Tag Archives: blood analysis

Blood Analysis: Part 2

Picking up from where we left off…

Once the blood arrived at the NIPHL from the field via the Blood Taxi, one of two things happened.

1. For the blood collected in the 4.5 mL tubes containing the EDTA:  a complete blood count (CBC) was conducted. This provides information on the total number of red blood cells, hemoglobin, white blood cells, and platelets present in a person’s bloodstream. The red portion undergoes gel electrophoresis  for hemoglobinopathy testing. This separates proteins in the blood so that the amount and type of hemoglobin can be determined. Low levels of hemoglobin indicate anemia.

2. For the blood collected in the 7 mL trace element free tube: the samples were prepped and sent to UBC. Researchers at UBC will be performing a serum analysis to trace ferritin, vitamin B12, folate, serum retinol (vitamin A) and carotenoids (another form of Vitamin A), and zinc. The data collected should provide us with a clear picture of how much iron, vitamin A, vitamin B12, folate, and zinc was present in a woman’s diet before and after we introduced HFP and aquaculture. These biochemical indicators will allow us to quantify some of the results of our intervention.

Here are some pictures I took in the lab:

Blood Analysis: Part 1

 As promised, I wrote about the “5Ws” of our blood collection: who, what, when, where, and why. I even threw in the “how”. Today’s post focuses on what happened in the field.

One of the most important (and expensive!) components of FoF is the blood collection and subsequent analysis. This process provides us with concrete evidence that there are more (or less) nutrients in a person’s body by looking at biochemical indicators of nutritional status. Obtaining this information is what sets FoF apart from previous homestead food production interventions.

During baseline blood was collected from 450 women. We will collect samples from the same 450 women during endline for a paired analysis. While we considered collecting blood from children as well, it was decided this would be too traumatic. Plus, we had other less-invasive methods available to assess whether or not children were anemic (finger-pricking to measure hemoglobin).

When we arrived in each village, a random lottery was held. Each house was assigned a number (1-10) and slips of paper with those numbers were randomly selected. In the end, we selected 5 women from each village. Women were given an ID number and asked to report to their local Health Center the day after our visit to their village.

The morning of collection, the local Village Health Volunteer (VHV) came with the women and their children to the Health Center. The VHV was responsible for making sure everyone who agreed to provide samples was present. Most mornings everyone was at the Health Center by 7:30 am, as they had to fast for a minimum of 3 hours before providing samples. When all the women assembled, they were briefed by Mr. Tee from HKI.

One at a time, the women entered a room where a technician was working. The technician drew 3 vials of blood. After, the women were given a sarong as a thank you for their participation.

Two of the vials collected were 4.5 mL test tubes containing Ethylenediaminetetraacetic acid(EDTA), which is an anticoagulant, that prevents blood from clotting. They were then packed on ice and transported via a car we dubbed the “Blood Taxi” to the National Institute of Public Health Laboratories (NIPHL) in Phnom Penh for further processing.

The third tube collected was a 7 mL trace element free tube. It was centrifuged in the field. This separated the blood into 3 components: the serum plasma, the buffy coat (most of the white blood cells and platelets), and the erythrocytes (red blood cells). The serum was packed on ice and sent to the NIPHL, where samples were stored at -70 degrees Celcius until the end of baseline.

Some pictures:

Check back tomorrow to find out what happened once the blood arrived at the NIPHL.

Updates

Unfortunately the blog has been neglected lately due to other projects, such as working on our 6-month Interim Technical Report for the IDRC. It’s a huge undertaking, involving several people in Vancouver and Phnom Penh. Thankfully it’s coming together and should be a wealth of well-organized information on what FoF has accomplished in the past 6 months.

Yesterday we took a time-out from working on the report to go visit the National Institute of Public Health Laboratories (NIPHL) in Toul Kork, which is at the northern end of Phnom Penh. It was interesting to see where the blood collected in the field has gone to be processed, and what kind of machinery is being used. I took pictures that will appear on the blog later.

