Question

Background:

As we discussed in class, there are several complement regulators on the surface of our cells and in our body fluids. These regulators are essential in preventing the activation of the complement cascade and eventual lysis of our self cells. In some cases, however, cells may become deficient in these regulatory proteins as a result of a random gene mutation in one of the pluripotent stem cells in the bone marrow. One such deficiency is called paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem-cell disorder caused by a somatic mutation in a gene known as phosphatidylinositol glycan class A (PIGA). The PIGA gene encodes a protein that constructs a structure known as a glycosylphosphatigyinositol (GPI) anchor. This anchor is created by a molecule of phosphatidylinositol (a lipid that associated with the cell membrane) and an additional structure to which a protein is attached. GPI-anchored proteins do not have a traditional transmembrane domain like most other membrane bound proteins. Proteins that attach to GPI anchors have signal sequences at their carboxy termini that allow the protein to attach to the anchor.

In PNH, the cells deficient in GPI anchored proteins generally all come from one single progenitor stem cell that has mutated. However, for some reason, these cells seem to have a survival advantage over cells derived from normal HSC. As a result, the majority of the cells in the body are derived from the single clone of defective progenitor cells.

In the case of PNH, two of the complement regulators fail to express on the surface of the red blood cells. As a result, complement attack is unregulated and leads to the release of free hemoglobin. This free hemoglobin is released into the urine, causing a dark red coloring.

The Case:

You are asked to see a 37-year-old male carpenter who was noted to be anemic and mildly thrombocytopenic during a routine physical examination. The patient has been married for 11 years and has a 5-year-old son and an 8-year-old daughter. He reports that he has been in good health but has noted increasing fatigue over the past 2 months. Two weeks ago, he and his son had viral gastroenteritis that manifested with nausea and diarrhea. His son recovered within 24 hours, but the patient had burgundy-colored urine that persisted for 2 additional days.

You decide to run a CBC and find the following:

White Blood Cells (WBC): 6,000 cells/μl (Normal 5,000-10,000)

Red Blood Cells (RBC): 1x10⁶ cells/μl (Normal 4.2-6.0x10⁶)

Platelet count: 72,000 platelets/μl (Normal 150,000-450,000)

Neut %: 65% (Normal 39.5-74%)

Abs Neut #: 4,000 cells/μl (Normal 1,450-7,500)

Lympho %: 30% (Normal 15.9-47.3)

Abs Lympho #: 3,600 cells/μl (Normal 1,000-4,000)

Monocyte %: 5% (Normal 0-12.5%)

Abs Mono #: 400 cells/μl (Normal 0-860)

You send a sample over to the biochemical lab and get a return email saying the sample was hemolyzed (all the red cells were lysed). Additionally, you send the blood off for a reticulocyte count. The results were:

Reticulocyte %: 1.1% (normal 0.4-2.0%)

Total Retic Count: 68,000 cells/uL (normal 18,000-100,000 cells/uL)

At this point, you suspect that your patient may have PNH. To get a final diagnosis, you send the patient’s blood for flow cytometry testing and order a FLAER test. The lab director is out of town and the tech running the assay does not know how to interpret the results so instead he sends you the printout from the machine and tells you that you can analyze it if you want the results so bad. After you call HR and have the tech fired, you look at the results (remember, in a flow histogram, the further right a peak is, the more fluorescent those cells are. Cells to the left side of the graph do not have any fluorescence indicating whatever you tested with did not bind to your cells):

Test from the FLAER assay indicate to you that your patient does indeed have PNH. You prescribe that the patient receives shots of i.v. eculizumab every other week to treat the condition.

Question: What is a reticulocyte? Why did you test for the % reticulocyte? What conclusion could you draw about the lack of RBCs from the reticulocyte count?

Answer 1

What is a reticulocyte?

Reticulocytes is a condition where you can get to see an immature red blood cell without a nucleus. When you stain these RBC's they will be granular or reticulated in their appearance.

Why did you test for the % reticulocyte?

Whenever patients with PNH will come to you, check them for elevated reticulocyte count and LDH. As patient will have decreased iron stores and he will lose all the iron in the urine, the bone marrow will be erythroid. The bone marrow will synthesize lots of new red blood cells.

What conclusion could you draw about the lack of RBCs from the reticulocyte count?

In PNH, you can see the RBCs breaking apart prematurely. The hematopoietic stem cells produced in bone marrow will develop into RBC and as these are defective, they will be destroyed by our own immune system (complement system). This leads to the reduction in the number of RBCs, this shows that the person is suffering with PNH condition.