, folate and iron
reference standards. The standard B12 and folate samples circulated by or sent to the reference. laboratory should be assayed in triplicate at
two dilutions in 3 different batcheso The individual assay results can -- then be analyzed statistically.
The standard iron samples should be assayed in duplicate in at least 2 different test batches.
Tissue iron. Liver specimens for iron analysis
are being collected from certain centers, some of which are taking part in the present study. Details of the procedures for collecting and dispatching:liver specimens are found below (Section 2).. lo7 Documentation
Duplicate forms based on Appendix A, B or C (whichever is relevant) should be used and should serve as the main record sheet for subjects included in the study.
e-v - 11 - RES 65.1
2. GUIDE TO THE PROTOCOL FOR STUDY OF TISSUE IRON STORES
For some years, a pilot study has been going on in different parts of the world in an attempt to define the incidence of various forms of nutritional anemia. As part of this study, liver samples have been collected and analyzed for their gtorage iron concentrationso The objective was to find out whether such analyses could be used as a simple means of assessing the iron status of different population groups. In most of these investigations the material was derived from subjects dying acute traumatic deaths since it is known that a number of dis- ease states may influence the distribution of iron in the body. In spite of this qualification, hospital material was used in certain instances when medico-legal material was not available. The results of this initial investigation have been promising enough to suggest
that meaningful data on the incidence of iron deficiency in different populations may be obtained by estimating the iron concentrations present in aliquots of liver.
It is now hoped that the initial program in the countries of
the Americas can be expandedo Requirements are as follows:
2.1 Two small pieces of liver, each weighing 3 to 5 grams
to be collected at autopsy and fixed in buffered formal-
saline. This is made up as follows: 100 ml. 40% formal-
dehyde, 900 ml. distilled water, 4 gm. NaHP04.H20 and 6.5 gm
Na2 HOP04 (anhydrous). Each aliquot of liver should then be
placed in a separate specimen bottle or in sealed plastic RES 65o1 - 12 -
tubing with some identification label on ito One
specimen should then be sent to Dr. T.B. Bothwell,
Department of Medicine, Medical School, Hospital
Street, Johannesburg, South Africa and the other
specimen should be sent to Dr. V. Ramalingaswami,
Professor of Pathology, All India Institute of Medical
Sciences, New Delhi 16, India. It is proposed that
Dr. Bothwell carry out the chemical estimations and
Professor Ramalingaswami the histological assessments.
2.2 It is desirable that the specimens from at least 100
males and at least 50 females be obtained by each
investigator from adult subjects dying acute traumatic
deaths. However, if medico-legal material is not :
available in any particular area hospital material
will also be suitable for analysis' . ..
2.3 The following additional information on each subject
is essential: (a) Age (b) Sex (c) Race (d) Cause of
death. In addition it would be helpful to know whether
the subject is of rural or urban origin and to know the
weight of the liver. However these last two points are
not essential° Each specimen should carry a reference
for identification purposeso -
2.4 Ideally specimens should be dispatched by air freight as
this has proved the most satisfactory means so far. Mailing
charges for the specimens will be borne by WHO/PARO. A
- r - 13 - RES 65o1
consolidated bill for all the specimens dispatched should
be sent to: Dr. Miguel Layrisse, PAHO Reference Laboratory
on Nutritional Anemias, Instituto Venezolano 'de Investigaciones
Científicas, (IVIC), Apartado 1827, Caracas, Venezuela.. In
addition PARO will supply specimen bottles if these are required.
2.5 Any further details concerning this phase of the study can be
obtainedfrom Dr. T.Ho Bothwell, Department of Medicine,
Medical School, Hospital Street, Johannesburg, South Africa. -j RES 65.1 -14-
3- LITERATURE CITATIONS
4-:
Anderson, B.Bo Investigations into the Euglena method for the assay of the vitamin B in serum. Jo Clin. Path., 17:14-26, -6 January 1964. 12 - - -
Bothwell, T.oHo and Mallett, Bo The determinations of iron in SI plasma or serum. Biochemo Jo, 59 (4): 599-602, April 19550
Bothwell, ToH. and Finch, Co.E. Iron metabolism, London, Churchill, -4 1962.