We also relaxed in the evening by watching a topically relevant film, “Salmon Fishing in the Yemen“. The plot focused on a wealthy sheik in Yemen who wants to introduce the sport of fishing to his country, despite the geography of the region. While there were some obvious differences between a film set in the Middle East and what we’re accomplishing in Cambodia, the sheik makes an astute observation when he says that his project will address the growing need for sustainable food sources and that a river will provide not just fish, but water to the region that can be used to grow fruit as well. Our fishponds will be working in tandem with homestead food production to create a symbiotic relationship between aquaculture and farming to ensure year-round sustainable production of nutritious food. Pretty smart, don’t you think?

Baseline: Day 7

I‘ll confess, I made a rookie mistake – I forgot to charge my camera battery. The only picture I managed to take before my battery ran out was of a recently slaughtered chicken next to a bowl of its own blood.

Ingredients for today's lunch.

I didn’t get to ask the cook what they intended to do with the blood, but since they went to great lengths to collect it in a bowl post-slaughter, I assume it was incorporated into the meal in some way. While this may sound unappetizing to a lot of readers (especially those in North America), it’s pretty common in other parts of the world — even in Western countries. The Brits are known for their blood sausage and blood pudding. For centuries the Maasai warriors of Kenya have consumed the blood of cows. In Cambodia, where so many are anemic, adding animal blood to the diet makes sense as it’s an excellent source of iron.

I’m spending the 8th day of baseline at a health center to watch the NIPH (National Institute of Public Health) technicians collect more blood samples, but I’m saving the details for a post on the blood collection process. So instead of a recap, check back tomorrow for another special feature!

Sampling Methodology

One of the issues that we have encountered during the first week of baseline is that some of the houses that were originally selected to be part of the study are no longer eligible.

In some instances, the mother went to Phnom Penh or the Thai border to seek work. The purpose of our study is to show the effects of HFP and aquaculture on household food security and the nutritional status of women and children. As such, the woman of the house must be present year-round to reap the benefits of these interventions. By migrating for work, a woman’s nutritional status at the end of the study would not reflect the addition of HFP and aquaculture in her life. Therefore, her household is now ineligible for participation in FoF.

In other instances, when we arrived at the house we found out that the children were over the cutoff age of 5 years old. This happened for a variety of reasons: some women couldn’t accurately remember the date of birth (the Khmer calendar is different from ours), the selection team didn’t check the proper documents to verify the child’s age, or the age on the village chief’s list was incorrect. It’s also possible that some women provided false ages for their children because they wanted to be part of a study that provides the expensive inputs needed for HFP and aquaculture.

In any event, our team has been scrambling to fill their spots so that we have the right sample size for the study. We’ve accounted for a possible 15% of houses lost due to follow up, but we want to start with the biggest sample possible to minimize that loss given how expensive and time-consuming the project is. 

This seems like a good opportunity to discuss the methodology behind household selection. In an earlier post (Household Selection), I briefly outlined the criteria that households needed to meet to be part of FoF. However, meeting the selection criteria does not mean a household is automatically enrolled in FoF. This is, after all, a scientific experiment; certain research principles must be upheld.

In order for this to be a valid and reliable scientific experiment, we need our sample to represent our target population as closely as possible while eliminating any potential biases or confounders. We achieve this by picking the proper sampling method. FoF is using a multi-stage sampling strategy. The first stage is cluster sampling, which is a form of probability sampling that examines naturally occurring groups such as villages. The second stage is systematic sampling, which is used to select the houses in the villages by picking a random point to start (eg the fourth house on the list) and continuing through the list in a systematic fashion (eg every fourth house on the list). A few key definitions are needed at this point:

  • Valid – we are measuring what we say we are going to measure
  • Reliable – our measurements are as accurate as possible
  • Probability sampling – the entire target population is known, and thus everyone in that population has an equal chance of being selected
  • Randomization – picking units (in our case, villages) at random to ensure the sample is representative of the target population
  • Target Population – the population we want to study, as defined by certain parameters (eg location, age, SES)

First, we looked at all of the villages in the province of Prey Veng on a list from the most recent census conducted by the Ministry of Planning in 2008. We excluded the villages that had already been part of a HFP program by HKI that was funded by the EU, the villages that are part of the ongoing ODOV (Organization for Development of Our Villages – one of our partner NGOs) food security project, and the villages that are taking part in other Cambodian NGO projects. This left us with 164 villages in 4 districts: Ba Phnum, Kamchay Mear, Me Sung, and Svay Antor. Then the villages were randomized, resulting in 120 villages with 40 villages per group (HFP, HFP + aquaculture, or comparison) being selected.  Finally, 30 out of 40 villages were selected after further randomization.

Workers from the ODOV went into the field and met with the village chiefs (and in some instances, a village council) to divide the households with children under the age of five into 3 categories: poorest, poor, and medium wealth. They wrote their wealth ranking assessment for each household on a slip of paper that was placed into a box to maintain anonymity. This was done because it was our intention to try to help those most in need.

The ODOV and the village chiefs met with the households categorized as poorest or poor to explain the project to them and to ask if they were interested in joining. If they responded “yes”, field staff went to the house to make sure it met the selection criteria. They made sure that each house had enough land to support HFP farms and fish ponds, and they assessed whether or not the house would be able to maintain these projects during the course of our study. They also inquired about the ages of the children, most often by looking at the village chief’s list of villagers, but as we’ve discovered this list isn’t always correct.

A list of all eligible households was sent back to HKI. The houses categorized as poor or poorest were listed, and from that list we began with the 4th house and picked every 4th house after that. We were able to find 10 eligible houses in each village by using this method. The ODOV received a list of selected households and went to the village chief to inform him of the date and time of the survey.

In the field, each supervisor brings his or her list of 10 households per village that have been selected for the study. Sometimes, something goes amiss and the household is no longer eligible (for all of the reasons I listed above). Then we have 2 options: 1, we pick another house categorized as poor on the ODOV list that wasn’t originally selected during the systematic sampling; or 2, we go back to the very first list that the ODOV produced (the one that listed all the households in the village before the wealth ranking) and we discuss with the chief whether or not picking a new house from that list is a good idea. This means that sometimes we will get houses that vary in socioeconomic status (SES). Ideally, we’d like to control for SES before we collect data, but our survey includes a module about household income and wealth that will allow us to control for SES after the data has been collected.

Once we have our 10 houses picked in the village, we hold a lottery to randomly select 5 houses to participate in the 24-hour recall and blood analysis components of our study. Slips of paper with the numbers 1 through 10 are placed faced down, and 5 slips are drawn. Those houses are highlighted on the list. If, for some reason, we have to replace a house that has been highlighted, the replacement house is automatically assigned to be part of the 24-hour recall and blood draw. The enumerator goes to the house to conduct the 24-hour recall and to obtain consent (very important) for the blood draw. The woman is given a slip of paper that has her unique identifier and the time and location of the blood draw. We are only conducting recalls and collecting samples from 450 women (half of the women in the study) because the recalls are time-consuming and the blood collection is invasive and expensive.

Households selected by lottery for the 24-hour recall and blood draw

And that is the method we used to recruit 900 households for FoF while adhering to the principles of sound research as best as we possibly can.

I’d like to give a special thank you to Sokhoing Ly from HKI for explaining all of this to me with great patience.

Baseline: Day 3

Day 3 of baseline started out differently than the past two days. Instead of heading directly to a village to survey, we went to the Svay Antor Health Center to watch the first day of our blood collection. This part of the project is significant because it will provide us with quantifiable data (in the form of biochemical analyses) that will show whether or not our interventions have had the desired impact. I will write a special post about the more technical aspects of this process in a few days.

Several mothers and their children were waiting patiently at 7:30 am for us to set up. The village health volunteers were also there to make sure that everyone was accounted for, and to go back to their village to pick up women who hadn’t made it to the health center yet. There were a lot of familiar faces, as the women who were having their blood drawn today were women we have interviewed over the past two days.

A special team from the National Institute of Public Health (NIPH) came to collect and process the blood samples. They were extremely efficient, so the morning went by quickly. The only troubleshooting that had to be done was tracking down the right kind of tubes for one of the samples. Thankfully the problem was solved quickly.