Cartwright, G.E. Diagnostic Laboratory Hematology, 2 ed. Grune & Stratton, N.Y., 1956.
Dacie, J.V. and Lewis, S.oM. Practical Haematology, 3 edo New York, - Grune & Stratton, 1964.
Grossowicz, N., Aronovitch, Jo and Rachmilewitz, M. Determination of vitamin B in human serum by a mutant of Escherichia coli. 3- Proc. Soc. po. Biol.(N.oYo.) 87 (3): 513-4, December 1954. 4., Herbert, V. The assay and nature of folic acid activity in human serum. J. Clino Invest., 40: 81-91, January 1961.
Herbert, V. Studies of folate deficiency in mano Proco Roy Soco Med., 57: 377-384, May 1964.
Mollin, DoL, Anderson, B.Bo and Waters, A.H. Vitamin B12 in serum. Brit. Med. J., i, 535, February 23, 1963.
Spray, G.H. An improved method for the rapid estimation of vitamin B12 in serum. Clin. Scio, 14, 661, 1955
Wintrobe, M.Mo Clinical Hematology. 5 ed., Lea and Fabiger, Phila. 1961. . i
i. RES 65.1 APENDICE A APPENDIX A
WHO/BPAHO ESTUDIO SOBRE LAS ANEMIAS NUTRICIONALES
WHO/PAHO. STUDY ON THE NUTRITIONAL ANEMIAS
PROTOCOLO DE EMBARAZO - PREGNANCY PROTOCOL
Norribi E dad Name Age
D,1 ecc,dn Grupo étnico Address ...... Ethnic group ...... Historia No. Hospital No ......
Análisis No. Survey No. Condiciones socio-económicas: Alta i Mediana i Baja - ! Socio-economic status: High Medium Low
Número de embarazos Fecha último parto Parity Date of last pregnancy Termination
Semanas de embarazo Embarazos múltiples Week of pregnancy Multiple pregnancy
Altura Peso Orina Fondo de ojo Height ...... Weight .". Urine ...... Optic fundus
Presión arterial Esplenorrmegalia Hemoglobina Blood pressure Splenomegaly Hemoglobin
MCV Recuento de gl6bulos blancos PCV .White blood cell count Hemoglobina fetal Recuento reticulocitario/100 y hemoglobina anormal Reticulocyte count ...... Fetal or abnormal hemoglobin - 2 - RES 65.1
Examen cualitativo sangre periférica: Peripheral blood film:
Anisocitosis Macrocitosis Poiquilocitosis Anisocytosis ...... Macrocytosis . . Poikilocytosis.... Microcitosis Hipocromia Anisocromia
Microcytosis Hypochromasia ... ' Anisochromasia Policromi'a Punteado bas6filo Células blancas primitivas Polychromasia Punctate basophilia Primitive white cells Células rojas nucleadas Plaquetas Nucleated red cells Plateletse......
Promedio de recuento de lóbulos Normal r[ Anormal Average lobe count Normal Abnormal
Porcentaje de células con más de 5 lóbulos Percentage with more than 5 lobes
Médula ósea (si es posible) Hierro sérico Bone-marrow (if possible) ...... Serum iron
: e Vitamina B12 en suero Folato sérico Albúmina sérica Serum vitamin B1 2 ...... Serum folate ...... Serum albumin
Capacidad de saturación del hierro con el suero Serum iron binding capacity
Observaciones: Remarks: RES 65.1 APENDICE B APPENDIX B
WHO/PAHO ESTUDIO SOBRE LAS ANEMIAS NUTRICIONALES
WHO/PAHO STUDY ON THE NUTRITIONAL ANEMIAS
PROTOCOLO PARA MUJERES NO EMBARAZADAS
PROTOCOL FOR NON-PREGNANT WOMEN
Nombre Edad Name Age
Dirección Grupo étnico Address * Ethnic group ...... Historia No. Hospital No. '...... Anaisis No. Survey No......
Condiciones socio-económicas: Alta r- - Mediana - Baja - Socio-economic status: High Medium Low '
Casada U Solter Ca No. de embarazos Married Single Parity ....·......
No. de hijos Fecha del Ultimo embarazo No. of children Date of last pregnancy
Fecha del último destete Date of last weaning ......