This afternoon we went to a new village to watch more surveys, 24 hour recalls, anthropometric measurements and hemoglobin analyses. My favourite place to sit is with the anthropometrists and blood analysts. They are usually set up at the village chief’s house (or the village health volunteer’s house), and every mother and child that is part of our study has to come over to have their measurements taken and their fingers pricked. Usually news of our arrival spreads throughout the whole village, and a lot of children come just to see the foreigners. This afternoon went according to plan, so I don’t have many stories. I do, however, have a lot of pictures that I will be sharing soon.

Test Survey

Preparations are underway as the countdown to the baseline survey continues. Yesterday morning we checked off one of the most important “to-do”s on our pre-baseline checklist: the test survey. After a week of training, our enumerators had the opportunity to put their new skills to use. This also gave us a chance to work out any “bugs” in the survey.

The day began bright and early, as two vans carrying 20+ people departed from HKI’s office in Phnom Penh to head to the province of Kampong Chhnang. Although FoF is taking place in the province of Prey Veng, Kampong Chhnang is closer to Phnom Penh, and was thus better suited for a day trip.

Our first stop was at the Longvek Health Center, where we picked up iron and folic acid tablets to distribute to the households we were going to visit.

Outside the Longvek Health Center, our first stop in Kampong Chhnang.

Our next stop was at the home of the village chief. We informed him of our plans to meet with different households and ask them questions about nutrition and health. He directed us to the house of the village health volunteer, who would be able to assist us in identifying suitable households to visit.

The final stop for our van was at the house of the village health volunteer. The second van went to the next village to find more households to survey. Outside of the volunteer’s house, the enumerators set up stations to conduct anthropometric measurements and blood analyses. Then the volunteer took us to different houses to meet with women and their families. As an incentive, every woman who agreed to speak with us received a bag of detergent.

Bags of the detergent we distributed as an incentive to participate in the test survey.

The first house we visited was the home of a husband and wife whose three daughters were present. Two of the daughters lived in adjacent houses, while one still lived at home. We interviewed the two daughters who had young children.

The enumerator going through his list of questions with a mother and her child.

As the enumerator worked his way through the survey, I had time to observe the family’s home. Houses in Kampong Chhnang are very different from houses in Vancouver! The houses are all raised so that the “house” part is actually the second story. Underneath the house is the area where most family activity occurs. There are cots, tables, and chairs underneath, taking advantage of the shade the raised portion of the house provides. The cooking is done in a separate area nearby on the property. The kitchen I observed consisted of a cupboard and a heat source. Most families eat from the same pot. This particular family also raised chickens who roamed the yard freely.

The kitchen, separate from the rest of the house.

The kitchen at the daughter's house, which was behind the parents' house.

Chickens that the family raises on its property.

The question portion of the test survey went fairly smoothly. One of the challenges was that the infant wanted breast milk before his nap. However, women aren’t comfortable breastfeeding in front of strange men. Unfortunately, it was hard to find enough female enumerators who are able to leave their homes for the 2+ weeks of baseline, so the majority of our enumerators are men.

The final portion of the test survey involved bringing the women and their children to the village health volunteer’s house to get measurements and blood. First the women were weighed, their height was measured, and their mid-upper arm circumference (MUAC) was recorded.

Recording a mother's weight

...her height

...and her MUAC.

Then their children went through the same process. Most of the small children cried when they were placed on the length board! It must have looked like a scary contraption to them.

Recording a girl's weight

...her length (she was not happy)

...and her MUAC.

Finally, the women and children had their fingers pricked. A drop of blood was placed on a slide that was fed to a machine to analyze the hemoglobin content of the sample. Based on the results, many women were given iron and folic acid tablets.

Pricking the mother's finger to draw blood.

Analyzing the blood sample.

The day concluded back at the HKI office in Phnom Penh. Everyone gathered to discuss their experiences and raise any questions or concerns. A few questions were further clarified on the survey, but overall the results of the test survey were encouraging. Our enumerators are now confident and prepared to head to Prey Veng for baseline next Wednesday!