Altur a Peso Orina Fondo de ojo ...... Height Weight Urine Optic fundus A. Esplenorre galiia Hemoglobina MC'V Splenomegaly eeeeeeeeeeet ··eeee e- Hemoglobin ...... PCV
Hemoglobina fetal O_ Recuento de glóbulos blancos y hemoglobina anormal White blood cell count Fetal or abnormal hemoglobin - 2 - RES 65.1
Examen cualitativo sangre periférica: Peripheral blood film:
Anisocitosis Macrocitosis Poiquilocitosis Anisocytosis - '...... Macrocytosis ...... Poikilocytosis Microcitosis Hipocrorria Anisocromia Microcytosis Hypochromasia Anisochromasia Policromi'a Punteado basófilo Células blancas primitivas Polychromasia'*"* Punctate basophilia Primitive white cells Células rojas nucleadas Plaquetas Nucleated red cells ...... e...... Platelets ......
Promedio de recuento de ldbulos Normal O Anormal C! Average lobe count Normal Abnormal
Porcentaje de células con más de 5 lóbulos Percentage with more than 5 lobes
Médula ósea (si es posible) Hierro sérico Bone-marrow (if possible) * .*.*--*** Serum iron
Vitamina B12 en suero Folato sérico Albúmina sérica Serum vitamin B12 Serum folate Serum...... album in ...
Capacidad de saturación del hierro con el suero Serum iron binding capacity
Observaciones: Remarks: RES 65.1 APENDICE C APPENDIX C
WHO/PAHO ESTUDIO SOBRE LAS ANEMIAS NUTRICIONALES
WHO/PAHO STUDY ON THE NUTRITIONAL ANEMIAS
PROTOCOLO PARA EL GRUPO CONTROL EN HOMBRES
PROTOCOL FOR CONTROL MALES
Nombre Edad Name ...... Age
Dirección Grupo étnico Address ...... ~Addrec~ss."....Ethnic ...... group ...... Historia No. Hospital No.- ---..... Análisis No. Survey No.
Condiciones socio-econórnicas: Alta 1I Medianaí--j Baja -- Socio-economic status: High Medium Low
Casado Soltero,--- Ocupac ion Salario sernanal Married ¿L Single ! Occupation' ...... Weekly salary
Altura Peso Orina Fondo de ojo Height Weight Urine Optic fundus
E splenome galia Hemoglobina Presión arterial ...... Splenomegaly Hemoglobin Blood pressure
MCV Recuento de glóbulos blancos PCV White blood cell count
Hemoglobina fetal Recuento reticulocitario/100 y hemoglobina anormal Reticulocyte count ...... Fetal or abnormal hemoglobin - 2 - RES 65.1
Examen cualitativo sangre periférica: Peripheral blood film:
Anisocitosis Mlacrocitosis Poiquilocitosis Anisocytosis . .... Macrocytosis """""Poikilocytosis *.' ' Microcitosis Hipocromia Anisocromia Microcytosis Hypochromasia ...... Anisochromasia ...... Policromia Punteado bas6filo Células blancas primitivas Polychromasia Punctate basophilia ... Primitive white cells Células rojas nucleadas Plaquetas Nucleated red cells ...... Platelets ......
Promedio de recuento de lóbulos Normal n Anormal a- Average lobe count Normal Abnormal
Porcentaje de células con más de 5 lóbulos Percentage with more than 5 lobes
Médula ósea (si es posible) Hierro sérico Bone-marrow (if possible) `--..... Serum iron
Vitamina B12 en suero Folato sérico Albúmina sérica Serum vitamin B12 ...... Serum folate ...... Serum albumin
Capacidad de saturación del hierro con el suero Serum iron binding capacity
Obs ervac ione s: Remarks: RES 65.1 APENDICE D APPENDIX D
ILLUSTRATION FOR
SUGGESTED METHOD OF PACKING SERUM IN
DRY YLCE FOR SENDING BY AIR FREIGHT
1'"/... /~/ ,ir------DRY ICE POLYETHYLENE , i CONTAINER
-- SERUM CONTAIN:
· i fs"l L Z ! i EXPANDED POLY`STYRENE CONTAINE. R
_ __ _ _ ~~~~ ~ ~~~.4.